<%BANNER%>

Insulin-Like Growth Factor Binding Protein-3 Regulates Hematopoietic Stem Cell and Endothelial Progenitor Cell Function ...

Permanent Link: http://ufdc.ufl.edu/UFE0022015/00001

Material Information

Title: Insulin-Like Growth Factor Binding Protein-3 Regulates Hematopoietic Stem Cell and Endothelial Progenitor Cell Function during Vascular Development
Physical Description: 1 online resource (120 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: development, epc, hsc, igfbp, retina, vessel
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Retinal blood vessels develop by a combination of vasculogenesis and angiogenesis, called neovascularization. Endothelial progenitor cells (EPCs) which originated from lateral and posterior mesoderm contribute to neovascularizaiton in response to certain cues such as cytokines and hypoxic gradient. Aberrant vessel growth in retina results in vascular retinopathies, the leading cause of visual disability and blindness worldwide. The pathological outgrowth of new blood vessels involves the recruitment and proliferation of circulating EPCs. This study was initiated to better understand a complex network regulating neovascularization involving EPCs function. Many factors have been identified to promote trafficking, mobilization, and homing of EPCs. We demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) has a critical function in postnatal vasculogenesis both in vitro and in vivo. IGFBP-3 showed enhanced migration, tube formation and proliferation of EPCs in vitro. In vivo, IGFBP-3 inhibited pathological neovascularization by protecting developmental retinal vessels from oxygen induced regression. In EPCs, nitric oxide (NO) regulates migration through redistribution and phosphorylation of the motor protein vasodilator-stimulated phosphoprotein (VASP). IGFBP-3 has been shown to trigger EPC mobilization by generating NO and subsequently activating VASP. The signaling mechanisms of IGFBP-3 on EPCs were also identified in this study. We have defined that down stream pathway of IGFBP-3 involve sphingosine kinase (SK)/sphingosine-1 phosphate (S1P) signal transduction. NO release from EPCs was reduced after treatment with a SK inhibitor, dimethylsphingosine (DMS) and could not be recovered by exposure to IGFBP-3. This result suggests that IGFBP-3 may act as an upstream mediator of SK/S1P signaling. We addressed the hypothesis that IGFBP-3 stimulates NO production and VASP phosphorylation through SK activation. Thus, IGFBP-3-induced angiogenesis of EPCs may be involved in a SK dependent signaling pathway. Whereas an inhibitory angiogenic role for IGFBP-3 has been widely reported, several other researchers have shown contradictory results in that IGFBP-3 also enhances angiogenic effects. Our results suggest that IGFBP-3 has an angiogenic/vasculogenic function in modulating the migratory ability of EPCs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Grant, Maria A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022015:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022015/00001

Material Information

Title: Insulin-Like Growth Factor Binding Protein-3 Regulates Hematopoietic Stem Cell and Endothelial Progenitor Cell Function during Vascular Development
Physical Description: 1 online resource (120 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: development, epc, hsc, igfbp, retina, vessel
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Retinal blood vessels develop by a combination of vasculogenesis and angiogenesis, called neovascularization. Endothelial progenitor cells (EPCs) which originated from lateral and posterior mesoderm contribute to neovascularizaiton in response to certain cues such as cytokines and hypoxic gradient. Aberrant vessel growth in retina results in vascular retinopathies, the leading cause of visual disability and blindness worldwide. The pathological outgrowth of new blood vessels involves the recruitment and proliferation of circulating EPCs. This study was initiated to better understand a complex network regulating neovascularization involving EPCs function. Many factors have been identified to promote trafficking, mobilization, and homing of EPCs. We demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) has a critical function in postnatal vasculogenesis both in vitro and in vivo. IGFBP-3 showed enhanced migration, tube formation and proliferation of EPCs in vitro. In vivo, IGFBP-3 inhibited pathological neovascularization by protecting developmental retinal vessels from oxygen induced regression. In EPCs, nitric oxide (NO) regulates migration through redistribution and phosphorylation of the motor protein vasodilator-stimulated phosphoprotein (VASP). IGFBP-3 has been shown to trigger EPC mobilization by generating NO and subsequently activating VASP. The signaling mechanisms of IGFBP-3 on EPCs were also identified in this study. We have defined that down stream pathway of IGFBP-3 involve sphingosine kinase (SK)/sphingosine-1 phosphate (S1P) signal transduction. NO release from EPCs was reduced after treatment with a SK inhibitor, dimethylsphingosine (DMS) and could not be recovered by exposure to IGFBP-3. This result suggests that IGFBP-3 may act as an upstream mediator of SK/S1P signaling. We addressed the hypothesis that IGFBP-3 stimulates NO production and VASP phosphorylation through SK activation. Thus, IGFBP-3-induced angiogenesis of EPCs may be involved in a SK dependent signaling pathway. Whereas an inhibitory angiogenic role for IGFBP-3 has been widely reported, several other researchers have shown contradictory results in that IGFBP-3 also enhances angiogenic effects. Our results suggest that IGFBP-3 has an angiogenic/vasculogenic function in modulating the migratory ability of EPCs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Grant, Maria A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022015:00001


This item has the following downloads:


Full Text





INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 REGULATES
HEMATOPOIETIC STEM CELL AND ENDOTHELIAL PROGENITOR CELL FUNCTION
DURING VASCULAR DEVELOPMENT





















By

KYUNG HEE CHANG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008

































O 2008 Kyung Hee Chang


































To my beloved family









ACKNOWLEDGMENTS

It is my pleasure to have a chance to express my gratitude to all those who helped me

complete this dissertation.

I would like to specially thank to my mentor, Dr. Maria Grant, who has constantly helped

me with patience. She has been an amazingly great teacher, researcher, and advisor. She taught

me how to solve the many problems I confronted. Her enthusiastic and inspiring guidance kept

me work even harder and helped me overcome many difficulties. I have been very fortunate to

have her as my mentor throughout this academic j ourney.

I would like to also thank my committee, Dr. Bryon Petersen, Dr. Daniel Purich, Dr,

Jeffrey Harrison, and Dr. Mark Segal. Without a doubt, their insightful comments and

constructive criticism strengthened me as a scientist.

Thanks to the members of the Grant Lab: Jennifer Kielczewski, Nilanj ana Sengupta,

Sergio Li Calzi, and Sergio Caballero, Jr. Interaction with my lab members was one of the ways I

relieved daily stresses. Particularly, I am grateful to Dr. Lynn Shaw for his valuable time and

efforts to teach me all the details of how to interpret experimental output. Special thanks also go

to Dr. Aqeela Afzal. She trained me from the first day I joined the Grant Lab. She taught me

everything I needed to fulfill this entire doctoral work. She has been always considerate and was

my friend, sister, and a great trainer.

In addition, I would like to thank to the men and women who donated their blood for this

research. Their thoughtful gift enabled this research to step forward.

I also warmly appreciate the generosity and understanding of my friends and my lovely

pet, Camus. I thank Don for his great support and valuable advice for my writing.

Most importantly, I would like to express my special thanks to my parents whose

unconditional love enabled me to complete this work. None of this would have been possible









without their belief in me. I also thank my dear brother, TaeHong, for all his consideration and

support, and my family in Korea who have been a great source of inspiration.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF FIGURES .............. ...............8.....


LI ST OF AB BREVIAT IONS .............. ....... ............... 10..


AB S TRAC T ............._. .......... ..............._ 14...


CHAPTER


1 BACKGROUND AND SIGNIFICANCE ................. ............_........16.........


The Eye ........_................. ..........._..........1
A natom y .............. ...............16....
The Retina .............. ...............20....
Retinal Blood Supply .............. ...............22....
Retinopathies .............. ...... ..... ..............2
Retinopathy of Prematurity .............. ...............24....
Diabetic Retinopathy .................. ...............27........ ......
Age-related Macular Degeneration .............. ...............29....
Neovascularization ................... ...............30..
Endothelial Progenitor Cells............... ...............30.
Hypoxia Inducible Factor-1 ....._.. ................ ........__. ........3
Vascular Endothelial Growth Factor ......... ......._.._.._ ......... ............3
Stromal Derived Factor-1 ........._.._... ......... .... ...............37......
Insulin-like Growth Factor Binding Protein-3 .............. ...............39....
Sphingosine 1-phosphate ........._..... ...._... ...............43....
Nitric Oxide ................. ...............45.................
Carbon M monoxide ................. .. ......... ...............47.......
Vasodilator Stimulated Phosphoprotein ...._.._.._ ........__. ...._.._ ............4
Significance (Specific Aims) ............_ ..... ..__ ...............51...

2 METHODS AND MATERIALS .............. ...............52....


Cell Preparation .............. ...............52....
Migration Assay............... ...............53.
EPC Tube Formation .............. ...............54....
Cell Proliferation Assay ............ ..... .._ ...............54...
Nitric Oxide Measurement .............. ...............55....
W western Blot Analysis .............. ...............55....
In-cell W western Assay ............... .... ...... ... .. ......_ .. .. .. .......5
Quantitative Transcription Analysis with Real Time Polymerase Chain Reaction (RT
PCR) ........._..... ...... ...............57....
Immunohistochemistry .............. ...............57....











Flow Cytometry Analysis .............. ........_ ...............58...
Hematopoietic Stem Cell (HSC) Transfection .............. ...............59....
Experimental Animals ..................... .... ............. .......6
Oxygen Induced Retinopathy (OIR) Mouse Model .............. ...............60....
Retinal Flat M ounts ............... ..................... ................6
GS Isolectin and GFP Double Labeled Immunohistochemi stry ................. ............. .......62
Microscopy and Mapping ........._..._........ .... ...._. ...............62....
Vascular Density Analysis and Statistical Analysis .............. ...............63....

3 RE SULT S .............. ...............64....


IGFBP-3 Induces Migration of CD34 cells and Endothelial Cells ................ ................ ..64
IGFBP-3 Increases Expression of VEGF Receptors on CD3 4 cells ........._..... ........._.._....64
IGFBP-3 Promotes CD34 cells Differentiation to Endothelial Cells............... ..................6
IGFBP-3 Enhances CD34 cells Proliferation............... .............6
Expression of Hypoxia-regulated Factors in Retina ........._.._........ ......._. ..... ...._.._...........6
IGFBP-3 Protects Neonatal Retinal Vessels from Oxygen Induced Vaso-obliteration .........66
Quantitative Analysis of Vascular Density in Vaso-obliteration Phase .............. ..............67
IGFBP-3 Decreases the Incidence of Pre-retinal Neovascularization .........._..._.. ................67
IGFBP-3 Expression in Transfected HSC .................. ............... ...............6
Co-localized IGFBP-3 Expressing gfp HSC within the Vasculature Inhibit
Neova scul ari zati on ............... ..... ..... ..._.. ... .. ...............68..
Hypoxia-regulated Factors and Nitric Oxide Signaling .............. ...............68....
NO and CO Promotes CD34 cells Migration............... ...............6
Different Phosphorylation Sites of VASP .....__ .................. ............ ... ............ .....69
NO Increases VASP Phosphorylation in Diabetic CD34 cells ................. ...................6
NO and CO Cause VASP Redistribution to the Leading Edge of the Cells. .......................70
IGFBP-3 Increases eNOS Phosphorylation............... ............7
IGFBP-3 Induces NO Production............... ...............7
IGFBP-3 Modulates VASP Phosphorylation ................ .. ...............71..
Inhibition of SK Activity Results in Reduced NO Production............... ...............7

4 DI SCUS SSION ............ ...... ..__ ............... 1...


Factors Influencing the EPC Studies .............. ...............91....
IGFBP-3 as a Hypoxia-regulated Factor .............. ...............93....
S1P: Possible Role in EPC Mobilization............... ..............9
VASP: New Perspectives and Open Questions .....__.....___ ..........._ ...........9
Conclusions............... ..............9

LIST OF REFERENCES ............ ..... ._ ...............101...

BIOGRAPHICAL SKETCH ............ ..... .__ ...............120...











LIST OF FIGURES


Figure page

1-1 Anatomy of human eye. ........... ..... ._ ...............18..

1-2 The ten layers of the retina. ............ ...............21.....

1-3 Hypoxia-regulated factors and BM-derived cells. .............. ...............33....

1-4 The VEGF (VEGF-R2) signaling pathway............... ...............36

1-5 The interaction between SDF-1 and CXCR4 ................. ...............38......_.__..

1-6 IGF-1R signaling pathway ................. ...............40...............

1-7 Schematic diagram of IGF system ........... ...... .__ ...............41.

3-1 IGFBP-3 induces CD3 4 cells and endotheli al cells migrati on ................. ............... ....72

3-2 Receptor levels in CD34 cells following IGFBP-3 exposure............... ................7

3-3 IGFBP-3 enhances CD34 cells and EPC differentiation. ............ .....................7

3-4 IGFBP-3 enhances CD34 cells proliferation. ......._................ ............... 75 ....

3-5 Hypoxia retina expresses IGFBP-3 ................. ...............76........... ...

3-6 IGFBP-3 protects from hyperoxia-induced vascular regression ................. ................. .77

3-8 Reduced preretinal neovascularization by expression of IGFBP-3. ........._.... ........._.....79

3-9 IGFBP-3 expression in plasmid transfected HSC. ....._._.__ .... .._.... ........_._........80

3-10 Localizati on of gfp+HS C express sing IGFBP-3 within the retinal vascul ature ..................8 1

3-11 eNOS phosphorylation by hypoxia-regulated factor. ............. ...............82.....

3-12 NO and CO stimulate CD34 cells migration............... ...............8

3-13 VASP, phosphorylated VASP 157, and 239 expression levels .............. .....................8

3-14 Diabetic CD34 cells show increased VASP phosphorylation following exposure of
N O donor .............. ...............85....

3-15 NO and CO mediates VASP redistribution within endothelial cells ................ ...............86

3-16 Phosphorylation of eNOS following exposure of IGFBP-3 ................. .......___ .........87










3-17 Increased intracellular NO production in CD34 cells .............. ...............88....

3-18 IGFBP-3 modulates site specific phosphorylation of VASP in CD34 cells ........._.._........89

3-19 Inhibition of SK activity results in reduced NO production. .............. ....................9









LIST OF ABBREVIATIONS

Acid-labile subunit

Age-related macular degeneration

Adenosine triphosphate

Blood-brain barrier

Tetrahydrobiopterin

bone marrow

Blood-retinal barrier

Bovine serum albumin

Adenosine 3' 5'-cyclic monophosphate

Cell division cycle 6

Guanosine 3', 5'-cyclic monophosphate

Choroidal neovascularization

Carbon monoxide-releasing molecules

Cell preparation tube

4-amino-5 -methylamino-2', 7' -difluorofluorescein diacetate

4', 6-di amino-2-phenylindol e

Diabetes mellitus

Dimethyl sphingosine

Deoxyribonucleic acid

Diabetic retinopathy

Endothelial cell basal medium-2

Extracellular matrix

Endothelial differentiation gene

Ethylenediamine tetraacetic acid


ALS

AMD

ATP

BBB

BH4

BM

BRB

BSA

cAMP

Cdc6

cGMP

CNV

CORMs

CPT

DAF-FM diacetate

DAPI

DM

DMS

DNA

DR

EBM-2

ECM

EDG

EDTA










Drosophila melan2oga~ster protein enabled

Endothelial nitric oxide synthase

Extraocular muscles

Endothelial progenitor cell

Erythropoietin

Endothelin enhancer

Ena/VASP homology

Fluorescence-activated cell sorting

Fetal bovine serum

Fluorescein isothiocyanate

Vascular endothelial growth factor receptor-2

Vascular endothelial growth factor receptor-1

Vascular endothelial growth factor receptor-3

Ganglion cell layer

Green fluorescent protein

Granulocyte/macrophage colony-stimulating factors

G protein-coupled receptor

Grifobnia Simplicifolia isolectic

Hypoxia inducible factor-1

Human immunodeficiency virus

Human lung derived microvascular endothelial cells

Heme oxygenase

Hematopoietic progenitor growth medium

Human retinal endothelial cells

Hypoxia response element


Ena

eNOS

EOMs

EPC

EPO

ET

EVH

FACS

FBS

FITC

Flk-1

Flt-1

Flt-4

GCL

GFP

GM-C SF

GPCR

GS isolectin

HIF-1

HIV

HMVEC-L

HO

HPGM

HREC

HRE









HSC Hematopoietic stem cell

IACUC Institutional animal care and use committee

IGF Insulin-like growth factor

IGF-1R Insulin-like growth factor-1 receptor

IGFBP Insulin-like growth factor binding protein

IL Interleukin

INL Inner nuclear layer

iNOS inducible/inflammatory nitric oxide synthase

IPL Inner plexiform layer

KDR Vascular endothelial growth factor receptor-2

IV1VP-9 Metall oprotei nas e- 9

mRNA messenger ribonucleic acid

NaCl Sodium chloride

NIH National institutes of health

nNOS neuronal nitric oxide synthase

NO Nitric oxide

NOS Nitric oxide synthase

NPDR Nonproliferative diabetic retinopathy

OIR Oxygen induced retinopathy

ONL Outer nuclear layer

OPL Outer plexiform layer

PBS Phosphate buffered saline

PDR Proliferative diabetic retinopathy

PECA1V-1 Platelet/endothelial cell adhesion molecule-1

PEI Polyethylenimine









Paraformaldehyde

Protein kinase A

Placenta growth factor

recombinant adeno-associated virus

Retinopathy of prematurity

Retinal pigment epithelium

Real time polymerase chain reaction

Sphingosine 1-phosphate

Stem cell factor

Stromal cell derived factor-1

Standard deviation

soluble guanylyl cyclase

Sphingosine kinase

Transforming growth factor

Thrombopoietin

Vasodilator stimulated phosphoprotein

Vascular endothelial growth factor receptor-1

Vascular endothelial growth factor receptor-2

Vascular endothelial growth factor

Vascular permeability factor

Z oul a-oc cluden s- 1


PFA

PKA

PlGF

rAAV

ROP

RPE

RT-PCR

S1P

SCF

SDF-1

SD

sGC

SK

TGF

TPO

VASP

VEGF R-1

VEGF R-2

VEGF

VPF

ZO-1









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 REGULATES
HEMATOPOIETIC STEM CELL AND ENDOTHELIAL PROGENITOR CELL FUNCTION
DURING VASCULAR DEVELOPMENT

By

Kyung Hee Chang

May 2008

Chair: Maria B. Grant
Major: Medical Sciences-Physiology and Pharmacology

Retinal blood vessels develop by a combination of vasculogenesis and angiogenesis, called

neovascularization. Endothelial progenitor cells (EPCs) which originated from lateral and

posterior mesoderm contribute to neovascularizaiton in response to certain cues such as

cytokines and hypoxic gradient. Aberrant vessel growth in retina results in vascular

retinopathies, the leading cause of visual disability and blindness worldwide. The pathological

outgrowth of new blood vessels involves the recruitment and proliferation of circulating EPCs.

This study was initiated to better understand a complex network regulating neovascularization

involving EPCs function. Many factors have been identified to promote trafficking, mobilization,

and homing of EPCs. We demonstrated that insulin-like growth factor binding protein-3

(IGFBP-3) has a critical function in postnatal vasculogenesis both in vitro and in vivo. IGFBP-3

showed enhanced migration, tube formation and proliferation of EPCs in vitro. In vivo, IGFBP-3

inhibited pathological neovascularization by protecting developmental retinal vessels from

oxygen induced regression. In EPCs, nitric oxide (NO) regulates migration through redistribution

and phosphorylation of the motor protein vasodilator-stimulated phosphoprotein (VASP).

IGFBP-3 has been shown to trigger EPC mobilization by generating NO and subsequently









activating VASP. The signaling mechanisms oflIGFBP-3 on EPCs were also identified in this

study. We have defined that down stream pathway oflIGFBP-3 involve sphingosine kinase

(SK)/sphingosine-1 phosphate (S1P) signal transduction. NO release from EPCs was reduced

after treatment with a SK inhibitor, dimethylsphingosine (DMS) and could not be recovered by

exposure to IGFBP-3. This result suggests that IGFBP-3 may act as an upstream mediator of

SK/S 1P signaling. We addressed the hypothesis that IGFBP-3 stimulates NO production and

VASP phosphorylation through SK activation. Thus, IGFBP-3-induced angiogenesis of EPCs

may be involved in a SK dependent signaling pathway. Whereas an inhibitory angiogenic role

for IGFBP-3 has been widely reported, several other researchers have shown contradictory

results in that IGFBP-3 also enhances angiogenic effects. Our results suggest that IGFBP-3 has

an angiogenic/vasculogenic function in modulating the migratory ability of EPCs.









CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Blood vessel development is mediated by angiogenesis as well as by vasculogenesis. In

vasculogenesis stem cells are mobilized from the bone marrow and differentiate into circulating

endothelial progenitor cells which are then integrated into the primary capillary plexus. This

process is responsible for the development of the vascular system during embryogenesis. In

contrast, angiogenesis is the formation of new blood vessels from sprouts on preexisting vessels,

and occurs both during development and in postnatal phase. In adults, neovascularization

involves the recruitment and proliferation of either endothelial cells from preexisting vessels or

circulating EPCs originating from bone marrow. This process is involved in many physiological

and pathological conditions such as tissue remodeling, regeneration, wound healing and

tumorigenesis. Vessel development is regulated by a complex network of mediators and cellular

interactions. Reduced oxygen tension, various cytokines, and angiogenic factors at least partially

regulate neovascularization. This study was intended to better understand the mutifactorial

processes of neovascularization.

The Eye

The eye exists in a relatively isolated compartment. The ocular tissue and vasculature are

highly differentiated for conducting the complex process of visual transduction with little

systemic exposure. The retina is an ideal model system to study molecular mechanisms of

angiogenesis due to the unique vascular supply of the eye, the ability to visualize this vasculature

in vivo and the ability to selectively express genes in the eye.

Anatomy

The eye is a complex organ composed of many parts. The ability to see is dependent on

the actions of several structures. Figure 1-1 shows many of the essential components of the eye.









Initially, light enters the eye through a lubricating tear film that covers the cornea.l The

cornea is the transparent outer covering of the eye and helps to focus incoming light. After light

rays pass the cornea, they travel through a clear, watery fluid, called the aqueous humor.2 The

aqueous humor transports nourishment for the surrounding lens and cornea as well as

maintaining a constant intraocular pressure.2 The aqueous humor is produced by the ciliary body

which also changes the shape of the lens for focusing.3 The iris is the colored part of the eye. It

separates the anterior chamber from the posterior chamber and regulates the amount of light

entering through the pupil.3 The size of the pupil is controlled by the dilator and sphincter

muscles of the iris and regulates the amount of light that enters the eye.3 After light travels

through the pupil, it passes through the lens. The lens is suspended by ligaments (called zonule

fibers) that are attached to the anterior portion of the ciliary body.4 As a consequence of ciliary

muscle actions, the contraction or relaxation of these ligaments changes the shape of the lens (a

process called accommodation allowing the formation of a sharp image on the retina).4 Light

then passes a clear, jelly-like substance called the vitreous before it Einally reaches the retina.

The vitreous is a viscous, transparent liquid that fills the center of the eye.' It is composed

mainly of water and comprises about 2/3 of the eye's volume, and helps to maintain eye shape.'

The retina is a multi-layered sensory tissue lining the back of the eye that operates similar to the

fi1m in a camera. At the retina, the light rays are converted to electrical impulses which are

transmitted via the optic nerve to the brain. The central portion of the human retina contains a

yellow pigment called the macular pigment.' This pigment helps protect the sensitive receptors

in the retina, particularly from the potentially harmful effects of blue light.5 The density of the

pigment has been shown to be linked to diet and can be reduced in a person who smokes.' The

macula is the area of the retina that contains the highest concentration of photoreceptor cells.







v i treo~u s hu mo~r
,/re tin a
/ optic nerve





m ac ula

r


can unctive


ciliary


lflS


aqueous



a nte rio~r-

c ry~stallin e
'lens


e xtra oc ul ar
mnuscle


Figure 1-1. Anatomy of human eye.









At the very center of the macula is the fovea, the site of our sharpest vision.6

The optic nerve is a bundle of nerve fibers that carries visual information from the eye to

the brain. The optic nerve runs from the optic disc through the optic foramen to the optic

chiasma where it becomes the optic tract.' It is 5cm in length and surrounded by 3 layered

membranes of the central nervous system: pia, arachnoid, and dura.7

The eye is comprised of three different layers and spatially divided into three chambers of

fluid. The external layer is formed by the sclera and comea.2 The cornea is a refracting surface,

providing 2/3 of the eye's focusing power.2 The cornea is extremely sensitive and contains more

nerve ending than anywhere else in the body.2 The sclera is composed of tough, fibrous tissue

that protects the inside of the eye.2 Extraocular muscles are attached to the sclera and maintain

the shape of the eye.8 The six tiny muscles, known as the extraocular muscles (EOMs), surround

the eye and control its movements.8 The primary function of the four rectus muscles is to control

the eye's movements from left to right and up and down.9 The two oblique muscles allow the

eye to rotate inward and outward.9 All six muscles of both eyes work in unison so that the eyes

are always aligned.8 The intermediate layer is divided into two parts: anterior (iris and ciliary

body) and posterior part, called the choroids.10 The choroid contains a layer of blood vessels and

lies between the retina and sclera.10 The choroid supplies oxygen and nutrients to the outer

layers of the retina." The choroid connects the ciliary body with the front of the eye and is

attached to edges of the optic nerve.ll The internal layer is the sensory part of the eye called

retina.

The eye consists of three chambers; the anterior chamber (between cornea and iris),

posterior chamber (between iris, zonule fibers, and lens), and the vitreous chamber (between the









lens and the retina). The first two chambers are filled with aqueous humor and the vitreous

chamber is filled with more viscous fluid, the vitreous humor.

The Retina

The retina is a multi-layered structure that is involved in signal transduction. It covers

about 65 % of interior surface of the vitreous chamber.2 The human retina is approximately 0.2

mm thick, and has an area of approximately 1 100 mm.2 Each retina is composed of about 200

million neurons.12 The retina captures the light and converts it into electrical impulse using

photoreceptors. There are two types of photoreceptors in the retina: rods and cones.13

Approximately 125 million rods exist in human retina.13 They are spread throughout the

peripheral retina and function best in dim lighting; therefore, the rods are responsible for

peripheral and night vision.13 The retina contains approximately 6 million cones.14 COnOS

function best in bright light and color perception.14 The highest density of cones is in the

macula. The macular contains a very different retinal configuration towards the center called

foveal region.l Cones are most densely packed within the fovea, the center portion of the

macular. The fovea is maximally thinned and mainly consists of photoreceptors and their

nuclei." The purpose of foveal thinning is to permit greater light absorption by the dense array

of photoreceptors.6, 15 Another interesting aspect of the fovea is the absence of blood vessels

over the photoreceptors. This absence of blood vessels contributes to increasing visual acuity in

the macular region. However, the vascularization in the rest of the macula is very dense and

thus increases possibility of many vascular related diseases. The retina is loosely attached to the

retinal pigment epithelium (RPE).10 These cells contain a great amount of pigment that is

necessary for light absorption and transportation of oxygen, nutrients, and cellular wastes

between the photoreceptors and the choroids.10 Bruch's membrane is tightly bound to the RPE,

stabilizing the RPE layer by separating it from the blood vessels of the choriod.l0








Inner Limmiting Membrane
V1 ~II(ILM)
Gagio el ae
i (GCL)

I Inner Plexiform Layer


(INL)


li I: 4IOuter Plexiform Layer


1~~1~ I i(OPL)

i' l)OuteH~sr NlcarLae
(ONL)



Photoreceptor Cells
(rods and cones)


Retinal Pigment Epithelium
,(RPE)
II_~----~-_--- ~- ..I~ -;~-~Choroid-~c

Sciera


Figure 1-2. The ten layers of the retina. This drawing shows retinal and choroidal cross-section.
From the most anterior layers of the retina, the ten layers of the retina consist of
sclera, choroids, retinal pigment epithelium, rod and cone layer, outer nuclear layer,
outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer,
and the inner limiting membrane.









Oxygen diffuses across the Bruch's membrane and this membrane grows thinker with age.10

Breaks in Bruch's membrane are the hallmark for choroidal neovascularization (CNV) into the

retina.'o Beneath the Bruch' s membrane the chroroid, containing its network of blood vessels,

nerves, immune cells, and fibroblasts, supplies all of nutritional needs of the RPE and the outer

part of the sensory retina.

Human retina consists of ten layers (Figure 1-2). Among them, three layers of nerve cell

bodies and two layers of synapses are mainly responsible for converting a light signal into neural

signal. The photoreceptor cell bodies form the outer nuclear layer (ONL).16 While the inner

nuclear layer (INL) contains cell bodies of the bipolar, horizontal and amacrine cells, the

ganglion cell layer (GCL) contains cell bodies of ganglion cells and displaced amacrine cells.16

The outer plexiform layer (OPL), the first area of neutrophil, is located in between the ONL and

the INL.17 In the OPL, the photoreceptors convey their information to the bipolar cells as well as

the horizontal cells. Afterward, bipolar cells relay information to the inner plexiform layer (IPL),

which separates the INL and the GCL.17 Bipolar cells are connected to the retinal ganglion cells

in addition to amacrine cells in the IPL.17 The ganglion cells are the output neurons of the retina

that transmit the signal from the eye to the brain.16

Retinal Blood Supply

The blood supply to the retina originates from the ophthalmic artery.l There are two

sources of blood supply to the mammalian retina: the central retinal artery and the choroidal

blood vessels.l The outer retina is supplied by the choriocapillaries. The choroidal arteries arise

from long and short posterior ciliary arteries and branches of Zinn' s circle around the optic

disc.10 Each of the posterior ciliary arteries is further classified into fan-shaped lobules of

capillaries that supply localized regions of the choroids.10,1s The arteries penetrate the sclera

around the optic nerve and spread out to form vascular layers in the choroids.10 The choroid









receives the greatest blood flow (65-85%), which is critical for the maintenance of the outer

retina, particularly the photoreceptors.l0 The central retinal artery supplies the remaining 20-

30% blood flow from the optic nerve head to nourish the inner retinal layers. The central retinal

artery supplies the blood as it branches into smaller segments upon leaving the optic disc.l The

vessels are further divided into either an artery or a vein ". The central retinal artery has 4 main

branches in the human retina. The arterial intraretinal branches then supply three layers of

capillary networks: the radial peripapillary capillaries, an inner layer of capillaries, and an outer

layer of capillaries.' The radial peripapillary capillaries are the most superficial layer of

capillaries lying in the inner part of the nerve fiber layer, and run along the paths of the major

superotemporal and inferotemporal vessels.l The inner capillaries are located in the ganglion

cell layer both under and parallel to the radial peripapillary capillaries.l The outer capillary

network runs from the inner plexiform layer to the outer plexiform layer though the inner nuclear

layer.18s

Retinal blood vessels that are similar to cerebral blood vessels maintain the blood-retinal

barrier (BRB). The BRB consists of two distinct monolayers of cells: the retinal pigment

epithelium (RPE: outer barrier) and the retinal capillary endothelial cells (inner barrier).19 Both

monolayers form tight junctions, which are operative in the maintenance of the barrier.

The concept of the BRB was first proposed by Schnaudigel in 1913 following the classical

work of Ehrlich and Goldman who discovered the blood-brain barrier (BBB). Similar to the

structure of BBB, the inner BRB is covered with pericytes and glial cells.19 Glial Midller cells

predominantly support retinal endothelial cells and glial astrocytes are partly responsible for

supporting endothelial functions at the inner BRB.20,21









The inner BRB plays an important role in supplying nutrients to the neural retina and is

responsible for the efflux of neurotransmitter metabolites from the retina to maintain neural

functions.20 The outer BRB consists of specialized nonfenestrated capillaries and tight junctions

within the RPE.21 The outer BRB forms a transport barrier between the retinal capillaries and

the retinal tissue.21 In addition, it prevents the passage of the large molecules from

choriocapillaries into the retina.21

The eye is extremely sensitive to any disruption of its blood supply. The BRB maintains

the ocular milieu and protects the neural retina from any pathological blood circulation. The

breakdown of the BRB is a clinical hallmark of early diabetic retinopathy.

Retinopathies

Vision impairment, disability, and blindness are maj or public health problems. Significant

suffering, disability, loss of productivity, and lower quality of life can affect millions of people.

In the United States, more than 11 million people have some degree of visual impairment.

Approximately 890,000 people in the US are legally blind. Retinopathies are ocular diseases in

which deterioration of the retina is initiated by abnormal neovascularization, resulting in vision

loss. Vascular retinopathies are the leading causes of visual disability and blindness worldwide.

Pathological growth of new blood vessels in pre-retinal region is the hallmark of retinopathies.

Retinopathies affect all age groups: retinopathy of prematurity (ROP) is a disease that occurs in

premature babies. Diabetic retinopathy (DR) primarily affects the working age population, and

age-related macular degeneration (ARMD) affects the aging population.

Retinopathy of Prematurity

ROP is the leading cause of blindness in children in both developing and developed

countries. ROP mainly affects premature infants weighing about 1.25 kg (approximately 2.751b)

or less that are born before 3 1 weeks of gestation. At 16 weeks of gestation, blood vessels









gradually grow over the surface of the retina.22 Active growth of the human fetal eye occurs

within the last 12 weeks of full term delivery (28 to 40 weeks of gestation).23 Vessels reach the

anterior edge of the retina and then stop progressing at about 40 weeks of gestation.22-2 A

premature baby is placed into an oxygen chamber to assist the still developing lungs. Once in

high oxygen the retinal vessel development is stopped. Upon removal from the high oxygen

environment the return to normal levels of oxygen is seen as a hypoxic environment in the eye,

and this stimulates the neovascularization within the retina.

There are approximately 3.9 million babies born in the U.S. annually. According to

National Eye institute, about 28,000 premature infants are born. About 14,000 to 16,000 of these

premature infants could potentially develop some degree of ROP. Although, approximately 90%

of all the infants with ROP are in the milder stage and do not need treatment, the rest of 10 %

(about 1,100 to 1,500) of the babies develop severe ROP and require medical treatment. As a

consequence, about 400 to 600 infants each year in the U.S. become legally blind from ROP.

Many factors are likely to cause ROP. Once a premature baby is born, excessive oxygen

supply is needed to help the development of the premature baby's lungs.25 ROP was first

described in 1942, but its cause was unknown at that time. Most of premature babies were

treated with high oxygen whether they were having breathing problems or not.22 After a while, it

was found that although supplemental oxygen helped the premature babies who were having

lung and breathing complications, the high oxygen destroyed blood vessels in the retina.22

Despite adjustment of oxygen delivery and other medical advances, the total number of infants

with ROP has not decreased because of the increased survival rates among the low birth weight

infants.22,26 ROP progresses in two phases.24 The hyperoxia extrauterine environment

surrounding the baby precedes the development of the first phase of ROP.24 The growth









inhibition of neural retina and retinal vasculature in the first phase is followed by a second phase

of ROP involving relative hypoxia-induced uncontrolled proliferative vessel growth.24 The

pathological growth of vessels produces a fibrous scar that extends from the retina to the vitreous

gel and lens.24 Retraction of this scar tissue can separate the retina from the retinal pigment

epithelium (RPE), resulting in a retinal detachment, bleeding and blindness.22,23

This biphasic disease is associated with unbalanced levels of growth factors. Low levels of

insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are detected

in phase I, whereas, excessively high levels of IGF-1 and VEGF are found in phase II.27 IGF-1

plays a critical role in ROP infants. Reducing IGF-1 levels inhibits vessel growth even in the

presence of VEGF.28 Low levels of IGF-1 directly reduces vascular density, which subsequently

causes early vessel degeneration in phase I. The mean serum levels of IGF-1 in age-matched

premature babies are directly correlated with the severity of ROP disease stages.28,29 In the

second phase of ROP, which is driven by hypoxia, VEGF expression is increased in the retina,

resulting in pathological neovascularization.30

Because the retinal vessel development of mice is incomplete at birth, Smith et al.

developed a mouse model of ROP to study the molecular mechanism in the disease.30 This

mouse model is intended to mimic the first and the second phases of ROP.

Traditional therapies such as cryotherapy and laser photocoagulation of other proliferative

retinapathies can also be used to prevent blindness in ROP infants.23 However, these methods

can reduce peripheral vision and include risks from the anesthesia.23 Therefore, preventive and

less invasive therapies for ROP are desirable. Likewise, efforts to understand diseases that

involve VEGF and IGF-1 are important to develop such medical treatments. The two phases of

ROP require apposite approaches. In phase I, the hyperoxia induced vessel loss can be partially









prevented by administrating exogenous VEGF or PlGF-1.31-33 While an injection of anti-VEGF

aptamer as well as anti-VEGF antibody fragment can be used to treat neovascularization

associated with phase II of ROP.34 Pharmacological intervention related to the prevention of

vessel loss may be more effective in the treatment of ROP since the extent of the second

destructive phase of ROP is determined by the amount of vessel loss in the first phase.

Diabetic Retinopathy

Approximately 100 million people worldwide have been affected by diabetes mellitus

(DM).35 In the United States, 16 million individuals are diabetic, and about 40,000 patients per

year are diagnosed with the ocular complications of DM in the U.S.36 Among them, 5 to 10

percent are known to be insulin-dependent type 1 DM and 90 % to 95 % is known to be insulin-

independent type 2 DM.36 Vascular diseases are the principal causes for death or disability in

people with diabetes.35 The metabolic abnormalities that characterize diabetes such as elevated

blood glucose levels, increased levels of free fatty acids, and insulin resistance cause vascular

dysfunction.35 According to the NIH, the microvascular complications of DR are the most

common complication of diabetes and thus a leading cause of blindness. DR is known to affect

approximately 75 % of diabetic patients within 15 years after onset of the disease.37 Although

the best way to prevent visual loss is to initiate treatment before symptoms develop, many

diabetic patients are only diagnosed after visual complications have already begun. DR is divided

into two stages: nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic

retinopathy (PDR).36 PDR typically develops in patients with type 1 diabetes, while NPDR is

more common in patients with type 2 diabetes.36

The progression of DR begins with apoptosis of pericytes and adhesion of leukocytes to

the vessel wall that lead to microvascular occlusion, basement membrane thickening, and

increased vascular permeability.38 At this stage, the blood vessels become leaky, allowing blood









and vascular fluids to accumulate in the retinal tissue and form exudate deposits.38 These

pathological processes then result in macular edema which is a common feature in patients with

NPDR.39 NPDR produces an increasing area of capillary non-perfusion which leads to hypoxia

in the retina.38,40 To compensate for the decreased oxygen supply, angiogenic factors are

released from the hypoxic retinal tissues that stimulate the growth of new blood vessels on the

surface of the retina.37,41 This stage is called PDR. The walls of the new blood vessels are fragile

and may break, allowing blood to leak out.38 This can cloud the vitreous and compromise

vision.38 In an advanced stage of PDR, newly formed fibro vascular tissue grows from the

retinal surface into the vitreous cavity.36 This can cause retinal detachment which can result in

kindness.

To date, a common therapy for DR, including advanced PDR or diabetic macular edema, is

laser photocoagulation. However, this method often causes common side effects such as neural

tissue loss, peripheral vision loss, impairment of night vision, and change in color perception.42

Moreover, in some patients, the retinopathy continues to progress after treatment.42 Thus, there

is a great need for the development of new therapies that treat diabetic retinal vascular disease.

Recently, pharmacological agents that directly inhibit angiogenesis have been developed to treat

DR.34 VEGF plays a pivotal role in the retinal microvascular complications of diabetes.37 The

overexpression of VEGF plays a key role in diabetes inducing retinal vascular dysfunction.37

The developments of agents that directly target VEGF and its receptors have been actively

studied in clinical research.43-4 The use of endothelial progenitor cells for drug delivery or

molecular and genetic manipulation is a technique that takes a new approach in the treatment of

DR.









Age-related Macular Degeneration

AMD is the most common cause of poor sight in people with age over 60.46 Recently, it is

reported that AMD affects approximately 11% of the U.S. population age 65 to 74.46 In the

western world, there are approximately 12 to 15 million cases of AMD.46

Two main types of AMD can be distinguished: the dry form (atrophic) and the wet form

exudativee), based on the absence or the presence of choroidal neovascularization (CNV).46 Dry

AMD is more common than the wet form.46 It develops very slowly and causes gradual loss of

central vision. Dry AMD is characterized by the presence of drusen in the macular region.47

The excreted materials, damaged photoreceptors and concentrated by-products of cellular

metabolism affect the formation of drusen, which looks like yellow-gray nodules localized

between the retinal pigment epithelium (RPE) and Bruch's membrane.47 Increased drusen

formation affects RPE function and eventually causes RPE alteration and depigmentation.47 As a

result of dry AMD, patients are likely to lose the central perception as well as color contrast

sensitivity. Although wet AMD only consists of approximately 10% of all AMD cases, about

80% of severe vision loss is caused by the wet form as compared to 20% that are caused by the

dry form. CNV determines the characteristics of the wet AMD. CNV is the process of the

growth of immature blood vessels in the choroid, and the pathological new vessels penetrate the

subretinal space. Over time, CNV causes hemorrhages, RPE detachment, scarring, and

kindness. 14

The main factor that causes AMD is not known. However, a number of risk factors have

been identified that partially contribute to AMD such as age, gender (women are more likely to

develop AMD), smoking, genetics and nutrition.48 There are also molecular factors that are

known to affect the development of AMD.48 It is found that VEGF expression is increased in

RPE cells of patients with AMD.11,41 In experimental animal models, VEGF levels were found









to be significantly higher in the vitreous of wet AMD than healthy controls.11,41,48 To date, the

most promising results of a treatment for ARMD has been achieved with anti-angiogenic

reagents that target VEGF.42

Neovascularization

Blood vessels are developed by vasculogenesis or angiogenesis. During vasculogenesis,

endothelial cells differentiate from progenitor cells and angioblasts, which are already present

throughout the tissue, and then link together to form vessels. During angiogenesis, sprouts form

from preexisting blood vessels and invade into surrounding tissue. Most organs are vascularized

by vasculogenesis, but brain and kidney are vascularizaed by angiogenesis. Retinal vascular

development occurs by a combination of vasculogenesis and angiogenesis, called

neovascularization. A variety of stimuli are known to contribute to neovascularization by

recruiting stem or progenitor cells and inducing adhesion to activated ECs.

Cytokines, chemotactic factors, and angiogenic factor have been implicated as positive

regulators of neovascularization. Some of these molecules are strongly induced by hypoxia in

cultured cells, including tumor cell lines, cardiac myocytes, and vascular smooth muscle cells as

well as in ischemic tissues.

Endothelial Progenitor Cells

Bone marrow (BM) is the maj or reservoir of stem cells in adults. The bone marrow

microenvironment, in which bone marrow stem cells remain quiescent, is comprised of stromal

cells and extracellular matrix (ECM) components. A special subtype of BM derived stem cells,

termed endothelial progenitor cells (EPCs) that are able to differentiate into mature endothelial

cells and incorporate into sites of neovascularization under physiological as well as pathological

conditions such as wound healing, organ regeneration, and tumor growth. EPCs can be isolated

from peripheral blood, fetal liver, or umbilical cord blood.49-51 EPCs are characterization by









specific antigens expressed on the surface of the cells. Stem cells maintain primitive

characteristics so that they can differentiate or transdifferentiate into a wide range of cell types.

This is called stem cell plasticity.52 The identification of true EPCs has been challenged by the

phenomenon of stem cell plasticity. Defining the validated EPCs has been debated because

several studies have demonstrated overlapping antigens among subtypes of bone marrow derived

cells including EPCs and mesenchymal stem cells.52-58 Although it is not clear what markers

define EPCs, it is widely accepted that CD34, vascular endothelial growth factor receptor-2

(VEGFR-2), and CD133 are the common antigens used in the enrichment of EPCs.59-61

Schatterman, et al. suggested that expression of endothelial nitric oxide synthase (eNOS) is a

reliable marker for EPCs.49,53 EPCs have other characteristics of endothelial cells including

acetylated low density lipoprotein incorporation and endothelial specific lectin binding in

vitro.49,59,62 Furthermore, EPCs also show typical endothelial functional characteristics like

formation of capillary tubes and production of nitric oxide (NO).63,64 With maturation, EPCs

begin to lose expression of CD34 and CD133 (i.e., early hematopoietic stem cell marker) or start

to express CD31, also known as PECAM-1 (platelet/endothelial cell adhesion molecule),

vascular endothelial cadherin, and von Willebrand factor.49,57 The differentiation and maturation

of EPCs occur when circulating EPCs move to the site of injured vessel or integrated into mature

endothelium.5o

Increasing evidences suggest that EPCs are preferentially recruited to sites of ischemia and

tumor formation and incorporated into functional vasculature.50'65'66 EPC recruitment as well as

the release from BM is influenced by various factors. Proangiogenic growth factors such as

VEGF, granulocyte/macrophage colony-stimulating factors (GM-CSF), SDF-1, and

erythropoietin (EPO) have been shown to modulate EPC functions that play a critical role in









embryo development as well as in homeostasis in adult.65,67 For instance, these factors are

essential for EPCs differentiation and blood vessel development during embryogenesis and also

contribute in increasing circulating numbers of EPCs in adults.65,67 Transplantation of cultured

EPCs successfully promotes therapeutic neovascularization in both ischemic hind limbs and

acute myocardial infarction modelS.50,65

Both the number of circulating EPCs and colony forming ability of EPCs are correlated

with some types of diseases. It was found that fewer CD34+ EPCs are circulating in patients with

diabetes, diabetic retinopathy, and peripheral artery disease.68 Increasing numbers of EPCs were

found in patients with limb ischemia or vessel wall damage after coronary thrombosis, burn

injury, or coronary bypass surgery to rescue the damaged vessels.65,68,69 Since new blood vessel

growth from mature ECs has rarely been found in adults, and turnover of the quiescent

endothelium is considerably low, the vascular repair may need the support of EPCs. The study

of EPC biology will help to better understand postnatal vasculogenesis and also help to find

novel therapies for the treatment of pathological neovascularization.

Hypoxia Inducible Factor-1

Hypoxia occurs when there is an imbalance between oxygen supply and demand in cancer

or ischemic tissues. In wounds, capillary injury generates a hypoxic environment, and altered

oxygenation induces the reconstructive angiogenic response.70 Hypoxia serves as a critical cue

for both physiological and pathological angiogenesis in the brain, heart, kidneys, lungs or

muscles.n In stem cell research, hypoxia is considered a potent trigger for mobilization of bone

marrow derived cells.

Hypoxia inducible factor-1 (HIF-1), a transcription factor, functions as a maj or regulator of

02 homeostasis or an adaptor of 02 deprivation. HIF-1 is a heterodimer composed of an oxygen

related HIF-la subunit and a constitutively expressed HIF-1P subunit.72 In Order to respond









Bone Marrow Self Renewal

67 ~Hemangioblast


~HSC


~Myoid |


Lymphocytes
Erythrocytes
Platelets


Figure 1-3. Hypoxia-regulated factors and BM-derived cells. HSC and EPC are originated from
common precursor, hemangioblast. These cells maintain primitive characteristics so
that they can differentiate themselves into a wide range of cell types. EPC has been
primary material for this study and it is well characterized as CD34+cells. EPC
mobilization is a complex process involving many mediators and cellular interactions.
Reduced oxygen tension has been widely believed to trigger this process. Once tissue
got an injury, the damaged tissue releases various cytokines and angiogenic factors.
Respond to Hypoxia-regulated factors such as EPO, SDF-1, VEGF, and IGFBP-3,
circulating EPCs migrate to ischemic area then re-endothelized damaged vessel


EPCca









rapidly to hypoxia, HIF-la is continuously synthesized and degraded under non-hypoxic

conditions. Under hypoxic conditions, however, the degradation of HIF-la is inhibited, so that

the expression is increased exponentially as 02 COncentration declines, resulting in dimerization

with HIF-10.73,74 Dimerized complex of HIF-1 binds to hypoxia response element (HRE) within

a target gene, and recruits coactivator proteins, which lead to increased transcription of the target

gene."

More than 40 genes are known to be directly activated by HIF-1 at the transcriptional

level.76 Genetic studies revealed the presence of a functionally essential HIF-1 binding site in

the target gene. The genes induced by HIF-1 regulate molecular mechanism for sensing and

responding to changes in Oz COncentration. HIF-1 regulated genes contain a cis-acting

transcriptional regulatory element, HRE, as a HIF-1 binding site. Recently, DNA microarray

analysis showed that over 2% of all human genes are either directly or indirectly regulated by

HIF-1 in endothelial cells.76,77

Physiological stimuli other than hypoxia can also induce HIF-1 activation and the

transcription of hypoxia-inducible genes under non-hypoxia conditions. IGF-1 induces HIF-la

synthesis through phosphatidylinositol 3- kinase and MAP kinase pathways ", and IGF-1

receptor tyrosine kinase induces HIF-la protein synthesis, independent from oxygen

concentration." HIF-1 has been shown to activate transcription of the gene encoding VEGF.79

HIF-1 induces VEGF secretion which subsequently induces upregulation of SDF-1 expression.

In turn, SDF-1 has a reciprocal effect on inducing VEGF. In addition to growth factors, nitric

oxide is also known to play a similar role enhancing HIF-1 activation under non-hypoxic

conditions mediating prolyl hydorxylase activitieS.80-8









Vascular Endothelial Growth Factor

VEGF was discovered in 1983 and called vascular permeability factor (VPF) due to its

blood vessel permeability increasing capacity.83,84 In 1989, it was determined that VPF and the

endothelial specific mitogen, VEGF was the same protein." VEGF plays an important role

both in normal physiological angiogenesis and in most of the pathological angiogenesis

associated with diseases such as diabetic retinopathy, rheumatoid arthritis, and solid tumors.

The VEGF family consists of seven structurally related homodimeric glycoproteins:

VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D, orf virs-encoded

VEGF-like proteins (called as VEGF-E), and a series of snake venoms (collectively called as

VEGF-F).88-91 Despite structural similarity, the VEGF homologs play distinct roles and bind to

specific subtypes of VEGF receptors. VEGF exerts its effects by binding to one of its three

receptors that belong to the superfamily of receptor tyrosine kinsase. VEGF-A binds to either

VEGF receptor 1 (VEGFR-1 / Flt-1) or VEGF receptor 2 (VEGFR-2 / KDR / Flk-1). However,

PlGF and VEGF-B exclusively bind to VEGFR-1. VEGF-C and VEGF-D are specific ligands

for VEGFR-2 and VEGFR-3 (Flt-4), regulating both blood and lymphatic vessel development.

Viral VEGF-Es and some snake venom VEGF-F variants exclusively activate VEGFR-2.84,88

Within the VEGF family, most of the research has focused on VEGF-A (usually referred

as VEGF). VEGF-A is regarded as the most potent mediator of angiogenesis. There are several

splice variants of VEGF-A including VEGF-121, -145, -165, -189, and -206, while VEGF-165 is

the predominant form.67

VEGF mobilization appears to be isoform specific. For instance, VEGF-165, rather than

VEGF-189, induces a rapid mobilization of VEGFR-2+ cells into circulation.92 VEGF initiates

embryonic vasculogenesis and triggers angiogenic sprouting by activating VEGF-R2 on vascular

endothelial cells.93,94. VEGFR-2 is also essential for the development of HSCs during early








VEGF g -

V~ EGFR




i J cA Ras



IMAPK
SPathway
AKT IIMP

Rearrangement

Cell Gene Expression
Survival Cell Proliferation



IAngiogen esisj


SDAG
PKC





Cell Proliferation
Vasoperrneability


Figure 1-4. The VEGF (VEGF-R2) signaling pathway. Upon binding of VEGF, VEGF-R2 is
activated by autophosphorylation, and initiates several signaling cascades all of which
lead to angiogenesis.









embryonic development.95 In addition, the activation of VEGFR-1 is sufficient to rescue HSC

survival in vitro and hematopoietic repopulation in vivo.96

VEGF regulates several endothelial cell functions, including proliferation, differentiation,

permeability, vascular tone, and the production of vasoactive molecules.95 VEGF is also a

chemoattractant and plays a role in EPC recruitment and induces in vitro differentiation of EPCs

into mature endothelial cells.97 Genetic modifications of VEGF have helped to understand its

biology related to EPCs. Overexpression of VEGF in nonischemic mouse hearts can lead to the

formation of endothelial cell-derived intramural vascular tumors.98 In addition, VEGF gene

transfer promotes EPC migration into ischemic regions. VEGF-deficient HSCs and bone

marrow mononuclear cells show lack of ability to repopulate lethally irradiated hosts.96

Stromal Derived Factor-1

Stromal cell derived factor-1 (SDF-1) belongs to the group of chemokine CXC subfamily,

originally isolated from murine bone marrow stromal cells.99 It is produced by multiple bone

marrow stromal cell types as well as epithelial cells in many organs.1oo

CXCR4, a 7-transmembrane spanning G protein-coupled receptor, is the only known

receptor for SDF-1 and is also a coreceptor for human immunodeficiency virus (HIV) type 1.101

SDF-1 is chemotactic for EPC. The chemokine SDF-1 or CXCL12 mediates homing of stem

cells to bone marrow by binding to its receptor CXCR4 on circulating cellS.102

The SDF-1/CXCR4 signaling pathway is critical during embryogenesis, vascular

development, and cardiac development. Blockade of SDF-1 in ischemic tissue or CXCR4 on

circulating cells inhibits progenitor cell recruitment to sites of injury.61 Overexpression of

CXCR4 on stem and progenitor cells promotes its proliferation, migration, and in vivo

engraftment of NOD/SCID mice.103 SDF-1 gene expression is regulated by the transcription

factor, HIF-1. Progenitor cell mobilization is triggered by hypoxia gradients through HIF-1








SD F-1




CXC R4







iii~~' 1;1 I VIEK11 H" (
i.ERK1 ERK2l

Figure1-5.The inercto bewe SDF-1 an CXR4 S F-1bnin to i tsrcpoG
protein ~ ~ j~ cope XR east ciaiono Rr 2 civtono MP

aciain hsineato eut i elmgain rpoieain









induction of SDF-1. HIF-1 induced secretion of SDF-1 in ischemic tissue has a direct correlation

with reduced oxygen tension.70

SDF-1 gene transfer induces EPC mobilization from bone marrow into peripheral blood and

also improved perfusion to ischemic limbs. It is proposed that SDF-1 induces upregulation of

metalloproteinase-9 (MMP-9) activity, which causes cleavage of membrane bound Kit-ligand

into soluble Kit-ligand, stem cell factor (SCF). As a consequence, SCF promotes stem cell

mobilization into the circulation. Recently, it was shown that SDF-1 is critical for the

development of proliferative retinopathy.104 Vitreous concentrations of SDF-1 and VEGF are

increased in diabetic patients. Compared to VEGF, exogenous SDF-1 has a greater effect in

causing retinal neovascularization in an animal model.104 Intravitreal injection of blocking

antibodies to SDF-1 disrupts retinal and choroidal neovascularization in mouse.47,103 Blockage

of SDF-1 is now being considered for potential treatment for ocular vascular diseases.

Insulin-like Growth Factor Binding Protein-3

Insulin-like growth factor-I (IGF-I) and II (IGF-II) modulate a diverse range of biological

activities including growth, differentiation, survival, and regulation of cell metabolism.'o' In

serum and the extracellular fluid, the maj ority of circulating IGFs are sequestered into 150 kDa

ternary complexes with IGF binding protein (IGFBP) and the liver-derived glycoprotein (acid-

labile subunit: ALS).106 This complex prolongs the half-life of IGFs in the circulation and

prevents them from crossing the capillary barrier.107,10s IGFBPs consist of six homologous

secreted proteins, which specifically bind to IGF-I with high affinity.

IGFBP-3, the most abundant binding protein in serum, is present in various glycosylated

forms between 40 and 44 kDa. A number of investigators have reported that IGFBP-3 has IGF-1

independent cellular actionS.109,110 For instance, independent of IGF-I, IGFBP-3 regulates cell

activities such as growth, proliferation, and apoptosis in both carcinoma cell lines and normal















IG F-1 R


e~3 ~ ~13K/A~ ~P


MAPK)


Jr
:I PKC~


Figure 1-6. IGF-1R signaling pathway. IGF-1R is a tetramer consisting of 2 extracellular a-
chains and 2 intracellular P-chains with the intracellular tyrosine kinase domain. The
activation of IGF-1R signaling pathways induces numerous physiologic actions of
IGF-1.


IG F-1 f
IGF1"-2
Insulin


IVIEK1)









High-affinity IGF binders
IGFBP-1 IGFBP-2 IGFBP-3


Low-affinity IGF binders


co o o



IGFBP-5 IGFBP-3 1


IGF-2 IGF-1









IRR IGFBP-R ??


Insulin


IGF-1


IGF-2


Figure 1-7. Schematic diagram of IGF system. Ligands (IGF-1, IGF-2 and insulin), IGFBPs (1
to 6) and receptors (IR, IGF-1R, hybrid IR/IGF-1R, IGF-2R, IRR and IGFBP-R) are
represented. IGF-I interacts with IGF-1R, IR, hybrid IR/IGF-1R and IGFBPs; IGF-2
interacts with IR (mainly with the IR from lacking the exon 11 sequence), IGF-1R,
hybrid IR/IGF-1R, IGF-2R and IGFBPs; insulin interacts with IR, IGF-IR and hybrid
IR/IGF-IR. Some IGFBPs are known to be cleaved by IGFBP proteases releasing
IGFBP proteolysed fragments, which have low-affinity for IGFs. IGFBP-related
proteins (IGFBP-rPs) which have low affinity for IGFs also exist. IGFBP-3 and
IGFBP-5 may act through their own receptor (IGFBP-R).


IGFBP-4 IGFBP-5 IGFBP-6


o ePo


IGF-1R IR/IGF-1R IG;F-2R
hybrid









Cells.11111 Whereas an inhibitory role for IGFBP-3 has been widely reported in the field of

cancer research, several other researchers have shown contradictory results that IGFBP-3

enhances angiogenic effects.120,121 IGFBP-3 also induces differentiation of chondrocytes and

human skeletal myoblasts.123

Liver and kidney are the main sources of IGFBP-3.124,125 According to the study by

Foulstone's group, skeletal muscle may be another source of autocrine tissue for production of

IGFBP-3.123 The level of IGFBP-3 in serum is modulated by not only its rate of synthesis but

also post-translational modification and proteolysis. While normal individuals have minimal

IGFBP-3 protease activity, IGFBP-3 protease activity is increased among individuals with

pregnancy, acute catabolic illness, or diabetes.116 IGFBP-3 proteases have been identified

including plasmin, matrix metallproteases, kallikreins, prostate-specific antigen, and cathepsin

D.126

IGFBP-3 concentration in serum is also regulated by other factors such as IGF-I, HIF-1,

VEGF, NO, and TGF-P. IGF-I affects HIF-1 which upregulates VEGF and IGFBP-3. IGFBP-3

has been identified as one of the hypoxia induced factors.71,127 Felser and colleagues

demonstrated that IGFBP-3 doesn't contain an HRE within its promoter, and that IGFBP-3 gene

expression was markedly reduced in HIF-la-deficient cells under hypoxic conditions.128 V/EGF

enhances upregulation of IGFBP-3 both HIF-1 dependent and independent ways.129 High

concentration of NO, induced by iNOS, decreases the levels of IGF-1 and IGFBP-3 by activating

IGFBP-3 protelysis in serum.130 TGF-P modulates IGF-independent IGFBP3 function.131-133

TGF-P l increases the secretion of IGFBP-3 in a variety of breast cancer cell lines and renal

carcinoma cells.122,134-136 IGFBP-3 is induced by TGF-P and is critical in mesenchymal cell









growth and podocyte apoptosis.122,137,138 Although IGFBP-3 has widely been studied for

decades, there are still many questions about the functions of IGFBP-3.

Sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) is a platelet derived sphingolipid that has been broadly

implicated in angiogenesis, platelet activation, inhibition of apoptosis, cytoskeletal organization,

adherens junction assembly, and morphogenesis. 139-141 Sphingosine kinase (SK) catalyzes the

formation of S1P by phosphorylation of sphingosine. Basal levels of S1P in mammalian cells

are generally low, but can increase rapidly or transiently when cells are exposed to mitogenic

agents or other stimuli. These signals activate SK which is responsible for increased level of

S1P. SK is an evolutionarily conserved lipid kinase which consists of Hyve conserved domains.

There are two isoforms of SK: the sphinosine kinase type 1 (SKl) and the sphinosine kinase type

2 (SK2). SK1 is mainly expressed in the cytosol, whereas SK2 is localized in the nucleus.142

SK1 and SK2 have different functions. Overexpression of SK1 protects against apoptosis

resulting in enhanced fibroblasts proliferation, tumor formation in NOD/SCID mice.143 In

contrast to pro-survival SKl, SK2 contains a functional putative BH3-only domain that induces

the inhibition of cell growth. Maceyka, et al. showed that SK2 has catalytic activity to induce

apoptosis. 142, 144

It is accepted that S1P is the ligand for plasma membrane localized G protein-coupled

receptors (GPCR) referred to as endothelial differentiation gene (EDG) receptors or S1PRs. S1P

binds to the Hyve members of this receptor family: S1P1, S1P2, S1P3, S1P4, and S1PS (previously

referred to as EDG-1, 5, 3, 6, and -8). These receptors are highly specific and only bind S1P.

The diverse biological processes that are triggered by S1P depend on the pattern of expression of

S1P receptors in each cell type as well as coupled G proteins.13 The study of S1P1 null mice

emphasized the importance of S1P1 on endothelial cell-pericyte communication in vascular









maturation and angiogenesis.145 For instance, Liu et al. demonstrated that S1P1 null mice are

embryonic lethal due to massive hemorrhage that is caused by incomplete vascular maturation in

arteries and capillaries.145,146

S1P binds to the S1P1 receptor which induces the activation of eNOS localized in caveolae

which poses a sphingolipid enriched domain in the plasma membrane.147,148 S1P/S1P1 pathway

acutely increases eNOS phosphorylation through PI3K and Akt activities in bovine aortic

endothelial cells.149

In addition, S1P has been identified as a potent signal-transducing molecule that may exert

diverse biological responses such as cellular differentiation, hypertrophy, proliferation and

migration.150-153

S1P activates the small GTPases Rac and Rho, functioning as a chemoattractant for

endothelial cells. S1P induced Rho-dependent integrin clustering into focal contact sites that

modulate cell adhesion, spreading, and migration."s To activate cell migration, S1P enhances

phosphorylation of protein kinase Akt in endothelial cells. S1P has been shown to have dual

effects on migration of early lymphocytes. In low levels, S1P induced chemoattractant migration

of CD4 and CD8 T cells and also enhanced chemotaxis to CCL-21 and CCL-5. However, at

higher levels, S1P had the opposite effect, reducing the migratory responses.154,155

S1P acts extracellularly by binding to members of the S1P receptors and regulating cell

movement. In addition, S1P also acts as a second messenger intracellularly to regulate calcium

homeostasis and apoptosis.88,141,156-158 However, the mechanism of S1P transport is unknown.

Recent studies in yeast showed that a member of ABC family of protein might be involved in

S1P translocation.159,160









Nitric Oxide

Nitric Oxide (NO) is a highly reactive, diffusible, and unstable radical and is involved in

signaling in the cardiovascular, gastrointestinal, genitourinary, respiratory, and nervous systems.

For instance, NO regulates cellular immunity, angiogenesis, neurotransmission, and platelet

aggregation and also promotes synaptic transmission and cytostatic/cytotoxic actions in

macrophages.64,82, 161,162

NO is generated by NO synthases (NOS). NOS is a heme-containing enzyme that is linked

to NADPH-derived electorn transport. NOS catalyzes the oxidation of L-arginine to L-citrulline

and NO, with tetrahydrobioterin and NADPH as essential cofactors.163

Three NOS isoforms have been identified and named after the cell type or conditions in

which they were first described: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible

or inflammatory NOS (iNOS). Because free NO is a transient species with a half-life of about 5

seconds, many investigations of this gaseous molecule have largely relied on studies of NOS.

All three isoforms of NOS are found in different cell types in the eye.164 nNOS is

responsible for producing NO in photoreceptors and bipolar cells, whereas eNOS is present in

vascular endothelial cells. iNOS, which is found in Muller cells and in retinal pigment

epithelium, is involved in inflammatory process and phagocytosis of the photoreceptor outer

segment. iNOS is also thought to be responsible for the pathogenesis of diabetic

retinopathy. 161,164-167

NO generation is important to maintain the vasculature in a relaxed state, inhibit the

adhesion of platelets and white cells, and suppress the replication of smooth muscle cells.168

eNOS derived NO diffuses into smooth muscle cells or pericytes then binds to the iron within the

heme-group of guanylyl cyclase and produces a conformational change that leads to enzyme

activation. NO has been observed to modulating vasculogenesis. NO has an important function









for the stem cell microenvironment in the bone marrow as a molecular mediator in controlling

the stem cell niche. eNOS is also important as it promotes angiogenesis and regulates the

expression of VEGF. eNOS deficient mice have an impaired capacity to mobilize cells from the

bone marrow.169 Guthrie, et al. found that NO/NOS pathway is a significant regulator of

neovascularization and can modulate hemangioblast activity by dictating the size and branching

characteristics of blood vessels that are formed in response to ischemic or chronic injury.170

Disordered NO generation has been implicated in a wide range of diseases. It is well

established that endothelial NO bioavailability is systemically reduced in patients with coronary

artery disease and heart failure.171,172 In patients with diabetes mellitus reduced NO

bioavailability may result from altered NO metabolism. In diabetic mice, vascular endothelial

dysfunction is associated with uncoupling of eNOS within the endothelium that is caused by

oxidation of its essential cofactor tetrahydrobiopterin (BH4), resulting in a specific loss of

endothelial NO bioavailability.173 The subsequent rise in cGMP accounts for many of the

physiological effects of NO. The NO dependent cGMP response is rapidly and selectively

reduced in diabetic rats, and the cGMP response to exogenous NO donor is progressively

reducedl74. NO and its signaling mechanism modulate various physiological processes,

however, relatively short half-life makes the study less feasible in this field.

Relatively stable NO donors with potential therapeutic value have been developed.

Synthetic chemical reagents that release NO continuously over a period of time under

physiological conditions have long been used in clinical management of cardiovascular diseases.

NO donors developed with therapeutic value must be able to control the amount of NO released,

produce byproducts with minimal side effects, and the release of NO should not be affected by

common biochemical factors. NO decomposes rapidly into nitrite (NO2) and nitrate (NO3) 18









biological solutions. These two stable compounds are indicators of NO activity in vivo and can

be used as an alternative way to analyze NO concentration in serum.17

Carbon Monoxide

Endogenous CO is a signaling molecule that regulates physiological vascular functions.

CO is generated from a family of heme oxygenases (HO), consisting of three isoforms. HO-1 is

an inducible stress enzyme, while HO-2 and HO-3 are constitutively expressed proteins. HO-1 is

inducible after the stimulation of cytokines, hypoxia and NO.176 HO-1 (32 kDa) was first

purified from the livers of CoCl2 Or heme-induced rats and from porcine spleen.177'soHO-1

catalyzes the rate-limiting step in the oxidative degradation of heme to generate CO, bilirubin (an

antioxidant derived from biliverdin) and iron (sequestered by ferritin).18118

Recent researchers have revealed that CO has profound effects on intracellular signaling

processes such as anti-inflammatory, antiproliferative, antiapoptotic, and anticoagulative effects.

Using a HO-1 knockout mice, Bak, et al. showed that CO generated from HO-1 has a protective

role in cardiovascular system from ischemia/reperfusion induced damage.176

The physiological signaling effects of CO involve relatively few defined mechanisms. The

modulations of PKG, PKA, and subsequent stimulation of cGMP or cAMP production are

commonly observed in CO related signaling pathways. There are many similarities between CO

and NO. Both gases are endogenously produced. Their synthetic enzymes, HO and NOS, are

both oxidative enzymes that use NADPH as an electron donor. CO and NO have similar

physiological functions (i.e., vasodilation, inhibition of platelet aggregation, and

neurotransmission), and can act as second messenger.185,186

Similar to NO, CO binds directly to the heme iron of soluble guanylyl cyclase (sGC),

leading to the stimulation of enzymatic activity. The vasoactive properties of CO rely on the

stimulation of sGC and the subsequent elevation of cGMP levels.'" CO-mediated activation of









sGC leads to the increase in cGMP production, with a potency of enzyme activation 30-100

times lower than that of NO.187-189 Other signaling mechanisms of CO include the modulation of

MAPK activation and the stimulation of Ca2+-dependent K+ channel activity.

HO-1 activity is correlated with angiogenic factors. VEGF activates HO-1 expression in

endothelial cells.190,191 In HOrmal tissue, depending on the amount of NO, HIF-1 modulates HO-

1 activity.192 Deshane, et al. demonstrated that SDF-1 directly regulates HO-1 activity which

promotes angiogenesis in different cell types including human and mouse aortic endothelial cells

as well as mouse EPCs.193

A novel class of compounds, termed carbon monoxide-releasing molecules (CORMs), are

stable carbonyl transition metal complex with the capacity of releasing CO in biological systems,

and are becoming a useful research tool to explore the mechanism of which CO exerts its

pharmacological activities.194,195 Several experiments of CORMs have provided mechanistic

insights in the behavior of CO in biological systems. CORM-1 (dimanganese decacarbonyl),

CORM-2 (tricarbonyldichloro ruthenium(II) dimmer), and CORM-3

((tricarbonylchloro(glycinato)ruthenium(I) simulate the bioactivities of gaseous CO including

vessel relaxationl96'197, prOtection against ischemia-reperfusion injury 194,198, and prevention of

organ rej section following transplantation and inhibition of the inflammatory response.

Vasodilator Stimulated Phosphoprotein

Nitric Oxide dependent, vasodilator stimulated phosphoprotein (VASP) plays a pivotal role

in cytoskeletal actin regulation. VASP belongs to a family of proline-rich proteins that includes

the Drosophila melan2oga~ster protein Enabled (Ena), its mammalian ortholog Mena, and the Ena-

Vasp-like protein Evl.199 All Ena/VASP family members share a highly conserved amino-

terminal Ena/VASP homology 1 (EVH1) domain followed by a proline-rich central region and a

carboxy-terminal Ena/VASP homology 2 (EVH2) domain.200 The structure of EVH1 domain of









Ena/VASP family has been identified by using X-ray crystallography and nuclear magnetic

resonance spectroscopy. The EVH1 domain serves as an Ena/VASP protein-binding site for the

focal adhesion proteins including vinculin, zyxin, and axon guidance proteins roundabout

(Robo). EVH1 domain-protein interactions are necessary for the localization of Ena/VASP

family to focal adhesions as well as to the periphery of protruding lamellipodia. 199,201,202 The

central proline-rich region has binding sites for several SH3 and WW domain-containing

proteins and profilin. The C-terminal EVH2 domain not only mediates tetramerization of

Ena/VASP proteins but also binds both monomeric (G) and polymerized (F) actin. The EVH2

domain functions appear to be important for both actin-filament bundling and

stabilizati on. 199,200, 203

VASP is a cytoskeletal actin filament promoting protein, which is involved in platelet

activation, cell adhesion, and migration.204,205 VASP mutant mice exhibit defects in the actin-

dependent process of platelet aggregation.206 The results from genetic approaches such as loss-

of- function experiment, site directed mutation, or overexpression study are suggesting that the

importance of Ena/VASP proteins in the developmental and physiological processes in various

cell types. For instance, VASP modulates T cell activation, phagocytosis, and epithelial

morphogenesis. It also induces migration of neutrophils, fibroblasts, and neurons.207-212

In mammalian cells, VASP is localized to focal adhesions and areas of dynamic membrane

activity in actin-filament assembly. In endothelial cells, for instance, VASP functions in

membrane ruffling, aggregation, and tethering of actin filaments during the formation of

endothelial cell-substrate and cell-cell contacts. VASP expression is increased in endothelial

cells during angiogenesis.213









Hypoxia has a direct influence on barrier function by decreasing VASP expression.

Rosenberger, et al. showed that VASP transcription was reduced in a HIF-1-dependent manner

(HIF-loc functioning as a transcriptional repressor). They further demonstrated hypoxia-

dependent binding of HIF-1 to the human VASP promoter by functional studies using chromatin

immunoprecipitation and site-directed mutagenesis. VASP expression during hypoxia is

involved in tissue permeability.214

Vertebrate VASP was discovered and characterized as a common substrate for both PKA

and PKG serine/threonine kinases.215 Elevated guanosine 3', 5'-cyclic monophosphate (cGMP)

or adenosine 3', 5'-cyclic monophosphate (cAMP) stimulates the phosphorylation of VASP. To

date, three phosphorylation sites (Serl57, Ser239, and Thr278) have been identified.

Phosphorylation of Ser 157 of VASP leads to a shift in apparent molecular mass in SDS-PAGE

from 46 to 50 kDa, indicating phosphorylation causing a change in secondary structure of the

molecule.216 Phosphorylation of serine 239 in VASP is a useful marker for monitoring PKG

activation as well as signaling pathway. Unlike phosphorylation of serine 157, it doesn't alter

the electrophoretic motility of VASP.217,218 During the subsequent cell moving, VASP becomes

heavily phosphorylated.219 Phosphorylated VASP has been localized to cell-cell junctions and

could be co-immunoprecipitated with the tight-junction marker zoula-occludens-1 (ZO-1)

protein from endothelial cells.215 There is a need for more research to identify whether such

phosphorylation reflects the overall phosphorylation of all VASP within the cell. The question

of whether VASP at the leading edge or VASP at focal adhesions are differently phosphorylated

also remains to be answered.20









Significance (Specific Aims)

The role of HSC and EPC in supporting postnatal vasculogenesis has been extensively

studied regarding many physiological and pathological situations. Blood vessel development is a

complex process, involving multiple proteins expressed by different cell types, all contributing to

an integrated sequence of events.

The goal of this study was to highlight IGFBP-3 among other angiogenic factors involved

in vessel development. IGFBP-3 regulates cell activity in various ways and exerts both pro-

angiogenic and anti-angiogenic actions. Characterization of its putative receptor which initiates

downstream cascade is thought to provide better description of IGF-independent effects.

Numerous investigators have tried to determine the characteristics of IGFBP receptors; however,

specific IGFBP receptors still remain unknown.

Here, we demonstrate that IGFBP-3 has a critical function in vessel development related

with NO and SK/S1P signaling pathways.

The underlying hypotheses are; (1) IGFBP-3 has an angiogenic effect on HSCs as well as

EPCs. (2) IGFBP-3 modulates EPC migration to participate in neovascularization by influencing

NO generation and VASP redistribution. (3) The down stream pathway of IGFBP-3 on EPCs is

related to SK signaling.









CHAPTER 2
METHODS AND MATERIALS

Cell Preparation

Mobilized peripheral blood derived human CD34+ cells and CD14+ cells were

commercially purchased (Lonza Walkersville, Inc. Walkersville, MD). A vial of frozen cells

were thawed in a 37oC water bath then washed with hematopoietic progenitor growth medium


(HPGM; Lonza Walkersville, Inc. Walkersville, MD) containing 10% fetal bovine serum (FBS)

and 20 U/ml of DNase I (Sigma-Aldrich, St. Louis, MO). CD34+ cells were cultured in HPGM

supplemented with 25 ng/ml of human stem cell factor (SCF; R&D Systems, Inc. Minneapolis,

MN), 50 ng/ml of human thrombopoietin (TPO; R&D Systems, Inc. Minneapolis, MN), and 50

ng/ml of human Flt/Flk2 ligand (FL; R&D Systems, Inc. Minneapolis, MN) for maintaining an

undifferentiated state. CD14+ cells were maintained an undifferentiated state in HPGM

supplemented with 10% FBS.

To expand CD34' cells, defined serum free medium (StemSpan SFEM; StemCell

Technologies, Inc. Vancouver, Canada) was used for culture. One ml of StemSpan SFEM with

the addition of cytokines cocktail (100 ng/ml FL, 100 ng/ml SCF, 20 ng/ml interleukin-3, and 20

ng/ml interleukin-6; StemCell Technologies, Inc. Vancouver, Canada) and 50 ng/ml TPO (R&D

Systems, Inc. Minneapolis, MN) enables 300,000 CD34' cells to proliferate and expand without

differentiation. The number of cells was determined with a hemacytometer (Hausser Scientific,

Horsham, PA) every 3 days when the medium was changed.

Cryo-preserved human lung derived microvascular endothelial cells (HMVEC-L) were

commercially purchased (Lonza Walkersville, Inc. Walkersville, MD). Once the cells were

thawed, endothelial cell basal medium-2 (EBM-2) supplemented with growth supplements (5%

FBS, 0.04% Hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% IGF-1, 0.1% ascorbic acid, 0. 1%









EGF, and 0. 1% GA-100) (EGM-2-MV singleQuots) (Lonza Walkersville, Inc. Walkersville,

MD) was used for optimal growth and proper maintenance. When plated cells were confluent

the cells were washed twice with PBS then 0.025% trypsin and 0.01% EDTA mix (Lonza

Walkersville, Inc. Walkersville, MD) was added. The plate was placed for 45 seconds at 370C in


humidified 5% CO2 incubator. The trypsin was neutralized using twice the volume of trypsin

neutralizing solution (Lonza Walkersville, Inc. Walkersville, MD) and then cells were

centrifuged at 1000 RPM in an Eppendorf CT 5810R. The pellet was resuspended with EBM-2

containing growth supplements for splitting into new plate.

Endothelial progenitor cells (EPC) were isolated from peripheral blood from healthy

individuals or diabetic patients. The blood was collected into cell preparation tubes (CPT; BD

Biosciences, San Jose, CA) and spun to obtain mononuclear cells. EPC was separated from the

mononuclear fraction using a CD34+ isolation kit (StemCell Technologies, Vancouver, CA).

Mononuclear cells (2 x 10 ) were incubated with a CD34+ selection cocktail for 15 minutes. 50

Cll of nanoparticles were then added to the cells and incubated for a further 10 minutes. The

suspension volume was increased to 2.5 ml and the tube containing the cells was placed in a

magnet for 5 minutes. The supernatant from the tube was poured off and the remaining CD34+

cells in the tube were resuspended in culture media (Endocult; StemCell Technologies,

Vancouver, Canada). These isolated EPCs were cultured for tube formation and differentiation

assay using Endocult containing 20% Endocult supplement (StemCell Technologies, Vancouver,

Canada) .

Migration Assay

CD34+ cells and CD14+ cells were stained with calcein-AM (Molecular Probes, Eugene,

OR), prior to loading them into the upper wells of a disposable chemotaxis chamber (Neuro









Probe, Gaithersburg, MD). The lower wells were filled with IGFBP3 at 0, 1, 10, and 100 ng/ml

dissolved in HPGM (negative control) or HPGM supplemented with 20% FBS (positive control).

The chamber was incubated at 37oC, 95% humidity, 5% CO2 for 4.5 hours. The number of

migrating cells was determined by relative fluorescence of the lower chamber using a SynergyTM

HT (Bio-Tek Instruments, Inc. Winooski, VT) with an excitation of 485 f 20 nm and emission of


528 f 20 nm.


EPC Tube Formation

Peripheral blood was collected into CPT tubes with heparin (BD Biosciences, San Jose,

CA) by routine venipuncture. The mononuclear cells were collected after centrifugation at room

temperature in a swinging bucket rotor at 1,800g for 20 minutes. These cells were then cultured

on fibronectin-coated culture dishes (BD Biosciences, San Jose, CA) with Endocult stem cell

liquid media (Stem Cell Technologies, Vancouver, CA) per manufacturer' s protocol. IGFBP3

was added to the cultures at day 3 at 0, 1, 10 and 100 ng/ml. Cells were imaged on day 5.

Images were captured with a fluorescence microscope (Axiovert 135; Carl Zeiss, Thornwood,

NY). The endothelial nature of the cells was confirmed by incorporation of Dil (1, 1'-

dioctadycl-3, 3, 3'3 '-tetramethyl-indocarbocyanin percholrate)-labeled acetylated-LDL

(Molecular Probes, Eugene, OR) at 50Cpg/ml final concentration.

Cell Proliferation Assay

A high sensitivity cell proliferation kit (ViaLight Plus Kit: Lonza Rockland, Inc. ME) was

used according to manufacture's protocol to assay for cell proliferation. Cell proliferation was

measured based on the bioluminescent cytoplasmic ATP level. Cultured cells on 96 well plates

were removed from the incubator and allowed to cool -to room temperature for about 5 minutes.

50Cl1 of the cell lysis reagent (Lonza Rockland, Inc. ME) was added to each well to extract ATP









from metabolically active cells. The cell lysate (100 Cl) was transferred to a white walled

luminometer plate (Lonza Rockland, Inc. ME). Then ATP monitoring reagent plus (AMR

PLUS: Lonza Rockland, Inc. ME) was added to each well to generate a luminescent signal. The

luminometer (BioTek Instruments, Inc. Winooski, VT) was programmed to take one second

integrated luminescence reading of each well.

Nitric Oxide Measurement

NO production was measured by the use of 4-amino-5-methylamino-2' 7'-

difluorofluorescein diacetate (DAF-FM diacetate: Invitrogen Carlsbad, CA). The cells were

washed with PBS containing calcium, magnesium, and 1 mg/ml glucose to remove phenol red in

the culture media. The cells were incubated with 5 CLM DAF-FM diacetate for 1 hour on ice in

the dark. Cells were washed with PBS containing calcium, magnesium, and 1 mg/ml glucose

then transferred to black 96 well plate (Nunc International, Rochester, NY). NO fluorescence

was measured using excitation and emission wavelengths of 488 f 20 nm and 520 f 20 nm


respectively.

Western Blot Analysis

For western blot analysis 8 Cpg of total protein was loaded on a 10-20% gradient Criterion

gel (BioRad, Richmond, CA). The samples were electrophoresed at 120 V for 20 minutes to

allow for stacking the samples and then 140 V for 65 minutes to separate proteins. The proteins

were transferred from the gel to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules,

CA) using a semi dry membrane apparatus (Bio-Rad Laboratories, Inc., Hercules, CA) at 20 V

for 40 minutes. The membrane was blocked in Odyssey blocking buffer (LI-COR Biosciences,

Lincoln, NE) for 1 hour at room temperature with gentle shaking. The blocked membrane was

incubated with diluted primary antibody at 4oC overnight in a cold room. The membrane was









washed 4 times for 5 minutes in PBS containing 0.1% Tween-20 (Fisher Scientifie, Pittsburgh,

PA) and then incubated with diluted fluorescently labeled secondary antibody for 1 hour at room

temperature with gentle shaking. The membrane was washed 4 times for 5 minutes each with

PBS containing 0.1% Tween-20 followed by Einal washing with PBS to remove excessive

Tween-20. The membrane then was scanned in the appropriate channels using Odyssey infrared

imaging system (LI-COR Biosciences, Lincoln, NE). The same membrane was used to detect

the internal protein control, cofilin (Sigma-Aldrich, St. Louis, MO).

In-cell Western Assay

HMVEC-L was grown in 96-well plate (Nalge Nunc International, Rochester, NY). When

the cells reached 75-80% of confluence, cells were washed twice with PBS. Instead of serum

starvation, cells were treated with IGFBP-3 in EBM-2 without cytokines during the treatment.

CD34' cells were cultured in defined serum free medium (StemSpan SFEM; StemCell

Technologies, Inc. Vancouver, Canada) to obtain the optimal number of cells. StemSpan SFEM

with the addition of cytokines cocktail (100 ng/ml FL, 100 ng/ml SCF, 20 ng/ml interleukin-3,

and 20 ng/ml interleukin-6; StemCell Technologies, Inc. Vancouver, Canada) and 50 ng/ml TPO

(R&D Systems, Inc. Minneapolis, MN) enables CD34' cells to proliferate and expand without

differentiation. The number of cells was determined with a hemacytometer (Hausser Scientifie,

Horsham, PA). The cells were transferred into 96-well plate (10,000 cells per well: BD Falcon,

San Jose, CA). After incubation with or without CO or NO donor, cells were Eixed with 4%

formaldehyde for 20 minutes at room temperature then pelleted by centrifugation. Fixing

solution was removed then triton washing solution (PBS containing 0. 1% triton X-100) was

added to the cells for permeabilization. After permeablization, cells were blocked in LI-COR

Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1.5 hour at room temperature

with moderate shaking. Blocking buffer was removed by aspiration and the cells were incubated









with 50 Cl1 of diluted primary antibody at 4oC overnight in a cold room. The cells were washed 4

times for 5 minutes in PBS containing 0.1% Tween-20 (Fisher Scientifie, Pittsburgh, PA) and

then incubated with diluted fluorescently labeled secondary antibody. After 1 hour the cells

were washed 4 times for 5 minutes each with PBS containing 0.1% Tween-20 followed by a

Einal washing with PBS to remove excessive Tween-20. The 96-well plate was scanned in the

appropriate channels using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln,

NE). Relative quantifieation was normalized and re-adjusted in cell number from well to well

using DNA staining.

Quantitative Transcription Analysis with Real Time Polymerase Chain Reaction (RT PCR)

Total mRNA from human CD34' cells, human CD14' cells or retina from the mouse pups

was isolated using the Total RNA Mini Kit (Bio-Rad Laboratories, Inc., Hercules, CA). The

mRNA was transcribed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules,

CA) and real-time PCR were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories,

Inc., Hercules, CA). Primers for the PCR were designed using vector NTI (Infomax, North

Bethesda, MD) and purchased (Intregrated DNA Technologies, Coralville, IA).

Immunohistochemistry

EPCs were cultured on 8-well tissue culture chamber slides coated with human fibronectin

(BD Biosciences San Jose, CA) and treated with 100 CLM diethylenetriamine/nitric oxide adduct

(DETA-NO) (Sigma-Al dri ch, St. Loui s, MO), 10 CLM CO donor (tri carb onyl di chlororuthenium

(II) dimer) (Sigma-Aldrich, St. Louis, MO), or 100 ng/ml IGFBP-3 (Upstate cell signaling

solution, Lake Placid, NY) for 15 minutes or 4 hours. After treatment, medium was removed

and fresh ice cold 4% paraformaldehyde (PFA) was added and the samples held overnight at

40C. Cells were then washed in PBS and permeabilized with 0.1% Triton X-100 (Fisher

Scientific, Pittsburgh, PA) for 30 minutes at room temperature. The cells were washed 3 times









with PB S and blocked in 10% normal goat serum (Jackson ImmunoResearch Labs, West Grove,

PA) or 1% bovine serum albumin (BSA; Sigma-Aldrich, St.Louis, MO) at room temperature to

block non specific antigens. After 1 hour the cells were incubated with 5 Cpg/ml mouse anti-

VASP antibody (BD Biosciences, San Jose, CA) in 5% normal goat serum overnight at 4og.


Specific secondary antibody, goat anti-mouse IgGl-fluorescein isothiocyanate (FITC; Southern

Biotech, Birmingham, AL) was diluted and added to each chamber for 1 hour at room

temperature. Then the cells were washed, dried, and mounted with Vectashield 9 4', 6-diamino-

2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA) for DNA labeling. Cells

were examined using a fluorescence microscope (Nikon Eclipse TE200) using a Nikon plan-

fluor 40 X 1.30 oil objective and a FITC-conjugated standard filter set (520 + 2 nm). Pictures

were captured using a SPOTT= digital camera 0.60X HRDO60 NIK (Diagnostic Instruments,

Inc. Sterling Heights, MI) and processed using SPOTT" Advanced software Version 2.2.1 for

Windows.

Flow Cytometry Analysis

Protein expression of VEGF receptor, phosphorylated eNOS and CD133 cell surface

antigen in CD34+ cells were evaluated using flow cytometry analysis. VEGF receptor expression

in CD34' cells was examined after incubation for 0 or 15 minutes in 5% CO2 at 370C with


exposure to 100ng/ml IGFBP3 (Upstate cell signaling solution, Lake Placid, NY). Following

treatment, the cells were permeabilized using a Cytofix/Cytoperm Kit (BD Bioscience, San Jose,

CA). The cells were blocked with 10% normal human serum (Jackson Immuno Reserch labs,

West Grove, PA) in PBS. Ten Cpg anti-VEGFR-1 (Santa Cruz Biotechnology, Inc. Santa Cruz,

CA) or 10 Cpg anti-VEGFR-2 antibody (NeoMarkers, Fremont, CA) was added to the cells and

subsequently incubated for 30 minutes on ice. The cells were washed with PBS and incubated









with 23 Cpg of FITC conjugated goat anti-mouse antibody (Jackson Immuno Research labs, West

Grove, PA) in the dark for 30 minutes on ice. Cells were then washed and analyzed by flow

cytometry. The isotype control for both VEGFR-1 and VEGFR-2 antibodies was anti-GFP

(Molecular probes, Carlsbad, CA) antibody (15 Cpg). To measure the eNOS protein expression,

the cells were incubated with 100 ng/ml of IGFBP-3 for up to 72 hours. IGFBP-3 was added

every 24 hours. When the cells were collected for analysis, cells were permeabilized and

blocked. Then 5 Cpg/ml of anti-eNOS antibody (BD Bioscience, San Jose, CA) was added for 30

minutes. As a secondary antibody, 23 Cpg of FITC conjugated goat anti-mouse IgG (Jackson

ImmunoResearch Labs, West Grove, PA) was used. The surface expression of CD 133 antigen

was assessed using a phycoerythrin (PE) conjugated anti-CD133 antibody (Miltenyi Biotec Inc.

Auburn, CA). CD34+ cells were incubated with or without IGFBP-3 for up to 72 hours as above.

Isotype control for this experiment was PE-conjugated mouse IgGa,K immunoglobulin isotype


control monoclonal antibody (BD Bioscience, San Diego, CA). Apoptotic dead cells were

removed before analysis by 7-aminoactinomycin D (Sigma-Aldrich, St. Louis, MO) positive

selection. Data were acquired with FACS Calibur flow cytometer (BD Biosciences, San Jose,

CA) and were analyzed with BD Cell QuestTM (BD Biosciences, San Jose, CA).

Hematopoietic Stem Cell (HSC) Transfection

Mouse HSCs were obtained from bone marrow isolated from homozygous transgenic gfp

mice. Highly enriched gfp Scal+ (stem cell antigen 1) and c-kit+ HSCs were obtained by

fluorescence-activated cell sorting (FACS). Cells were transfected with a recombinant adeno-

associated virus (rAAV) vector encoding IGFBP-3. The rAAV vector was chosen for long term

IGFBP-3 overexpression in the eye. Expression of IGFBP-3 was selectively increased in

proliferating endothelial cell by a specific promoter composed of 7 x 46-mer multimerized









endothelin enhancer (ET) upstream of a human Cdc6 (cell division cycle 6) promoter. Gfp+

HSCs were transfected using polyethylenimine (PEI) plasmidd complexes. 4.2 mg/ml stock PEI

(Sigma-Aldrich, St. Louis, MO) solution was made in acidified distilled water (pH 5.0). The PEI

plasmidd complexes were prepared by adding branched PEI to the plasmid DNA. 100 Cl1 PEI

plasmidd complex were composed of 150 mM sodium chloride (NaC1) 1 Cpg DNA, and 24 Cl~ PEI

stock solution. The complexes were then mixed by vortex mixer set on high for 10 seconds and

incubated for 30 minutes at room temperature then added to the cells.

Experimental Animals

All animal procedures conducted in this study were in agreement with the National

Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (DHEW

Publication No. NIH 80-23, Offices of Science and Health Reports, DRR/NIH, Bethesda, MD

20205). All protocols were approved by the University of Florida Institutional Animal Care and

Use Committee (IACUC). Timed pregnant C57BL/6 mice were obtained from Jackson

Laboratories (Bar Harbor, ME) and housed in a temperature controlled room with a 12-hour

light/dark cycle in the University of Florida Health Science Center Animal Resources facilities.

Oxygen Induced Retinopathy (OIR) Mouse Model

The OIR mouse model was used to induce the formation of preretinal neovascularization in

mouse pups. On postnatal day 7 (P7), the pups and the nursing dam were placed into an oxygen

chamber and exposed to hyperoxic levels of 02 (75%) for 5 days. On Pl2 the mice were

returned to room air. The return to normoxic conditions (21% 02) after being held at 75% 02

simulates a hypoxic stimulus that initiates the formation of the preretinal vasculature. The mice

were sacrificed at Pl2.5 and Pl7 and their eyes removed for analysis.

One cohort of animals (n=12) underwent the OIR model and were euthanized at Pl2.5 to

examine the expression of VEGF-A and IGFBP-3 in combined posterior poles and neural retinas.









A second cohort of mice was intravitreally inj ected on Pl with either the plasmid expressing

mouse IGFBP-3 under the control of the proliferating endothelial cell-specific promoter or the

cloning vector as an injection control. For intravitreal injection into Pl mouse pups, ice-induced

anesthesia was performed by placing the neonate on a plastic shield over a layer of crushed ice

for 1 to 2 minutes. By utilizing a proliferating endothelial cell-specific promoter IGFBP-3

expression was targeted to areas of neovascularization. Selected animals were euthanized

immediately upon removal from hyperoxia at Pl2.5. Eyes from mice injected with IGFBP-3

plasmid (n=9) were compared with eyes from mice inj ected with empty plasmid vector (n=9), or

the uninj ected eye of the same animal (contralateral eye). A third cohort of mice (n=18) was

inj ected on Pl with mouse gfp+ HSCs (5 x 103 CellS per eye in 1 Cll inj section volume) transfected

with the identical plasmid as described above. The mice were subjected to the OIR model and

euthanized at Pl7. Data from these mice were compared with the uninjected eye of the same

animal or with mice inj ected with mouse gfp' HSCs transfected with empty plasmid (n=18).

Retinal Flat Mounts

The mouse thoracic cavity was opened, the right atrium was punctured with a 27-gauge

needle and the left ventricle was cannulated with a 22-gauge angiocatheter, and the mouse was

perfused with rhodamine-labeled dextran for preparation of retinal flat mounts. One whole eye

from the animal was removed and fixed in 10% PFA overnight. The retina was washed

overnight in running water and then incubated for 3 hours at 370C in 0.1M Tris buffer (pH 7.8)

supplemented with DIFCO BACTO 1:250 Trypsin (Invitrogen, Carlsbad, CA). The incubation

was terminated after the internal limiting membrane had been removed. All nonvascular cells

were brushed away from the vasculature. The retina was then mounted onto a glass slide.









GS Isolectin and GFP Double Labeled Immunohistochemistry

Retinal whole-mounts were prepared as described above. GS isolectin B4 (Sigma-

Aldrich, St. Louis, MO) and chicken anti-GFP or mouse anti-GFP/EGFP (Chemicon, Temecula,

CA) were used to co-visualize gfp' cells with the vasculature. Retinal whole mounts (n=6) were

permeabilized with 0.1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) in PBS. Samples were

then blocked in 1% BSA in PBS for 30 minutes and retina was incubated with biotinylated GS

isolectin B4 (Sigma, St Louis, MO) overnight. The retinas were washed twice for 10 minutes in

PBS supplemented with 0.1% Triton X-100 followed by a last 10 minute wash in PBS. The

retinas were then incubated with streptavidin Cy-3 (1:100) for 2 hours at room temperature.

Retinas were incubated overnight at 40C with diluted anti-GFP antibody then washed with PBS

supplemented with 0.1% Triton X-100. The retinas were allowed to incubate for 4 hours at room

temperature with the appropriately diluted secondary antibodies followed by washing. Finally

the whole retinas were flat mounted with the ganglion cell layer up in Prolong Anti-Fade

(Molecular Probes, Eugene, OR).

Microscopy and Mapping

Retinal whole-mounts (n=6) were examined by both deconvolutional and confocal

microscopy. Zeiss microscope (model Axioplan 2 attachment HBO 100; Carl Zeiss,

Inc.Germany) and Axiocam HRm camera (Carl Zeiss, Inc, Germany) were used for

deconvolutional analysis. Confocal microscopy was conducted with an argon-krypton laser

(Leica Microsystems, Wetzlar, Germany) mounted on a Leica DMRBE epifluorescence

photomicroscope. Alexa Fluor 488 and Cy3 fluorescence was excited sequentially at 488 and

550 nm respectively. Images were processed with Adobe Photoshop software (Adobe Systems,

Inc. San Jose, CA).









Vascular Density Analysis and Statistical Analysis

To validate vascular density, morphological analysis needed to be changed to a

quantitative measurement. After retina whole mounts were immunostained, vasculartues in

retina were then subj ect to vascular density analysis. The retina was divided into 3

representative Hields of views from each of the central, mid-peripheral and peripheral retinas.

Fields of view selected as peripheral retinas included regions of capillary sized vessels directly

adj acent to radial arterioles. The area of central and mid-peripheral retina included the radial

arteriole. Then each field was captured using the 40x objective. A 10 x 10 grid was

superimposed onto the micrograph and the incidence of presence or absence of vessels at the

intersection points of each grid was determined so that vascular density expressed as a number

from 0 to 100. The mean vascular density incidence was determined for each area and compared

with its control. The data is presented as means + standard deviation (SD), and the statistical

significance of differences among mean values was determined by one-way ANOVA and the

Tukey HSD multiple comparison post hoc tests for the hyperoxia experiments, and a two-tailed

t-test utilized for the hypoxic experiment. ANOVA statistical analysis was performed with SPSS

13.0 software (SPSS, Chicago, Illinois), and two-tailed t-test statistical analysis was performed

with a P value of <0.05.









CHAPTER 3
RESULTS

IGFBP-3 Induces Migration of CD34 cells and Endothelial Cells

CD34+ cells are known to leave the circulation and migrate along a hypoxic gradient into

sites of ischemia and it is believed that CD34 cell trafficking is mainly regulated by hypoxia-

regulated factors. To determine whether IGFBP-3 functions as a hypoxia-regulated factor, the

effect of IGFBP-3 on migration of different cell types was examined. Migration assays for

circulating cells were performed using modified Boyden chamber assay. Figure 3-1A

demonstrates that IGFBP-3 stimulated the migration of circulating CD34' cells in a

concentration-dependent manner, whereas circulating CD14+ monocytes showed a blunt

response to increasing concentration of IGFBP-3.

Human retinal endothelial cells (HREC) were also exposed to varying concentrations of

IGFBP-3 for 12 hours using the Boyden chamber assay for adhered cells. As depicted in Figure

3-1B, primary cultured HREC migrated toward IGFBP-3, but the response was not as robust as

seen with the circulating CD34 population. These results (Figure 3-1A and B) demonstrate that

IGFBP-3 has a cell-type specific chemotactic function.

Compared to mature endothelial cells, the response of CD34 cell to IGFBP-3 was

exceptionally sensitive even at the lowest concentration (Ing/ml) supporting that mobilization of

bone marrow derived cells can be triggered by subtle changes in a hypoxia regulated factor such

as IGFBP-3.

IGFBP-3 Increases Expression of VEGF Receptors on CD34 cells

To examine possible was in which IGFBP-3 could modulate CD34 cell response to

hypoxia, we examined whether IGFBP-3 could influence VEGF and SDF-1 receptor expression

on CD34 cells. VEGF and SDF-1 are well known stem cell homing factors that are also









hypoxia-regulated factors. Exposure of CD34 cells to varying concentrations of IGFBP-3

resulted in increased expression of VEGFR-1 (Figure 3-2A) and VEGFR-2 (Figure 3-2B) on

CD34 cells. By contrast, IGFBP-3 did not have an effect on CXCR-4 expression in these cells

(Figure 3-2C). Figure 3-2 suggested that IGFBP-3 has a VEGF-dependent, SDF-1-independent

function on CD34 cells.

IGFBP-3 Promotes CD34 cells Differentiation to Endothelial Cells

Exposure to IGFBP-3 for 72 hrs resulted in a reduction of CD 133 surface expression in

CD34 cells (Figure 3-3A), which is associated with promoting differentiation of immature cells

to a more committed phenotype. EPC grown on fibronectin showed a dose-dependent tube

formation and acetylated LDL incorporation compared to control untreated cells (Figure 3-3B),

supporting that IGFBP-3 can influence multiple steps that are relevant to angiogenesis.

IGFBP-3 Enhances CD34 cells Proliferation

To determine whether IGFBP-3 modulates proliferation of CD34 cells in vitro, CD34 cells

were cultured in the presence of 100 ng/ml IGFBP-3. After 24 hour, 3 days, and 5 days

suspension culture, cytoplasmic ATP was detected. IGFBP-3 increased the proliferation of

CD34 cells by 36. 11% (day 3) and 56.03% (day 5) compare to untreated control (Figure 3-4).

Corroborated with the result observed in Figure 3-3B, IGFBP-3 enhances the proliferation of

CD34 cells as well as EPC.

Expression of Hypoxia-regulated Factors in Retina

Messenger RNA was extracted from posterior cups including neural retinas of mouse pups

to check the expression level of IGFBP-3 in ischemic retina. Neonatal mice were divided into

two groups. One group was exposed to high oxygen for 5 days then returned to normal oxygen

tension and the other group was subjected to normoxia control. VEGF and IGFBP-3 mRNA

levels were determined using reverse transcription on total mRNA followed by real time PCR on









the cDNA products using specific primer pairs for VEGF and IGFBP-3. As shown in Figure 3-5,

the mRNA expression of VEGF and IGFBP-3 was significantly increased in hypoxic retina. The

fold-change in IGFBP-3 expression was markedly greater that the fold-change in VEGF. It

supports the importance of IGFBP-3 response following hypoxia.

IGFBP-3 Protects Neonatal Retinal Vessels from Oxygen Induced Vaso-obliteration

To validate the function of IGFBP-3 in vivo, mouse pups were injected with rAAV

protein expression vectors expressing IGFBP-3 on postnatal day 1 (Pl). The expression of the

IGFBP-3 was driven by a proliferating endothelial cell specific promoter (ET/cdc6). The

uninj ected eyes (contra-lateral) of IGFBP-3 plasmid inj ected mice were used as one of the

control conditions and empty plasmid injected eyes served as the other control. Vessels positive

for Griffonia simplicifolia isoletin B4 (GS isolectin) show the changes in the vasculature

following high oxygen exposure (Figure 3-6 A to L). The GS isoletin conjugated to HRP

provided a low magnifieation view of the entire retinal vasculatures (Figure 3-6 M and N). The

vessels in the IGFBP-3 treated eye had a more normal and mature vascular tree (Figure 3-6 A-D

and M), than eyes treated with plasmid control. Vessel growth was shown in the IGFBP-3

inj ected eyes as evidenced by the presence of lectin positive vascular extensions migrating

towards the avascular peripheral retina (arrows in Figure 3-6 D).

In contrast, massive oxygen-induced vaso-obliteration was shown in empty plasmid

inj ected retina (Figure 3-6 E-H). The remaining vascular remnants, shown at higher

magnification in Figure 3-6 F-H, lacked effective vascular perfusion and had a highly aberrant

branching pattern (Figure 3-6 N). The morphology of vascular remnants in the peripheral retina

of the uninj ected eyes (the contra-lateral eye of the pups inj ected with IGFBP-3 containing

plasmid) had regions with vascular abnormalities including reduced capillary density (Figure 3-6

I-L) and closure of capillary segments (arrow in Figure 3-6 K). As depicted in Figure 3-6, over-










expressing IGFBP-3 in vivo resulted in the maintenance of a vascular bed with a more normal

morphology under hyperoxia condition.

Quantitative Analysis of Vascular Density in Vaso-obliteration Phase

To determine vascular density, morphological analysis was performed. Figure 3-7 shows

representative retinas from IGFBP-3 expressing plasmid inj ected eyes (Figure 3-7 A-C), contra-

lateral uninjected eyes (D-F), and control plasmid injected eyes (G-I). A 10 x10 grid was

superimposed onto the 40 X obj ectives from each field of retina and the vessels found at

intersection points of each grid were determined. The number counted as a vascular density was

expressed as a percentage from 0 to 100 (bottom right corner of the image).


Figure 3-7 J summarizes quantitative measurement of vascular density results from

central, mid peripheral, and peripheral regions of retinas in each group. As revealed in Figure 3-

7 J, IGFBP-3 protected the retinal vasculatures from hyperoxia-induced vessel regression in mid

peripheral and peripheral regions of the retina.

IGFBP-3 Decreases the Incidence of Pre-retinal Neovascularization

Abnormal vessel growth in pre-retinal region is a hallmark of proliferative retinopathies.

Pre-retinal blood vessels grow outside the retinal inner limiting membrane into the vitreous space

under pathological circumstances. Neovascularization in the eye of OIR model was evaluated by

the average number of pre-retinal endothelial nuclei per H&E stained retinal section. Figure 3-8

shows reduced aberrant neovascularization by induction of IGFBP-3 in mouse retina.


IGFBP-3 Expression in Transfected HSC

To examine IGFBP-3 expression by the plasmid, HSC were transfected with IGFBP-3

expressing plasmid and total mRNA was isolated. The promoter (ET/cdc6) has been previously

characterized both in vitro and in vivo. 220,221 Low molecular weight PEI was used for effective









transfection of HSC. Transfection efficiency of 40% was typically observed. The fold-change

of IGFBP-3 in transfected cells was significantly increased compared to untransfected HSC

(Figure 3-9).

Co-localized IGFBP-3 Expressing gfp HSC within the Vasculature Inhibit
Neovascularization

To evaluate the effect of endogenous IGFBP-3 on HSC behavior in vivo, Pl mouse pups

underwent intravitreal inj section with IGFBP-3 plasmid-transfected HSC. The effect was observed

by incorporation of gfp' HSC into the retinal vasculature (Figure 3-10 A-C). Gfp HSC localized

vascular endothelial cells were evident in radial arterioles (Figure 3-10A) and hemangiomas (B).

In addition, filopodial extensions were seen originating from the neovascular clump towards

avascular retina (C). Eyes injected with IGFBP-3 transfected HSC showed less pathological

neovascularization compared to uninj ected eyes or eyes inj ected with control transfected HSC.

Figures 3-10D and 3-10E demonstrate quantitative vascular density from the retinas during

hyperoxia and hypoxia phase, respectively. IGFBP-3 overexpressing HSC inhibit abnormal

neovascularization in hypoxia phase by protecting neonatal retinal vessels from oxygen induced

regression in hyperoxia phase.


Hypoxia-regulated Factors and Nitric Oxide Signaling

To support hypothesis that hypoxia-regulated factors modulate EPC mobilization by

increasing nitric oxide and activation of its downstream signaling pathways, we examined the

downstream signaling of two well known EPC chemoattractant, VEGF and SDF-1. CD34 cells

were treated with either 25 ng/ml VEGF or 100 nM SDF-1. Anti-phoshporylated eNOS

antibody was used to demonstrate whether these hypoxia-regulated factors activate eNOS in

CD34 cells. VEGF induced eNOS phosphorylation in 15minutes (Figure 3-11A), however SDF-

1 showed no significant effect on eNOS phosphorylation in CD34 cells (Figure 3-11B). SDF-1









mediated its effects through generation of CO rather than NO. SDF-1 increased HO-1

expression to generate CO which in turn triggers cell migration.19

NO and CO Promotes CD34 cells Migration

Direct stimulation of CD34 cells by NO has been reported.170,222 To determine the effect

of CO on cell motility, migration assay was performed with CO-treated CD34 cells. Exposure to

exogenous CO acutely increased cell migration in response to chemotactic stimulus, SDF-1

(Figure 3-12). The NO donor (DETA-NO) was compared to the CO donor (Ru(II)Cl2(CO)3

dimer) and were used for pretreatment of CD34 cells. Figure 3-12 shows both NO and CO has

an effect on CD34 cells migration.

Different Phosphorylation Sites of VASP

VASP function is initiated by phosphorylation. VASP was originally characterized as a

substrate of both PKA and PKG. PKA and PKG phosphorylate VASP on residues-serine 157

and serine 239, respectively. The effects of NO and CO on cell migration is shown in Figure 3-

12. CD34+ cells were incubated for 15 minutes in the presence of either the NO or CO donor.

Both NO and CO exposure enhanced VASP phoshporylation, however, site of phoshporylation

was different (Figure 3-13). As described in Figure 3-13, NO donor increased VASP

phosphorylation at serine 239, whereas CO increased phosphorylation at serinel57 in CD34

cells.

NO Increases VASP Phosphorylation in Diabetic CD34 cells

CD34+ cells from diabetic individuals have been shown to have reduced NO

bioavailability. Previously it was found that exogenous NO administration could correct

decreased EPC migratory response in diabetic CD34 cells.222 To evaluate this observation

further and to determine whether this improvement was due to increased VASP phosphorylation,

the level of phospho-VASP in diabetic CD34 cells was measured by flow cytometry analysis.









Diabetic CD34 cells were isolated from patients with type 1 and type 2 diseases and the cells

were pretreated with the NO donor. There was considerable patient to patient variation in the

level of VASP expression; however NO treatment resulted in stimulation of VASP

phosphorylation at serine 239 in all the diabetic cells (Figure 3-14).

NO and CO Cause VASP Redistribution to the Leading Edge of the Cells

Phospho-VASP is localized to focal adhesions and areas of dynamic membrane activity.

Redistribution of VASP to the leading edge of the endothelial cells in response to exogenous NO

or CO was observed (Figure 3-15). As shown in Figure 3-15 A and D, VASP is evenly

distributed within the cytoplasm under basal conditions. VASP was redistributed to the

advancing edge of the cell following 15 minutes stimulation with NO donor (3-15 B and E). CO

donor also causes VASP redistribution as same pattern as NO donor (3-15C and F).

IGFBP-3 Increases eNOS Phosphorylation

Like the other hypoxia-regulated factors, SDF-1 and VEGF, IGFBP-3 modulates

CD34 cells mobilization (Figure 3-1). We next examined whether IGFBP-3 stimutaled NO

generation in CD34 cells. Two different western blotting analyses were conducted to determine

eNOS activity.

In-Cell western assay is a quantitative analysis that is extremely sensitive. This assay

utilizes an infrared fluorescence antibody. However, the validity of the In-Cell western assay

needs to be examined by comparing it to standard western blotting. Figure 3-16A shows the

result of the In-Cell western assay and 3-16B illustrates the result of standard western blotting

analysis. Both results support that IGFBP-3 increases eNOS phosphorylation at Ser 1177 in

CD34 cells.









IGFBP-3 Induces NO Production

CD34 cells were exposed to IGFBP-3 for 30 minutes then intracellular NO was monitored

with DAF-FM diacetate to confirm increased NO generation in IGFBP-3 treated cells. As shown

in Figure 3-17, IGFBP-3 increased NO production in CD34 cells. NO release from IGFBP-3

treated cells was 3.7 fold greater than untreated cells.

IGFBP-3 Modulates VASP Phosphorylation

Increased NO generation by IGFBP-3 subsequently induced phosphorylation of VASP

(Figure 3-18). CD34 cells were exposed to IGFBP-3 (100 ng/ml) for 0, 10, 30, and 60 minutes.

To obtain whole cell lysate including cytoplasmic protein as well as membrane-bound protein,

2X SDS-PAGE buffer was added to the cells. Two anti-phospho-VASP (Ser 157 and Ser 239)

antibodies were used to detect the different sites of phosphorylation on VASP. Phospho-VASP

at serine 239 was significantly increased by IGFBP-3 treatment.

Inhibition of SK Activity Results in Reduced NO Production

Our preliminary data shows IGFBP-3 enhances SK activity in CD34 cells (not shown

here). To confirm SK is a downstream signaling mediator of IGFBP-3, the level of NO

production from SK inhibitor pretreated cells was measured. CD34 cells were exposed to

IGFBP-3 following pretreatment of SK blocker, dimethylsphingosine (DMS) for 30 minutes. As

depicted in Figure 3-19, NO generation was inhibited in SK blocked cells and it was not restored

by addition of IGFBP-3. This result suggests that IGFBP-3 modulates SK and that the S 1P/SK

pathway is involved in IGFBP-3's modulation of CD34 cells.










1200


" 1000


.5



400

200

0L


Controls


10 100 1000
IGFBP3 (ng/mi)


HRECs


.0300
.E *C 250

S 200
I Positive
0 ,,150 o Negative
.c* 0 GFBP3
.a .9 100 -




Controls 1 10 100
IGFBP3 (ng/ml)


Figure 3-1. IGFBP-3 induces CD34 cells and endothelial cells migration. Modified Boyden
chamber assay was used for circulating cells (A) and the Boyden chamber assay was
performed for adhered cells (B). Statistically significance were presented *P < 0.05
vs. negative control.


[7 positive
I negative
SCD14
C1 CD34





IO "~ "l ""'"' 010 """"' "' "" rg'-'


B



a

r


D D.25 4
Tim~e~ (hours)

O NT 1 ng/mi 0 1D ng/mi 0 100 ngirni


O0 NT 1111 ng/rni O10 ngfrrl E"I100 ng/rn I


A
350

~7-300



200



e 50


50


1I-


1I-
"100 -
80
6D-

21-
E-


~3


0.~25
Tirne (hours)


C.


120


1 OO


Tirne (hours)


Figure 3-2. Receptor levels in CD34 cells following IGFBP-3 exposure. CD34 cells were
exposed to IGFBP-3 for 15 min, 4 h, and 12 h (for CXCR4). A) VEGFR-1. *P <
0.05 vs. medium alone (non treatment, NT) for 15 min and *P <0.001 for 4 h. B)
VEGFR-2. *P < 0.001 vs. NT for both 15 min and 4 h. C) CXCR-4.


3~r

L~t~











A """"r~t G P


80







3 20-



24~hr 481hr 72hr
Tr~eaknent with IGFBP3 (100D ngirnl)


Figure 3-3. IGFBP-3 enhances CD34 cells and EPC differentiation. A) CD34 cell were
exposed to IGFBP-3 (white bars) for 72 hours. Statistically significance were
presented *P < 0.05 vs. control nontreated cells. B) Representative images for
growing EPC in the presence of different concentration of IGFBP-3. Magnification:
X100. Scale bars: 150 Clm for left and center panel and 100 Clm for right panel.










O Untreated O IGFBP3


80000
70000
60000
50000


JF


JF


JF


JF


40000
30000
20000
10000
0


II I


Day 1


Day 3


Day 5


Figure 3-4. IGFBP-3 enhances CD34 cells proliferation. Cells were cultured in StemSpan
SFEM with addition of cytokines cocktail for 5 days. IGFBP3 signifies StemSpan
SFEM with cytokines and 100 ng/ml IGFBP3. *P < 0.001 vs. Day 1 untreated cells
and untreated cells at each time points.





45-


40-


s- 35-


> 30-

cr O Normnoxia
S25-
m Hypoxia

S20-


-a 15-


-10-






VEGF IGFBP3



Figure 3-5. Hypoxia retina expresses IGFBP-3. Neonatal mouse pups (n=6 in each group) was
euthanized at Pl2.5. Under hypoxic condition, both VAGF (*P < 0.05) and IGFBP-3
(*P < 0.0001) mRNA were significantly increased when compared to normoxia
control retina





































































I


r

t


'J


.~n

.~c; ";'"

r


Fiur 3-6 IGFP- prtcsfo yeoi-nucdvsua ersin etiafo teee







Figu~retia 3-.IfB-romt IFBP-3 hplasmid injected eyes (M)and ceeontro vetorinjecomted eyes ()












































m IGFBP-3 injected
W Control plasrnid injected
1 contra lateral un injected


60

50

40

30

20

10
-


peripheral


central rnid-periphera
Position in retina


Figure 3-7. Quantitative analysis of vascular density. IGFBP-3 significantly protected the
retinal vasculature from hyperoxia-induced vessel regression in mid peripheral (*P <
0.001) and peripheral regions (*P < 0.001), but did not have any significant effect on
the central region of the retina (*P >0.05)











O Vector a mulGFBP3

100


90-


80-


c 70-








20-



40






Uninjected Eye Injected Eye


Figure 3-8. Reduced preretinal neovascularization by expression of IGFBP-3. IGFBP-3
expressing plasmid inj ected mice (n=9, black bars) and control plasmid inj ected mice
(n=9, white bars) were subjected to the OIR model. Statistically significance were
presented *P < 0.005 vs. control vector injected eyes.










3000

~ 2500 -1 *

2000

1 500

1000





NT IGFBP3


Figure 3-9. IGFBP-3 expression in plasmid transfected HSC. Transfection of GFP HSC with
IGFBP-3 expressing plasmid results in a 25-fold increase in IGFBP-3 expression in
vitro compared with nontransfected (NT) controls. *P= 0.02 vs. nontransfected HSC
(NT) .





HSC expressing IGFBP3 protects intratratinal HSC expresing IGFBP3 reduces preretinal
D vasculature during hyperoxia E neovaeulranzaton

C 60 80 a IGFBP-3 injected





central~ ~~~~ mi-eihrl prpea etrl mdprpea epe





vs.unnjctd cntol i D *P ~0.0 nE
























Nontreat


15rrin


VEGF (25ng/ml) treatment


Non


15min


4hrs


SDF (100nM) treatment


Figure 3-11. eNOS phosphorylation by hypoxia-regulated factor. CD34 cells were treated with
either 25 ng/ml VEGF or 100 nM SDF-1. Anti-phoshporylated eNOS (Ser 1177)
antibody was used to examine the activation of eNOS in CD34 cells. A) eNOS
phosphorylation following exposure of VEGF (*P <0.05 vs. nontreated control
cells). B) eNOS phosphorylation following exposure of SDF-1.









O Untreated O CO NO

350
300 -1 *"

a 250





10


1 min 15 min


Figure 3-12. NO and CO stimulate CD34 cells migration. Pretreatment with either the CO
donor (Ru(II)Cl2(CO)3 dimer) or the NO donor (DETA-NO) for either 1 minute or
15 minutes increases the cells responsiveness to SDF-1. EPC were obtained from 3
healthy control subjects. Values represent means +SD. *P<0.05 vs. untreated control.











S50 IO Ser-157 Ser-239


o 40
*! 35
o






20




Untreated NO-Treated CO-Treated



Figure 3-13. VASP, phosphorylated VASP 157, and 239 expression levels. VASP function is
regulated by its phosphorylation on serine 157 and serine 239. CD34 cells were
incubated for 15 minutes in the presence of either the NO or CO donor. The NO
donor increased the VASP phosphorylation at serine 239, whereas CO increased
phosphorylation at serinel57 in these cells. Values represent means +SD. *P <0.05
vs. untreated control










O Control O DM1 H DM2
200-
a 180-


e 140-



80-



20-


O hour 0.25 hour 4 hour


Figure 3-14. Diabetic CD34 cells show increased VASP phosphorylation following exposure of
NO donor. In CD34 cells from two diabetic individuals one with type 1 and the other
with type 2 disease, NO treatment resulted in stimulation of VASP phosphorylation at
serine 239. CD34 cells from diabetic individuals demonstrate reduced levels of
pVASP but phosphorylation increases in response to NO exposure. Values represent
means +SD.








































Figure 3-15. NO and CO mediates VASP redistribution within endothelial cells. A) Untreated
cells showing equal VASP immunoreactivity throughout the cytoplasm. B) CO-
induced redistribution of VASP to filopodia at the leading edge of the cells. C) NO-
induced redistribution of VASP to filopodia. Green channel represents VASP
redistribution. Blue channel shows DAPI stained nuclei. 100X magnification. D),
E), and F) Details of A), B), and C), respectively.















201









o 200






250-

S20
; 10




0 10 30 60

Time (minutes)


Figure 3-16. Phosphorylation of eNOS following exposure of IGFBP-3. Anti-phoshporylated
eNOS (Ser 1 177) antibody was used to examine the activation of eNOS in both
analysis. A) In-Cell western analysis (*p=0.0006 vs. untreated cells). B) Western
blotting analysis (*p<0.05 vs. O min treated cells). Values represent means +SD.











1800-


1600-


1400-


~L1200-


S1000-


.5 800-


S 600-


400-



200


Untreated BP3 Treated


Figure 3-17. Increased intracellular NO production in CD34 cells. CD34 cells were exposed to
IGFBP-3 for 30 minutes. 3.7-fold greater increase of intracellular NO in IGFBP-3
treated cells was confirmed by using DAF-FM diacetate. Values represent means
+SD. *P < 0.05 vs. untreated cells










O' 10' 30' 60
VASP Ser-157
Cofi lin


MMW VASP Ser-239
-Cofi lin


Relative to VASP (0min: set to 100%)


<350

o 300
o
250

2,00

v. 150
o
aL100


g pV-157
g pV-239


Figure 3-18. IGFBP-3 modulates site specific phosphorylation ofVASP in CD34 cells. IGFBP-
3 (100 ng/ml) was exposed to CD34 cells for 0, 10, 30, and 60 minutes. Two anti-
phospho-VASP (Ser 157 and Ser 239) antibodies were used to detect the different
sites of phosphorylation on VASP. Phospho-VASP at serine 239 was significantly
increased by IGFBP-3 treatment. Values represent means +SD.











2000 -
1800-
1600-
LL1400-
a 1200-
S1000-
X 800-
600-
O
2 400 *i
200-


UNT DMS DMS->BP3 IGFBP-3



Figure 3-19. Inhibition of SK activity results in reduced NO production. DMS signifies
CD34 cells were incubated with 20 CIM Dimethylsphingosine for 30 minutes. DMS~
BP3 signifies CD34 cells were pretreated with 20 CIM Dimethylsphingosine for 30
minutes followed by addition of 100 ng/ml IGFBP-3. Values represent means +SD.
*P<0.05 vs. untreated cells.









CHAPTER 4
DISCUSSION

This main purpose of this study was to address IGFBP-3 functions on EPC and its involved

signaling pathways. The primary focus was depicting the effect of IGFBP-3 on stem cells and

progenitor cells in retinal vasculatures.

Factors Influencing the EPC Studies

A difficulty for accomplishing this study was to obtain sufficient number of CD34 cells

from the peripheral blood to complete the experiments. These cells are an extremely rare

population of cells representing less than 0.01% of cells in the circulation. Furthermore

significant differences in circulating EPC numbers exist depending on the general health of the

individual providing the cells. Pathological, pharmacological and physiological factors influence

mobilization of CD34+ EPC. Numbers of EPCs are inversely related to factors such as presence

of coronary artery disease and endothelial dysfunction.60 More specifically, increased levels of

oxidative stress, inflammatory cytokines and asymmetric dimethylarginine, an endogenous

inhibitor of endothelial nitric oxide synthase, have been linked to diminished mobilization and

function of EPCs.65 Indeed, cytokines such as granulocyte-macrophage colony-stimulating

factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), VEGF, SDF-1 and EPO, as

well as therapeutic interventions with stations and estrogen are able to increase EPC numbers in

circulation.'o The contribution of BM derived cells to the endothelium of injured tissue or

hypoxic areas ranges from 1% up to 26% of vessels53,66. The magnitude of recruitment of

circulating endothelial cells is organ specific and dependent on the extent of vascular injury and

remodeling.53,66

It is important to emphasize that identification of putative EPC is complicated due to not

only small numbers of cells but also antigen plasticity, overlapping phenotype or antigen










expression among other subsets. Investigators have struggled to Eind an antigen or characteristic

that is unique to EPC. Recently many studies have demonstrated that there is overlap in antigenic

expression between EC and monocyte phenotypes. LDL uptake, lectin binding, and

CD3 1/CD 105/CD144 expression are inherent features of monocytes, making them often

phenotypically indistinguishable from EPCs.5 Furthermore, monocytes and their progeny can

function as EPCs in various experimental models.52,54,56

The properties of these cells were examined using flow cytometry, a powerful technique

for the analysis of multiple characteristics of a single cell. Flow cytometry was used to determine

the characteristics of cells including cell size, granularity, and relative fluorescences. In this

study, fluorescent antibodies were used to detect the densities of specific receptors and activities

of specific enzymes.

We further attempted to overcome the lack of cells with In-Cell western assay, a

quantitative immunofluorescence based technique. This technique uses infrared labeled

secondary antibodies to directly detect protein in the cellular environment. One of the advantages

in using infrared fluorescence is it reduces interference caused by background autofluorescence

from cell, culture environment, proteins and test compounds. Two separate detection channels

(700 and 800 nm) result in more accurate evaluation. One channel can probe target protein of

interest, the second channel can be used for normalization of cell number or protein

concentration in these same cells. Quantifieation accuracy is maximized by normalization

because adjustments can be made for differences in cell number from well to well. In order to

test the suitability of In-Cell western assay in our system, we established a standard western blot

analysis with same set of experimental groups. The results from In-Cell western and standard

western blot analysis were showing both 44% increases in phospho-eNOS expression following









IGFBP-3 exposure (100 ng/ml, 30 min), supporting that In-Cell western represents a reliable way

to tracking subtle changes of protein among small population of cells such as CD34 cells.

More interestingly, this assay can quantify proteins in a 96-or 384-well microplate in less

time and fewer proteins than a gel-based traditional western blot assay. The optimal cell number

to run the In-Cell western was determined to be 10,000 cells per well. In contrast, traditional

western blot analysis required 1,000,000 cells per lane.

IGFBP-3 as a Hypoxia-regulated Factor

EPCs support postnatal vasculogenesis by incorporating into the vascular lumen as well as

delivering bioavailable angiogenic factors including VEGF, matrix metalloproteases (MMPs),

and angiopoietins to new vessels.92,223-225 Hypoxia especially HIF-1 either directly or indirectly

regulates the cell type specific expression of multiple angiogenic growth factors and cytokines.

Numerous hypoxia-regulated factors have been implicated in vasculogenesis associated with

EPCs.

The mechanism of how HIF-1 modulates cellular response to hypoxia has been relatively

well studied. HIF-1 is a heterodimeric transcription factor that consists of both HIF- la and HIF-

10. The amino-terminal half of each subunit contains both basic helix-loop-helix (bHLH) and

PAS (Per, ARNT, Sim) motifs that are required for dimerization and DNA binding.74 HIF-1P is

also known as aryl hydrocarbon receptor nuclear translocator (ARNT) because it dimerizes with

the aryl hydrocarbon receptor as well as with HIF- la. The carboxy-terminal half of HIF- la

mediates nuclear localization, protein stabilization, and transactivation.74 Under hypoxic

conditions, HIF-la protein accumulates, the heterodimer translocates to the nucleus and binds to

a family of genes containing a specific sequence motif (HRE).73 The target genes are expressed

in most cell types, such as those encoding glucose transporters, glycolytic enzymes, and VEGF,









as well as genes that are expressed in a cell type-specific manner, such as EPO, iNOS and IGF-

2.76

VEGF is commonly expressed as one of the most potent regulator of both vasculogensis

and angiogenesis. Compared to VEGF, the effects of IGFBPs on EPCs are greater. IGFBP-3

mRNA is abundantly expressed in hypoxia-related inflammatory angiogenesis.226 Hypoxia-

induced expression of IGFBP-3 was also validated by Northern blot analysis.227 An anti-

angiogenic role of IGFBP-3 has been reported in cancer research.109,112,18,121 For instance, the

growth-inhibitory effects of various anti-proliferative agents including TGF-P, retinoic acid,

antiestrogens, vitamin D analogs, and TNF-a are associated with increases in IGFBP3 mRNA

and protein expression.122,132,133,135 IGFBP-3 has also been shown to stimulate angiogenesis,

however prior to our studies the vasculogenic effects of IGFBP-3 had not been appreciated.

The expression level and various functional differences of IGFBP-3 are cell type specific

and thus context dependent. We have previously demonstrated that mature human retinal

endothelial cells express high levels of IGFBP-3. In contrast, CD34' cells released undetectable

levels of IGFBP-3. This finding suggests that the immature CD34 cells are more susceptible to

changes in IGFBP-3 concentrations than mature endothelial cells. Furthermore, the

concentrations of IGFBP-3 required to stimulate migration of CD34 cells is extremely low. In

support of our results, Liu et al. reported using in vitro cell proliferation assays and

immunophenotype analysis that addition of nanomolar concentration of IGFBP-3 on human

umbilical cord blood-derived CD34+ CD3 8- Lin cells resulted in their proliferation.228 These

studies combining our Eindings have been conclusively proved that IGFBP-3 supports expansion

of HSC in vitro. In our studies, we further extended these observations by showing that IGFBP-3









stimulates differentiation of CD34 cells to endothelial cells by loss of immature HSC marker and

tube formation assay.

Understanding of IGFBP-3 mechanism has been difficult due to its uncharacterized

receptor. Several candidate receptors of IGFBP-3 have been described but their signaling

functions are poorly understood. Granata et al. suggested possible interaction of IGFBP-3

signaling with SK related angiogenesis by endothelial cells.120 Granata group suggested dual

functions of IGFBP-3 on human endothelial cells. For instance, they revealed pro-apoptosis and

anti-apoptosis action of IGFBP-3 by regulating intracellular ceramide levels. Our results also

indicate that the possible relationship between SK/S 1P and IGFBP-3. mRNA level of SK1 has

been stimulated by IGFBP-3 whereas reduced mRNA level of SK2 was shown in CD34 cells.

Once SK activity was inhibited by SK blocker, dimethylsphingosine (DMS), IGFBP-3 was not

able to exert its function on CD34 cells. Based on our observations, together with the growing

amount of published evidence, IGFBP-3 exerts its vasculogenic actions on EPC though SK/S 1P

signaling pathways.

Our in vivo studies have been showing the protective role of IGFBP-3 in vasculature

following high oxygen stress with subsequent reduction of preretinal neovascularization. Thus,

we postulate that IGFBP-3 expression may represent a physiological adaptation to ischemia and

potentially a novel therapeutic target for treatment of ischemia conditions.

Corroborated with the work of Lofqvist et al, exogenous administration of IGFBP-3 may

represent a novel approach for the treatment of conditions associated with pathological

neovascularization such as retinopathy of prematurity or PDR29. However, there are potential

obstacles for IGFBP-3 study: (1) The volume and the wide diversity of biological activity in

which IGFBP-3 is involved, (2) IGFBP-3 is very closely related to IGF system, and (3) various









laboratories utilize different cell systems in which response to IGFBP-3 may not only be

different but contradictory.

S1P: Possible Role in EPC Mobilization

Sphingosine was discovered by J.L.W. Thudichum in 1884. At that time, he suggested the

name "sphingo" that were derived from the Greek mythology "Sphinx" for a new lipid due to its

chemical nature containing both amine and alcohol groups, but insoluble in water.229 Further

investigations are required to examine intracellular targets of S1P, the mechanism of its transport

in and out of the cells, and the modulation of S 1P levels. Related to S 1P receptors, future

challenges include better characterization of the patho-physiological role of the various S1P

receptors, what regulates their expression and their activity, and which genes are in turn

regulated upon receptor activation.

SIP stimulates human umbilical vein endothelial cells in vitro and in vivo angiogenesis in

the matrigel plug assay in mice.230 Gene knock out studies of S1PI receptor in mice revealed that

this receptor on endothelial cells has an important role in vessel stabilization during

embryogenesis.145 In addition, recent studies have expanded the sphingolipid signaling to the

regulation of eNOS. eNOS pathway (eNOS induced endothelium-dependent vasodilation) has

been suggested as a downstream target for the biological effects of S1P.231 Furthermore, S1P-

induced signaling in human lung EC resulted in cytoskeletal rearrangement (cortical F-actin) and

barrier enhancement through PI3K.232 Our immunohistochemistry result support these findings

by showing that S1P rapidly induced VASP redistribution in human lung EC.

To be considered as a significant intracellular messenger, more experiments remain to be

conducted. Transgenic models, gene targeting approaches, and in vivo use of small interference

RNA will further help understand the physiological role of S1P and its receptors. We are

currently exploring the underlying mechanism by which IGFBP-3 regulates EPC mobilization









and carefully examining the signal transduction cascades activated by IGFBP-3 will ultimately

help understand the how it modulates EPC function .

VASP: New Perspectives and Open Questions

Hypoxia stimulates IGFBP-3 expression and also generates gaseous molecules, CO and

NO by activating HO-1 and NOS respectively. In the current study, two maj or serine

phosphorylation sites of VASP were evaluated on CD34 cells in response to NO, CO, and

IGFBP-3. We have observed that IGFBP-3 mediates increases in phosphorylation, and

redistribution of VASP which subsequently supports EPC migration. To distinguish the

difference of VASP phosphorylation induced by CO, NO, or IGFBP-3, we used two

phosphospecific VASP antibodies targeting the Serl57 and Ser239 residue phosphorylation sites.

We observed that IGFBP-3 (as well as NO) exposure to CD34 cells induced VASP

phosphorylation on serine 239. In contrast, VASP was phosphorylated through serine 157 by

exogenous CO administration to CD34 cells.

All vertebrate Ena/VASP proteins are substrates for the cyclic nucleotide-dependent

kinases PKA/PKG.203,206 Both PKA and PKG recognize and phosphorylate all three sites of

VASP, but with different specifieities and kinetics. Serl57 is the site preferred by PKA, whereas

Ser239 is phosphorylated by PKG.217 Thr23 8 is phosphorylated last by both PKA and PKG.

Phosphorylation at additional sites in VASP might add additional levels of regulation, but it

appears that the conserved N-terminal PKA/PKG site is the maj or site of phospho-regulation in

vertebrate Ena/VASP proteins. Consistent with this, Loureuro et al proved that mutation of this

N-terminal phosphorylation site in Mena was sufficient to block function in fibroblasts.233

Phosphorylation by different kinases has shown cell type specifieity. Studies of platelets

derived from knockout mice revealed a requirement for VASP in this cyclic-nucleotide-mediated

signaling cascade, suggesting that VASP plays a key role in mediating this PKA-dependent









function.206,234 Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-

induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of

cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of

VASP and platelet inhibition in human platelets.23 PKA-dependent phosphorylation of

Ena/VASP proteins correlates with changes in cell adhesion in fibroblasts.233 Under basal

conditions, the maj ority of VASP (more than 95%) is in the unphoshporylated state in human

micro vascular endothelial cells. However, PKA induced VASP phosphorylation changed its

localization to cell-cell junctions and regulated endothelial permeability.215

The role of NO/PKG/ pathway has been tested by many researchers. PKG plays an

important role in smooth muscle cell relaxation, inhibition of platelet aggregation, retinal signal

transduction and synaptic transmission.211 In rat aorta phosphor-VASP ser239 correlates with

relaxation of VSMC layer.206 SGC is the intracellular mediator for the ubiquitous biological

messenger NO.163,187 However, NO-independent stimulators of sGC are very desirable as both

pharmacological tools to proof the NO/cyclic GMP pathway and as potential therapeutic agents.

Organic nitrates like GTN or ISDN have been used for decades as a treatment for coronary heart

disease. However, the maj or drawbacks of this therapy are the development of tolerance and the

negligible anti-platelet effect. This obstacle could now be overcome by the discovery of potent

and specific NO-independent sGC stimulators. As discussed above, PKA/PKG phosphorylation

plays a crucial role in various cell types and in numerous animal models. In this study, we

suggested the role of PKA/PKG in the regulation of VASP function in human progenitor cells

and EC. We found out NO and IGFBP-3 phosphorylates VASP-Ser239 through PKG whereas

CO activates phosphor-VASP-Serl57 through PKA. As depicted in this study, both

physiological gases and IGFBP-3 regulate human mature EC and progenitor cell dynamics by









VASP phoshporylation and localization. Thus, our findings may explain what initiates progenitor

cell migration from BM and how EC migrates into ischemic tissues, although the kinetic analysis

and detailed structural changes that were derived by different site of phosphorylation are still

unknown.

Although a great deal of information about VASP proteins is available there are still a

number of very important questions that remain unanswered. For example, a question concerning

how VASP proteins interact with the barbed ends of actin filaments and what allows filament

elongation need to be answered. Identification of whether such phosphorylation reflects the

overall phosphorylation of all VASP within the cell will be the direction of future studies in our

lab. The question of whether VASP at the leading edge or VASP at focal adhesions are

differently phosphorylated also remains to be determined.

Conclusions

Circulating bone marrow-derived stem cells and progenitor cells home to areas of hypoxia

and participate in vessel development and re-vascularization to facilitate vascular repair. In this

study, we asked whether the hypoxia-regulated factor IGFBP-3 could serve as a homing factor

for EPCs and stimulate their vasculogenic functions. We examined the effect of IGFBP-3 on NO

generation, consequent VASP activation and redistribution. We also evaluated the role of SK in

IGFBP-3 modulating EPC vasculogenesis.


Exposure of CD34' EPC population to nanomolar concentrations of IGFBP-3 resulted in

rapid differentiation into endothelial cells, dose-dependent migration, and capillary tube

formation. For in vivo study, a plasmid expressing IGFBP-3 under the control of a proliferating

endothelial-specific promoter was designed. This plasmid was inj ected either alone or HSC

transfected form into the vitreous of neonatal pups undergoing the oxygen-induced retinopathy









model. Endogeneously delivered IGFBP-3 resulted in reduced areas of vaso-obliteration,

protection of the developing vasculature from hyperoxia, and reduction in preretinal

neovascularization compared to control conditions.

This study supports that IGFBP-3 promotes EPC mobilization by cytoskeletal changes

through NO signaling pathway related phosphorylation and redistribution of VASP. In EPCs,

IGFBP-3 induced eNOS phoshporylation and NO generation. Similar to NO induced PKG

related VASP phosphorylation, IGFBP-3 selectively phoshporylated VASP on serine 239.

Granata group suggested pro-apoptosis and anti-apoptosis action of IGFBP-3 is regulated by

intracellular ceramide/S 1P levels. Our results also indicate that the possible relationship between

SK/S 1P and IGFBP-3. SK1 mRNA was upregulated by IGFBP-3. IGFBP-3 induced intracellular

NO production in EPCs was significantly reduced by SK inhibitor. These findings provide a

mechanism for EPC mobilization and angiogenic function of IGFBP-3. IGFBP-3 expression may

represent a physiological adaptation to ischemia, and IGFBP-3 could be considered as a novel

therapeutic target for treatment of ischemic conditions.









LIST OF REFERENCES


1. Kaplan, H.J. Anatomy and function of the eye. Chentical inanunology and'allergy 92, 4-
10 (2007).

2. Mc~aa, C.S. The eye and visual nervous system: anatomy, physiology and toxicology.
Environmental health perspectives 44, 1-8 (1982).

3. Brubaker, R.F. The flow of aqueous humor in the human eye. Transactions of the
American Ophthalmological Society 80, 391-474 (1982).

4. Koretz, J.F. & Handelman, G.H. How the human eye focuses. Scientific American 259,
92-99 (1988).

5. Bone, R.A., Landrum, J.T. & Cains, A. Optical density spectra of the macular pigment in
vivo and in vitro. Vision research 32, 105-110 (1992).

6. Dowling, J.E. Foveal Receptors of the Monkey Retina: Fine Structure. Science (New
York, N Y 147, 57-59 (1965).

7. Watanabe, T. & Raff, M.C. Retinal astrocytes are immigrants from the optic nerve.
Nature 332, 834-837 (1988).

8. Moschovakis, A.K. & Highstein, S.M. The anatomy and physiology of primate neurons
that control rapid eye movements. Annual review ofneuroscience 17, 465-488 (1994).

9. Pelphrey, K.A., Morris, J.P., Michelich, C.R., Allison, T. & Mc~arthy, G. Functional
anatomy of biological motion perception in posterior temporal cortex: an FMRI study of
eye, mouth and hand movements. Cereb Cortex 15, 1866-1876 (2005).

10. Hayreh, S.S. Segmental nature of the choroidal vasculature. The British journal of
ophthalmology 59, 631-648 (1975).

11. Schwesinger, C., et al. Intrachoroidal neovascularization in transgenic mice
overexpressing vascular endothelial growth factor in the retinal pigment epithelium. The
American journal of pathology 158, 1161-1172 (2001).

12. Zhang, D. & Eldred, W.D. Anatomical characterization of retinal ganglion cells that
proj ect to the nucleus of the basal optic root in the turtle (Pseudemys scripta elegans).
Neuroscience 61, 707-718 (1994).

13. Curcio, C.A., Sloan, K.R., Kalina, R.E. & Hendrickson, A.E. Human photoreceptor
topography. The Journal of comparative neurology 292, 497-523 (1 990).

14. Dacey, D.M., Lee, B.B., Stafford, D.K., Pokorny, J. & Smith, V.C. Horizontal cells of the
primate retina: cone specificity without spectral opponency. Science (New York, N. Y 271,
656-659 (1996).











15. Bouman, M.A. The simple perfection of quantum correlation in human vision. Progress
in neurobiology 78, 38-60 (2006).

16. Wassle, H. & Boycott, B.B. Functional architecture of the mammalian retina.
Physiological reviews 71, 447-480 (1991).

17. Hopkins, J.M. & Boycott, B.B. Synapses between cones and diffuse bipolar cells of a
primate retina. Journal ofneurocytology 24, 680-694 (1995).

18. Zhang, J.J., et al. Tamoxifen blocks chloride channels. A possible mechanism for cataract
formation. The Journal of clinical investigation 94, 1690-1697 (1994).

19. Rubin, L.L. & Staddon, J.M. The cell biology of the blood-brain barrier. Annual review
ofneuroscience 22, 11-28 (1999).

20. Vinores, S.A. Assessment of blood-retinal barrier integrity. Histology and histopathology
10, 141-154 (1995).

21. Vinores, S.A., et al. Blood-ocular barrier breakdown in eyes with ocular melanoma. A
potential role for vascular endothelial growth factor/vascular permeability factor. The
American journal ofpathology 147, 1289-1297 (1995).

22. Weinberger, B., Laskin, D.L., Heck, D.E. & Laskin, J.D. Oxygen toxicity in premature
infants. Toxicology and applied pharmacology 181, 60-67 (2002).

23. McColm, J.R. & Fleck, B.W. Retinopathy of prematurity: causation. Sensin Neonatol 6,
453-460 (2001).

24. Wheatley, C.M., Dickinson, J.L., Mackey, D.A., Craig, J.E. & Sale, M.M. Retinopathy of
prematurity: recent advances in our understanding. The British journal of ophthalmology
86, 696-700 (2002).

25. Kotecha, S. & Allen, J. Oxygen therapy for infants with chronic lung disease. Archives of
disease in childhood 87, F11-14 (2002).

26. Flynn, J.T. Acute proliferative retrolental fibroplasia: multivariate risk analysis.
Transactions of the American Ophthalmological Society 81, 549-591 (1983).

27. Hellstrom, A., et al. IGF-I is critical for normal vascularization of the human retina. The
Journal of clinical endocrinology and nzetabolisn; 87, 3413-3416 (2002).

28. Hellstrom, A., et al. Low IGF-I suppresses VEGF-survival signaling in retinal endothelial
cells: direct correlation with clinical retinopathy of prematurity. Proceedings of the
National Academy of Sciences of the thrited States ofAnzerica 98, 5 804-5 808 (200 1).









29. Lofqvist, C., et al. Postnatal head growth deficit among premature infants parallels
retinopathy of prematurity and insulin-like growth factor-1 deficit. Pediatrics 117, 1930-
1938 (2006).

30. Smith, L.E., et al. Oxygen-induced retinopathy in the mouse. investigative
ophthalmology & visual science 35, 101-111 (1994).

31. Alon, T., et al. Vascular endothelial growth factor acts as a survival factor for newly
formed retinal vessels and has implications for retinopathy of prematurity. Nature
medicine 1, 1024-1028 (1995).

32. Pierce, E.A., Foley, E.D. & Smith, L.E. Regulation of vascular endothelial growth factor
by oxygen in a model of retinopathy of prematurity. Archives of ophthalmology 114,
1219-1228 (1996).

33. Shih, S.C., Ju, M., Liu, N. & Smith, L.E. Selective stimulation of VEGFR-1 prevents
oxygen-induced retinal vascular degeneration in retinopathy of prematurity. The Journal
of clinical investigation 112, 50-57 (2003).

34. Chen, J. & Smith, L.E. Retinopathy of prematurity. Angiogenesis 10, 133-140 (2007).

35. Amos, A.F., McCarty, D.J. & Zimmet, P. The rising global burden of diabetes and its
complications: estimates and proj sections to the year 2010. Diabet2~ed 14 Suppl 5, S1-85
(1997).

36. Aiello, L.P., et al. Diabetic retinopathy. Diabetes care 21, 143-156 (1998).

37. Caldwell, R.B., et al. Vascular endothelial growth factor and diabetic retinopathy:
pathophysiological mechanisms and treatment perspectives. Diabetes nzetabolisn;
research and reviews 19, 442-455 (2003).

38. Miyamoto, K., Hiroshiba, N., Tsujikawa, A. & Ogura, Y. In vivo demonstration of
increased leukocyte entrapment in retinal microcirculation of diabetic rats. investigative
ophthalmology & visual science 39, 2190-2194 (1998).

39. Gardner, T.W., Antonetti, D.A., Barber, A.J., LaNoue, K.F. & Levison, S.W. Diabetic
retinopathy: more than meets the eye. Survey of ophthalmology 47 Suppl 2, S253-262
(2002).

40. Kern, T.S. & Engerman, R.L. Comparison of retinal lesions in alloxan-diabetic rats and
galactose-fed rats. Current eye research 13, 863-867 (1994).

41. Wells, J.A., et al. Levels of vascular endothelial growth factor are elevated in the vitreous
of patients with subretinal neovascularisation. The British journal of ophthalmology 80,
363-366 (1996).









42. Frank, R.N. Potential new medical therapies for diabetic retinopathy: protein kinase C
inhibitors. American journal of ophthalmology 133, 693-698 (2002).

43. Haritoglou, C., et al. Intravitreal bevacizumab (Avastin) therapy for persistent diffuse
diabetic macular edema. Retina (Philadelphia, Pa 26, 999-1005 (2006).

44. Arevalo, J.F., et al. Intravitreal bevacizumab (avastin) for proliferative diabetic
retinopathy: 6-months follow-up. Eye (2007).

45. Arevalo, J.F., et al. Primary intravitreal bevacizumab (Avastin) for diabetic macular
edema: results from the Pan-American Collaborative Retina Study Group at 6-month
follow-up. Ophthalmology 114, 743-750 (2007).

46. Kalina, R.E. Seeing into the future. Vision and aging. The Western journal of medicine
167, 253-257 (1997).

47. Sengupta, N., et al. The role of adult bone marrow-derived stem cells in choroidal
neovascularization.1Investigative ophthalmology & visual science 44, 4908-4913 (2003).

48. Kliffen, M., Sharma, H.S., Mooy, C.M., Kerkvliet, S. & de Jong, P.T. Increased
expression of angiogenic growth factors in age-related maculopathy. The British journal
ofophthalmology 81, 154-162 (1997).

49. Asahara, T., et al. Isolation of putative progenitor endothelial cells for angiogenesis.
Science (New York, N. Y 275, 964-967 (1997).

50. Asahara, T. & Kawamoto, A. Endothelial progenitor cells for postnatal vasculogenesis.
American journal ofphysiology 287, C572-579 (2004).

51. Grant, M.B., et al. Adult hematopoietic stem cells provide functional hemangioblast
activity during retinal neovascularization. Nature medicine 8, 607-612 (2002).

52. Fernandez Puj ol, B., et al. Endothelial-like cells derived from human CD14 positive
monocytes. Differentiation; research in biological diversity 65, 287-300 (2000).

53. Harraz, M., Jiao, C., Hanlon, H.D., Hartley, R.S. & Schatteman, G.C. CD34- blood-
derived human endothelial cell progenitors. Stent cells (Dayton, Ohio) 19, 304-312
(2001).

54. Schmeisser, A., et al. Monocytes coexpress endothelial and macrophagocytic lineage
markers and form cord-like structures in Matrigel under angiogenic conditions.
Cardiovascular research 49, 671-680 (2001).

55. Schmeisser, A. & Strasser, R.H. Phenotypic overlap between hematopoietic cells with
suggested angioblastic potential and vascular endothelial cells. Journal ofhentatotherapy
& stem cell research 11, 69-79 (2002).










56. Rohde, E., et al. Blood monocytes mimic endothelial progenitor cells. Stem cells
(Dayton, Ohio) 24, 357-367 (2006).

57. Walenta, K., Friedrich, E.B., Sehnert, F., Werner, N. & Nickenig, G. In vitro
differentiation characteristics of cultured human mononuclear cells-implications for
endothelial progenitor cell biology. Biochemical and biophysical research
communications 333, 476-482 (2005).

58. Woj akowski, W., et al. The pro- and anti-inflammatory markers in patients with acute
myocardial infarction and chronic stable angina. International journal ofmolecular
medicine 14, 317-322 (2004).

59. Peichev, M., et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+)
cells identifies a population of functional endothelial precursors. Blood 95, 952-958
(2000).

60. Gill, M., et al. Vascular trauma induces rapid but transient mobilization of
VEGFR2(+)AC133(+) endothelial precursor cells. Circulation research 88, 167-174
(2001).

61. Woj akowski, W., et al. Mobilization of CD34/CXCR4+, CD34/CD 117+, c-met+ stem
cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers
into peripheral blood in patients with acute myocardial infarction. Circulation 110, 3213-
3220 (2004).

62. Assmus, B., et al. HMG-CoA reductase inhibitors reduce senescence and increase
proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes.
Circulation research 92, 1049-1055 (2003).

63. Hoetzer, G.L., Irmiger, H.M., Keith, R.S., Westbrook, K.M. & DeSouza, C.A.
Endothelial nitric oxide synthase inhibition does not alter endothelial progenitor cell
colony forming capacity or migratory activity. Journal of cardiovascular pharmacology
46, 387-389 (2005).

64. Murohara, T. & Asahara, T. Nitric oxide and angiogenesis in cardiovascular disease.
Antioxidantsd~~~~~dddd~~~~ & redox signaling 4, 825-831 (2002).

65. Takahashi, T., et al. Ischemia- and cytokine-induced mobilization of bone marrow-
derived endothelial progenitor cells for neovascularization. Nature medicine 5, 434-438
(1999).

66. Crosby, J.R., et al. Endothelial cells of hematopoietic origin make a significant
contribution to adult blood vessel formation. Circulation research 87, 728-730 (2000).









67. Kalka, C., et al. Vascular endothelial growth factor(165) gene transfer augments
circulating endothelial progenitor cells in human subjects. Circulation research 86, 1198-
1202 (2000).

68. Shintani, S., et al. Mobilization of endothelial progenitor cells in patients with acute
myocardial infarction. Circulation 103, 2776-2779 (2001).

69. Shintani, S., et al. Augmentation of postnatal neovascularization with autologous bone
marrow transplantation. Circulation 103, 897-903 (2001).

70. Ceradini, D.J., et al. Progenitor cell trafficking is regulated by hypoxic gradients through
HIF-1 induction of SDF-1. Nature medicine 10, 858-864 (2004).

71. Koong, A.C., et al. Candidate genes for the hypoxic tumor phenotype. Cancer research
60, 883-887 (2000).

72. Wang, G.L. & Semenza, G.L. Purification and characterization of hypoxia-inducible
factor 1. The Journal of biological chemistry 270, 1230-1237 (1995).

73. Semenza, G.L. HIF-1 and mechanisms of hypoxia sensing. Current opinion in cell
biology 13, 167-171 (2001).

74. Semenza, G.L. Hydroxylation of HIF-1 : oxygen sensing at the molecular level.
Physiology (Blelr~thd, M~d 19, 176-182 (2004).

75. Laughner, E., Taghavi, P., Chiles, K., Mahon, P.C. & Semenza, G.L. HER2 (neu)
signaling increases the rate of hypoxia-inducible factor lalpha (HIF-lalpha) synthesis:
novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.
Molecular and cellular biology 21, 3995-4004 (2001).

76. Semenza, G.L., Shimoda, L.A. & Prabhakar, N.R. Regulation of gene expression by HIF-
1. Novartis Foundation symposium 272, 2-8; discussion 8-14, 33-16 (2006).

77. Manalo, D.J., et al. Transcriptional regulation of vascular endothelial cell responses to
hypoxia by HIF-1. Blood 105, 659-669 (2005).

78. Fukuda, R., et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-
mediated vascular endothelial growth factor expression, which is dependent on MAP
kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. The Journal of
biological chemistry 277, 38205-38211 (2002).

79. Forsythe, J.A., et al. Activation of vascular endothelial growth factor gene transcription
by hypoxia-inducible factor 1. Molecular and cellular biology 16, 4604-4613 (1996).









80. Kasuno, K., et al. Nitric oxide induces hypoxia-inducible factor 1 activation that is
dependent on MAPK and phosphatidylinositol 3-kinase signaling. The Journal of
biological chentistry 279, 2550-2558 (2004).

81. Metzen, E., Zhou, J., Jelkmann, W., Fandrey, J. & Brune, B. Nitric oxide impairs
normoxic degradation of HIF-lalpha by inhibition of prolyl hydroxylases. Molecular
biology of the cell 14, 3470-3481 (2003).

82. Hagen, T., Taylor, C.T., Lam, F. & Moncada, S. Redistribution of intracellular oxygen in
hypoxia by nitric oxide: effect on HIF lalpha. Science (New York, N. Y 302, 1975-1978
(2003).

83. Senger, D.R., et al. Tumor cells secrete a vascular permeability factor that promotes
accumulation of as cites fluid. Science (New York, N. Y 219, 983-985 (1983).

84. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. & Heldin, C.H.
Different signal transduction properties of KDR and Fltl, two receptors for vascular
endothelial growth factor. The Journal of biological chentistry 269, 26988-26995 (1 994).

85. Keck, P.J., et al. Vascular permeability factor, an endothelial cell mitogen related to
PDGF. Science (New York, N. Y246, 1309-1312 (1989).

86. Leung, D.W., Cachianes, G., Kuang, W.J., Goeddel, D.V. & Ferrara, N. Vascular
endothelial growth factor is a secreted angiogenic mitogen. Science (New York, N. Y 246,
1306-1309 (1989).

87. Gospodarowicz, D., Abraham, J.A. & Schilling, J. Isolation and characterization of a
vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells.
Proceedings of the National Academy of Sciences of the thrited States of America 86,
7311-7315 (1989).

88. Meyer, M., et al. A novel vascular endothelial growth factor encoded by Orf virus,
VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not
VEGFR-1 (Flt-1) receptor tyrosine kinases. The EM~BO journal 18, 363-374 (1999).

89. Ogawa, S., et al. A novel type of vascular endothelial growth factor, VEGF-E (NZ-7
VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity
without heparin-binding domain. The Journal of biological chentistry 273, 31273-31282
(1998).

90. Wise, L.M., et al. Vascular endothelial growth factor (VEGF)-like protein from orf virus
NZ2 binds to VEGFR2 and neuropilin-1. Proceedings of the National Academy of
Sciences of the thrited States of Anerica 96, 3071-3076 (1999).









91. Junqueira de Azevedo, I.L., Farsky, S.H., Oliveira, M.L. & Ho, P.L. Molecular cloning
and expression of a functional snake venom vascular endothelium growth factor (VEGF)
from the Bothrops insularis pit viper. A new member of the VEGF family of proteins.
The Journal of biological chemistry 276, 3 983 6-3 9842 (200 1).

92. Hattori, K., et al. Vascular endothelial growth factor and angiopoietin-1 stimulate
postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells.
The Journal of experimental medicine 193, 1 005- 10O14 (200 1).

93. Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N. & Nishikawa, S. Maturation
of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.
Blood 93, 1253-1263 (1999).

94. Ferrara, N., et al. Differential expression of the angiogenic factor genes vascular
endothelial growth factor (VEGF) and endocrine gland-derived VEGF in normal and
polycystic human ovaries. The American journal of pathology 162, 1881-1893 (2003).

95. Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nature
medicine 9, 669-676 (2003).

96. Gerber, H.P., et al. VEGF regulates haematopoietic stem cell survival by an internal
autocrine loop mechanism. Nature 417, 954-958 (2002).

97. Carmeliet, P., et al. Abnormal blood vessel development and lethality in embryos lacking
a single VEGF allele. Nature 380, 435-439 (1996).

98. Lee, H.T. & Emala, C.W. Protective effects of renal ischemic preconditioning and
adenosine pretreatment: role of A(1) and A(3) receptors. Am JPhysiol Penal Physiol 278,
F380-387 (2000).

99. Nagasawa, T., Kikutani, H. & Kishimoto, T. Molecular cloning and structure of a pre-B-
cell growth-stimulating factor. Proceedings of the National Academy of Sciences of the
United States ofAmerica 91, 23 05-23 09 (1994).

100. Nagasawa, T., Tachibana, K. & Kishimoto, T. A novel CXC chemokine PBSF/SDF-1
and its receptor CXCR4: their functions in development, hematopoiesis and HIV
infection. Seminars in immunology 10, 179-185 (1998).

101. Bleul, C.C., et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin
and blocks HIV-1 entry. Nature 382, 829-833 (1996).

102. Pituch-Noworolska, A., et al. Circulating CXCR4-positive stem/progenitor cells compete
for SDF-1-positive niches in bone marrow, muscle and neural tissues: an alternative
hypothesis to stem cell plasticity. Folia histochemica et cytobiologica /Polish Academy
of Sciences, Polish Histochemical and Cytochemical Society 41, 13-21 (2003).









103. Kahn, J., et al. Overexpression of CXCR4 on human CD34+ progenitors increases their
proliferation, migration, and NOD/SCID repopulation. Blood 103, 2942-2949 (2004).

104. Butler, J.M., et al. SDF-1 is both necessary and sufficient to promote proliferative
retinopathy. The Journal of clinical investigation 115, 86-93 (2005).

105. Russell-Jones, D.L., et al. A comparison of the effects of IGF-I and insulin on glucose
metabolism, fat metabolism and the cardiovascular system in normal human volunteers.
European journal of clinical investigation 25, 403-411 (1995).

106. Baxter, R.C. & Martin, J.L. Structure of the Mr 140,000 growth hormone-dependent
insulin-like growth factor binding protein complex: determination by reconstitution and
affinity-labeling. Proceedings of the National Academy of Sciences of the United States
ofAmerica 86, 6898-6902 (1989).

107. Firth, S.M., McDougall, F., McLachlan, A.J. & Baxter, R.C. Impaired blockade of
insulin-like growth factor I (IGF-I)-induced hypoglycemia by IGF binding protein-3
analog with reduced ternary complex-forming ability. Endocrinology 143, 1669-1676
(2002).

108. Spagnoli, A. & Rosenfeld, R.G. The mechanisms by which growth hormone brings about
growth. The relative contributions of growth hormone and insulin-like growth factors.
Endocrinology and metabolism clinics of North America 25, 615-63 1 (1996).

109. Butt, A.J. & Williams, A.C. IGFBP-3 and apoptosis--a license to kill? Apoptosis 6, 199-
205 (2001).

110. Butt, A.J., Fraley, K.A., Firth, S.M. & Baxter, R.C. IGF-binding protein-3-induced
growth inhibition and apoptosis do not require cell surface binding and nuclear
translocation in human breast cancer cells. Endocrinology 143, 2693-2699 (2002).

111. Cohen, P., Lamson, G., Okajima, T. & Rosenfeld, R.G. Transfection of the human
IGFBP-3 gene into Balb/c fibroblasts: a model for the cellular functions of IGFBPs.
Gi ,~ ron t regulation 3, 23-26 (1993).

112. Oh, Y., Gucev, Z., Ng, L., Muller, H.L. & Rosenfeld, R.G. Antiproliferative actions of
insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells.
Progress in gain th~ factor research 6, 503-512 (1995).

113. Valentinis, B., Bhala, A., DeAngelis, T., Baserga, R. & Cohen, P. The human insulin-like
growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted
disruption of the IGF-I receptor gene. Molecular endocrinology (Baltimore, M~d 9, 361-
367 (1995).









114. Lalou, C., Lassarre, C. & Binoux, M. A proteolytic fragment of insulin-like growth factor
(IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and
insulin. Endocrinology 137, 3206-3212 (1996).

115. Spagnoli, A., et al. Antiproliferative effects of insulin-like growth factor-binding protein-
3 in mesenchymal chondrogenic cell line RCJ3.1C5.18. relationship to differentiation
stage. The Journal of biological chentistry 276, 5533-5540 (200 1).

116. Firth, S.M. & Baxter, R.C. Cellular actions of the insulin-like growth factor binding
proteins. Endocrine reviews 23, 824-854 (2002).

117. Longobardi, L., et al. A novel insulin-like growth factor (IGF)-independent role for IGF
binding protein-3 in mesenchymal chondroprogenitor cell apoptosis. Endocrinology 144,
1695-1702 (2003).

118. Raj ah, R., Valentinis, B. & Cohen, P. Insulin-like growth factor (IGF)-binding protein-3
induces apoptosis and mediates the effects of transforming growth factor-betal on
programmed cell death through a p53- and IGF-independent mechanism. The Journal of
biological chentistry 272, 12181-12188 (1997).

119. Rajah, R., Khare, A., Lee, P.D. & Cohen, P. Insulin-like growth factor-binding protein-3
is partially responsible for high-serum-induced apoptosis in PC-3 prostate cancer cells.
The Journal of endocrinology 163, 487-494 ( 1999).

120. Granata, R., et al. Dual effects of IGFBP-3 on endothelial cell apoptosis and survival:
involvement of the sphingolipid signaling pathways. Faseb J 18, 1456-1458 (2004).

121. Liu, B., et al. Combination therapy of insulin-like growth factor binding protein-3 and
retinoid X receptor ligands synergize on prostate cancer cell apoptosis in vitro and in
vivo. Clin Cancer Res 11, 4851-4856 (2005).

122. O'Rear, L., et al. Signaling cross-talk between IGF-binding protein-3 and transforming
growth factor-(beta) in mesenchymal chondroprogenitor cell growth. Journal of
molecular endocrinology 34, 723-737 (2005).

123. Foulstone, E.J., Savage, P.B., Crown, A.L., Holly, J.M. & Stewart, C.E. Role of insulin-
like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human
adult skeletal myoblasts. Journal of cellularphysiology 195, 70-79 (2003).

124. Feld, S. & Hirschberg, R. Growth hormone, the insulin-like growth factor system, and the
kidney. Endocrine reviews 17, 423-480 (1996).

125. Landau, D., et al. Expression of insulin-like growth factor binding proteins in the rat
kidney: effects of long-term diabetes. Endocrinology 136, 183 5-1842 (1995).









126. Lamson, G., et al. Proteolysis of IGFBP-3 may be a common regulatory mechanism of
IGF action in vivo. G; 1,n thr regulation 3, 91-95 (1993).

127. Tazuke, S.I., et al. Hypoxia stimulates insulin-like growth factor binding protein 1
(IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in
fetal hypoxia. Proceedings of the National Academy of Sciences of the United States of
America 95, 10188-10193 (1998).

128. Feldser, D., et al. Reciprocal positive regulation of hypoxia-inducible factor lalpha and
insulin-like growth factor 2. Cancer research 59, 3915-3918 (1999).

129. Slomiany, M.G. & Rosenzweig, S.A. Autocrine effects of IGF-I-induced VEGF and
IGFBP-3 secretion in retinal pigment epithelial cell line ARPE-19. American journal of
physiology 287, C746-753 (2004).

130. Ibanez de Caceres, I., et al. Effect of inducible nitric oxide synthase inhibition by
aminoguanidine on insulin-like growth factor binding protein-3 in adjuvant-induced
arthritic rats. European journal of pharmacology 481, 293-299 (2003).

131. Fanayan, S., Firth, S.M. & Baxter, R.C. Signaling through the Smad pathway by insulin-
like growth factor-binding protein-3 in breast cancer cells. Relationship to transforming
growth factor-beta 1 signaling. The Journal of biological chemistry 277, 7255-726 1
(2002).

132. Fanayan, S., Firth, S.M., Butt, A.J. & Baxter, R.C. Growth inhibition by insulin-like
growth factor-binding protein-3 in T47D breast cancer cells requires transforming growth
factor-beta (TGF-beta) and the type II TGF-beta receptor. The Journal of biological
chemistry 275, 39146-39151 (2000).

133. Leal, S.M., Huang, S.S. & Huang, J. S. Interactions of high affinity insulin-like growth
factor-binding proteins with the type V transforming growth factor-beta receptor in mink
lung epithelial cells. The Journal of biological chemistry 274, 67 1 1-67 17 (1 999).

134. Rosendahl, A.H. & Forsberg, G. IGF-I and IGFBP-3 augment transforming growth
factor-beta actions in human renal carcinoma cells. Kidney international 70, 1584-1590
(2006).

135. Oh, Y., Muller, H.L., Ng, L. & Rosenfeld, R.G. Transforming growth factor-beta-induced
cell growth inhibition in human breast cancer cells is mediated through insulin-like
growth factor-binding protein-3 action. The Journal of biological chemistry 270, 13589-
13592 (1995).

136. McCaig, C., et al. Differential interactions between IGFBP-3 and transforming growth
factor-beta (TGF-beta) in normal vs cancerous breast epithelial cells. British journal of
cancer 86, 1963-1969 (2002).









137. Izumi, K., et al. Involvement of insulin-like growth factor-I and insulin-like growth factor
binding protein-3 in corneal fibroblasts during corneal wound healing. investigative
ophthalmology & visual science 47, 591-598 (2006).

138. Peters, I., et al. IGF-binding protein-3 modulates TGF-b eta/BMP-signaling in glomerular
podocytes. JAnz Soc Nephrol 17, 1644-1656 (2006).

139. Spiegel, S. Sphingosine 1-phosphate: a ligand for the EDG-1 family of G-protein-coupled
receptors. Annals of the New York Academy of Sciences 905, 54-60 (2000).

140. Hisano, N., et al. Induction and suppression of endothelial cell apoptosis by
sphingolipids: a possible in vitro model for cell-cell interactions between platelets and
endothelial cells. Blood 93, 4293-4299 (1999).

141. Cuvillier, O., et al. Suppression of ceramide-mediated programmed cell death by
sphingosine-1 -phosphate. Nature 381, 800-803 (1996).

142. Maceyka, M., et al. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing
functions in sphingolipid metabolism. The Journal of biological chentistry 280, 37118-
37129 (2005).

143. Xia, P., et al. An oncogenic role of sphingosine kinase. Curr Biol 10, 1527-1530 (2000).

144. Hait, N.C., et al. Role of sphingosine kinase 2 in cell migration toward epidermal growth
factor. The Journal of biological chentistry 280, 29462-29469 (2005).

145. Allende, M.L., Yamashita, T. & Proia, R.L. G-protein-coupled receptor S1PI acts within
endothelial cells to regulate vascular maturation. Blood 102, 3665-3667 (2003).

146. Liu, Y., et al. Edg-1, the G protein-coupled receptor for sphingosine-1 -phosphate, is
essential for vascular maturation. The Journal of clinical investigation 106, 951-961
(2000).

147. Igarashi, J. & Michel, T. Agonist-modulated targeting of the EDG-1 receptor to
plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of
caveolin- 1 in sphingolipid signal transduction. The Journal of biological chentistry 275,
32363-32370 (2000).

148. Shaul, P.W., et al. Acylation targets emdothelial nitric-oxide synthase to plasmalemmal
caveolae. The Journal of biological chentistry 271, 65 18-6522 ( 1996).

149. Igarashi, J., Bernier, S.G. & Michel, T. Sphingosine 1-phosphate and activation of
endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase
pathways by EDG and bradykinin receptors in vascular endothelial cells. The Journal of
biological chentistry 276, 12420-12426 (2001).









150. Lee, M.J., et al. Sphingosine-1 -phosphate as a ligand for the G protein-coupled receptor
EDG-1. Science (New York, N.Y279, 1552-1555 (1998).

151. Hla, T. & Maciag, T. An abundant transcript induced in differentiating human endothelial
cells encodes a polypeptide with structural similarities to G-protein-coupled receptors.
The Journal of biological chentistry 265, 9308-93 13 (1 990).

152. Olivera, A. & Spiegel, S. Sphingosine-1 -phosphate as second messenger in cell
proliferation induced by PDGF and FCS mitogens. Nature 365, 557-560 (1993).

153. Lee, H., Goetzl, E.J. & An, S. Lysophosphatidic acid and sphingosine 1-phosphate
stimulate endothelial cell wound healing. American journal ofphysiology 278, C612-618
(2000).

154. Graeler, M. & Goetzl, E.J. Activation-regulated expression and chemotactic function of
sphingosine 1-phosphate receptors in mouse splenic T cells. Faseb J 16, 1874-1878
(2002).

155. Graeler, M., Shankar, G. & Goetzl, E.J. Cutting edge: suppression of T cell chemotaxis
by sphingosine 1-phosphate. Jlnanunol l69, 4084-4087 (2002).

156. Mattie, M., Brooker, G. & Spiegel, S. Sphingosine-1 -phosphate, a putative second
messenger, mobilizes calcium from internal stores via an inositol trisphosphate-
independent pathway. The Journal of biological chentistry 269, 31 8 1-3 188 (1 994).

157. Morita, Y., et al. Oocyte apoptosis is suppressed by disruption of the acid
sphingomyelinase gene or by sphingosine-1 -phosphate therapy. Nature medicine 6, 1109-
1114 (2000).

158. Spiegel, S. & Milstien, S. Sphingosine-1 -phosphate: signaling inside and out. FEBS
letters 476, 55-57 (2000).

159. van Meer, G. & Lisman, Q. Sphingolipid transport: rafts and translocators. The Journal of
biological chentistry 277, 25855-25858 (2002).

160. Bouj aoude, L.C., et al. Cystic fibrosis transmembrane regulator regulates uptake of
sphingoid base phosphates and lysophosphatidic acid: modulation of cellular activity of
sphingosine 1-phosphate. The Journal of biological chentistry 276, 3 5258-3 5264 (2001i).

161. Becquet, F., Courtois, Y. & Goureau, O. Nitric oxide in the eye: multifaceted roles and
diverse outcomes. Survey of ophthalmology 42, 71-82 (1997).

162. Rassaf, T., et al. Evidence for in vivo transport of bioactive nitric oxide in human plasma.
The Journal of clinical investigation 109, 1241-1248 (2002).









163. Hanafy, K.A., Krumenacker, J.S. & Murad, F. NO, nitrotyrosine, and cyclic GMP in
signal transduction. M~ed Sci 2onit 7, 801-819 (2001).

164. Yoshida, A., et al. Nitric oxide synthesis in retinal photoreceptor cells. Visual
neuroscience 12, 493-500 (1995).

165. Chakravarthy, U., et al. Nitric oxide synthase activity and expression in retinal capillary
endothelial cells and pericytes. Current eye research 14, 285-294 (1995).

166. Becquet, F., Courtois, Y. & Goureau, O. Nitric oxide decreases in vitro phagocytosis of
photoreceptor outer segments by bovine retinal pigmented epithelial cells. Journal of
cellular physiology 159, 256-262 (1994).

167. Dighiero, P., et al. Expression of inducible nitric oxide synthase in cytomegalovirus-
infected glial cells of retinas from AIDS patients. Neuroscience letters 166, 31-34 (1994).

168. Scalera, F., et al. Erythropoietin increases asymmetric dimethylarginine in endothelial
cells: role of dimethylarginine dimethylaminohydrolase. JAm Soc Nephrol 16, 892-898
(2005).

169. Laufs, U., et al. Physical training increases endothelial progenitor cells, inhibits
neointima formation, and enhances angiogenesis. Circulation 109, 220-226 (2004).

170. Guthrie, S.M., et al. The nitric oxide pathway modulates hemangioblast activity of adult
hematopoietic stem cells. Blood 105, 1916-1922 (2005).

171. Schachinger, V., Britten, M.B. & Zeiher, A.M. Prognostic impact of coronary vasodilator
dysfunction on adverse long-term outcome of coronary heart disease. Circulation 101,
1899-1906 (2000).

172. Zeiher, A.M. Endothelial vasodilator dysfunction: pathogenetic link to myocardial
ischaemia or epiphenomenon? Lancet 348 Suppl 1, sl0-12 (1996).

173. Pannirselvam, M., Verma, S., Anderson, T.J. & Triggle, C.R. Cellular basis of
endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -
/-) mice: role of decreased tetrahydrobiopterin bioavailability. British journal of
pharmacology 136, 255-263 (2002).

174. Craven, P.A., Studer, R.K. & DeRubertis, F.R. Impaired nitric oxide-dependent cyclic
guanosine monophosphate generation in glomeruli from diabetic rats. Evidence for
protein kinase C-mediated suppression of the cholinergic response. The Journal of
clinical investigation 93, 311-320 (1994).









175. Rosselli, M., Imthurn, B., Keller, P.J., Jackson, E.K. & Dubey, R.K. Circulating nitric
oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 beta-estradiol
and norethisterone acetate. A two-year follow-up study. Hypertension 25, 848-853
(1995).

176. Bak, I., et al. Heme oxygenase-1-related carbon monoxide production and ventricular
fibrillation in isolated ischemic/reperfused mouse myocardium. Faseb J 17, 2133-2135
(2003).

177. Yoshida, T. & Kikuchi, G. Purifieation and properties of heme oxygenase from pig
spleen microsomes. The Journal of biological chemistry 253, 4224-4229 (1978).

178. Maines, M.D., Ibrahim, N.G. & Kappas, A. Solubilization and partial purification of
heme oxygenase from rat liver. The Journal of biological chemistry 252, 5900-5903
(1977).

179. Yoshida, T. & Kikuchi, G. Features of the reaction of heme degradation catalyzed by the
reconstituted microsomal heme oxygenase system. The Journal of biological chemistry
253, 4230-4236 (1978).

180. Yoshida, T. & Kikuchi, G. Reaction of the microsomal heme oxygenase with cobaltic
protoporphyrin IX, and extremely poor substrate. The Journal of biological chemistry
253, 8479-8482 (1978).

181. Carter, E.P., et al. Regulation of heme oxygenase-1 by nitric oxide during
hepatopulmonary syndrome. Am JPhysiol Lung Cell2~ol Physiol 283, L346-3 53 (2002).

182. Miyazono, M., Garat, C., Morris, K.G., Jr. & Carter, E.P. Decreased renal heme
oxygenase-1 expression contributes to decreased renal function during cirrhosis. Am J
Physiol Renal Physiol 283, F1123-1131 (2002).

183. Ozawa, N., et al. Leydig cell-derived heme oxygenase-1 regulates apoptosis of
premeiotic germ cells in response to stress. The Journal of clinical investigation 109,
457-467 (2002).

184. Johnson, F.K., Durante, W., Peyton, K.J. & Johnson, R.A. Heme oxygenase inhibitor
restores arteriolar nitric oxide function in dahl rats. Hypertension 41, 149-155 (2003).

185. Brann, D.W., Bhat, G.K., Lamar, C.A. & Mahesh, V.B. Gaseous transmitters and
neuroendocrine regulation. Neuroendocrinology 65, 385-395 (1997).

186. Carvajal, J.A., Germain, A.M., Huidobro-Toro, J.P. & Weiner, C.P. Molecular
mechanism of cGMP-mediated smooth muscle relaxation. Journal of cellularphysiology
184, 409-420 (2000).









187. Furchgott, R.F. & Jothianandan, D. Endothelium-dependent and -independent
vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide
and light. Blood vessels 28, 52-61 (1991).

188. Kajimura, M., Goda, N. & Suematsu, M. Organ design for generation and reception of
CO: lessons from the liver. Antioxidantsd~~~~~dddd~~~~ & redox signaling 4, 633-637 (2002).

189. Stone, J.R. & Marletta, M.A. Soluble guanylate cyclase from bovine lung: activation with
nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric
states. Biochemistry 33, 5636-5640 (1994).

190. Bussolati, B., et al. Bifunctional role for VEGF-induced heme oxygenase-1 in vivo:
induction of angiogenesis and inhibition of leukocytic infiltration. Blood 103, 761-766
(2004).

191. Cisowski, J., et al. Role of heme oxygenase-1 in hydrogen peroxide-induced VEGF
synthesis: effect of HO-1 knockout. Biochemical and biophysical research
communications 326, 670-676 (2005).

192. Kimura, H. & Esumi, H. Reciprocal regulation between nitric oxide and vascular
endothelial growth factor in angiogenesis. Acta biochimica Polonica 50, 49-59 (2003).

193. Deshane, J., et al. Stromal cell-derived factor 1 promotes angiogenesis via a heme
oxygenase 1-dependent mechanism. The Journal of experimental medicine 204, 605-618
(2007).

194. Foresti, R., et al. Vasoactive properties of CORM-3, a novel water-soluble carbon
monoxide-releasing molecule. British journal of pharmacology 142, 453-460 (2004).

195. Motterlini, R., et al. CORM-Al: a new pharmacologically active carbon monoxide-
releasing molecule. Faseb J 19, 284-286 (2005).

196. Clark, J.E., et al. Cardioprotective actions by a water-soluble carbon monoxide-releasing
molecule. Circulation research 93, e2-8 (2003).

197. Foresti, R., Hoque, M., Bains, S., Green, C.J. & Motterlini, R. Haem and nitric oxide:
synergism in the modulation of the endothelial haem oxygenase-1 pathway. The
Biochemical journal 372, 381-390 (2003).

198. Guo, Y., et al. Administration of a CO-releasing molecule at the time of reperfusion
reduces infarct size in vivo. Am JPhysiol Heart Circ Physiol 286, H1649-1653 (2004).

199. Gertler, F.B., Niebuhr, K., Reinhard, M., Wehland, J. & Soriano, P. Mena, a relative of
VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell 87, 227-239 (1996).









200. Bachmann, C., Fischer, L., Walter, U. & Reinhard, M. The EVH2 domain of the
vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and
actin bundle formation. The Journal of biological chentistry 274, 23 549-23 557 ( 1999).

201. Niebuhr, K., et al. A novel proline-rich motif present in ActA of Listeria monocytogenes
and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in
the Ena/VASP family. The EM~BO journal 16, 5433-5444 (1997).

202. Ahern-Dj amali, S.M., et al. Mutations in Drosophila enabled and rescue by human
vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for
Ena/VASP homology domain 1 (EVH1) and EVH2 domains. Molecular biology of the
cell 9, 2157-2171 (1998).

203. Lambrechts, A., et al. cAMP-dependent protein kinase phosphorylation of EVL, a
Mena/VASP relative, regulates its interaction with actin and SH3 domains. The Journal
of biological chentistry 275, 36143-36151 (2000).

204. Massb erg, S., et al. Enhanced in vivo pl atelet adhe si on in vasodil ator-stimul ated
phosphoprotein (VASP)-deficient mice. Blood 103, 136-142 (2004).

205. Krause, M., Dent, E.W., Bear, J.E., Loureiro, J.J. & Gertler, F.B. Ena/VASP proteins:
regulators of the actin cytoskeleton and cell migration. Annual review of cell and
developmental biology 19, 541-564 (2003).

206. Aszodi, A., et al. The vasodilator-stimulated phosphoprotein (VASP) is involved in
cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is
dispensable for smooth muscle function. The EM~BO journal 18, 37-48 (1999).

207. Anderson, S.I., Behrendt, B., Machesky, L.M., Insall, R.H. & Nash, G.B. Linked
regulation of motility and integrin function in activated migrating neutrophils revealed by
interference in remodelling of the cytoskeleton. Cellnzotility and the cytoskeleton 54,
135-146 (2003).

208. Bear, J.E., et al. Negative regulation of fibroblast motility by Ena/VASP proteins. Cell
101, 717-728 (2000).

209. Bear, J.E., et al. Antagonism between Ena/VASP proteins and actin filament capping
regulates fibroblast motility. Cell 109, 509-521 (2002).

210. Coppolino, M.G., et al. Evidence for a molecular complex consisting of Fyb/SLAP, SLP-
76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor
signalling during phagocytosis. Journal of cell science 114, 43 07-43 18 (200 1).

211. Goh, K.L., Cai, L., Cepko, C.L. & Gertler, F.B. Ena/VASP proteins regulate cortical
neuronal positioning. Curr Biol 12, 565-569 (2002).









212. Krause, M., et al. Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP),
Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link
T cell receptor (TCR) signaling to the actin cytoskeleton. The Journal of cell biology 149,
181-194 (2000).

213. Price, C.J. & Brindle, N.P. Vasodilator-stimulated phosphoprotein is involved in stress-
fiber and membrane ruffle formation in endothelial cells. Arteriosclerosis, thirlidesti\i,
and vascular biology 20, 2051-2056 (2000).

214. Rosenberger, P., et al. Identification of vasodilator-stimulated phosphoprotein (VASP) as
an HIF-regulated tissue permeability factor during hypoxia. Faseb J21, 2613-2621
(2007).

215. Comerford, K.M., Lawrence, D.W., Synnestvedt, K., Levi, B.P. & Colgan, S.P. Role of
vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional
permeability. Faseb J 16, 583-585 (2002).

216. Halbrugge, M. & Walter, U. Purifieation of a vasodilator-regulated phosphoprotein from
human platelets. European journal of biochemistry /FEBS 185, 41-50 (1989).

217. Oelze, M., et al. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a
sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction.
Circulation research 87, 999-1005 (2000).

218. Lawrence, D.W. & Pryzwansky, K.B. The vasodilator-stimulated phosphoprotein is
regulated by cyclic GMP-dependent protein kinase during neutrophil spreading. J
Immunol l66, 5550-5556 (2001).

219. Howe, A.K., Hogan, B.P. & Juliano, R.L. Regulation of vasodilator-stimulated
phosphoprotein phosphorylation and interaction with Abl by protein kinase A and cell
adhesion. The Journal of biological chemistry 277, 38121-38126 (2002).

220. Shaw, L.C., et al. Proliferating endothelial cell-specific expression of IGF-I receptor
ribozyme inhibits retinal neovascularization. Gene therapy 13, 752-760 (2006).

221. Szymanski, P., Anwer, K. & Sullivan, S.M. Development and characterization of a
synthetic promoter for selective expression in proliferating endothelial cells. The journal
of gene medicine 8, 514-523 (2006).

222. Segal, M.S., et al. Nitric oxide cytoskeletal-induced alterations reverse the endothelial
progenitor cell migratory defect associated with diabetes. Diabetes 55, 102-109 (2006).

223. Hiratsuka, S., et al. MMP9 induction by vascular endothelial growth factor receptor-1 is
involved in lung-specific metastasis. Cancer cell 2, 289-300 (2002).









224. Cursiefen, C., et al. Roles of thrombospondin-1 and -2 in regulating corneal and iris
angiogenesis. Investigative ophthalmology & visual science 45, 1117-1124 (2004).

225. Luttun, A., et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition
of tumor angiogenesis, arthritis and atherosclerosis by anti-Fltl. Nature medicine 8, 83 1-
840 (2002).

226. Li, H., et al. Hypoxia-inducible factor-lalpha (HIF-lalpha) gene polymorphisms,
circulating insulin-like growth factor binding protein (IGFBP)-3 levels and prostate
cancer. The Prostate 67, 1354-1361 (2007).

227. Le Jan, S., et al. Characterization of the expression of the hypoxia-induced genes
neuritin, TXNIP and IGFBP3 in cancer. FEBS letters 580, 3395-3400 (2006).

228. Liu, L.Q., et al. Functional cloning of IGFBP-3 from human microvascular endothelial
cells reveals its novel role in promoting proliferation of primitive CD34+CD3 8-
hematopoietic cells in vitro. Oncology research 13, 359-371 (2003).

229. Kee, T.H., Vit, P. & Melendez, A.J. Sphingosine kinase signalling in immune cells.
Clinical and experimentalpha~rmacology & physiology 32, 153-161 (2005).

230. Lee, O.H., et al. Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and
signaling mechanism in human umbilical vein endothelial cells. Biochemical and
biophysical research communications 264, 743-750 (1999).

231. Roviezzo, F., et al. Essential requirement for sphingosine kinase activity in eNOS-
dependent NO release and vasorelaxation. Faseb J 20, 340-342 (2006).

232. Igarashi, J. & Michel, T. Sphingosine 1-phosphate and isoform-specific activation of
phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-
regulated endothelial nitric-oxide synthase signaling pathways. The Journal of biological
chemistry 276, 36281-36288 (2001).

233. Loureiro, J.J., et al. Critical roles of phosphorylation and actin binding motifs, but not the
central proline-rich region, for Ena/vasodil ator-stimulated phosphoprotein (VASP)
function during cell migration. Molecular biology of the cell 13, 2533-2546 (2002).

234. Hauser, W., et al. Megakaryocyte hyperplasia and enhanced agonist-induced platelet
activation in vasodilator-stimulated phosphoprotein knockout mice. Proceedings of the
National Academy of Sciences of the United States ofAmerica 96, 8 1 20-8 125 ( 1999).

235. Li, Z., Aj dic, J., Eigenthaler, M. & Du, X. A predominant role for cAMP-dependent
protein kinase in the cGMP-induced phosphorylation of vasodilator- stimulated
phosphoprotein and platelet inhibition in humans. Blood 101, 4423-4429 (2003).









BIOGRAPHICAL SKETCH

Kyung Hee Chang was born in Seoul, South Korea. She received her Bachelor of Science

degree in animal science from Seoul National University in 2001. She continued her graduate

education in Seoul National University and obtained her Master degree maj oring embryology in

March 2003. In 2003, she j oined the Interdisciplinary Program in Biomedical Sciences at

College of Medicine, University of Florida, where in May 2008 she received the degree of

Doctor of Philosophy.





PAGE 1

1 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 REGULATES HEMATOPOIETIC STEM CELL AND ENDOTH ELIAL PROGENITOR CELL FUNCTION DURING VASCULAR DEVELOPMENT By KYUNG HEE CHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Kyung Hee Chang

PAGE 3

3 To my beloved family

PAGE 4

4 ACKNOWLEDGMENTS It is m y pleasure to have a chance to expr ess my gratitude to all those who helped me complete this dissertation. I would like to specially thank to my mentor, Dr. Maria Grant, who has constantly helped me with patience. She has been an amazingly gr eat teacher, researcher, and advisor. She taught me how to solve the many problems I confronted. Her enthusiastic and inspiring guidance kept me work even harder and helped me overcome many difficulties. I have been very fortunate to have her as my mentor throughout this academic journey. I would like to also thank my committee, Dr. Bryon Petersen, Dr. Daniel Purich, Dr, Jeffrey Harrison, and Dr. Mark Segal. W ithout a doubt, their insightful comments and constructive criticism strengthe ned me as a scientist. Thanks to the members of the Grant Lab: Jennifer Kielczewski, Nilanjana Sengupta, Sergio Li Calzi, and Sergio Caballero, Jr. Intera ction with my lab members was one of the ways I relieved daily stresses. Particul arly, I am grateful to Dr. Lynn Shaw for his valuable time and efforts to teach me all the details of how to inte rpret experimental output Special thanks also go to Dr. Aqeela Afzal. She trained me from the first day I joined the Grant Lab. She taught me everything I needed to fulfill this entire doctora l work. She has been always considerate and was my friend, sister, and a great trainer. In addition, I would like to th ank to the men and women who donated their blood for this research. Their thoughtful gift enabled this research to step forward. I also warmly appreciate the generosity and understanding of my friends and my lovely pet, Camus. I thank Don for his great s upport and valuable advice for my writing. Most importantly, I would like to express my special thanks to my parents whose unconditional love enabled me to complete this work. None of this would have been possible

PAGE 5

5 without their belief in me. I al so thank my dear brother, TaeH ong, for all his consideration and support, and my family in Korea who have been a great source of inspiration.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES.........................................................................................................................8 LIST OF ABBREVIATIONS........................................................................................................ 10 ABSTRACT...................................................................................................................................14 CHAP TER 1 BACKGROUND AND SIGNIFICANCE ..............................................................................16 The Eye...................................................................................................................................16 Anatomy..........................................................................................................................16 The Retina.......................................................................................................................20 Retinal Blood Supply......................................................................................................22 Retinopathies..........................................................................................................................24 Retinopathy of Prematurity............................................................................................. 24 Diabetic Retinopathy.......................................................................................................27 Age-related Macular Degeneration................................................................................. 29 Neovascularization.................................................................................................................30 Endothelial Progenitor Cells............................................................................................ 30 Hypoxia Inducible Factor-1............................................................................................. 32 Vascular Endothelial Growth Factor...............................................................................35 Stromal Derived Factor-1................................................................................................ 37 Insulin-like Growth Factor Binding Protein-3................................................................ 39 Sphingosine 1-phosphate.................................................................................................43 Nitric Oxide.....................................................................................................................45 Carbon Monoxide............................................................................................................47 Vasodilator Stimulated Phosphoprotein.......................................................................... 48 Significance (Specific Aims)..................................................................................................51 2 METHODS AND MATERIALS........................................................................................... 52 Cell Preparation............................................................................................................... .......52 Migration Assay......................................................................................................................53 EPC Tube Formation............................................................................................................. .54 Cell Proliferation Assay....................................................................................................... ...54 Nitric Oxide Measurement.....................................................................................................55 Western Blot Analysis.......................................................................................................... ..55 In-cell Western Assay.......................................................................................................... ...56 Quantitative Transcription Analysis with R eal Tim e Polymerase Chain Reaction (RT PCR)........................................................................................................................... .........57 Immunohistochemistry........................................................................................................... 57

PAGE 7

7 Flow Cytometry Analysis.......................................................................................................58 Hematopoietic Stem Cell (HSC) Transfection.......................................................................59 Experimental Animals........................................................................................................... .60 Oxygen Induced Retinopathy (OIR) Mouse Model............................................................... 60 Retinal Flat Mounts............................................................................................................ ....61 GS Isolectin and GFP Double La beled Imm unohistochemistry............................................. 62 Microscopy and Mapping....................................................................................................... 62 Vascular Density Analysis and Statistical Analysis ............................................................... 63 3 RESULTS...............................................................................................................................64 IGFBP-3 Induces Migration of CD34+cells and Endothelial Cells........................................ 64 IGFBP-3 Increases Expression of VEGF Receptors on CD34+cells...................................... 64 IGFBP-3 Promotes CD34+cells Differentiation to Endothelial Cells..................................... 65 IGFBP-3 Enhances CD34+cells Proliferation......................................................................... 65 Expression of Hypoxia-regulat ed Factors in Retina ............................................................... 65 IGFBP-3 Protects Neonatal Retinal Vessels from Oxygen Induced Vaso-obliteration ......... 66 Quantitative Analysis of Vascular Density in Vaso-obliteration Phase................................. 67 IGFBP-3 Decreases the Incidence of Pre-retinal Neovascularization .................................... 67 IGFBP-3 Expression in Transfected HSC .............................................................................. 67 Co-localized IGFBP-3 Expressing gfp+HSC within the Vasculature Inhibit Neovascularization..............................................................................................................68 Hypoxia-regulated Factors and Nitric Oxide Signaling......................................................... 68 NO and CO Promotes CD34+cells Migration......................................................................... 69 Different Phosphorylation Sites of VASP..............................................................................69 NO Increases VASP Phosphorylation in Diabetic CD34+cells.............................................. 69 NO and CO Cause VASP Redistribution to the L eading Edge of the Cells........................... 70 IGFBP-3 Increases eNOS Phosphorylation............................................................................70 IGFBP-3 Induces NO Production...........................................................................................71 IGFBP-3 Modulates VASP Phosphorylation.........................................................................71 Inhibition of SK Activity Results in Reduced NO Production............................................... 71 4 DISCUS SION .........................................................................................................................91 Factors Influencing the EPC Studies...................................................................................... 91 IGFBP-3 as a Hypoxia-regulated Factor................................................................................93 S1P: Possible Role in EPC Mobilization................................................................................96 VASP: New Perspectives and Open Questions...................................................................... 97 Conclusions.............................................................................................................................99 LIST OF REFERENCES.............................................................................................................101 BIOGRAPHICAL SKETCH.......................................................................................................120

PAGE 8

8 LIST OF FIGURES Figure page 1-1 Anatomy of hum an eye...................................................................................................... 181-2 The ten layers of the retina. ............................................................................................ ..211-3 Hypoxia-regulated factors and BM-derived cells.............................................................. 331-4 The VEGF (VEGF-R2) signaling pathway........................................................................361-5 The interaction between SDF-1 and CXCR4..................................................................... 381-6 IGF-1R signaling pathway................................................................................................. 401-7 Schematic diagram of IGF system..................................................................................... 413-1 IGFBP-3 induces CD34+cells and endothelial cells migration.......................................... 723-2 Receptor levels in CD34+cells following IGFBP-3 exposure............................................ 733-3 IGFBP-3 enhances CD34+cells and EPC differentiation. ................................................ 743-4 IGFBP-3 enhances CD34+cells proliferation..................................................................... 753-5 Hypoxia retina expresses IGFBP-3....................................................................................763-6 IGFBP-3 protects from hyperoxia-induced vascular regression........................................773-8 Reduced preretinal neovascula rization by expression of IGFBP-3................................... 793-9 IGFBP-3 expression in plasmid transfected HSC.............................................................. 803-10 Localization of gfp+HSC expressing IGFBP-3 within the retinal vasculature..................813-11 eNOS phosphorylation by hypoxia-regulated factor. ........................................................823-12 NO and CO stimulate CD34+cells migration..................................................................... 833-13 VASP, phosphorylated VASP 157, and 239 expression levels.........................................843-14 Diabetic CD34+cells show increased VASP phosphor ylation following exposure of NO donor....................................................................................................................... ....853-15 NO and CO mediates VASP redistri bution within endothelial cells.................................863-16 Phosphorylation of eNOS following exposure of IGFBP-3..............................................87

PAGE 9

9 3-17 Increased intracellu lar N O production in CD34+cells....................................................... 883-18 IGFBP-3 modulates site specifi c phosphorylation of VASP in CD34+cells..................... 893-19 Inhibition of SK activity re sults in reduced NO production.............................................. 90

PAGE 10

10 LIST OF ABBREVIATIONS ALS Acid-labile subunit AMD Age-related macular degeneration ATP Adenosine triphosphate BBB Blood-brain barrier BH4 Tetrahydrobiopterin BM bone marrow BRB Blood-retinal barrier BSA Bovine serum albumin cAMP Adenosine 3 5-cyclic monophosphate Cdc6 Cell division cycle 6 cGMP Guanosine 3, 5-cyclic monophosphate CNV Choroidal neovascularization CORMs Carbon monoxide-releasing molecules CPT Cell preparation tube DAF-FM diacetate 4-amino-5-methylamino-2 7-difluorofluorescein diacetate DAPI 4, 6-diamino-2-phenylindole DM Diabetes mellitus DMS Dimethylsphingosine DNA Deoxyribonucleic acid DR Diabetic retinopathy EBM-2 Endothelial cell basal medium-2 ECM Extracellular matrix EDG Endothelial differentiation gene EDTA Ethylenediamine tetraacetic acid

PAGE 11

11 Ena Drosophila melanogaster protein enabled eNOS Endothelial nitr ic oxide synthase EOMs Extraocular muscles EPC Endothelial progenitor cell EPO Erythropoietin ET Endothelin enhancer EVH Ena/VASP homology FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FITC Fluorescein isothiocyanate Flk-1 Vascular endothelial growth factor receptor-2 Flt-1 Vascular endothelial growth factor receptor-1 Flt-4 Vascular endothelial growth factor receptor-3 GCL Ganglion cell layer GFP Green fluorescent protein GM-CSF Granulocyte/macrophage colony-stimulating factors GPCR G protein-coupled receptor GS isolectin Griffonia Simplicifolia isolectic HIF-1 Hypoxia inducible factor-1 HIV Human immunodeficiency virus HMVEC-L Human lung derived micr ovascular endothelial cells HO Heme oxygenase HPGM Hematopoietic progenitor growth medium HREC Human retinal endothelial cells HRE Hypoxia response element

PAGE 12

12 HSC Hematopoietic stem cell IACUC Institutional animal care and use committee IGF Insulin-like growth factor IGF-1R Insulin-like grow th factor-1 receptor IGFBP Insulin-like growth factor binding protein IL Interleukin INL Inner nuclear layer iNOS inducible/inflammator y nitric oxide synthase IPL Inner plexiform layer KDR Vascular endothelial growth factor receptor-2 MMP-9 Metalloproteinase-9 mRNA messenger ribonucleic acid NaCl Sodium chloride NIH National institutes of health nNOS neuronal nitric oxide synthase NO Nitric oxide NOS Nitric oxide synthase NPDR Nonproliferative diabetic retinopathy OIR Oxygen induced retinopathy ONL Outer nuclear layer OPL Outer plexiform layer PBS Phosphate buffered saline PDR Proliferative diabetic retinopathy PECAM-1 Platelet/endothelial cell adhesion molecule-1 PEI Polyethylenimine

PAGE 13

13 PFA Paraformaldehyde PKA Protein kinase A PlGF Placenta growth factor rAAV recombinant adeno-associated virus ROP Retinopathy of prematurity RPE Retinal pigment epithelium RT-PCR Real time polymerase chain reaction S1P Sphingosine 1-phosphate SCF Stem cell factor SDF-1 Stromal cell derived factor-1 SD Standard deviation sGC soluble guanylyl cyclase SK Sphingosine kinase TGF Transforming growth factor TPO Thrombopoietin VASP Vasodilator stimulated phosphoprotein VEGF R-1 Vascular endothelial growth factor receptor-1 VEGF R-2 Vascular endothelial growth factor receptor-2 VEGF Vascular endothe lial growth factor VPF Vascular permeability factor ZO-1 Zoula-occludens-1

PAGE 14

14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 REGULATES HEMATOPOIETIC STEM CELL AND ENDOTH ELIAL PROGENITOR CELL FUNCTION DURING VASCULAR DEVELOPMENT By Kyung Hee Chang May 2008 Chair: Maria B. Grant Major: Medical Sciences-Physiology and Pharmacology Retinal blood vessels develop by a combination of vasculogenesis and angiogenesis, called neovascularization. Endothelial progenitor cells (EPCs) which originated from lateral and posterior mesoderm contribute to neovascularizaiton in response to ce rtain cues such as cytokines and hypoxic gradient. Aberrant vessel growth in retina results in vascular retinopathies, the leading cause of visual disability and blindn ess worldwide. The pathological outgrowth of new blood vessels i nvolves the recruitment and pro liferation of circulating EPCs. This study was initiated to better understand a complex network regulat ing neovascularization involving EPCs function. Many factors have been identified to promote trafficking, mobilization, and homing of EPCs. We demonstrated that in sulin-like growth factor binding protein-3 (IGFBP-3) has a critical function in postnatal vasculogenesis both in vitro and in vivo IGFBP-3 showed enhanced migration, tube formation and proliferation of EPCs in vitro In vivo IGFBP-3 inhibited pathological neovascularization by protecting developm ental retinal vessels from oxygen induced regression. In EPCs, nitric oxide (NO) regulates migratio n through redistribution and phosphorylation of the motor protein va sodilator-stimulated phosphoprotein (VASP). IGFBP-3 has been shown to trigger EPC mobilization by generating NO and subsequently

PAGE 15

15 activating VASP. The signaling mechanisms of IG FBP-3 on EPCs were also identified in this study. We have defined that down stream path way of IGFBP-3 involve sphingosine kinase (SK)/sphingosine-1 phosphate (S1P ) signal transduction. NO rele ase from EPCs was reduced after treatment with a SK inhibitor, dimethyl sphingosine (DMS) and could not be recovered by exposure to IGFBP-3. This result suggests that IGFBP-3 may act as an upstream mediator of SK/S1P signaling. We addressed the hypothesis that IGFBP-3 stimulates NO production and VASP phosphorylation through SK activation. Thus IGFBP-3-induced a ngiogenesis of EPCs may be involved in a SK dependent signaling path way. Whereas an inhib itory angiogenic role for IGFBP-3 has been widely reported, several other researchers have shown contradictory results in that IGFBP-3 also enhances angiogeni c effects. Our results suggest that IGFBP-3 has an angiogenic/vasculogenic function in m odulating the migratory ability of EPCs.

PAGE 16

16 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Blood vessel developm ent is mediated by angiog enesis as well as by vasculogenesis. In vasculogenesis stem cells are mobilized from th e bone marrow and differentiate into circulating endothelial progenitor cells which are then inte grated into the primary capillary plexus. This process is responsible for the development of the vascular system during embryogenesis. In contrast, angiogenesis is the formation of ne w blood vessels from sprouts on preexisting vessels, and occurs both during development and in pos tnatal phase. In adults, neovascularization involves the recruitment and prolif eration of either endothelial ce lls from preexisting vessels or circulating EPCs originating from bone marrow. This process is involved in many physiological and pathological conditions such as tissue remodeling, regeneration, wound healing and tumorigenesis. Vessel development is regulated by a complex network of mediators and cellular interactions. Reduced oxygen tension, various cytoki nes, and angiogenic fact ors at least partially regulate neovascularization. This study was in tended to better understand the mutifactorial processes of neovascularization. The Eye The eye exis ts in a relatively isolated compar tment. The ocular tissue and vasculature are highly differentiated for conducting the complex pr ocess of visual transduction with little systemic exposure. The retina is an ideal model system to study molecular mechanisms of angiogenesis due to the unique vascul ar supply of the eye, the ability to visualize this vasculature in vivo and the ability to selectively express genes in the eye. Anatomy The eye is a com plex organ composed of many pa rts. The ability to see is dependent on the actions of several structures. Figure 1-1 show s many of the essential components of the eye.

PAGE 17

17 Initially, light enters the eye through a lubr icating tear film that covers the cornea.1 The cornea is the transparent outer covering of the ey e and helps to focus incoming light. After light rays pass the cornea, they travel through a cl ear, watery fluid, called the aqueous humor.2 The aqueous humor transports nourishment for th e surrounding lens and cornea as well as maintaining a constant intraocular pressure.2 The aqueous humor is produced by the ciliary body which also changes the shape of the lens for focusing.3 The iris is the colored part of the eye. It separates the anterior chamber from the posterior chamber and regulates the amount of light entering through the pupil.3 The size of the pupil is controlled by the dilator and sphincter muscles of the iris and regulates the amount of light that enters the eye.3 After light travels through the pupil, it passes through the lens. Th e lens is suspended by ligaments (called zonule fibers) that are attached to the anterior portion of the ciliary body.4 As a consequence of ciliary muscle actions, the contraction or relaxation of th ese ligaments changes the shape of the lens (a process called accommodation allowing the formation of a sharp image on the retina).4 Light then passes a clear, jelly-like subs tance called the vitreous before it finally reaches the retina. The vitreous is a viscous, transparent liquid that fills the center of the eye.1 It is composed mainly of water and comprises about 2/3 of the eye's volume, and helps to maintain eye shape.1 The retina is a multi-layered sensory tissue lining the back of the eye that operates similar to the film in a camera. At the retina, the light rays are converted to electr ical impulses which are transmitted via the optic nerve to the brain. Th e central portion of the human retina contains a yellow pigment called the macular pigment.5 This pigment helps protect the sensitive receptors in the retina, particularly from the pot entially harmful eff ects of blue light.5 The density of the pigment has been shown to be linked to diet and can be reduced in a person who smokes.5 The macula is the area of the retina that contains the highest concentration of photoreceptor cells.

PAGE 18

18 Figure 1-1. Anatomy of human eye.

PAGE 19

19 At the very center of the macula is the fovea, the site of our sharpest vision.6 The optic nerve is a bundle of nerve fibers that carries visual information from the eye to the brain. The optic nerve runs from the optic disc through the optic foramen to the optic chiasma where it becomes the optic tract.7 It is 5cm in length and surrounded by 3 layered membranes of the central nervous system: pia, arachnoid, and dura.7 The eye is comprised of three different layers and spatially divided into three chambers of fluid. The external layer is formed by the sclera and cornea.2 The cornea is a refracting surface, providing 2/3 of the eyes focusing power.2 The cornea is extremely sensitive and contains more nerve ending than anywhere else in the body.2 The sclera is composed of tough, fibrous tissue that protects the inside of the eye.2 Extraocular muscles are attach ed to the sclera and maintain the shape of the eye.8 The six tiny muscles, known as th e extraocular muscles (EOMs), surround the eye and control its movements.8 The primary function of the four rectus muscles is to control the eye's movements from left to right and up and down.9 The two oblique muscles allow the eye to rotate inward and outward.9 All six muscles of both eyes work in unison so that the eyes are always aligned.8 The intermediate layer is divided into two parts: anterior (iris and ciliary body) and posterior part, called the choroids.10 The choroid contains a layer of blood vessels and lies between the retina and sclera.10 The choroid supplies oxygen and nutrients to the outer layers of the retina.11 The choroid connects th e ciliary body with the front of the eye and is attached to edges of the optic nerve.11 The internal layer is the se nsory part of the eye called retina. The eye consists of three chambers; the ante rior chamber (between cornea and iris), posterior chamber (between iris, zonule fibers, a nd lens), and the vitreous chamber (between the

PAGE 20

20 lens and the retina). The first two chambers are filled with aqueous humor and the vitreous chamber is filled with more viscous fluid, the vitreous humor. The Retina The retina is a m ulti-layered structure that is involved in signal transduction. It covers about 65 % of interior surf ace of the vitreous chamber.2 The human retina is approximately 0.2 mm thick, and has an area of approximately 1100 mm.2 Each retina is composed of about 200 million neurons.12 The retina captures the light and converts it into electrical impulse using photoreceptors. There are two types of photoreceptors in the retina: rods and cones.13 Approximately 125 million rods exist in human retina.13 They are spread throughout the peripheral retina and function best in dim lighting; therefore, the rods are responsible for peripheral and night vision.13 The retina contains approximately 6 million cones.14 Cones function best in bright light and color perception.14 The highest density of cones is in the macula.15 The macular contains a very different re tinal configuration towards the center called foveal region.15 Cones are most densely packed within the fovea, the center portion of the macular. The fovea is maximally thinned and mainly consists of phot oreceptors and their nuclei.15 The purpose of foveal thinning is to perm it greater light absorption by the dense array of photoreceptors.6, 15 Another interesting as pect of the fovea is th e absence of blood vessels over the photoreceptors. This absence of blood vessels contributes to increasing visual acuity in the macular region.15 However, the vascularization in the rest of the macula is very dense and thus increases possibility of many vascular related diseases. The reti na is loosely attached to the retinal pigment epithelium (RPE).10 These cells contain a great amount of pigment that is necessary for light absorption and transporta tion of oxygen, nutrients, and cellular wastes between the photoreceptors and the choroids.10 Bruchs membrane is tightly bound to the RPE, stabilizing the RPE layer by separating it from the blood vessels of the choriod.10

PAGE 21

21 Figure 1-2. The ten layers of the re tina. This drawing shows retinal a nd choroidal cross-se ction. From the most anterior layers of the retina the ten layers of the retina consist of sclera, choroids, retinal pigment epithelium, rod and cone layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, and the inner limiting membrane.

PAGE 22

22 Oxygen diffuses across the Bruchs membrane and this membrane grows thinker with age.10 Breaks in Bruchs membrane are the hallmark fo r choroidal neovasculariz ation (CNV) into the retina.10 Beneath the Bruchs membrane the chroroid, containing its network of blood vessels, nerves, immune cells, and fibroblas ts, supplies all of nutritional needs of the RPE and the outer part of the sensory retina. Human retina consists of ten layers (Figur e 1-2). Among them, three layers of nerve cell bodies and two layers of synapses are mainly res ponsible for converting a light signal into neural signal. The photoreceptor cell bodies form the outer nuclear layer (ONL).16 While the inner nuclear layer (INL) contains cell bodies of the bipolar, horiz ontal and amacrine cells, the ganglion cell layer (GCL) contains cell bodies of ganglion cells and displaced amacrine cells.16 The outer plexiform layer (OPL), the first area of neutrophil, is located in between the ONL and the INL.17 In the OPL, the photoreceptors convey their information to the bipolar cells as well as the horizontal cells. Afterward, bipol ar cells relay information to the inner plexiform layer (IPL), which separates the INL and the GCL.17 Bipolar cells are connected to the retinal ganglion cells in addition to amacrine cells in the IPL.17 The ganglion cells are the output neurons of the retina that transmit the signal from the eye to the brain.16 Retinal Blood Supply The blood supply to the retina origin ates from the ophthalmic artery.1 There are two sources of blood supply to the mammalian retina: the central retinal artery and the choroidal blood vessels.1 The outer retina is supplied by the chorio capillaries. The chor oidal arteries arise from long and short posterior ciliary arteries and branches of Zinns circle around the optic disc.10 Each of the posterior ciliar y arteries is further classified into fan-shaped lobules of capillaries that supply locali zed regions of the choroids.10,18 The arteries penetrate the sclera around the optic nerve and spread out to fo rm vascular layers in the choroids.10 The choroid

PAGE 23

23 receives the greatest blood flow (65-85%), which is critical for the maintenance of the outer retina, particularly the photoreceptors.10 The central retinal artery supplies the remaining 2030% blood flow from the optic nerve head to nouris h the inner retinal layers. The central retinal artery supplies the blood as it branches into smaller segments upon leaving the optic disc.18 The vessels are further divided into either an artery or a vein18. The central retinal artery has 4 main branches in the human retina.18 The arterial intraretinal branch es then supply three layers of capillary networks: the radial peripapillary capillari es, an inner layer of capillaries, and an outer layer of capillaries.18 The radial peripapillary capillarie s are the most superficial layer of capillaries lying in the inner pa rt of the nerve fiber layer, an d run along the paths of the major superotemporal and inferotemporal vessels.18 The inner capillaries are located in the ganglion cell layer both under and parallel to the radial peripapi llary capillaries.18 The outer capillary network runs from the inner plex iform layer to the outer plexif orm layer though the inner nuclear layer.18 Retinal blood vessels that are similar to cer ebral blood vessels main tain the blood-retinal barrier (BRB). The BRB consists of two distinct monolayers of cells: the retinal pigment epithelium (RPE: outer barrier) and the retinal capillary endothelial cells (inner barrier).19 Both monolayers form tight junctions, which are ope rative in the maintena nce of the barrier. The concept of the BRB was first proposed by Schnaudigel in 1913 following the classical work of Ehrlich and Goldman who di scovered the blood-brain barrier (BBB). Similar to the structure of BBB, the inner BRB is covered with pericyte s and glial cells.19 Glial Mller cells predominantly support retinal endot helial cells and glial astrocytes are partly responsible for supporting endothelial func tions at the inner BRB.20,21

PAGE 24

24 The inner BRB plays an important role in supp lying nutrients to the neural retina and is responsible for the efflux of neurotransmitter me tabolites from the retina to maintain neural functions.20 The outer BRB consists of specialized nonf enestrated capillaries and tight junctions within the RPE.21 The outer BRB forms a transport barrie r between the retinal capillaries and the retinal tissue.21 In addition, it prevents the passa ge of the large molecules from choriocapillaries into the retina.21 The eye is extremely sensitive to any disr uption of its blood supply. The BRB maintains the ocular milieu and protects the neural retina from any pathological blood circulation. The breakdown of the BRB is a clinical hallmark of early diabetic retinopathy. Retinopathies Vision impairment, disability, and blindness ar e major public health problems. Significant suffering, disability, loss of productivity, and lower quality of life can affect millions of people. In the United States, more than 11 million people have some degree of visual impairment. Approximately 890,000 people in the US are legally blind. Retinopathies are ocular diseases in which deterioration of the retina is initiated by abnormal neovasc ularization, resulting in vision loss. Vascular retinopathies are the leading causes of visual disa bility and blindness worldwide. Pathological growth of new blood ve ssels in pre-retinal region is the hallmark of retinopathies. Retinopathies affect all age groups: retinopathy of prematurity (ROP) is a disease that occurs in premature babies. Diabetic re tinopathy (DR) primarily affects the working age population, and age-related macular degeneration (A RMD) affects the aging population. Retinopathy of Prematurity ROP is the leading cause of blindness in children in both developing and developed countries. R OP mainly affects premature in fants weighing about 1.25 kg (approximately 2.75lb) or less that are born before 31 weeks of gestation. At 16 w eeks of gestation, blood vessels

PAGE 25

25 gradually grow over the surface of the retina.22 Active growth of the human fetal eye occurs within the last 12 weeks of full term delivery (28 to 40 weeks of gestation).23 Vessels reach the anterior edge of the retina and then stop progressing at about 40 weeks of gestation.22-25 A premature baby is placed into an oxygen chamber to assist the still developing lungs. Once in high oxygen the retinal vessel development is stopped. Upon removal from the high oxygen environment the return to normal levels of oxyge n is seen as a hypoxic environment in the eye, and this stimulates the neovasc ularization within the retina. There are approximately 3.9 million babies bor n in the U.S. annually. According to National Eye institute, about 28,000 premature in fants are born. About 14,000 to 16,000 of these premature infants could potential ly develop some degree of ROP. Although, approximately 90% of all the infants with ROP are in the milder st age and do not need treatment, the rest of 10 % (about 1,100 to 1,500) of the babies develop severe ROP and require medical treatment. As a consequence, about 400 to 600 infants each year in the U.S. become legally blind from ROP. Many factors are likely to cause ROP. On ce a premature baby is born, excessive oxygen supply is needed to help the develo pment of the premature babys lungs.25 ROP was first described in 1942, but its cause was unknown at th at time. Most of premature babies were treated with high oxygen whether they we re having breathing problems or not.22 After a while, it was found that although supplemental oxygen helped the premature babies who were having lung and breathing complications, the high oxyge n destroyed blood vessels in the retina.22 Despite adjustment of oxygen de livery and other medical advances the total number of infants with ROP has not decreased because of the incr eased survival rates among the low birth weight infants.22,26 ROP progresses in two phases.24 The hyperoxia extrauterine environment surrounding the baby precedes the development of th e first phase of ROP.24 The growth

PAGE 26

26 inhibition of neural retina and re tinal vasculature in the first ph ase is followed by a second phase of ROP involving relative hypoxia-induced unc ontrolled proliferative vessel growth.24 The pathological growth of vessels produces a fibrous scar that extends from the retina to the vitreous gel and lens.24 Retraction of this scar tissue can sepa rate the retina from the retinal pigment epithelium (RPE), resulting in a reti nal detachment, bleeding and blindness.22,23 This biphasic disease is associat ed with unbalanced levels of gr owth factors. Low levels of insulin-like growth facto r-1 (IGF-1) and vascular endothelial growth factor (VEGF) are detected in phase I, whereas, excessively high levels of IGF-1 and VEGF are found in phase II.27 IGF-1 plays a critical role in ROP infants. Reducing IGF-1 levels inhibits vessel growth even in the presence of VEGF.28 Low levels of IGF-1 directly reduces vascular density, which subsequently causes early vessel degeneration in phase I. Th e mean serum levels of IGF-1 in age-matched premature babies are directly correlated with the severity of ROP disease stages.28,29 In the second phase of ROP, which is driven by hypoxia, VEGF expression is incr eased in the retina, resulting in pathologi cal neovascularization.30 Because the retinal vessel development of mice is incomplete at birth, Smith et al developed a mouse model of ROP to study the molecular mechanism in the disease.30 This mouse model is intended to mimic the fi rst and the second ph ases of ROP. Traditional therapies such as cryotherapy and laser photocoagulation of other proliferative retinapathies can also be used to prevent blindness in ROP infants.23 However, these methods can reduce peripheral vision and include risks from the anesthesia.23 Therefore, preventive and less invasive therapies for ROP are desirable. Likewise, efforts to understand diseases that involve VEGF and IGF-1 are important to develop such medical treatments. The two phases of ROP require apposite approaches. In phase I, th e hyperoxia induced vessel loss can be partially

PAGE 27

27 prevented by administrating exogenous VEGF or PlGF-1.31-33 While an injection of anti-VEGF aptamer as well as anti-VEGF antibody fragment can be used to treat neovascularization associated with phase II of ROP.34 Pharmacological intervention related to the prevention of vessel loss may be more effective in the treat ment of ROP since the extent of the second destructive phase of ROP is determined by the amount of ve ssel loss in the first phase. Diabetic Retinopathy Approxim ately 100 million people worldwide ha ve been affected by diabetes mellitus (DM).35 In the United States, 16 million individu als are diabetic, and about 40,000 patients per year are diagnosed with the ocular complications of DM in the U.S.36 Among them, 5 to 10 percent are known to be insulin-d ependent type 1 DM and 90 % to 95 % is known to be insulinindependent type 2 DM.36 Vascular diseases are the principa l causes for death or disability in people with diabetes.35 The metabolic abnormalities that char acterize diabetes such as elevated blood glucose levels, increased leve ls of free fatty acids, and insu lin resistance cause vascular dysfunction.35 According to the NIH, the microvascul ar complications of DR are the most common complication of diabetes and thus a lead ing cause of blindness. DR is known to affect approximately 75 % of diabetic patients with in 15 years after onset of the disease.37 Although the best way to prevent visual loss is to in itiate treatment before symptoms develop, many diabetic patients are only diagnosed after visual complications have already begun. DR is divided into two stages: nonproliferative diabetic re tinopathy (NPDR) and pr oliferative diabetic retinopathy (PDR).36 PDR typically develops in patients with type 1 diabetes, while NPDR is more common in patients with type 2 diabetes.36 The progression of DR begins with apoptosis of pericytes and adhe sion of leukocytes to the vessel wall that lead to microvascular occlusion, basement membrane thickening, and increased vascular permeability.38 At this stage, the blood vesse ls become leaky, allowing blood

PAGE 28

28 and vascular fluids to accumulate in th e retinal tissue and form exudate deposits.38 These pathological processes then resu lt in macular edema which is a common feature in patients with NPDR.39 NPDR produces an increasing area of capillary non-perfusion which leads to hypoxia in the retina.38,40 To compensate for the decreased oxygen supply, angiogenic factors are released from the hypoxic retinal tissues that stimulate the growth of new blood vessels on the surface of the retina.37,41 This stage is called PDR. The walls of the new blood vessels are fragile and may break, allowing blood to leak out.38 This can cloud the vitreous and compromise vision.38 In an advanced stage of PDR, newly fo rmed fibro vascular tissue grows from the retinal surface into the vitreous cavity.36 This can cause retinal detachment which can result in blindness.36,39 To date, a common therapy for DR, including ad vanced PDR or diabetic macular edema, is laser photocoagulation. However, this method of ten causes common side effects such as neural tissue loss, peripheral vision loss, impairment of night vision, and change in color perception.42 Moreover, in some patients, the retinopa thy continues to progress after treatment.42 Thus, there is a great need for the development of new therapie s that treat diabetic retinal vascular disease. Recently, pharmacological agents that directly in hibit angiogenesis have been developed to treat DR.34 VEGF plays a pivotal role in the retinal microvascular complications of diabetes.37 The overexpression of VEGF plays a key role in diabetes induci ng retinal vascular dysfunction.37 The developments of agents that directly targ et VEGF and its receptors have been actively studied in clinical research.43-45 The use of endothelial progen itor cells for drug delivery or molecular and genetic manipulation is a technique that takes a new approach in the treatment of DR.

PAGE 29

29 Age-related Macular Degeneration AMD is the most common cause of poor sight in people with age over 60.46 Recently, it is reported that AMD affects approximately 11% of the U.S. population age 65 to 74.46 In the western world, there are approximately 12 to 15 million cases of AMD.46 Two main types of AMD can be distinguished : the dry form (atroph ic) and the wet form (exudative), based on the absence or the pres ence of choroidal neova scularization (CNV).46 Dry AMD is more common than the wet form.46 It develops very slowly and causes gradual loss of central vision. Dry AMD is characterized by the presence of drusen in the macular region.47 The excreted materials, damage d photoreceptors and concentrated by-products of cellular metabolism affect the formation of drusen, which looks like yellow-gray nodules localized between the retinal pigment epithe lium (RPE) and Bruchs membrane.47 Increased drusen formation affects RPE function and eventually causes RPE alteration and depigmentation.47 As a result of dry AMD, patients are likely to lose the central percep tion as well as color contrast sensitivity. Although wet AMD only consists of approximately 10% of all AMD cases, about 80% of severe vision loss is caused by the wet fo rm as compared to 20% that are caused by the dry form. CNV determines the characteristics of the wet AMD. CNV is the process of the growth of immature blood vessels in the choroid, and the pathol ogical new vessels penetrate the subretinal space. Over time, CNV causes hemorrhages, RPE detachment, scarring, and blindness.11,47 The main factor that causes AMD is not known. However, a nu mber of risk factors have been identified that partially contribute to AMD such as age, gender (women are more likely to develop AMD), smoking, genetics and nutrition.48 There are also mol ecular factors that are known to affect the development of AMD.48 It is found that VEGF expression is increased in RPE cells of patients with AMD.11,41 In experimental animal models, VEGF levels were found

PAGE 30

30 to be significantly higher in the vitre ous of wet AMD than healthy controls.11,41,48 To date, the most promising results of a treatment for ARMD has been achieve d with anti-angiogenic reagents that target VEGF.42 Neovascularization Blood vessels are developed by vasculogenesis or angiogenesis. Du ring vasculogenesis, endothelial cells differentiate from progenitor cells and angioblasts, which are already present throughout the tissue, and then link together to form vessels. During angiogenesis, sprouts form from preexisting blood vessels and invade into su rrounding tissue. Most or gans are vascularized by vasculogenesis, but brain and kidney are vasc ularizaed by angiogenesis. Retinal vascular development occurs by a combination of vasculogenesis and angiogenesis, called neovascularization. A variety of stimuli are known to contribute to neovascularization by recruiting stem or progenitor cells and inducing adhesion to activated ECs. Cytokines, chemotactic factors, and angiogeni c factor have been implicated as positive regulators of neovascularization. Some of th ese molecules are strongly induced by hypoxia in cultured cells, including tumo r cell lines, cardiac myocytes, and vascular smooth muscle cells as well as in ischemic tissues. Endothelial Progenitor Cells Bone marrow (BM) is the major reservoir of stem cells in adults. The bone marrow microenvironment, in which bone marrow stem cells remain quiescent, is comprised of stromal cells and extracellular matrix (ECM) components. A special subtype of BM derived stem cells, termed endothelial progenitor cells (EPCs) that ar e able to differentiate into mature endothelial cells and incorporate into sites of neovasculari zation under physiological as well as pathological conditions such as wound healing, organ regenerati on, and tumor growth. EPCs can be isolated from peripheral blood, fetal liver, or umbilical cord blood.49-51 EPCs are characterization by

PAGE 31

31 specific antigens expressed on the surface of the cells. Stem cells maintain primitive characteristics so that they can differentiate or transdifferentiate into a wide range of cell types. This is called stem cell plasticity.52 The identification of true EPCs has been challenged by the phenomenon of stem cell plasticity. Defining the validated EPCs has been debated because several studies have demonstrated overlapping antig ens among subtypes of bone marrow derived cells including EPCs and mesenchymal stem cells.52-58 Although it is not clear what markers define EPCs, it is widely accepted that CD34, va scular endothelial growth factor receptor-2 (VEGFR-2), and CD133 are th e common antigens used in the enrichment of EPCs.59-61 Schatterman, et al. suggested that expression of endotheli al nitric oxide synthase (eNOS) is a reliable marker for EPCs.49,53 EPCs have other characterist ics of endothelial cells including acetylated low density lipoprotein incorporati on and endothelial specif ic lectin binding in vitro.49,59,62 Furthermore, EPCs also show typical endothelial functional characteristics like formation of capillary tubes and production of nitric oxide (NO).63,64 With maturation, EPCs begin to lose expression of CD 34 and CD133 (i.e., early hematopoietic stem cell marker) or start to express CD31, also known as PECAM-1 (platelet/endothelial cell adhesion molecule), vascular endothelial cadherin, and von Willebrand factor.49,57 The differentiation and maturation of EPCs occur when circulating EPCs move to the site of injured vessel or integrated into mature endothelium.50 Increasing evidences suggest that EPCs are preferentially recruited to sites of ischemia and tumor formation and incorporated into functional vasculature.50,65,66 EPC recruitment as well as the release from BM is influenced by various fa ctors. Proangiogenic growth factors such as VEGF, granulocyte/macrophage colony-stim ulating factors (GM-CSF), SDF-1, and erythropoietin (EPO) have been sh own to modulate EPC functions th at play a critical role in

PAGE 32

32 embryo development as well as in homeostasis in adult.65,67 For instance, these factors are essential for EPCs differentiation and blood vess el development during embryogenesis and also contribute in increasing circulati ng numbers of EPCs in adults.65,67 Transplantation of cultured EPCs successfully promotes therapeutic neovas cularization in both ischemic hind limbs and acute myocardial infarction models.50,65 Both the number of circulating EPCs and colony forming ability of EPCs are correlated with some types of diseases. It was found that fewer CD34+ EPCs are circulating in patients with diabetes, diabetic retinopathy, and peripheral artery disease.68 Increasing numbers of EPCs were found in patients with limb ischemia or vesse l wall damage after coronary thrombosis, burn injury, or coronary bypass surgery to rescue the damaged vessels.65,68,69 Since new blood vessel growth from mature ECs has rarely been f ound in adults, and turnover of the quiescent endothelium is considerably low, the vascular repair may need the support of EPCs. The study of EPC biology will help to better understand po stnatal vasculogenesis and also help to find novel therapies for the treatment of pathological neovascularization. Hypoxia Inducible Factor-1 Hypoxia occurs when there is an im balan ce between oxygen supply and demand in cancer or ischemic tissues. In wounds, capillary inju ry generates a hypoxic environment, and altered oxygenation induces the reconstr uctive angiogenic response.70 Hypoxia serves as a critical cue for both physiological and patholog ical angiogenesis in the brai n, heart, kidneys, lungs or muscles.71 In stem cell research, hypoxia is consider ed a potent trigger for mobilization of bone marrow derived cells. Hypoxia inducible factor-1 (HIF-1), a transcript ion factor, functions as a major regulator of O2 homeostasis or an adaptor of O2 deprivation. HIF-1 is a hete rodimer composed of an oxygen related HIF-1 subunit and a constitu tively expressed HIF-1 subunit.72 In order to respond

PAGE 33

33 Figure 1-3. Hypoxia-regulated fact ors and BM-derived cells. HSC and EPC are originated from common precursor, hemangioblast. These cells maintain primitive characteristics so that they can differentiate themselves into a wide range of cell types. EPC has been primary material for this study and it is well characterized as CD34+cells. EPC mobilization is a complex process involving many mediators and cellular interactions. Reduced oxygen tension has been widely believ ed to trigger this process. Once tissue got an injury, the damaged ti ssue releases various cytoki nes and angiogenic factors. Respond to Hypoxia-regulated factors such as EPO, SDF-1, VEGF, and IGFBP-3, circulating EPCs migrate to ischemic area then re-endothelized damaged vessel

PAGE 34

34 rapidly to hypoxia, HIF-1 is continuously synthesized and degraded under non-hypoxic conditions. Under hypoxic conditions, how ever, the degradation of HIF-1 is inhibited, so that the expression is increased exponentially as O2 concentration declines, resulting in dimerization with HIF-1 .73,74 Dimerized complex of HIF-1 binds to hypoxia response el ement (HRE) within a target gene, and recruits coactiv ator proteins, which lead to incr eased transcription of the target gene.75 More than 40 genes are known to be directly activated by HIF-1 at the transcriptional level.76 Genetic studies revealed the presence of a functionally essential HIF-1 binding site in the target gene. The genes induced by HIF-1 regulate molecular mechanism for sensing and responding to changes in O2 concentration. HIF-1 regulated genes contain a cis -acting transcriptional regulato ry element, HRE, as a HIF-1 bindi ng site. Recently, DNA microarray analysis showed that over 2% of all human genes are either directly or indirectly regulated by HIF-1 in endothelial cells.76,77 Physiological stimuli other than hypoxia can also induce HIF-1 activation and the transcription of hypoxia-inducible genes under non-hypoxia conditions. IGF-1 induces HIF-1 synthesis through phosphatidylinositol 3kinase and MAP kinase pathways78, and IGF-1 receptor tyrosine kinase induces HIF-1 protein synthesis, independent from oxygen concentration.75 HIF-1 has been shown to activate tr anscription of the gene encoding VEGF.79 HIF-1 induces VEGF secretion which subsequen tly induces upregulation of SDF-1 expression. In turn, SDF-1 has a reciprocal effect on inducin g VEGF. In addition to growth factors, nitric oxide is also known to play a similar ro le enhancing HIF-1 activation under non-hypoxic conditions mediating prol yl hydorxylase activities.80-82

PAGE 35

35 Vascular Endothelial Growth Factor VEGF was discovered in 1983 and called vascu l ar permeability factor (VPF) due to its blood vessel permeability increasing capacity.83,84 In 1989, it was determined that VPF and the endothelial specific mitogen, VEGF was the same protein.85-87 VEGF plays an important role both in normal physiological angiogenesis and in most of the pathological angiogenesis associated with diseases such as diabetic re tinopathy, rheumatoid arthritis, and solid tumors. The VEGF family consists of seven struct urally related homodimeric glycoproteins: VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D, orf virus-encoded VEGF-like proteins (called as VEGF-E), and a series of snake venoms (collectively called as VEGF-F).88-91 Despite structural similarity, the VEGF homologs play distin ct roles and bind to specific subtypes of VEGF receptors. VEGF exerts its effects by binding to one of its three receptors that belong to the superfamily of recepto r tyrosine kinsase. VEGF-A binds to either VEGF receptor 1 (VEGFR-1 / Flt1) or VEGF receptor 2 (VEGFR-2 / KDR / Flk-1). However, PlGF and VEGF-B exclusively bind to VEGFR1. VEGF-C and VEGF-D are specific ligands for VEGFR-2 and VEGFR-3 (Flt-4) regulating both blood and lymphatic vessel development. Viral VEGF-Es and some snake venom VEGF-F variants exclusively activate VEGFR-2.84,88 Within the VEGF family, most of the resear ch has focused on VEGF-A (usually referred as VEGF). VEGF-A is regarded as the most pot ent mediator of angiogene sis. There are several splice variants of VEGF-A including VEGF121, -145, -165, -189, and -206, while VEGF-165 is the predominant form.67 VEGF mobilization appears to be isoform specific. For instance, VEGF-165, rather than VEGF-189, induces a rapid mobilization of VEGFR-2+ cells into circulation.92 VEGF initiates embryonic vasculogenesis and trigge rs angiogenic sprouting by ac tivating VEGF-R2 on vascular endothelial cells.93,94. VEGFR-2 is also essential for the development of HSCs during early

PAGE 36

36 Figure 1-4. The VEGF (VEGF-R2) signaling pa thway. Upon binding of VEGF, VEGF-R2 is activated by autophosphorylation, and initiates several signaling cascades all of which lead to angiogenesis.

PAGE 37

37 embryonic development.95 In addition, the activation of VE GFR-1 is sufficient to rescue HSC survival in vitro and hematopoietic repopulation in vivo.96 VEGF regulates several endothelial cell functions, including proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules.95 VEGF is also a chemoattractant and plays a role in EPC recruitment and induces in vitro differentiation of EPCs into mature endothelial cells.97 Genetic modifications of VEGF have helped to understand its biology related to EPCs. Overexpression of VEGF in nonischemic mouse hearts can lead to the formation of endothelial cell-derived intramural vascular tumors.98 In addition, VEGF gene transfer promotes EPC migration into ischem ic regions. VEGF-deficient HSCs and bone marrow mononuclear cells show lack of ability to repopulate lethally irradiated hosts.96 Stromal Derived Factor-1 Strom al cell derived factor-1 (SDF-1) belongs to the group of chemokine CXC subfamily, originally isolated from mu rine bone marrow stromal cells.99 It is produced by multiple bone marrow stromal cell types as well as epithelial cells in many organs.100 CXCR4, a 7-transmembrane spanning G protein-coupled receptor, is the only known receptor for SDF-1 and is also a coreceptor fo r human immunodeficiency virus (HIV) type 1.101 SDF-1 is chemotactic for EPC. The chemokine SDF-1 or CXCL12 mediates homing of stem cells to bone marrow by binding to its receptor CXCR4 on circulating cells.102 The SDF-1/CXCR4 signaling pathway is cr itical during embryogenesis, vascular development, and cardiac development. Blocka de of SDF-1 in ischemic tissue or CXCR4 on circulating cells inhibits progenitor cell recruitment to sites of injury.61 Overexpression of CXCR4 on stem and progenitor cells prom otes its proliferation, migration, and in vivo engraftment of NOD/SCID mice.103 SDF-1 gene expression is re gulated by the transcription factor, HIF-1. Progenitor cell mobilization is triggered by hypoxia gr adients through HIF-1

PAGE 38

38 Figure 1-5. The interaction between SDF-1 a nd CXCR4. SDF-1 binding to its receptor, Gprotein coupled CXCR4 leads to activa tion of ERK1/2 activation or MAPK activation. This interaction results in cell migration or proliferation.

PAGE 39

39 induction of SDF-1. HIF-1 induced secretion of SD F-1 in ischemic tissue has a direct correlation with reduced oxygen tension.70 SDF-1 gene transfer induces EPC mobiliz ation from bone marrow into peripheral blood and also improved perfusion to ischemic limbs. It is proposed that SDF-1 induces upregulation of metalloproteinase-9 (MMP-9) ac tivity, which causes cleavage of membrane bound Kit-ligand into soluble Kit-ligand, stem cell factor (SCF). As a consequence, SCF promotes stem cell mobilization into the circulation. Recently, it was shown that SDF-1 is critical for the development of proliferative retinopathy.104 Vitreous concentrations of SDF-1 and VEGF are increased in diabetic patients. Compared to VEGF, exogenous SDF-1 has a greater effect in causing retinal neovascularization in an animal model.104 Intravitreal inj ection of blocking antibodies to SDF-1 disrupts retinal and choroidal neovascularization in mouse.47,103 Blockage of SDF-1 is now being considered for potentia l treatment for ocular vascular diseases. Insulin-like Growth Fact or Bindin g Protein-3 Insulin-like growth factor-I (IGF-I) and II (IGF-II) modulate a diverse range of biological activities including growth, differentiation, su rvival, and regulation of cell metabolism.105 In serum and the extracellular fluid, the majority of circulating IGFs are sequestered into 150 kDa ternary complexes with IGF binding protein (IGFBP) and the liver-derived glycoprotein (acidlabile subunit: ALS).106 This complex prolongs the half-lif e of IGFs in the circulation and prevents them from cros sing the capillary barrier.107,108 IGFBPs consist of six homologous secreted proteins, which specifically bind to IGF-I with high affinity. IGFBP-3, the most abundant binding protein in serum, is present in various glycosylated forms between 40 and 44 kDa. A number of inves tigators have reported that IGFBP-3 has IGF-1 independent cellular actions.109,110 For instance, independent of IGF-I, IGFBP-3 regulates cell activities such as grow th, proliferation, and apoptosis in both carcinoma cell lines and normal

PAGE 40

40 Figure 1-6. IGF-1R signaling path way. IGF-1R is a tetramer consisting of 2 extracellular chains and 2 intracellular -chains with the intracellular tyrosine kinase domain. The activation of IGF-1R signaling pathways induces numerous physiologic actions of IGF-1.

PAGE 41

41 Figure 1-7. Schematic diagram of IGF system. Ligands (IGF-1, IGF-2 and insulin), IGFBPs (1 to 6) and receptors (IR, IGF-1R, hybrid IR /IGF-1R, IGF-2R, IRR and IGFBP-R) are represented. IGF-I interact s with IGF-1R, IR, hybrid IR /IGF-1R and IGFBPs; IGF-2 interacts with IR (mainly with the IR from lacking the exon 11 sequence), IGF-1R, hybrid IR/IGF-1R, IGF-2R and IGFBPs; insu lin interacts with IR, IGF-IR and hybrid IR/IGF-IR. Some IGFBPs are known to be cleaved by IGFBP proteases releasing IGFBP proteolysed fragments, which have low-affinity for IGFs. IGFBP-related proteins (IGFBP-rPs) which ha ve low affinity for IGFs also exist. IGFBP-3 and IGFBP-5 may act through th eir own receptor (IGFBP-R).

PAGE 42

42 Cells.111-119 Whereas an inhibitory role for IGFBP-3 has been widely reported in the field of cancer research, several other researchers have shown contradictory results that IGFBP-3 enhances angiogenic effects.120,121 IGFBP-3 also induces differe ntiation of chondrocytes and human skeletal myoblasts.123 Liver and kidney are the main sources of IGFBP-3.124,125 According to the study by Foulstones group, skeletal muscle may be another source of autocrine tissue for production of IGFBP-3.123 The level of IGFBP-3 in serum is modulat ed by not only its rate of synthesis but also post-translational modification and proteoly sis. While normal individuals have minimal IGFBP-3 protease activity, IGFBP3 protease activity is incr eased among individuals with pregnancy, acute catabolic illness, or diabetes.116 IGFBP-3 proteases have been identified including plasmin, matrix metallproteases, kallik reins, prostate-specific antigen, and cathepsin D.126 IGFBP-3 concentration in serum is also regul ated by other factors such as IGF-I, HIF-1, VEGF, NO, and TGF. IGF-I affects HIF-1 which upregul ates VEGF and IGFBP-3. IGFBP-3 has been identified as one of the hypoxia induced factors.71,127 Felser and colleagues demonstrated that IGFBP-3 doesnt contain an HRE within its prom oter, and that IGFBP-3 gene expression was markedly reduced in HIF-1 -deficient cells under hypoxic conditions.128 VEGF enhances upregulation of IGFBP-3 both HIF-1 dependent and independent ways.129 High concentration of NO, induced by iNOS, decreases the levels of IGF-1 and IGFBP-3 by activating IGFBP-3 protelysis in serum.130 TGFmodulates IGF-independent IGFBP3 function.131-133 TGF1 increases the secretion of IGFBP-3 in a variety of breast cancer cell lines and renal carcinoma cells.122,134-136 IGFBP-3 is induced by TGFand is critical in mesenchymal cell

PAGE 43

43 growth and podocyte apoptosis.122,137,138 Although IGFBP-3 has widely been studied for decades, there are still many questions about the functions of IGFBP-3. Sphingosine 1-phosphate Sphingosine 1-phosphate (S1P) is a platelet derived sphingolipid that has been broadly im plicated in angiogenesis, plat elet activation, inhibi tion of apoptosis, cyto skeletal organization, adherens junction assembly, and morphogenesis.139-141 Sphingosine kinase (SK) catalyzes the formation of S1P by phosphorylation of sphingosine. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly or tr ansiently when cells are exposed to mitogenic agents or other stimuli. These signals activate SK which is responsible for increased level of S1P. SK is an evolutionarily conserved lipid ki nase which consists of five conserved domains. There are two isoforms of SK: the sphinosine kina se type 1 (SK1) and the sphinosine kinase type 2 (SK2). SK1 is mainly expressed in the cytosol, whereas SK2 is localized in the nucleus.142 SK1 and SK2 have different functions. Overexpression of SK1 protec ts against apoptosis resulting in enhanced fibroblasts proliferation, tumor formation in NOD/SCID mice.143 In contrast to pro-survival SK1, SK2 contains a functional putativ e BH3-only domain that induces the inhibition of cell growth. Maceyka, et al showed that SK2 has cat alytic activity to induce apoptosis.142,144 It is accepted that S1P is the ligand for plasma membrane localized G protein-coupled receptors (GPCR) referred to as endothelial differ entiation gene (EDG) receptors or S1PRs. S1P binds to the five members of this receptor family: S1P1, S1P2, S1P3, S1P4, and S1P5 (previously referred to as EDG-1, 5, 3, 6, and -8). These recep tors are highly specific and only bind S1P. The diverse biological processes that are triggered by S1P depend on the pattern of expression of S1P receptors in each cell type as well as coupled G proteins.139 The study of S1P1 null mice emphasized the importance of S1P1 on endothelial cell-pericyte communication in vascular

PAGE 44

44 maturation and angiogenesis.145 For instance, Liu et al. demonstrated that S1P1 null mice are embryonic lethal due to massive hemorrhage that is caused by incomplete vascular maturation in arteries and capillaries.145,146 S1P binds to the S1P1 receptor which induces the activation of eNOS localized in caveolae which poses a sphingolipid enriched domain in the plasma membrane.147,148 S1P/S1P1 pathway acutely increases eNOS phosphorylation through PI3K and Akt activities in bovine aortic endothelial cells.149 In addition, S1P has been identified as a poten t signal-transducing molecule that may exert diverse biological responses such as cellular differentiation, hypertr ophy, proliferation and migration.150-153 S1P activates the small GTPases Rac and Rho, functioning as a chemoattractant for endothelial cells. S1P induced R ho-dependent integrin clustering into focal contact sites that modulate cell adhesion, sp reading, and migration.150 To activate cell migration, S1P enhances phosphorylation of protein kinase Akt in endothelial cells. S1P has been shown to have dual effects on migration of early lym phocytes. In low levels, S1P induced chemoattractant migration of CD4 and CD8 T cells and also enhanced ch emotaxis to CCL-21 and CCL-5. However, at higher levels, S1P had the opposite eff ect, reducing the migratory responses.154,155 S1P acts extracellularly by binding to members of the S1P receptors and regulating cell movement. In addition, S1P also acts as a seco nd messenger intracellularly to regulate calcium homeostasis and apoptosis.88,141,156-158 However, the mechanism of S1P transport is unknown. Recent studies in yeast showed that a member of ABC family of protein might be involved in S1P translocation.159,160

PAGE 45

45 Nitric Oxide Nitric Oxide (NO) is a highly reactive, diffusible, and unstable radical and is involved in signaling in the cardiovascular, ga strointestinal, genitourinary, respiratory, and nervous system s. For instance, NO regulates cellu lar immunity, angiogenesis, ne urotransmission, and platelet aggregation and also promotes synaptic transmission and cytostatic/cytotoxic actions in macrophages.64,82,161,162 NO is generated by NO synthases (NOS). NOS is a heme-containing enzyme that is linked to NADPH-derived electorn transport. NOS catalyzes the oxidation of L-ar ginine to L-citrulline and NO, with tetrahydrobioterin a nd NADPH as essential cofactors.163 Three NOS isoforms have been identified a nd named after the cell type or conditions in which they were first described: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible or inflammatory NOS (iNOS). Because free NO is a transient species with a half-life of about 5 seconds, many investigations of this gaseous molecu le have largely relied on studies of NOS. All three isoforms of NOS are found in different cell types in the eye.164 nNOS is responsible for producing NO in phot oreceptors and bipolar cells, whereas eNOS is present in vascular endothelial cells. iNOS, which is found in Muller cells and in retinal pigment epithelium, is involved in inflammatory process and phagocytosis of the photoreceptor outer segment. iNOS is also thought to be res ponsible for the pathogenesis of diabetic retinopathy.161,164-167 NO generation is important to maintain the va sculature in a relaxe d state, inhibit the adhesion of platelets and white cells, and suppr ess the replication of smooth muscle cells.168 eNOS derived NO diffuses into smooth muscle cells or pericytes then binds to the iron within the heme-group of guanylyl cyclase and produces a conformational ch ange that leads to enzyme activation. NO has been observed to modulating vasculogenesis. NO has an important function

PAGE 46

46 for the stem cell microenvironment in the bone marrow as a molecular mediator in controlling the stem cell niche. eNOS is also important as it promotes angioge nesis and regulates the expression of VEGF. eNOS defici ent mice have an impaired capaci ty to mobilize cells from the bone marrow.169 Guthrie, et al found that NO/NOS pathway is a significant regulator of neovascularization and can modulate hemangioblast activity by dictating the size and branching characteristics of blood vessels that are formed in response to ischemic or chronic injury.170 Disordered NO generation has been implicated in a wide range of diseases. It is well established that endothelial NO bi oavailability is systemically reduced in patients with coronary artery disease and heart failure.171,172 In patients with diabetes mellitus reduced NO bioavailability may result from altered NO metabolis m. In diabetic mice, vascular endothelial dysfunction is associated with uncoupling of eNOS within the endothelium that is caused by oxidation of its essential cofact or tetrahydrobiopterin (BH4), re sulting in a specific loss of endothelial NO bioavailability.173 The subsequent rise in cGMP accounts for many of the physiological effects of NO. The NO dependent cGMP response is rapidly and selectively reduced in diabetic rats, and the cGMP re sponse to exogenous NO donor is progressively reduced174. NO and its signaling mechanism modul ate various physiological processes, however, relatively short half-life makes the study less feasible in this field. Relatively stable NO donors with potential th erapeutic value have been developed. Synthetic chemical reagents that releas e NO continuously over a period of time under physiological conditions have long been used in cl inical management of cardiovascular diseases. NO donors developed with therapeutic value must be able to control the amount of NO released, produce byproducts with minimal si de effects, and the release of NO should not be affected by common biochemical factors. NO decomposes rapidly into nitrite (NO2) and nitrate (NO3) in

PAGE 47

47 biological solutions. These two stable compounds are indicators of NO activity in vivo and can be used as an alternative way to analyze NO concentration in serum.175 Carbon Monoxide Endogenous CO is a signaling m olecule that regulates physiological vascular functions. CO is generated from a family of heme oxygenase s (HO), consisting of three isoforms. HO-1 is an inducible stress enzyme, while HO-2 and HO-3 are constitutively expre ssed proteins. HO-1 is inducible after the stimulati on of cytokines, hypoxia and NO.176 HO-1 (32 kDa) was first purified from the livers of CoCl2 or heme-induced rats and from porcine spleen.177-180 HO-1 catalyzes the rate-limiting step in the oxidative degradation of heme to generate CO, bilirubin (an antioxidant derived from biliverdin) and iron (sequestered by ferritin).181-184 Recent researchers have revealed that CO has profound effects on intracellular signaling processes such as anti-inflammatory, antiproliferative, antiapoptotic, and anticoagulative effects. Using a HO-1 knockout mice, Bak, et al. showed that CO generated from HO-1 has a protective role in cardiovascular system from ischemia/reperfusion induced damage.176 The physiological signaling effects of CO involve relatively few defined mechanisms. The modulations of PKG, PKA, and subsequent stimulation of cGMP or cAMP production are commonly observed in CO related signaling pathways. There are many similarities between CO and NO. Both gases are endogenously produced. Their synthetic enzymes, HO and NOS, are both oxidative enzymes that use NADPH as an electron donor. CO and NO have similar physiological functions (i.e., vasodilation, inhibition of platel et aggregation, and neurotransmission), and can act as second messenger.185,186 Similar to NO, CO binds directly to the heme iron of soluble guanylyl cyclase (sGC), leading to the stimulation of enzymatic activity. The vasoactive properties of CO rely on the stimulation of sGC and the subse quent elevation of cGMP levels.187 CO-mediated activation of

PAGE 48

48 sGC leads to the increase in cGMP production, with a poten cy of enzyme activation 30 times lower than that of NO.187-189 Other signaling mechanisms of CO include th e modulation of MAPK activation and th e stimulation of Ca2+-dependent K+ channel activity. HO-1 activity is correlated with angiogenic f actors. VEGF activates HO-1 expression in endothelial cells.190,191 In normal tissue, depending on th e amount of NO, HIF-1 modulates HO1 activity.192 Deshane, et al. demonstrated that SDF-1 direc tly regulates HO-1 activity which promotes angiogenesis in differe nt cell types including human and mouse aortic endothelial cells as well as mouse EPCs.193 A novel class of compounds, termed carbon monoxide-releasing molecules (CORMs), are stable carbonyl transition metal complex with the capacity of releasing CO in biological systems, and are becoming a useful resear ch tool to explore the mechan ism of which CO exerts its pharmacological activities.194,195 Several experiments of CORMs have provided mechanistic insights in the behavior of CO in biological systems. CORM-1 (dimanganese decacarbonyl), CORM-2 (tricarbonyldichloro ruthenium(II) dimmer), and CORM-3 ((tricarbonylchloro(glycinato)ruthenium(II)) simulate the bioactivities of gaseous CO including vessel relaxation196,197, protection against ischemiareperfusion injury 194,198, and prevention of organ rejection following transplantation and inhibition of the inflammatory response. Vasodilator Stimulated Phosphoprotein Nitric Oxide dependent, vasodilator stim ulated phosphoprotein (VASP) plays a pivotal role in cytoskeletal actin regulation. VASP belongs to a family of pro line-rich proteins that includes the Drosophila melanogaster protein Enabled (Ena), its mammalian ortholog Mena, and the EnaVasp-like protein Evl.199 All Ena/VASP family members share a highly conserved aminoterminal Ena/VASP homology 1 (EVH1) domain fo llowed by a proline-rich central region and a carboxy-terminal Ena/VASP homology 2 (EVH2) domain.200 The structure of EVH1 domain of

PAGE 49

49 Ena/VASP family has been identified by using X-ray crystallography and nuclear magnetic resonance spectroscopy. The EVH1 domain serves as an Ena/VA SP protein-binding site for the focal adhesion proteins incl uding vinculin, zyxin, and a xon guidance proteins roundabout (Robo). EVH1 domain-protein interactions are necessary for the localization of Ena/VASP family to focal adhesions as well as to the periphery of protruding lamellipodia.199,201,202 The central proline-rich region has binding site s for several SH3 and WW domain-containing proteins and profilin. The C-terminal EVH2 do main not only mediates tetramerization of Ena/VASP proteins but also bi nds both monomeric (G) and pol ymerized (F) actin. The EVH2 domain functions appear to be impor tant for both actin-filament bundling and stabilization.199,200,203 VASP is a cytoskeletal actin filament promo ting protein, which is i nvolved in platelet activation, cell adhesion, and migration.204,205 VASP mutant mice exhibit defects in the actindependent process of platelet aggregation.206 The results from genetic approaches such as lossoffunction experiment, site directed mutati on, or overexpression study are suggesting that the importance of Ena/VASP proteins in the develo pmental and physiological processes in various cell types. For instance, VA SP modulates T cell activation, phagocytosis, and epithelial morphogenesis. It also induces migrati on of neutrophils, fibroblasts, and neurons.207-212 In mammalian cells, VASP is localized to focal adhesions and areas of dynamic membrane activity in actin-filament assembly. In endot helial cells, for instance, VASP functions in membrane ruffling, aggregation, and tethering of actin filaments during the formation of endothelial cell-substrate and cel l-cell contacts. VASP expression is increased in endothelial cells during angiogenesis.213

PAGE 50

50 Hypoxia has a direct influence on barrie r function by decreasing VASP expression. Rosenberger, et al. showed that VASP transcription was reduced in a HIF-1-dependent manner (HIF-1 functioning as a transcriptional represso r). They further demonstrated hypoxiadependent binding of HIF-1 to the human VASP pr omoter by functional studies using chromatin immunoprecipitation and site-directed mutage nesis. VASP expression during hypoxia is involved in tissue permeability.214 Vertebrate VASP was discovered and characte rized as a common substrate for both PKA and PKG serine/threonine kinases.215 Elevated guanosine 3', 5'-cyclic monophosphate (cGMP) or adenosine 3', 5'-cyclic monophosphate (cAMP) stimulates the phosphorylation of VASP. To date, three phosphorylation s ites (Ser157, Ser239, and Thr278) have been identified. Phosphorylation of Ser 157 of VA SP leads to a shift in apparent molecular mass in SDS-PAGE from 46 to 50 kDa, indicating phosphorylation caus ing a change in secondary structure of the molecule.216 Phosphorylation of serine 239 in VASP is a useful marker for monitoring PKG activation as well as signali ng pathway. Unlike phosphorylation of serine 157, it doesnt alter the electrophoretic motility of VASP.217,218 During the subsequent cell moving, VASP becomes heavily phosphorylated.219 Phosphorylated VASP has been localized to cell-cell junctions and could be co-immunoprecipitated wi th the tight-junction marker zoula-occludens-1 (ZO-1) protein from e ndothelial cells.215 There is a need for more re search to identify whether such phosphorylation reflects the overall phosphorylation of all VASP with in the cell. The question of whether VASP at the leading edge or VASP at focal adhesions are differently phosphorylated also remains to be answered.205

PAGE 51

51 Significance (Specific Aims) The role of HSC and EPC in supporting postn atal vasculogenesis has been extensively studied regarding m any physiological and pathologi cal situations. Blood vessel development is a complex process, involving multiple proteins expressed by different cell types, all contributing to an integrated sequence of events. The goal of this study was to highlight IGFB P-3 among other angiogenic factors involved in vessel development. IGFBP-3 regulates cell ac tivity in various ways and exerts both proangiogenic and anti-angiogenic actio ns. Characterization of its put ative receptor which initiates downstream cascade is thought to provide better description of IGF-independent effects. Numerous investigators have trie d to determine the characteristics of IGFBP receptors; however, specific IGFBP receptors still remain unknown. Here, we demonstrate that IG FBP-3 has a critical function in vessel development related with NO and SK/S1P signaling pathways. The underlying hypotheses are; (1) IGFBP-3 has an angiogenic effect on HSCs as well as EPCs. (2) IGFBP-3 modulates EPC migration to participate in ne ovascularization by influencing NO generation and VASP redistribution. (3) The dow n stream pathway of IGFBP-3 on EPCs is related to SK signaling.

PAGE 52

52 CHAPTER 2 METHODS AND MATERIALS Cell Preparation Mobilized peripheral blood derived hum an CD34+ cells and CD14+ cells were commercially purchased (Lonza Walkersville, Inc. Walkersville, MD). A vial of frozen cells were thawed in a 37 water bath then washed with hema topoietic progenitor growth medium (HPGM; Lonza Walkersville, Inc. Walkersville MD) containing 10% fe tal bovine serum (FBS) and 20 U/ml of DNase I (Sigma-Aldrich, St. Louis, MO). CD34+ cells were cultured in HPGM supplemented with 25 ng/ml of human stem cell factor (SCF; R&D Systems, Inc. Minneapolis, MN), 50 ng/ml of human throm bopoietin (TPO; R&D Systems, Inc. Minneapolis, MN), and 50 ng/ml of human Flt/Flk2 ligand (FL; R&D System s, Inc. Minneapolis, MN) for maintaining an undifferentiated state. CD14+ cells were maintained an undifferentiated state in HPGM supplemented with 10% FBS. To expand CD34+ cells, defined serum free medi um (StemSpan SFEM; StemCell Technologies, Inc. Vancouver, Canada) was used for culture. One ml of StemSpan SFEM with the addition of cytokines cocktail (100 ng/ml FL, 100 ng/ml SCF, 20 ng/ml interleukin-3, and 20 ng/ml interleukin-6; StemCell Technologies, In c. Vancouver, Canada) and 50 ng/ml TPO (R&D Systems, Inc. Minneapolis, MN) enables 300,000 CD34+ cells to proliferate and expand without differentiation. The number of cells was determin ed with a hemacytometer (Hausser Scientific, Horsham, PA) every 3 days when the medium was changed. Cryo-preserved human lung derived microvasc ular endothelial ce lls (HMVEC-L) were commercially purchased (Lonza Walkersville, Inc. Walkersville, MD). Once the cells were thawed, endothelial cell basal medium-2 (EBM-2) supplemented with growth supplements (5% FBS, 0.04% Hydrocortisone, 0.4% hFGF-B, 0.1% VE GF, 0.1% IGF-1, 0.1% ascorbic acid, 0.1%

PAGE 53

53 EGF, and 0.1% GA-100) (EGM-2-MV singleQuots) (Lonza Walkersville, Inc. Walkersville, MD) was used for optimal growth and proper main tenance. When plated cells were confluent the cells were washed twice with PBS th en 0.025% trypsin and 0.01% EDTA mix (Lonza Walkersville, Inc. Walkersville, MD) was adde d. The plate was placed for 45 seconds at 37 in humidified 5% CO2 incubator. The trypsin was neutraliz ed using twice the volume of trypsin neutralizing solution (Lonza Walkersville, Inc. Walkersville, MD) and then cells were centrifuged at 1000 RPM in an Eppendorf CT 5810R. The pellet was resuspended with EBM-2 containing growth supplements for splitting into new plate. Endothelial progenitor cells (EPC) were is olated from periphe ral blood from healthy individuals or diabetic patients. The blood was collected into cell preparation tubes (CPT; BD Biosciences, San Jose, CA) and spun to obtain mononuclear cells. EPC was separated from the mononuclear fraction using a CD34+ isolation kit (StemCell T echnologies, Vancouver, CA). Mononuclear cells (2 x 107) were incubated with a CD34+ selection cocktail for 15 minutes. 50 l of nanoparticles were then added to the ce lls and incubated for a further 10 minutes. The suspension volume was increased to 2.5 ml and th e tube containing the cells was placed in a magnet for 5 minutes. The supernatant from the tube was poured off and the remaining CD34+ cells in the tube were resuspended in culture media (Endocult; StemCell Technologies, Vancouver, Canada). These isolated EPCs were cultured for tube formation and differentiation assay using Endocult containing 20% Endocult supplement (StemCell Technologies, Vancouver, Canada). Migration Assay CD34+ cells and CD14+ cells were stained with calcein-AM (Molecular Probes, Eugene, OR), prior to loading them into the upper wells of a disposable chemotaxis chamber (Neuro

PAGE 54

54 Probe, Gaithersburg, MD). The lower wells were filled with IGFBP3 at 0, 1, 10, and 100 ng/ml dissolved in HPGM (negative control) or HPGM supplemented with 20% FBS (positive control). The chamber was incubated at 37oC, 95% humidity, 5% CO2 for 4.5 hours. The number of migrating cells was determined by relative fluo rescence of the lower ch amber using a SynergyTM HT (Bio-Tek Instruments, Inc. Wino oski, VT) with an excitation of 485 20 nm and emission of 528 20 nm. EPC Tube Formation Peripheral blood was collected into C PT tubes with heparin (BD Bios ciences, San Jose, CA) by routine venipuncture. The mononuclear cells were collected after centrifugation at room temperature in a swinging bucket rotor at 1,800g fo r 20 minutes. These cells were then cultured on fibronectin-coated culture dishes (BD Biosci ences, San Jose, CA) with Endocult stem cell liquid media (Stem Cell Technologies, Vancouver, CA) per manufacturers protocol. IGFBP3 was added to the cultures at day 3 at 0, 1, 10 and 100 ng/ml. Cells were imaged on day 5. Images were captured with a fluorescence microscope (Axiovert 135; Carl Zeiss, Thornwood, NY). The endothelial nature of the cells wa s confirmed by incorporation of Dil (1, 1dioctadycl-3, 3, 3-tetrame thyl-indocarbocyanin percholra te)-labeled acetylated-LDL (Molecular Probes, Eugene, OR) at 50 g/ml final concentration. Cell Proliferation Assay A high sensitivity ce ll proliferation kit (ViaLight Plus Kit: Lonza Rockland, Inc. ME) was used according to manufactures protocol to assa y for cell proliferation. Cell proliferation was measured based on the bioluminescent cytoplasmic ATP level. Cultured cells on 96 well plates were removed from the incubator and allowed to cool -to room temperature for about 5 minutes. 50 l of the cell lysis reagent (Lon za Rockland, Inc. ME) was added to each well to extract ATP

PAGE 55

55 from metabolically active cel ls. The cell lysate (100 l) was transferred to a white walled luminometer plate (Lonza Rockland, Inc. ME). Then ATP monitoring reagent plus (AMR PLUS: Lonza Rockland, Inc. ME) was added to each well to generate a lu minescent signal. The luminometer (BioTek Instruments, Inc. Winoos ki, VT) was programmed to take one second integrated luminescence reading of each well. Nitric Oxide Measurement NO production was m easured by the use of 4-amino-5-methylamino-2, 7difluorofluorescein diacetate (DAF-FM diacetate: Invitrogen Carlsbad, CA). The cells were washed with PBS containing calci um, magnesium, and 1 mg/ml glucose to remove phenol red in the culture media. The cells were incubated with 5 M DAF-FM diacetate for 1 hour on ice in the dark. Cells were washed with PBS containing calcium, magnesium and 1 mg/ml glucose then transferred to black 96 well plate (Nunc In ternational, Rochester, NY). NO fluorescence was measured using excitation and emission wavelengths of 488 20 nm and 520 20 nm respectively. Western Blot Analysis For western blot analysis 8 g of total protein was loaded on a 10-20% gradient Criterion gel (BioRad, Richm ond, CA). The samples we re electrophoresed at 120 V for 20 minutes to allow for stacking the samples and then 140 V for 65 minutes to separate pr oteins. The proteins were transferred from the gel to a nitrocellulose membrane (Bio-Rad Labor atories, Inc., Hercules, CA) using a semi dry membrane apparatus (Bio-R ad Laboratories, Inc., Hercules, CA) at 20 V for 40 minutes. The membrane was blocked in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour at room temperature with gentle shaki ng. The blocked membrane was incubated with diluted primary antibody at 4oC overnight in a cold room. The membrane was

PAGE 56

56 washed 4 times for 5 minutes in PBS containi ng 0.1% Tween-20 (Fisher Scientific, Pittsburgh, PA) and then incubated with d iluted fluorescently labeled secondary antibody for 1 hour at room temperature with gentle shaking. The membrane was washed 4 times for 5 minutes each with PBS containing 0.1% Tween-20 followed by final washing with PBS to remove excessive Tween-20. The membrane then was scanned in th e appropriate channels using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE ). The same membrane was used to detect the internal protein control, cofilin (Sigma-Aldrich, St. Louis, MO). In-cell Western Assay HMVEC-L was grown in 96-well plate (Nalge N unc International, Rochester, NY). When the cells reached 75-80% of conflu ence, cells were washed twice with PBS. Instead of serum starvation, cells were treated w ith IGFBP-3 in EBM-2 without cy tokines during the treatment. CD34+ cells were cultured in defined seru m free medium (StemSpan SFEM; StemCell Technologies, Inc. Vancouver, Canada) to obtain the optimal number of cells. StemSpan SFEM with the addition of cytokines cocktail (100 ng/ml FL, 100 ng/ml SCF, 20 ng/ml interleukin-3, and 20 ng/ml interleukin-6; StemCell Technologi es, Inc. Vancouver, Canada) and 50 ng/ml TPO (R&D Systems, Inc. Minneapolis, MN) enables CD34+ cells to proliferate and expand without differentiation. The number of cells was determin ed with a hemacytometer (Hausser Scientific, Horsham, PA). The cells were transferred in to 96-well plate (10,000 cells per well: BD Falcon, San Jose, CA). After incubation with or wit hout CO or NO donor, cells were fixed with 4% formaldehyde for 20 minutes at room temperat ure then pelleted by centrifugation. Fixing solution was removed then triton washing solu tion (PBS containing 0.1% triton X-100) was added to the cells for permeabilization. After pe rmeablization, cells were blocked in LI-COR Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1.5 hour at room temperature with moderate shaking. Blocking buffer was rem oved by aspiration and the cells were incubated

PAGE 57

57 with 50 l of diluted primary antibody at 4oC overnight in a cold room. The cells were washed 4 times for 5 minutes in PBS containing 0.1% Tw een-20 (Fisher Scientific, Pittsburgh, PA) and then incubated with diluted fluorescently la beled secondary antibody. After 1 hour the cells were washed 4 times for 5 minutes each w ith PBS containing 0.1% Tween-20 followed by a final washing with PBS to remove excessive Tween-20. The 96-well plate was scanned in the appropriate channels using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Relative quantification wa s normalized and re-adjusted in cell number from well to well using DNA staining. Quantitative Transcription Analysis with Real Time Poly merase Chai n Reaction (RT PCR) Total mRNA from human CD34+ cells, human CD14+ cells or retina from the mouse pups was isolated using the Total RNA Mini Kit (Bio -Rad Laboratories, Inc., Hercules, CA). The mRNA was transcribed using iScript cDNA Synthesi s Kit (Bio-Rad Laborator ies, Inc., Hercules, CA) and real-time PCR were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA). Primers for the PCR we re designed using vector NTI (Infomax, North Bethesda, MD) and purchased (Intregrate d DNA Technologies, Coralville, IA). Immunohistochemistry EPCs were cultur ed on 8-well tissue culture cham ber slides coated with human fibronectin (BD Biosciences San Jose, CA) and treated with 100 M diethylenetriamine/nitric oxide adduct (DETA-NO) (Sigma-Aldrich, St. Louis, MO), 10 M CO donor (tricarbon yldichlororuthenium (II) dimer) (Sigma-Aldrich, St. Louis, MO), or 100 ng/ml IGFBP-3 (Upstate cell signaling solution, Lake Placid, NY) for 15 minutes or 4 h ours. After treatment, medium was removed and fresh ice cold 4% paraformaldehyde (PFA) was added and the samples held overnight at 4C. Cells were then washed in PBS and permeabilized with 0.1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) for 30 minutes at room temperature. The cells were washed 3 times

PAGE 58

58 with PBS and blocked in 10% normal goat seru m (Jackson ImmunoResearch Labs, West Grove, PA) or 1% bovine serum albumin (BSA; Sigma-Aldrich, St.Louis, MO) at room temperature to block non specific antigens. After 1 hour the cells were incubated with 5 g/ml mouse antiVASP antibody (BD Biosciences, San Jose, CA) in 5% normal goat serum overnight at 4 Specific secondary antibody, goat an ti-mouse IgG1-fluorescein isot hiocyanate (FITC; Southern Biotech, Birmingham, AL) was diluted and a dded to each chamber for 1 hour at room temperature. Then the cells were washed, drie d, and mounted with Vect ashield 4, 6-diamino2-phenylindole (DAPI) (Vector Laboratories Inc. Burlingame, CA) for DNA labeling. Cells were examined using a fluorescence microscope (Nikon Eclipse TE200) using a Nikon planfluor 40 X 1.30 oil objective and a FITC-conjugated st andard filter set (520 2 nm). Pictures were captured using a SPOT digital camera 0.60X HRD060 NIK (Diagnostic Instruments, Inc. Sterling Heights, MI) and processed usi ng SPOT Advanced software Version 2.2.1 for Windows. Flow Cytometry Analysis Protein expression of VEGF recepto r, phosphorylated eNOS and CD133 cell surface antigen in CD34+ cells were evaluated using flow cytome try analysis. VEGF receptor expression in CD34+ cells was examined after incubation for 0 or 15 minutes in 5% CO2 at 37 with exposure to 100ng/ml IGFBP3 (Upstate cell sign aling solution, Lake Placid, NY). Following treatment, the cells were permeabilized using a Cytofix/Cytoperm Kit (BD Bioscience, San Jose, CA). The cells were blocked with 10% nor mal human serum (Jackson Immuno Reserch labs, West Grove, PA) in PBS. Ten g anti-VEGFR-1 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or 10 g anti-VEGFR-2 antibody (NeoMarkers, Fr emont, CA) was added to the cells and subsequently incubated for 30 minutes on ice. The cells were washed with PBS and incubated

PAGE 59

59 with 23 g of FITC conjugated goat anti-mouse anti body (Jackson Immuno Research labs, West Grove, PA) in the dark for 30 minutes on ice. Ce lls were then washed and analyzed by flow cytometry. The isotype control for both VE GFR-1 and VEGFR-2 antibodies was anti-GFP (Molecular probes, Carl sbad, CA) antibody (15 g). To measure the eNOS protein expression, the cells were incubated with 100 ng/ml of IGFBP-3 for up to 72 hours. IGFBP-3 was added every 24 hours. When the cells were collected for analysis, cells were permeabilized and blocked. Then 5 g/ml of anti-eNOS antibody (BD Biosci ence, San Jose, CA) was added for 30 minutes. As a secondary antibody, 23 g of FITC conjugated goat anti-mouse lgG (Jackson ImmunoResearch Labs, West Grove, PA) was use d. The surface expression of CD133 antigen was assessed using a phycoeryth rin (PE) conjugated anti-CD133 antibody (Miltenyi Biotec Inc. Auburn, CA). CD34+ cells were incubated with or without IGFBP-3 for up to 72 hours as above. Isotype control for this experiment was PE-conjugated mouse IgGa, immunoglobulin isotype control monoclonal antibody (BD Bi oscience, San Diego, CA). Apoptotic dead cells were removed before analysis by 7-aminoactinomycin D (Sigma-Aldrich, St. Louis, MO) positive selection. Data were acquired with FACS Cali bur flow cytometer (BD Biosciences, San Jose, CA) and were analyzed with BD Cell QuestTM (BD Biosciences, San Jose, CA). Hematopoietic Stem Cell (HSC) Transfection Mouse HSCs were obtained from bone marrow isolated from homozygous transgenic gfp mice. Highly enriched gfp+, Sca1+ (stem cell antigen 1) and c-kit+ HSCs were obtained by fluorescence-activated cell sorting (FACS). Cells were transfected with a recombinant adenoassociated virus (rAAV) vector encoding IGFBP-3. The rAAV vector was chosen for long term IGFBP-3 overexpression in the eye. Expression of IGFBP-3 was selectively increased in proliferating endothelial cell by a specific pr omoter composed of 7 x 46-mer multimerized

PAGE 60

60 endothelin enhancer (ET) upstream of a human Cdc6 (cell division cycle 6) promoter. Gfp+ HSCs were transfected using polyethylenimine (P EI) /plasmid complexes. 4.2 mg/ml stock PEI (Sigma-Aldrich, St. Louis, MO) solution was made in acidified distilled water (pH 5.0). The PEI /plasmid complexes were prepared by adding branched PEI to the plasmid DNA. 100 l PEI /plasmid complex were composed of 150 mM sodium chloride (NaCl) 1 g DNA, and 24 l PEI stock solution. The complexes were then mixed by vortex mixer set on high for 10 seconds and incubated for 30 minutes at room temperature then added to the cells. Experimental Animals All anim al procedures conducted in this study were in agreemen t with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (DHEW Publication No. NIH 80-23, Offices of Science and Health Reports, DRR/NIH, Bethesda, MD 20205). All protocols were approved by the Universi ty of Florida Institutional Animal Care and Use Committee (IACUC). Timed pregnant C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in a temperature controlled room with a 12-hour light/dark cycle in the University of Florida Heal th Science Center Animal Resources facilities. Oxygen Induced Retinopathy (OIR) Mouse Model The OIR m ouse model was used to induce the fo rmation of preretinal neovascularization in mouse pups. On postnatal day 7 (P7), the pups and the nursing dam were placed into an oxygen chamber and exposed to hyperoxic levels of O2 (75%) for 5 days. On P12 the mice were returned to room air. The retu rn to normoxic conditions (21% O2) after being held at 75% O2 simulates a hypoxic stimulus that initiates the forma tion of the preretinal va sculature. The mice were sacrificed at P12.5 and P17 and their eyes removed for analysis. One cohort of animals (n=12) underwent the OI R model and were euthanized at P12.5 to examine the expression of VEGF-A and IGFBP-3 in combined posterior poles and neural retinas.

PAGE 61

61 A second cohort of mice was intravitreally inject ed on P1 with either the plasmid expressing mouse IGFBP-3 under the control of the proliferating e ndothelial cell -specific promoter or the cloning vector as an injection cont rol. For intravitreal injectio n into P1 mouse pups, ice-induced anesthesia was performed by placing the neonate on a plastic shield over a layer of crushed ice for 1 to 2 minutes. By utilizing a proliferati ng endothelial cell-specific promoter IGFBP-3 expression was targeted to areas of neovascular ization. Selected anim als were euthanized immediately upon removal from hyperoxia at P12. 5. Eyes from mice injected with IGFBP-3 plasmid (n=9) were compared with eyes from mice injected with empty plasmid vector (n=9), or the uninjected eye of the same animal (contralat eral eye). A third cohort of mice (n=18) was injected on P1 with mouse gfp+ HSCs (5 x 103 cells per eye in 1 l injection volume) transfected with the identical plasmid as described above. The mice were subjected to the OIR model and euthanized at P17. Data from these mice were compared with the uninjected eye of the same animal or with mice injected with mouse gfp+ HSCs transfected with empty plasmid (n=18). Retinal Flat Mounts The m ouse thoracic cavity was opened, the right atrium was punctured with a 27-gauge needle and the left ventricle was cannulated wi th a 22-gauge angiocatheter, and the mouse was perfused with rhodamine-labeled dextran for preparation of retinal flat mounts. One whole eye from the animal was removed and fixed in 10% PFA overnight. The retina was washed overnight in running water and then incubated fo r 3 hours at 37C in 0.1M Tris buffer (pH 7.8) supplemented with DIFCO BACTO 1:250 Trypsin (Invitrogen, Carlsbad, CA). The incubation was terminated after the intern al limiting membrane had been removed. All nonvascular cells were brushed away from the vasculature. The retina was then mounted onto a glass slide.

PAGE 62

62 GS Isolectin and GFP Double La beled Immunohistochemistry Retinal whole-m ounts were prepared as described above. GS isolectin B4 (SigmaAldrich, St. Louis, MO) and chicken anti-GFP or mouse anti-GFP/EGFP (Chemicon, Temecula, CA) were used to co-visualize gfp+ cells with the vasculature. Retinal whole mounts (n=6) were permeabilized with 0.1% Triton X-100 (F isher Scientific, Pittsburgh, PA) in PBS. Samples were then blocked in 1% BSA in PBS for 30 minutes and retina was incubate d with biotinylated GS isolectin B4 (Sigma, St Louis, MO) overnight. The retinas were washed twice for 10 minutes in PBS supplemented with 0.1% Triton X-100 followed by a last 10 minute wash in PBS. The retinas were then incubated with streptavidin Cy-3 (1:100) for 2 hours at room temperature. Retinas were incubated overnight at 4C with diluted anti-GFP antibody then washed with PBS supplemented with 0.1% Triton X-100. The retinas were allowed to incubate for 4 hours at room temperature with the appropriate ly diluted secondary antibodies followed by washing. Finally the whole retinas were flat mounted with the ganglion cell layer up in Prolong Anti-Fade (Molecular Probes, Eugene, OR). Microscopy and Mapping Retinal whole-m ounts (n=6) were examined by both deconvolutional and confocal microscopy. Zeiss microscope (model Axioplan 2 attachment HBO 100; Carl Zeiss, Inc.Germany) and Axiocam HRm camera (Carl Zeiss, Inc, Germany) were used for deconvolutional analysis. Confocal microscopy was conducted with an argon-krypton laser (Leica Microsystems, Wetzlar, Germany) mounted on a Leica DMRBE epifluorescence photomicroscope. Alexa Fluor 488 and Cy3 fluor escence was excited sequentially at 488 and 550 nm respectively. Images were processed with Adobe Photoshop software (Adobe Systems, Inc. San Jose, CA).

PAGE 63

63 Vascular Density Analysis and Sta tistical Analysis To validate vascular density, morphological analysis needed to be changed to a quantitative measurement. Af ter retina whole mounts were immunostained, vasculartues in retina were then subject to vascular density analysis. The retina was divided into 3 representative fields of views from each of the central, midperipheral and pe ripheral retinas. Fields of view selected as peripheral retinas in cluded regions of capillary sized vessels directly adjacent to radial arterioles. The area of centr al and mid-peripheral retina included the radial arteriole. Then each field was captured using the 40x objective. A 10 x 10 grid was superimposed onto the micrograph and the inciden ce of presence or absence of vessels at the intersection points of each grid wa s determined so that vascular density expressed as a number from 0 to 100. The mean vascular density incidence was determined for each area and compared with its control. The data is presented as m eans standard deviation (SD), and the statistical significance of differences among mean values was determined by one-way ANOVA and the Tukey HSD multiple comparison post hoc tests for the hyperoxia experime nts, and a two-tailed t-test utilized for the hypoxic experiment. ANO VA statistical analysis was performed with SPSS 13.0 software (SPSS, Chicago, Illinois), and two-taile d t-test statistical an alysis was performed with a P value of <0.05.

PAGE 64

64 CHAPTER 3 RESULTS IGFBP-3 Induces Migration of CD34+cells and Endothelial Cells CD34+ cells are known to leave the circulati on and migrate along a hypoxic gradient into sites of ischemia and it is believed that CD34+cell trafficking is mainly regulated by hypoxiaregulated factors. To determine whether IGFB P-3 functions as a hypoxia -regulated factor, the effect of IGFBP-3 on migration of different cell types was exam ined. Migration assays for circulating cells were performed using m odified Boyden chamber assay. Figure 3-1A demonstrates that IGFBP-3 stimulated the migration of circulating CD34+ cells in a concentration-dependent manne r, whereas circulating CD14+ monocytes showed a blunt response to increasing concen tration of IGFBP-3. Human retinal endothelial cells (HREC) were also exposed to varying concentrations of IGFBP-3 for 12 hours using the Boyden chamber assay for adhered cells. As depicted in Figure 3-1B, primary cultured HREC mi grated toward IGFBP-3, but the response was not as robust as seen with the circulating CD34+population. These results (Figure 3-1A and B) demonstrate that IGFBP-3 has a cell-type speci fic chemotactic function. Compared to mature endothelial cells, the response of CD34+cell to IGFBP-3 was exceptionally sensitive even at the lowest concen tration (1ng/ml) supporting that mobilization of bone marrow derived cells can be triggered by subtle changes in a hypoxia regulated factor such as IGFBP-3. IGFBP-3 Increases Expression of VEGF Receptors on CD34+cells To examine possible was in wh ich IGFBP-3 could modulate CD34+cell response to hypoxia, we examined whether IGFBP-3 could in fluence VEGF and SDF1 receptor expression on CD34+cells. VEGF and SDF-1 are well known stem cell homing factors that are also

PAGE 65

65 hypoxia-regulated factors. Exposure of CD34+cells to varying concentrations of IGFBP-3 resulted in increased expression of VEGFR1 (Figure 3-2A) and VEGFR-2 (Figure 3-2B) on CD34+cells. By contrast, IGFBP-3 di d not have an effect on CXCR-4 expression in these cells (Figure 3-2C). Figure 3-2 suggested that IG FBP-3 has a VEGF-dependent, SDF-1-independent function on CD34+cells. IGFBP-3 Promotes CD34+cells Differentiation to Endothelial Cells Exposure to IGFBP-3 for 72 hrs resulted in a reduction of CD133 surface expression in CD34+cells (Figure 3-3A), which is associated with promoting differentiation of immature cells to a more committed phenotype. EPC grown on fibronectin showed a dose-dependent tube formation and acetylated LDL incorporation compar ed to control untreated cells (Figure 3-3B), supporting that IGFBP-3 can influence multiple steps that are relevant to angiogenesis. IGFBP-3 Enhances CD34+cells Proliferation To determine whether IGFBP-3 modulates proliferation of CD34+cells in vitro CD34+cells were cultured in the presence of 100 ng/ml IGFBP-3. After 24 hour, 3 days, and 5 days suspension culture, cytoplasmic ATP was detect ed. IGFBP-3 increased the proliferation of CD34+cells by 36.11% (day 3) and 56.03% (day 5) co mpare to untreated control (Figure 3-4). Corroborated with the result observed in Figure 3-3B, IGFBP-3 enhances the proliferation of CD34+cells as well as EPC. Expression of Hypoxia-regulated Factors in Retina Messenger RNA was extracted from posterior cu ps including neural retinas of m ouse pups to check the expression level of IGFBP-3 in isch emic retina. Neonatal mice were divided into two groups. One group was exposed to high oxyge n for 5 days then returned to normal oxygen tension and the other group was subjected to normoxia control. VEGF and IGFBP-3 mRNA levels were determined using reverse transcri ption on total mRNA follo wed by real time PCR on

PAGE 66

66 the cDNA products using specific primer pairs for VEGF and IGFBP-3. As shown in Figure 3-5, the mRNA expression of VEGF and IGFBP-3 was si gnificantly increased in hypoxic retina. The fold-change in IGFBP-3 expressi on was markedly greater that the fold-change in VEGF. It supports the importance of IGFB P-3 response following hypoxia. IGFBP-3 Protects Neonatal Retinal Vessels from Oxygen Induced Va so-obliteration To validate the function of IGFBP-3 in vivo mouse pups were injected with rAAV protein expression vectors expre ssing IGFBP-3 on postnatal day 1 (P1). The expression of the IGFBP-3 was driven by a proliferating endothelia l cell specific promoter (ET/cdc6). The uninjected eyes (contra-lateral) of IGFBP-3 plasmid injected mi ce were used as one of the control conditions and empty plasmi d injected eyes served as the other control. Vessels positive for Griffonia simplicifolia isoletin B4 ( GS isolectin) show the cha nges in the vasculature following high oxygen exposure (Figure 3-6 A to L). The GS isoletin conjugated to HRP provided a low magnification view of the entire retinal vasculatures (Figure 3-6 M and N). The vessels in the IGFBP-3 treated eye had a more normal and mature vascular tree (Figure 3-6 A-D and M), than eyes treated with plasmid contro l. Vessel growth was shown in the IGFBP-3 injected eyes as evidenced by the presence of lectin positive vascular extensions migrating towards the avascular peripheral retina (arrows in Figure 3-6 D). In contrast, massive oxygen-induced vasoobliteration was shown in empty plasmid injected retina (Figure 3-6 E-H). The rema ining vascular remnants, shown at higher magnification in Figure 3-6 F-H, lacked effectiv e vascular perfusion and had a highly aberrant branching pattern (Figure 3-6 N). The morphology of vascular remnants in the peripheral retina of the uninjected eyes (the contra-lateral ey e of the pups injected with IGFBP-3 containing plasmid) had regions with vascular abnormalities including reduced capillary density (Figure 3-6 I-L) and closure of capillary segments (arrow in Figure 3-6 K). As depicted in Figure 3-6, over-

PAGE 67

67 expressing IGFBP-3 in vivo resulted in the maintenance of a vascular bed with a more normal morphology under hyperoxia condition. Quantitative Analysis of Vascular Density in Vaso-oblite ration Phase To determine vascular density, morphological analysis was performe d. Figure 3-7 shows representative retinas from IGFBP3 expressing plasmid injected eyes (Figure 3-7 A-C), contralateral uninjected eyes (D-F), and control plasmid inject ed eyes (G-I). A 10 x10 grid was superimposed onto the 40 X objectives from each field of retina and the vessels found at intersection points of each grid we re determined. The number count ed as a vascular density was expressed as a percentage from 0 to 100 (bottom right corner of the image). Figure 3-7 J summarizes quantit ative measurement of vascul ar density results from central, mid peripheral, and periphe ral regions of retinas in each group. As revealed in Figure 37 J, IGFBP-3 protected the retinal vasculatures from hyperoxia-induced ve ssel regression in mid peripheral and peripheral regions of the retina. IGFBP-3 Decreases the Incidence of Pre-retinal Neovascularization Abnor mal vessel growth in pre-re tinal region is a hallmark of proliferative retinopathies. Pre-retinal blood vessels grow outside the retinal inner limiting membrane into the vitreous space under pathological circumstances. Neovascularization in the eye of OIR model was evaluated by the average number of pre-retina l endothelial nuclei per H&E stai ned retinal section. Figure 3-8 shows reduced aberrant neovascularization by induction of IGFBP-3 in mouse retina. IGFBP-3 Expression in Transfected HSC To exam ine IGFBP-3 expression by the plas mid, HSC were transfected with IGFBP-3 expressing plasmid and total mRNA was isolated. The promoter (ET/cdc6) has been previously characterized both in vitro and in vivo. 220,221 Low molecular weight PEI was used for effective

PAGE 68

68 transfection of HSC. Transfection efficiency of 40% was typi cally observed. The fold-change of IGFBP-3 in transfected cells was significan tly increased compared to untransfected HSC (Figure 3-9). Co-localized IGFBP-3 Expressing gfp+HSC within the Vasculature Inhibit Neovascularization To evaluate the effect of endogenous IGFB P-3 on HSC behavior in vivo, P1 mouse pups underwent intravitreal injection wi th IGFBP-3 plasmid-transfected HSC. The effect was observed by incorporation of gfp+ HSC into the retinal vasculature (Figure 3-10 A-C). Gfp+HSC localized vascular endothelial cells were evident in radial arterioles (Figure 3-10A) and hemangiomas (B). In addition, filopodial extensions were seen originating from the neovascular clump towards avascular retina (C). Eyes injected with IGFBP-3 transfected HSC showed less pathological neovascularization compared to uninjected eyes or eyes injected with control transfected HSC. Figures 3-10D and 3-10E demonstrate quantitative vascular density from the retinas during hyperoxia and hypoxia phase, respectively. IG FBP-3 overexpressing HSC inhibit abnormal neovascularization in hypoxia pha se by protecting neonatal retinal vessels from oxygen induced regression in hyperoxia phase. Hypoxia-regulated Factors and Nitric Oxide Signaling To support h ypothesis that hypoxia-regulated factors modulate EPC mobilization by increasing nitric oxide and activation of its downstream signali ng pathways, we examined the downstream signaling of two well known EPC chemoattractant, VEGF and SDF-1. CD34+cells were treated with either 25 ng/ml VEGF or 100 nM SDF-1. Anti-phoshporylated eNOS antibody was used to demonstrate whether thes e hypoxia-regulated factor s activate eNOS in CD34+cells. VEGF induced eNOS phosphorylation in 15minutes (Figure 3-11A), however SDF1 showed no significant effect on eNOS phosphorylation in CD34+cells (Figure 3-11B). SDF-1

PAGE 69

69 mediated its effects through generation of CO rather than NO. SDF-1 increased HO-1 expression to generate CO which in turn triggers cell migration.193 NO and CO Promotes CD34+cells Migration Direct stimulation of CD34+cells by NO has been reported.170,222 To determine the effect of CO on cell motility, migration assay was performed with CO-treated CD34+cells. Exposure to exogenous CO acutely increased cell migration in response to chemotactic stimulus, SDF-1 (Figure 3-12). The NO donor (DETA-NO) wa s compared to the CO donor (Ru(II)Cl2(CO)3 dimer) and were used for pretreatment of CD34+cells. Figure 3-12 shows both NO and CO has an effect on CD34+cells migration. Different Phosphorylati on Sites of VASP VASP function is initiated by phosphorylation. VASP was origin ally characterized as a substrate of both PKA and PKG. PKA a nd PKG phosphorylate VASP on residues-serine 157 and serine 239, respectively. The effects of NO and CO on cell m igration is shown in Figure 312. CD34+ cells were incubated for 15 minutes in the presence of either the NO or CO donor. Both NO and CO exposure enhanced VASP phos hporylation, however, site of phoshporylation was different (Figure 3-13). As descri bed in Figure 3-13, NO donor increased VASP phosphorylation at serine 239, whereas CO incr eased phosphorylation at serine157 in CD34+ cells. NO Increases VASP Phosphorylation in Diabetic CD34+cells CD34+ cells from diabetic individuals ha ve been shown to have reduced NO bioavailability. Previously it was found that exogenous NO administration could correct decreased EPC migratory response in diabetic CD34+cells.222 To evaluate this observation further and to determine whether this improve ment was due to increased VASP phosphorylation, the level of phospho-VASP in diabetic CD34+cells was measured by flow cytometry analysis.

PAGE 70

70 Diabetic CD34+cells were isolated from patients with t ype 1 and type 2 diseases and the cells were pretreated with the NO donor. There was considerable patient to patient variation in the level of VASP expression; however NO treat ment resulted in stimulation of VASP phosphorylation at serine 239 in all the diabetic cells (Figure 3-14). NO and CO Cause VASP Redistribution to the Leading Edge of the Cells Phospho-VASP is localized to focal adhesions and areas of dynam ic membrane activity. Redistribution of VASP to the leading edge of the endothelial cells in response to exogenous NO or CO was observed (Figure 3-15). As shown in Figure 3-15 A and D, VASP is evenly distributed within the cytoplasm under basal conditions. VASP was redistributed to the advancing edge of the cell following 15 minutes stimulation with NO donor (3-15 B and E). CO donor also causes VASP redistribution as sa me pattern as NO donor (3-15C and F). IGFBP-3 Increases eNOS Phosphorylation Like the other hypoxia-regulated factors, SDF-1 and VEGF, IGFBP-3 modulates CD34+cells mobilization (Figure 3-1). We next examined whether IGFBP-3 stimutaled NO generation in CD34+cells. Two different western blotting an alyses were conducted to determine eNOS activity. In-Cell western assay is a quant itative analysis that is extr emely sensitive. This assay utilizes an infrared fluorescen ce antibody. However, the validity of the In-Cell western assay needs to be examined by comparing it to standard western blotting. Figure 3-16A shows the result of the In-Cell western a ssay and 3-16B illustrates the result of standard western blotting analysis. Both results suppor t that IGFBP-3 increases eNOS phosphorylation at Ser 1177 in CD34+cells.

PAGE 71

71 IGFBP-3 Induces NO Production CD34+cells were exposed to IGFBP-3 for 30 minut es then intracellular NO was monitored with DAF-FM diacetate to confirm increased NO gene ration in IGFBP-3 treated cells. As shown in Figure 3-17, IGFBP-3 increased NO production in CD34+cells. NO release from IGFBP-3 treated cells was 3.7 fold gr eater than untreated cells. IGFBP-3 Modulates VASP Phosphorylation Increased NO generation by IGFBP-3 subs equently induced phosphorylation of VASP (Figure 3-18). CD34+cells were exposed to IGFBP-3 (100 ng/ml) for 0, 10, 30, and 60 minutes. To obtain whole cell lysate including cytoplas mic protein as well as membrane-bound protein, 2X SDS-PAGE buffer was added to the cells. Two anti-phospho-VASP (Ser 157 and Ser 239) antibodies were used to detect the different sites of phosphorylation on VASP. Phospho-VASP at serine 239 was significantly in creased by IGFBP-3 treatment. Inhibition of SK Activity Resu lts in Reduced NO Production Our preliminary data shows IGFBP3 enhances SK activity in CD34+cells (not shown here). To confirm SK is a downstream signaling mediator of IGFBP-3, the level of NO production from SK inhibitor pretre ated cells was measured. CD34+cells were exposed to IGFBP-3 following pretreatment of SK blocker, dimethylsphingosine (DMS) for 30 minutes. As depicted in Figure 3-19, NO genera tion was inhibited in SK blocke d cells and it was not restored by addition of IGFBP-3. This result suggests that IGFBP-3 modulates SK and that the S1P/SK pathway is involved in IG FBP-3s modulation of CD34+cells.

PAGE 72

72 Figure 3-1. IGFBP-3 induces CD34+cells and endothelial cells mi gration. Modified Boyden chamber assay was used for circulating cells (A) and the Boyden chamber assay was performed for adhered cells (B). Statisti cally significances were presented *P < 0.05 vs. negative control.

PAGE 73

73 Figure 3-2. Receptor levels in CD34+cells following IGFBP-3 exposure. CD34+cells were exposed to IGFBP-3 for 15 min, 4 h, and 12 h (for CXCR4). A) VEGFR-1. *P < 0.05 vs. medium alone (non treatment, NT) for 15 min and *P <0.001 for 4 h. B) VEGFR-2. *P < 0.001 vs. NT for both 15 min and 4 h. C) CXCR-4.

PAGE 74

74 Figure 3-3. IGFBP-3 enhances CD34+cells and EPC differentiation. A) CD34+cell were exposed to IGFBP-3 (white bars) for 72 hours. Statistically significances were presented *P < 0.05 vs. control nontreated cells. B) Representative images for growing EPC in the presence of different concentration of IGFBP-3. Magnification: X100. Scale bars: 150 m for left and cen ter panel and 100 m for right panel. A B

PAGE 75

75 Figure 3-4. IGFBP-3 enhances CD34+cells proliferation. Cells were cultured in StemSpan SFEM with addition of cytokines cocktail for 5 days. IGFBP3 signifies StemSpan SFEM with cytokines and 100 ng/ml IGFBP3. *P < 0.001 vs. Day 1 untreated cells and untreated cells at each time points. 0 10000 20000 30000 40000 50000 60000 70000 80000 D a y 1D a y 3D a y 5Prolliferation of CD34 cells (RFU) Untreated IGFBP3 *

PAGE 76

76 Figure 3-5. Hypoxia retina expres ses IGFBP-3. Neonatal mouse pups (n=6 in each group) was euthanized at P12.5. Under hypoxic condition, both VAGF (*P < 0.05) and IGFBP-3 (*P < 0.0001) mRNA were significantly in creased when compared to normoxia control retina

PAGE 77

77 Figure 3-6. IGFBP-3 protects from hyperoxia-induced vascular regr ession. Retina from the eyes injected with IGFBP-3 expressing plasmid (n=9; A-D), control plasmid (n=9; E-H), and contra-lateral eyes (I-L). Low magnification views of GS isolectin HRP-labeled retinas from IGFBP-3 plasmid injected eyes (M) and control vector injected eyes (N).

PAGE 78

78 Figure 3-7. Quantitative analysis of vascular density. IGFBP-3 significantly protected the retinal vasculature from hype roxia-induced vessel regressi on in mid peripheral (*P < 0.001) and peripheral regions (*P < 0.001), but did not have any significant effect on the central region of the retina (*P >0.05) J

PAGE 79

79 Figure 3-8. Reduced preretinal neovascula rization by expression of IGFBP-3. IGFBP-3 expressing plasmid injected mice (n=9, black bars) and control plasmid injected mice (n=9, white bars) were subjected to the OIR model. Statistically significances were presented *P < 0.005 vs. contro l vector injected eyes.

PAGE 80

80 0 500 1000 1500 2000 2500 3000 NT IGFBP3IGFBP3 mRNA/BA (%) Figure 3-9. IGFBP-3 expressi on in plasmid transfected HS C. Transfection of GFP+HSC with IGFBP-3 expressing plasmid results in a 25fold increase in IGFBP-3 expression in vitro compared with nontran sfected (NT) controls. *P= 0.02 vs. nontransfected HSC (NT).

PAGE 81

81 Figure 3-10. Localization of gfp+HSC expressing IGFBP-3 within the retinal vasculature. A) Representative image of redial arterioles. B) Hemangiomas. C) Filopodia. Merged green (gfp+HSC) and red (resident vasculatur e) channels represent incorporation of HSC in vasculature, indicating endogenous ly delivered IGFBP-3 in vivo. P< 0.001 vs. uninjected controls in D. *P< 0.01 in E. D E

PAGE 82

82 0 10 20 30 40 50Non 15min 4hrsSDF (100nM) treatment %of cell expressing phospho-eNOS Figure 3-11. eNOS phosphorylation by hypoxia-regulated factor. CD34+cells were treated with either 25 ng/ml VE GF or 100 nM SDF-1. Anti-phoshporylated eNOS (Ser 1177) antibody was used to examine the activation of eNOS in CD34+cells. A) eNOS phosphorylation follo wing exposure of VEGF (*P <0.05 vs. nontreated control cells). B) eNOS phosphorylation fo llowing exposure of SDF-1. 0 10 20 30 40 50 Nontreat 15min VEGF (25ng/ml) treatment %of cell expressing phosphoeNOS

PAGE 83

83 Figure 3-12. NO and CO stimulate CD34+cells migration. Pretreatme nt with either the CO donor (Ru(II)Cl2(CO)3 dimer) or the NO donor (DETA-NO) for either 1 minute or 15 minutes increases the cells responsivene ss to SDF-1. EPC were obtained from 3 healthy control subjects. Values represent means SD. *P<0.05 vs. untreated control. *

PAGE 84

84 Figure 3-13. VASP, phosphoryl ated VASP 157, and 239 expression levels. VASP function is regulated by its phosphorylation on serine 157 and serine 239. CD34+cells were incubated for 15 minutes in the presence of either the NO or CO donor. The NO donor increased the VASP phosphorylation at serine 239, whereas CO increased phosphorylation at serine157 in these cells. Values represent means SD. *P <0.05 vs. untreated control

PAGE 85

85 0 20 40 60 80 100 120 140 160 180 200 O hour 0.25 hour 4 hour% pVASP relative to Control at 0 ho u Control DM1 DM2 Figure 3-14. Diabetic CD34+cells show increased VASP phosphorylation following exposure of NO donor. In CD34+cells from two diabetic individuals one with type 1 and the other with type 2 disease, NO treatment resulte d in stimulation of VASP phosphorylation at serine 239. CD34+cells from diabetic individuals demonstrate reduced levels of pVASP but phosphorylation increa ses in response to NO exposure. Values represent means SD.

PAGE 86

86 Figure 3-15. NO and CO mediates VASP redistribution within endot helial cells. A) Untreated cells showing equal VASP immunoreactivity throughout the cytoplasm. B) COinduced redistribution of VASP to filopodia at the leading edge of the cells. C) NOinduced redistribution of VASP to fil opodia. Green channel represents VASP redistribution. Blue channel shows DAPI st ained nuclei. 100X magnification. D), E), and F) Details of A), B), and C), respectively.

PAGE 87

87 0 50 100 150 200 250 01 03 06 0 Time (minutes)eNOS levels (0 min. set to 100%) Figure 3-16. Phosphorylation of eNOS followi ng exposure of IGFBP-3. Anti-phoshporylated eNOS (Ser 1177) anti body was used to examine the activation of eNOS in both analysis. A) In-Cell western analysis (*p=0.0006 vs. unt reated cells). B) Western blotting analysis (*p< 0.05 vs. 0 min treated cells). Values represent means SD.

PAGE 88

88 0 200 400 600 800 1000 1200 1400 1600 1800 Untreated BP3 TreatedIntracellular NO levels (RFU) Figure 3-17. Increased intr acellular NO production in CD34+cells. CD34+cells were exposed to IGFBP-3 for 30 minutes. 3.7-fold greater increase of intracellular NO in IGFBP-3 treated cells was confirmed by using DAF-F M diacetate. Values represent means SD. *P < 0.05 vs. untreated cells

PAGE 89

89 Relative to VASP (0min: set to 100%)0 50 100 150 200 250 300 350 0103060site specific phosphorylation of VASP (%) pV-157 pV-239 Figure 3-18. IGFBP-3 modulates site sp ecific phosphorylation of VASP in CD34+cells. IGFBP3 (100 ng/ml) was exposed to CD34+cells for 0, 10, 30, and 60 minutes. Two antiphospho-VASP (Ser 157 and Ser 239) antibodies were used to detect the different sites of phosphorylation on VASP. PhosphoVASP at serine 239 was significantly increased by IGFBP-3 treatment. Values represent means SD.

PAGE 90

90 Figure 3-19. Inhibition of SK activity results in reduced NO production. DMS signifies CD34+cells were incubated with 20 M Di methylsphingosine for 30 minutes. DMS BP3 signifies CD34+cells were pretreated with 20 M Dimethylsphingosine for 30 minutes followed by addition of 100 ng/ml IG FBP-3. Values represent means SD. *P<0.05 vs. untreated cells. 0 200 400 600 800 1000 1200 1400 1600 1800 2000 UNT DMS DMS BP3 IGFBP-3NO production (RFU)

PAGE 91

91 CHAPTER 4 DISCUSSION This m ain purpose of this study was to addres s IGFBP-3 functions on EPC and its involved signaling pathways. The primary focus was depicting the effect of IGFBP-3 on stem cells and progenitor cells in retinal vasculatures. Factors Influencing the EPC Studies A difficulty for accom plishing this study was to obtain sufficient number of CD34+cells from the peripheral blood to complete the experiments. These cells are an extremely rare population of cells representing less than 0.01% of cells in the circulation. Furthermore significant differences in circul ating EPC numbers exist depending on the general health of the individual providing the cells. Pathological, pharmacological and physiological factors influence mobilization of CD34+ EPC. Numbers of EPCs are inversely related to factors such as presence of coronary artery diseas e and endothelial dysfunction.60 More specifically, increased levels of oxidative stress, inflammatory cytokines and asymmetric dimethylarginine, an endogenous inhibitor of endothelial nitric oxide synthase, have been linked to diminished mobilization and function of EPCs.65 Indeed, cytokines such as gra nulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulatin g factor (G-CSF), VEGF, SDF-1 and EPO, as well as therapeutic interventions with statins a nd estrogen are able to increase EPC numbers in circulation.50 The contribution of BM derived cells to the endothelium of injured tissue or hypoxic areas ranges from 1% up to 26% of vessels53,66. The magnitude of recruitment of circulating endothelial cells is or gan specific and dependent on the extent of vascular injury and remodeling.53,66 It is important to emphasize that identificati on of putative EPC is complicated due to not only small numbers of cells but also antigen plasticity, overlapping phenotype or antigen

PAGE 92

92 expression among other subsets. Inve stigators have struggled to find an antigen or characteristic that is unique to EPC. Recently many studies have demonstrated that there is overlap in antigenic expression between EC and m onocyte phenotypes. LDL uptake, lectin binding, and CD31/CD105/CD144 expr ession are inherent features of monocytes, making them often phenotypically indistinguishable from EPCs.55 Furthermore, monocytes and their progeny can function as EPCs in various experimental models.52,54,56 The properties of these cells were examined using flow cytometry, a powerful technique for the analysis of multiple characteristics of a single cell. Flow cytometry was used to determine the characteristics of cells in cluding cell size, granularity, and relative fluorescences. In this study, fluorescent antibodies were used to detect th e densities of specific receptors and activities of specific enzymes. We further attempted to overcome the lack of cells with In-C ell western assay, a quantitative immunofluorescence based technique This technique uses infrared labeled secondary antibodies to directly detect protein in the cellular environment. One of the advantages in using infrared fluorescence is it reduces in terferences caused by background autofluorescence from cell, culture environment, proteins and test compounds. Two separate detection channels (700 and 800 nm) result in more accurate evalua tion. One channel can probe target protein of interest, the second channel can be used for normalization of cell number or protein concentration in these same cells. Quantific ation accuracy is maximized by normalization because adjustments can be made for differences in cell number from well to well. In order to test the suitability of In-Cell western assay in ou r system, we established a standard western blot analysis with same set of experimental groups. The results from In-Ce ll western and standard western blot analysis were showing both 44% increases in p hospho-eNOS expression following

PAGE 93

93 IGFBP-3 exposure (100 ng/ml, 30 min), supporting that In-Cell western represents a reliable way to tracking subtle changes of protein among small populati on of cells such as CD34+cells. More interestingly, this assa y can quantify proteins in a 96-or 384-well microplate in less time and fewer proteins than a gel-based tradi tional western blot assay. The optimal cell number to run the In-Cell western was determined to be 10,000 cells per well. In contrast, traditional western blot analysis required 1,000,000 cells per lane. IGFBP-3 as a Hypoxia-regulated Factor EPCs support postnatal vasculoge nesis by incorporating into th e vascular lum en as well as delivering bioavailable angiogenic factors incl uding VEGF, matrix me talloproteases (MMPs), and angiopoietins to new vessels.92,223-225 Hypoxia especially HIF-1 either directly or indirectly regulates the cell type specific expression of multi ple angiogenic growth factors and cytokines. Numerous hypoxia-regulated factor s have been implicated in va sculogenesis associated with EPCs. The mechanism of how HIF-1 modulates cellular response to hypoxia has been relatively well studied. HIF-1 is a heterodimeric transc ription factor that c onsists of both HIF-1 and HIF1 The amino-terminal half of each subunit cont ains both basic helix-loop-helix (bHLH) and PAS (Per, ARNT, Sim) motifs that are required for dimerization and DNA binding.74 HIF-1 is also known as aryl hydrocarbon receptor nuclear tr anslocator (ARNT) because it dimerizes with the aryl hydrocarbon receptor as well as with HIF-1 The carboxy-terminal half of HIF-1 mediates nuclear localization, protei n stabilization, and transactivation.74 Under hypoxic conditions, HIF-1 protein accumulates, the heterodimer translocates to the nucleus and binds to a family of genes containing a specific sequence motif (HRE).73 The target genes are expressed in most cell types, such as those encoding gluc ose transporters, glycolytic enzymes, and VEGF,

PAGE 94

94 as well as genes that are expressed in a cell t ype-specific manner, such as EPO, iNOS and IGF2.76 VEGF is commonly expressed as one of the most potent regulator of both vasculogensis and angiogenesis. Compared to VEGF, the eff ects of IGFBPs on EPCs are greater. IGFBP-3 mRNA is abundantly expressed in hypoxi a-related inflammatory angiogenesis.226 Hypoxiainduced expression of IGFBP-3 was also validated by Northern blot analysis.227 An antiangiogenic role of IGFBP-3 has b een reported in cancer research.109,112,118,121 For instance, the growth-inhibitory effects of various an ti-proliferative agents including TGF, retinoic acid, antiestrogens, vitamin D analogs, and TNFare associated with increases in IGFBP3 mRNA and protein expression.122,132,133,135 IGFBP-3 has also been shown to stimulate angiogenesis, however prior to our studies the vasculogenic ef fects of IGFBP-3 had not been appreciated. The expression level and various functional differences of IG FBP-3 are cell type specific and thus context dependent. We have previous ly demonstrated that mature human retinal endothelial cells expres s high levels of IGFBP3. In contrast, CD34+ cells released undetectable levels of IGFBP-3. This finding suggests that the immature CD34+cells are more susceptible to changes in IGFBP-3 concentrations than ma ture endothelial cells. Furthermore, the concentrations of IGFBP-3 require d to stimulate migration of CD34+cells is extremely low. In support of our results, Liu et al. reported us ing in vitro cell pro liferation assays and immunophenotype analysis that addition of na nomolar concentration of IGFBP-3 on human umbilical cord blood-derived CD34+ CD38Lincells resulted in their proliferation.228 These studies combining our findings have been conclusively proved that IGFBP-3 supports expansion of HSC in vitro. In our studies, we further exte nded these observations by showing that IGFBP-3

PAGE 95

95 stimulates differentiation of CD34+cells to endothelial cells by lo ss of immature HSC marker and tube formation assay. Understanding of IGFBP-3 mechanism has b een difficult due to its uncharacterized receptor. Several candidate r eceptors of IGFBP-3 have been described but their signaling functions are poorly understood. Granata et al. suggested possible inte raction of IGFBP-3 signaling with SK related a ngiogenesis by endothelial cells.120 Granata group suggested dual functions of IGFBP-3 on human endothelial cells. For instance, they revealed pro-apoptosis and anti-apoptosis action of IGFBP-3 by regulating intracellular cerami de levels. Our results also indicate that the possible relationship between SK/S1P and IGFBP-3. mRNA level of SK1 has been stimulated by IGFBP-3 whereas redu ced mRNA level of SK2 was shown in CD34+cells. Once SK activity was inhibited by SK blocker, dimethylsphingosine (DMS), IGFBP-3 was not able to exert its function on CD34+cells. Based on our observations together with the growing amount of published evidence, IGFBP-3 exerts its vasculogenic actions on EPC though SK/S1P signaling pathways. Our in vivo studies have been showing the protective role of IG FBP-3 in vasculature following high oxygen stress with subsequent reduc tion of preretinal ne ovascularization. Thus, we postulate that IGFBP-3 expr ession may represent a physiologi cal adaptation to ischemia and potentially a novel therapeutic target for treatment of ischemia conditions. Corroborated with the work of Lofqvist et al, exogenous administ ration of IGFBP-3 may represent a novel approach for the treatment of conditions associated with pathological neovascularization such as reti nopathy of prematurity or PDR29. However, there are potential obstacles for IGFBP-3 study: (1) The volume and th e wide diversity of bi ological activity in which IGFBP-3 is involved, (2) IGFBP-3 is very closely related to IGF system, and (3) various

PAGE 96

96 laboratories utilize different cell systems in which response to IGFBP-3 may not only be different but contradictory. S1P: Possible Role in EPC Mobilization Sphingosine was discovered by J.L.W. Thudichum in 1884. At that tim e, he suggested the name sphingo that were derived from the Gree k mythology Sphinx for a new lipid due to its chemical nature containing both amine a nd alcohol groups, but insoluble in water.229 Further investigations are required to examine intracellular targets of S1P, the mechanism of its transport in and out of the cells, and the modulation of S1P levels. Related to S1P receptors, future challenges include better characterization of th e patho-physiological role of the various S1P receptors, what regulates their expression and their activity, and which genes are in turn regulated upon receptor activation. SIP stimulates human umbilical vein endothelial cells in vitro and in vivo angiogenesis in the matrigel plug assay in mice.230 Gene knock out studies of S1P1 receptor in mice revealed that this receptor on endothelial cells has an impor tant role in vessel stabilization during embryogenesis.145 In addition, recent studies have expanded the sphingolipid signaling to the regulation of eNOS. eNOS pathway (eNOS indu ced endothelium-dependent vasodilation) has been suggested as a downstream target for the biological effects of S1P.231 Furthermore, S1Pinduced signaling in human lung EC resulted in cytoskeletal rearrangemen t (cortical F-actin) and barrier enhancement through PI3K.232 Our immunohistochemistry result support these findings by showing that S1P rapidly induced VA SP redistribution in human lung EC. To be considered as a significant intracellula r messenger, more experiments remain to be conducted. Transgenic models, ge ne targeting approaches, and in vivo use of small interference RNA will further help understand the physiological role of S1P and its receptors. We are currently exploring th e underlying mechanism by which IG FBP-3 regulates EPC mobilization

PAGE 97

97 and carefully examining the signal transduction cascades activated by IGFBP-3 will ultimately help understand the how it modulates EPC function VASP: New Perspectives and Open Questions Hypoxia stimulates IGF BP-3 expression and also generates gaseous molecules, CO and NO by activating HO-1 and NOS respectively. In the current study, two major serine phosphorylation sites of VASP were evaluated on CD34+cells in response to NO, CO, and IGFBP-3. We have observed that IGFBP-3 mediates increases in phosphorylation, and redistribution of VASP which subsequently su pports EPC migration. To distinguish the difference of VASP phosphorylation induced by CO, NO, or IGFBP-3, we used two phosphospecific VASP antibodies ta rgeting the Ser157 and Ser239 residue phosphorylation sites. We observed that IGFBP-3 (as well as NO) exposure to CD34+cells induced VASP phosphorylation on serine 239. In contrast, VASP was phosphorylated through serine 157 by exogenous CO administration to CD34+cells. All vertebrate Ena/VASP proteins are substr ates for the cyclic nucleotide-dependent kinases PKA/PKG.203,206 Both PKA and PKG recognize and phosphorylate all three sites of VASP, but with different specificities and kinetics. Ser157 is the site preferred by PKA, whereas Ser239 is phosphorylated by PKG.217 Thr238 is phosphorylated last by both PKA and PKG. Phosphorylation at additional si tes in VASP might add additiona l levels of regulation, but it appears that the conserved N-term inal PKA/PKG site is the major site of phospho-regulation in vertebrate Ena/VASP proteins. C onsistent with this, Loureuro et al proved that mutation of this N-terminal phosphorylation site in Mena was sufficient to bl ock function in fibroblasts.233 Phosphorylation by different kinase s has shown cell type specificity. Studies of platelets derived from knockout mice revealed a requirement for VASP in this cyclic-nucleotide-mediated signaling cascade, suggesting that VASP plays a key role in mediati ng this PKA-dependent

PAGE 98

98 function.206,234 Furthermore, PKA inhibitors reverse cGMP-induced i nhibition of thrombininduced platelet aggregation, whereas PKG inhibitors further enhan ce the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.235 PKA-dependent phosphorylation of Ena/VASP proteins correlates with changes in cell adhesion in fibroblasts.233 Under basal conditions, the majority of VASP (more than 95%) is in the unphoshporyl ated state in human micro vascular endothelial cells. However, PKA induced VASP phosphorylation changed its localization to cell-cell junctions and regulated endothelial permeability.215 The role of NO/PKG/ pathway has been te sted by many researchers. PKG plays an important role in smooth muscle cell relaxation, i nhibition of platelet aggr egation, retinal signal transduction and synaptic transmission.211 In rat aorta phosphor-VASP ser239 correlates with relaxation of VSMC layer.206 sGC is the intracellular mediator for the ubiquitous biological messenger NO.163,187 However, NO-independent stimulators of sGC are very desirable as both pharmacological tools to proof th e NO/cyclic GMP pathway and as potential therapeutic agents. Organic nitrates like GTN or ISDN have been used for decades as a treatment for coronary heart disease. However, the major drawbacks of this therapy are the development of tolerance and the negligible anti-platelet effect. This obstacle could now be overcome by the discovery of potent and specific NO-independent sGC stimulators. As discussed above, PKA/PKG phosphorylation plays a crucial role in various cell types and in numerous animal models. In this study, we suggested the role of PKA/PKG in the regula tion of VASP function in human progenitor cells and EC. We found out NO and IGFBP-3 phos phorylates VASP-Ser239 through PKG whereas CO activates phosphor-VASP-Ser157 through P KA. As depicted in this study, both physiological gases and IGFBP-3 regulate human mature EC and progenitor cell dynamics by

PAGE 99

99 VASP phoshporylation and localization. Thus, our fi ndings may explain what initiates progenitor cell migration from BM and how EC migrates into ischemic tissues although the kinetic analysis and detailed structural changes th at were derived by di fferent site of phosp horylation are still unknown. Although a great deal of information about VA SP proteins is availa ble there are still a number of very important questi ons that remain unanswered. Fo r example, a question concerning how VASP proteins interact with the barbed ends of actin filame nts and what allows filament elongation need to be answere d. Identification of whether su ch phosphorylation reflects the overall phosphorylation of all VASP w ithin the cell will be the direction of future studies in our lab. The question of whether VASP at the lead ing edge or VASP at focal adhesions are differently phosphorylated also remains to be determined. Conclusions Circulating bone m arrow-derived stem cells and progenitor cells home to areas of hypoxia and participate in vessel development and re-vascula rization to facilitate vascular repair. In this study, we asked whether the hypoxia-regulated fact or IGFBP-3 could serve as a homing factor for EPCs and stimulate their vasculogenic func tions. We examined the effect of IGFBP-3 on NO generation, consequent VASP activa tion and redistribution. We also evaluated the role of SK in IGFBP-3 modulating EPC vasculogenesis. Exposure of CD34+ EPC population to nanomolar concentrations of IGFBP-3 resulted in rapid differentiation into endot helial cells, dose-dependent mi gration, and capillary tube formation. For in vivo study, a plasmid expressing IGFBP-3 under the contro l of a proliferating endothelial-specific promoter wa s designed. This plasmid was in jected either alone or HSC transfected form into the vitreous of neona tal pups undergoing the oxygen-induced retinopathy

PAGE 100

100 model. Endogeneously delivered IGFBP-3 result ed in reduced areas of vaso-obliteration, protection of the developing va sculature from hyperoxia, and reduction in preretinal neovascularization compared to control conditions. This study supports that IGFBP-3 promotes EPC mobilization by cytoskeletal changes through NO signaling pathway related phosphorylati on and redistribution of VASP. In EPCs, IGFBP-3 induced eNOS phoshporylation and NO generation. Similar to NO induced PKG related VASP phosphorylation, IGFBP-3 selec tively phoshporylated VASP on serine 239. Granata group suggested pro-apoptosis and anti-apoptosis action of IGFBP-3 is regulated by intracellular ceramide/S1P levels. Our results also indicate that the possible relationship between SK/S1P and IGFBP-3. SK1 mRNA was upregulated by IGFBP-3. IGFBP-3 induced intracellular NO production in EPCs was signifi cantly reduced by SK inhibitor. These findings provide a mechanism for EPC mobilization and angiogenic function of IG FBP-3. IGFBP-3 expression may represent a physiological adapta tion to ischemia, and IGFBP-3 could be considered as a novel therapeutic target for treatment of ischemic conditions.

PAGE 101

101 LIST OF REFERENCES 1. Kaplan, H.J. Anatomy and function of the eye. Chemical immunology and allergy 92 410 (2007). 2. McCaa, C.S. The eye and visual nervous system: anatomy, physiology and toxicology. Environmental health perspectives 44 1-8 (1982). 3. Brubaker, R.F. The flow of aqueous humor in the human eye. Transactions of the American Ophthalmological Society 80, 391-474 (1982). 4. Koretz, J.F. & Handelman, G.H. How the human eye focuses. Scientific American 259, 92-99 (1988). 5. Bone, R.A., Landrum, J.T. & Cains, A. Optical density spectra of the macular pigment in vivo and in vitro. Vision research 32 105-110 (1992). 6. Dowling, J.E. Foveal Receptors of the Monkey Retina: Fine Structure. Science (New York, N.Y 147, 57-59 (1965). 7. Watanabe, T. & Raff, M.C. Retinal astrocyt es are immigrants from the optic nerve. Nature 332, 834-837 (1988). 8. Moschovakis, A.K. & Highstein, S.M. The anatomy and physiology of primate neurons that control rapid eye movements. Annual review of neuroscience 17, 465-488 (1994). 9. Pelphrey, K.A., Morris, J.P., Michelich, C.R., Allison, T. & McCarthy, G. Functional anatomy of biological motion pe rception in posterior temporal cortex: an FMRI study of eye, mouth and hand movements. Cereb Cortex 15, 1866-1876 (2005). 10. Hayreh, S.S. Segmental nature of the choroidal vasculature. The British journal of ophthalmology 59, 631-648 (1975). 11. Schwesinger, C. et al. Intrachoroidal neovasculari zation in transgenic mice overexpressing vascular endotheli al growth factor in the retinal pigment epithelium. The American journal of pathology 158, 1161-1172 (2001). 12. Zhang, D. & Eldred, W.D. Anatomical char acterization of retina l ganglion cells that project to the nucleus of the basal optic root in the turtle (Pseudemys scripta elegans). Neuroscience 61, 707-718 (1994). 13. Curcio, C.A., Sloan, K.R., Kalina, R.E. & Hendrickson, A.E. Human photoreceptor topography. The Journal of comparative neurology 292, 497-523 (1990). 14. Dacey, D.M., Lee, B.B., Stafford, D.K., Pokor ny, J. & Smith, V.C. Horizontal cells of the primate retina: cone specific ity without spectral opponency. Science (New York, N.Y 271, 656-659 (1996).

PAGE 102

102 15. Bouman, M.A. The simple perfection of quantum correlation in human vision. Progress in neurobiology 78, 38-60 (2006). 16. Wassle, H. & Boycott, B.B. Functiona l architecture of the mammalian retina. Physiological reviews 71 447-480 (1991). 17. Hopkins, J.M. & Boycott, B.B. Synapses between cones and diffuse bipolar cells of a primate retina. Journal of neurocytology 24, 680-694 (1995). 18. Zhang, J.J. et al. Tamoxifen blocks chloride channels A possible mechanism for cataract formation. The Journal of clinical investigation 94 1690-1697 (1994). 19. Rubin, L.L. & Staddon, J.M. The cell biology of the blood-brain barrier. Annual review of neuroscience 22, 11-28 (1999). 20. Vinores, S.A. Assessment of blood-retinal barrier integrity. Histology and histopathology 10, 141-154 (1995). 21. Vinores, S.A. et al. Blood-ocular barrier breakdown in eyes with ocular melanoma. A potential role for vascular endothelial growth factor/v ascular permeability factor. The American journal of pathology 147, 1289-1297 (1995). 22. Weinberger, B., Laskin, D.L., Heck, D.E. & Laskin, J.D. Oxygen toxicity in premature infants. Toxicology and applied pharmacology 181 60-67 (2002). 23. McColm, J.R. & Fleck, B.W. Retin opathy of prematurity: causation. Semin Neonatol 6, 453-460 (2001). 24. Wheatley, C.M., Dickinson, J.L., Mackey, D.A ., Craig, J.E. & Sale, M.M. Retinopathy of prematurity: recent advances in our understanding. The British journal of ophthalmology 86, 696-700 (2002). 25. Kotecha, S. & Allen, J. Oxygen therapy for infants with chronic lung disease. Archives of disease in childhood 87, F11-14 (2002). 26. Flynn, J.T. Acute proliferative retrolental fibroplasia: multivariate risk analysis. Transactions of the American Ophthalmological Society 81, 549-591 (1983). 27. Hellstrom, A. et al. IGF-I is critical for normal vascularization of the human retina. The Journal of clinical e ndocrinology and metabolism 87, 3413-3416 (2002). 28. Hellstrom A. et al. Low IGF-I suppresses VEGF-surviva l signaling in retinal endothelial cells: direct correlation with clin ical retinopathy of prematurity. Proceedings of the National Academy of Sciences of the United States of America 98, 5804-5808 (2001).

PAGE 103

103 29. Lofqvist, C. et al. Postnatal head growth deficit among premature infants parallels retinopathy of prematurity and insuli n-like growth factor-1 deficit. Pediatrics 117, 19301938 (2006). 30. Smith, L.E. et al. Oxygen-induced retinopathy in the mouse. Investigative ophthalmology & visual science 35, 101-111 (1994). 31. Alon, T., et al. Vascular endothelial gr owth factor acts as a su rvival factor for newly formed retinal vessels and has impli cations for retinopathy of prematurity. Nature medicine 1, 1024-1028 (1995). 32. Pierce, E.A., Foley, E.D. & Smith, L.E. Regul ation of vascular endot helial growth factor by oxygen in a model of retinopathy of prematurity. Archives of ophthalmology 114, 1219-1228 (1996). 33. Shih, S.C., Ju, M., Liu, N. & Smith, L.E. Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degene ration in retinopathy of prematurity. The Journal of clinical investigation 112, 50-57 (2003). 34. Chen, J. & Smith, L.E. Retinopathy of prematurity. Angiogenesis 10, 133-140 (2007). 35. Amos, A.F., McCarty, D.J. & Zimmet, P. Th e rising global burden of diabetes and its complications: estimates and projections to the year 2010. Diabet Med 14 Suppl 5 S1-85 (1997). 36. Aiello, L.P., et al. Diabetic retinopathy. Diabetes care 21, 143-156 (1998). 37. Caldwell, R.B., et al. Vascular endothelial growth f actor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Diabetes/metabolism research and reviews 19 442-455 (2003). 38. Miyamoto, K., Hiroshiba, N., Tsujikawa, A. & Ogura, Y. In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. Investigative ophthalmology & visual science 39, 2190-2194 (1998). 39. Gardner, T.W., Antonetti, D.A., Barber, A. J., LaNoue, K.F. & Levison, S.W. Diabetic retinopathy: more than meets the eye. Survey of ophthalmology 47 Suppl 2 S253-262 (2002). 40. Kern, T.S. & Engerman, R.L. Comparison of re tinal lesions in alloxan-diabetic rats and galactose-fed rats. Current eye research 13, 863-867 (1994). 41. Wells, J.A. et al. Levels of vascular endot helial growth factor are elevated in the vitreous of patients with subret inal neovascularisation. The British journal of ophthalmology 80, 363-366 (1996).

PAGE 104

104 42. Frank, R.N. Potential new medical therapies for diabetic retinopat hy: protein kinase C inhibitors. American journal of ophthalmology 133 693-698 (2002). 43. Haritoglou, C. et al. Intravitreal bevacizumab (Avastin ) therapy for persistent diffuse diabetic macular edema. Retina (Philadelphia, Pa 26, 999-1005 (2006). 44. Arevalo, J.F. et al. Intravitreal bevacizumab (avastin) for proliferative diabetic retinopathy: 6-months follow-up. Eye (2007). 45. Arevalo, J.F. et al. Primary intravitreal bevacizumab (Avastin) for diabetic macular edema: results from the Pan-American Collaborative Retina Study Group at 6-month follow-up. Ophthalmology 114, 743-750 (2007). 46. Kalina, R.E. Seeing into the future. Vision and aging. The Western journal of medicine 167, 253-257 (1997). 47. Sengupta, N. et al. The role of adult bone marrow-derived stem cells in choroidal neovascularization. Investigative ophthalmol ogy & visual science 44, 4908-4913 (2003). 48. Kliffen, M., Sharma, H.S., Mooy, C.M., Ke rkvliet, S. & de Jong, P.T. Increased expression of angiogenic growth f actors in age-related maculopathy. The British journal of ophthalmology 81, 154-162 (1997). 49. Asahara, T., et al. Isolation of putative progenitor endothelial cel ls for angiogenesis. Science (New York, N.Y 275, 964-967 (1997). 50. Asahara, T. & Kawamoto, A. Endothelial pr ogenitor cells for postnatal vasculogenesis. American journal of physiology 287, C572-579 (2004). 51. Grant, M.B. et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nature medicine 8, 607-612 (2002). 52. Fernandez Pujol, B. et al. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation; research in biological diversity 65 287-300 (2000). 53. Harraz, M., Jiao, C., Hanlon, H.D., Har tley, R.S. & Schatteman, G.C. CD34bloodderived human endothelial cell progenitors. Stem cells (Dayton, Ohio) 19, 304-312 (2001). 54. Schm eisser, A. et al. Monocytes coexpress endothe lial and macropha gocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovascular research 49, 671-680 (2001). 55. Schmeisser, A. & Strasser, R.H. Phenotypi c overlap between hema topoietic cells with suggested angioblastic potential and vascular endothelial cells. Journal of hematotherapy & stem cell research 11, 69-79 (2002).

PAGE 105

105 56. Rohde, E., et al. Blood monocytes mimic e ndothelial progenitor cells. Stem cells (Dayton, Ohio) 24, 357-367 (2006). 57. Walenta, K., Friedrich, E.B., Sehnert, F ., Werner, N. & Nickenig, G. In vitro differentiation characteristics of cultured human mononuclear cells-implications for endothelial progenitor cell biology. Biochemical and biophysical research communications 333, 476-482 (2005). 58. Wojakowski, W. et al. The proand anti-inflammatory markers in patients with acute myocardial infarction and chronic stable angina. International journal of molecular medicine 14, 317-322 (2004). 59. Peichev, M., et al. Expression of VEGFR-2 and AC 133 by circulating human CD34(+) cells identifies a population of f unctional endothelial precursors. Blood 95 952-958 (2000). 60. Gill, M., et al. Vascular trauma induces rapi d but transient mobilization of VEGFR2(+)AC133(+) endothe lial precursor cells. Circulation research 88 167-174 (2001). 61. Wojakowski, W. et al. Mobilization of CD34/CXCR4+ CD34/CD117+, c-met+ stem cells, and mononuclear cells e xpressing early cardiac, muscle, and endothelial markers into peripheral blood in patients wi th acute myocardial infarction. Circulation 110, 32133220 (2004). 62. Assmus, B. et al. HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial pr ogenitor cells via regul ation of cell cycle regulatory genes. Circulation research 92, 1049-1055 (2003). 63. Hoetzer, G.L., Irmiger, H.M., Keith, R.S., Westbrook, K.M. & DeSouza, C.A. Endothelial nitric oxide synt hase inhibition does not alte r endothelial progenitor cell colony forming capacity or migratory activity. Journal of cardiovascular pharmacology 46, 387-389 (2005). 64. Murohara, T. & Asahara, T. Nitric oxide and angiogenesis in cardiovascular disease. Antioxidants & redox signaling 4, 825-831 (2002). 65. Takahashi, T. et al. Ischemiaand cytokine-induced mobilization of bone marrowderived endothelial progenitor cells for neovascularization. Nature medicine 5, 434-438 (1999). 66. Crosby, J.R. et al. Endothelial cells of hematopoi etic origin make a significant contribution to adult blood vessel formation. Circulation research 87, 728-730 (2000).

PAGE 106

106 67. Kalka, C. et al. Vascular endothelial growth fact or(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circulation research 86, 11981202 (2000). 68. Shintani, S. et al. Mobilization of endothelial progenitor cells in pati ents with acute myocardial infarction. Circulation 103 2776-2779 (2001). 69. Shintani, S. et al. Augmentation of postnatal neovas cularization with autologous bone marrow transplantation. Circulation 103, 897-903 (2001). 70. Ceradini, D.J. et al. Progenitor cell trafficking is re gulated by hypoxic gradients through HIF-1 induction of SDF-1. Nature medicine 10, 858-864 (2004). 71. Koong, A.C. et al. Candidate genes for the hypoxic tumor phenotype. Cancer research 60, 883-887 (2000). 72. Wang, G.L. & Semenza, G.L. Purificati on and characterizatio n of hypoxia-inducible factor 1. The Journal of biological chemistry 270 1230-1237 (1995). 73. Semenza, G.L. HIF-1 and mechanisms of hypoxia sensing. Current opinion in cell biology 13, 167-171 (2001). 74. Semenza, G.L. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda, Md 19, 176-182 (2004). 75. Laughner, E., Taghavi, P., Chiles, K., Mahon, P.C. & Semenza, G.L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Molecular and cellular biology 21, 3995-4004 (2001). 76. Semenza, G.L., Shimoda, L.A. & Prabhakar, N.R. Regulation of gene expression by HIF1. Novartis Foundation symposium 272, 2-8; discussion 8-14, 33-16 (2006). 77. Manalo, D.J. et al. Transcriptional regulati on of vascular endothel ial cell responses to hypoxia by HIF-1. Blood 105, 659-669 (2005). 78. Fukuda, R. et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1mediated vascular endothelial growth fact or expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. The Journal of biological chemistry 277 38205-38211 (2002). 79. Forsythe, J.A. et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Molecular and cellular biology 16, 4604-4613 (1996).

PAGE 107

107 80. Kasuno, K. et al. Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MAPK and phosphatidylinositol 3-kinase signaling. The Journal of biological chemistry 279 2550-2558 (2004). 81. Metzen, E., Zhou, J., Jelkma nn, W., Fandrey, J. & Brune, B. Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases. Molecular biology of the cell 14, 3470-3481 (2003). 82. Hagen, T., Taylor, C.T., Lam, F. & Moncada, S. Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha. Science (New York, N.Y 302, 1975-1978 (2003). 83. Senger, D.R. et al. Tumor cells secrete a vascular pe rmeability factor that promotes accumulation of ascites fluid. Science (New York, N.Y 219, 983-985 (1983). 84. Waltenberger, J., Claesson-Welsh, L., Si egbahn, A., Shibuya, M. & Heldin, C.H. Different signal transduction properties of KDR and Flt1, two recep tors for vascular endothelial growth factor. The Journal of biological chemistry 269, 26988-26995 (1994). 85. Keck, P.J., et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (New York, N.Y 246, 1309-1312 (1989). 86. Leung, D.W., Cachianes, G., Kuang, W.J., Goeddel, D.V. & Ferrara, N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (New York, N.Y 246, 1306-1309 (1989). 87. Gospodarowicz, D., Abraham, J.A. & Schilli ng, J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proceedings of the National Academy of Sc iences of the United States of America 86, 7311-7315 (1989). 88. Meyer, M., et al. A novel vascular endot helial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via si gnalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. The EMBO journal 18, 363-374 (1999). 89. Ogawa, S., et al. A novel type of vascular endothe lial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain. The Journal of biological chemistry 273 31273-31282 (1998). 90. W ise, L.M., et al. Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1. Proceedings of the National Academy of Sciences of the United States of America 96, 3071-3076 (1999).

PAGE 108

108 91. Junqueira de Azevedo, I.L., Farsky, S.H., Oliv eira, M.L. & Ho, P.L. Molecular cloning and expression of a functional snake venom va scular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A ne w member of the VEGF family of proteins. The Journal of biological chemistry 276 39836-39842 (2001). 92. Hattori, K. et al. Vascular endothelial growth fa ctor and angiopoietin-1 stimulate postnatal hematopoiesis by r ecruitment of vasculogenic and hematopoietic stem cells. The Journal of experimental medicine 193, 1005-1014 (2001). 93. Hirashima, M., Kataoka, H., Nishikawa, S ., Matsuyoshi, N. & Nishikawa, S. Maturation of embryonic stem cells into e ndothelial cells in an in vi tro model of vasculogenesis. Blood 93, 1253-1263 (1999). 94. Ferrara, N. et al. Differential expression of the a ngiogenic factor genes vascular endothelial growth factor (VEGF) and endocrine glandderived VEGF in normal and polycystic human ovaries. The American journal of pathology 162, 1881-1893 (2003). 95. Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nature medicine 9, 669-676 (2003). 96. Gerber, H.P. et al. VEGF regulates haematopoietic st em cell survival by an internal autocrine loop mechanism. Nature 417, 954-958 (2002). 97. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439 (1996). 98. Lee, H.T. & Emala, C.W. Protective e ffects of renal ischemic preconditioning and adenosine pretreatment: role of A(1) and A(3) receptors. Am J Physiol Renal Physiol 278 F380-387 (2000). 99. Nagasawa, T., Kikutani, H. & Kishimoto, T. Molecular cloning and structure of a pre-Bcell growth-stimulating factor. Proceedings of the National Ac ademy of Sciences of the United States of America 91, 2305-2309 (1994). 100. Nagasawa, T., Tachibana, K. & Kishim oto, T. A novel CXC ch emokine PBSF/SDF-1 and its receptor CXCR4: their functions in development, hematopoiesis and HIV infection. Seminars in immunology 10, 179-185 (1998). 101. Bleul, C.C. et al. The lymphocyte chemoattractant SD F-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382, 829-833 (1996). 102. Pituch-Noworolska, A. et al. Circulating CXCR4-positive stem/progenitor cells compete for SDF-1-positive niches in bone marrow, mu scle and neural tissues: an alternative hypothesis to stem cell plasticity. Folia histochemica et cytob iologica / Polish Academy of Sciences, Polish Histochemi cal and Cytochemical Society 41 13-21 (2003).

PAGE 109

109 103. Kahn, J., et al. Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migrati on, and NOD/SCID repopulation. Blood 103, 2942-2949 (2004). 104. Butler, J.M. et al. SDF-1 is both necessary and suffi cient to promote proliferative retinopathy. The Journal of clinical investigation 115, 86-93 (2005). 105. Russell-Jones, D.L. et al. A comparison of the effects of IGF-I and insulin on glucose metabolism, fat metabolism and the cardiovasc ular system in normal human volunteers. European journal of c linical investigation 25, 403-411 (1995). 106. Baxter, R.C. & Martin, J.L. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling. Proceedings of the National Academy of Sciences of the United States of America 86, 6898-6902 (1989). 107. Firth, S.M., McDougall, F., McLachlan, A.J. & Baxter, R.C. Impaired blockade of insulin-like growth factor I (IGF-I)-induced hypoglycemia by IGF binding protein-3 analog with reduced ternary complex-forming ability. Endocrinology 143, 1669-1676 (2002). 108. Spagnoli, A. & Rosenfeld, R.G. The mechan isms by which growth hormone brings about growth. The relative contributions of growth hormone and insulin-like growth factors. Endocrinology and metabolism clinics of North America 25, 615-631 (1996). 109. Butt, A.J. & Williams, A.C. IGFBP3 and apoptosis--a license to kill? Apoptosis 6, 199205 (2001). 110. Butt, A.J., Fraley, K.A., Firth, S.M. & Baxter, R.C. IGF-binding protein-3-induced growth inhibition and apoptosis do not require cell surface binding and nuclear translocation in human breast cancer cells. Endocrinology 143 2693-2699 (2002). 111. Cohen, P., Lamson, G., Okajima, T. & Rose nfeld, R.G. Transfection of the human IGFBP-3 gene into Balb/c fibroblasts: a m odel for the cellular f unctions of IGFBPs. Growth regulation 3, 23-26 (1993). 112. Oh, Y., Gucev, Z., Ng, L., Muller, H.L. & Rosenfeld, R.G. Antiproliferative actions of insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells. Progress in growth factor research 6 503-512 (1995). 113. Valentinis, B., Bhala, A., DeAngelis, T., Ba serga, R. & Cohen, P. The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene. Molecu lar endocrinology (Baltimore, Md 9, 361367 (1995).

PAGE 110

110 114. Lalou, C., Lassarre, C. & Binoux, M. A proteoly tic fragment of insulin -like growth factor (IGF) binding protein-3 that fa ils to bind IGFs inhibits the mitogenic effects of IGF-I and insulin. Endocrinology 137 3206-3212 (1996). 115. Spagnoli, A. et al. Antiproliferative effects of insulin -like growth factor-binding protein3 in mesenchymal chondrogenic cell line RCJ3.1C5.18. relationship to differentiation stage. The Journal of biological chemistry 276, 5533-5540 (2001). 116. Firth, S.M. & Baxter, R.C. Cellular actions of the insulin-like growth factor binding proteins. Endocrine reviews 23, 824-854 (2002). 117. Longobardi, L., et al. A novel insulin-like growth factor (IGF)-independent role for IGF binding protein-3 in mesenchymal chondroprogenitor cell apoptosis. Endocrinology 144 1695-1702 (2003). 118. Rajah, R., Valentinis, B. & Cohen, P. Insulin -like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effect s of transforming growth factor-beta1 on programmed cell death through a p53and IGF-independent mechanism. The Journal of biological chemistry 272 12181-12188 (1997). 119. Rajah, R., Khare, A., Lee, P.D. & Cohen, P. Insulin-like growth factor-binding protein-3 is partially responsible for high-serum-induced apoptosis in PC-3 prostate cancer cells. The Journal of endocrinology 163, 487-494 (1999). 120. Granata, R. et al. Dual effects of IGFBP-3 on endothe lial cell apoptosis and survival: involvement of the sphingolipid signaling pathways. Faseb J 18, 1456-1458 (2004). 121. Liu, B. et al. Combination therapy of insulin-like growth factor bind ing protein-3 and retinoid X receptor ligands synergize on prosta te cancer cell apoptosis in vitro and in vivo. Clin Cancer Res 11 4851-4856 (2005). 122. O'Rear, L., et al. Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-(beta) in mesenchymal chondroprogenitor cell growth. Journal of molecular endocrinology 34, 723-737 (2005). 123. Foulstone, E.J., Savage, P.B., Crown, A.L., Ho lly, J.M. & Stewart, C.E. Role of insulinlike growth factor binding pr otein-3 (IGFBP-3) in the diffe rentiation of primary human adult skeletal myoblasts. Journal of cellular physiology 195, 70-79 (2003). 124. Feld, S. & Hirschberg, R. Growth hormone, the insulin-like growth factor system, and the kidney. Endocrine reviews 17, 423-480 (1996). 125. Landau, D. et al. Expression of insulin-like growth factor binding proteins in the rat kidney: effects of long-term diabetes. Endocrinology 136, 1835-1842 (1995).

PAGE 111

111 126. Lamson, G. et al. Proteolysis of IGFBP-3 may be a common regulatory mechanism of IGF action in vivo. Growth regulation 3, 91-95 (1993). 127. Tazuke, S.I. et al. Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia. Proceedings of the National Academy of Sciences of the United States of America 95, 10188-10193 (1998). 128. Feldser, D. et al. Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2. Cancer research 59, 3915-3918 (1999). 129. Slomiany, M.G. & Rosenzweig, S.A. Auto crine effects of IGF-I-induced VEGF and IGFBP-3 secretion in retinal pigm ent epithelial cell line ARPE-19. American journal of physiology 287 C746-753 (2004). 130. Ibanez de Caceres, I. et al. Effect of inducible nitric oxide synthase inhibition by aminoguanidine on insulin-like growth fact or binding protein-3 in adjuvant-induced arthritic rats. European journal of pharmacology 481, 293-299 (2003). 131. Fanayan, S., Firth, S.M. & Baxter, R.C. Signaling through the Smad pathway by insulinlike growth factor-binding prot ein-3 in breast cancer cells. Relationship to transforming growth factor-beta 1 signaling. The Journal of biological chemistry 277, 7255-7261 (2002). 132. Fanayan, S., Firth, S.M., Butt, A.J. & Baxt er, R.C. Growth inhibition by insulin-like growth factor-binding protein-3 in T47D breas t cancer cells requires transforming growth factor-beta (TGF-beta ) and th e type II TGF-beta receptor. The Journal of biological chemistry 275 39146-39151 (2000). 133. Leal, S.M., Huang, S.S. & Huang, J.S. Intera ctions of high affinity insulin-like growth factor-binding proteins with the type V tran sforming growth factor-beta receptor in mink lung epithelial cells. The Journal of biological chemistry 274, 6711-6717 (1999). 134. Rosendahl, A.H. & Forsberg, G. IGF-I and IGFBP-3 augment transforming growth factor-beta actions in human renal carcinoma cells. Kidney international 70 1584-1590 (2006). 135. Oh, Y., Muller, H.L., Ng, L. & Rosenfeld, R. G. Transforming growth factor-beta-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-bindi ng protein-3 action. The Journal of biological chemistry 270, 1358913592 (1995). 136. McCaig, C. et al. Differential interactions betwee n IGFBP-3 and transforming growth factor-beta (TGF-beta) in normal vs cancerous breast epithelial cells. British journal of cancer 86, 1963-1969 (2002).

PAGE 112

112 137. Izumi, K. et al. Involvement of insulin-like growth fact or-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing. Investigative ophthalmology & visual science 47, 591-598 (2006). 138. Peters, I. et al. IGF-binding protein-3 modulates TGF-beta/BMP-signaling in glomerular podocytes. J Am Soc Nephrol 17, 1644-1656 (2006). 139. Spiegel, S. Sphingosine 1-phosphate: a ligand for the EDG-1 family of G-protein-coupled receptors. Annals of the New York Academy of Sciences 905, 54-60 (2000). 140. Hisano, N. et al. Induction and suppression of en dothelial cell apoptosis by sphingolipids: a possible in vitro model for cell-cell interactions between platelets and endothelial cells. Blood 93, 4293-4299 (1999). 141. Cuvillier, O. et al. Suppression of ceramide-med iated programmed cell death by sphingosine-1-phosphate. Nature 381, 800-803 (1996). 142. Maceyka, M. et al. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. The Journal of biological chemistry 280, 3711837129 (2005). 143. Xia, P. et al. An oncogenic role of sphingosine kinase. Curr Biol 10, 1527-1530 (2000). 144. Hait, N.C. et al. Role of sphingosine kinase 2 in ce ll migration toward epidermal growth factor. The Journal of biological chemistry 280, 29462-29469 (2005). 145. Allende, M.L., Yamashita, T. & Proia, R.L. G-protein-coupled receptor S1P1 acts within endothelial cells to regu late vascular maturation. Blood 102, 3665-3667 (2003). 146. Liu, Y. et al. Edg-1, the G protein-coupled recepto r for sphingosine-1-phosphate, is essential for vascular maturation. The Journal of clinical investigation 106, 951-961 (2000). 147. Igarashi, J. & Michel, T. Agonist-modulat ed targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction. The Journal of biological chemistry 275 32363-32370 (2000). 148. Shaul, P.W. et al. Acylation targets emdothelial nitric -oxide synthase to plasmalemmal caveolae. The Journal of biological chemistry 271 6518-6522 (1996). 149. Igarashi, J., Bernier, S.G. & Michel, T. Sphingosine 1-phosphate and activation of endothelial nitric-oxide s ynthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin rece ptors in vascular endothelial cells. The Journal of biological chemistry 276 12420-12426 (2001).

PAGE 113

113 150. Lee, M.J. et al. Sphingosine-1-phosphate as a ligand for the G pr otein-coupled receptor EDG-1. Science (New York, N.Y 279, 1552-1555 (1998). 151. Hla, T. & Maciag, T. An abundant transcri pt induced in differentiating human endothelial cells encodes a polypeptide with structural si milarities to G-protein-coupled receptors. The Journal of biological chemistry 265 9308-9313 (1990). 152. Olivera, A. & Spiegel, S. Sphingosin e-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens. Nature 365 557-560 (1993). 153. Lee, H., Goetzl, E.J. & An, S. Lyso phosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing. American journal of physiology 278, C612-618 (2000). 154. Graeler, M. & Goetzl, E.J. Activation-regul ated expression and ch emotactic function of sphingosine 1-phosphate receptors in mouse splenic T cells. Faseb J 16, 1874-1878 (2002). 155. Graeler, M., Shankar, G. & Goetzl, E.J. Cutting edge: suppression of T cell chemotaxis by sphingosine 1-phosphate. J Immunol 169, 4084-4087 (2002). 156. Mattie, M., Brooker, G. & Spiegel, S. Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from intern al stores via an inositol trisphosphateindependent pathway. The Journal of biological chemistry 269 3181-3188 (1994). 157. Morita, Y. et al. Oocyte apoptosis is suppres sed by disruption of the acid sphingomyelinase gene or by sphi ngosine-1-phosphate therapy. Nature medicine 6, 11091114 (2000). 158. Spiegel, S. & Milstien, S. Sphingosin e-1-phosphate: signaling inside and out. FEBS letters 476, 55-57 (2000). 159. van Meer, G. & Lisman, Q. Sphingolipid transport: rafts and translocators. The Journal of biological chemistry 277 25855-25858 (2002). 160. Boujaoude, L.C. et al. Cystic fibrosis transmembran e regulator regulates uptake of sphingoid base phosphates and ly sophosphatidic acid: modulation of cellular activity of sphingosine 1-phosphate. The Journal of biological chemistry 276, 35258-35264 (2001). 161. Becquet, F., Courtois, Y. & Goureau, O. N itric oxide in the eye: multifaceted roles and diverse outcomes. Survey of ophthalmology 42, 71-82 (1997). 162. Rassaf, T. et al. Evidence for in vivo transport of bi oactive nitric oxide in human plasma. The Journal of clinical investigation 109, 1241-1248 (2002).

PAGE 114

114 163. Hanafy, K.A., Krumenacker, J.S. & Murad, F. NO, nitrotyrosine, and cyclic GMP in signal transduction. Med Sci Monit 7, 801-819 (2001). 164. Yoshida, A. et al. Nitric oxide synthesis in retinal photoreceptor cells. Visual neuroscience 12, 493-500 (1995). 165. Chakravarthy, U. et al. Nitric oxide synthase activity and expressi on in retinal capillary endothelial cells and pericytes. Current eye research 14, 285-294 (1995). 166. Becquet, F., Courtois, Y. & Goureau, O. Nitr ic oxide decreases in vitro phagocytosis of photoreceptor outer segments by bovine retinal pigm ented epithelial cells. Journal of cellular physiology 159, 256-262 (1994). 167. Dighiero, P. et al. Expression of inducible nitric oxi de synthase in cytomegalovirusinfected glial cells of re tinas from AIDS patients. Neuroscience letters 166 31-34 (1994). 168. Scalera, F. et al. Erythropoietin increases asymmetric dimethylarginine in endothelial cells: role of dimethylargi nine dimethylaminohydrolase. J Am Soc Nephrol 16, 892-898 (2005). 169. Laufs, U. et al. Physical training increases endot helial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 109, 220-226 (2004). 170. Guthrie, S.M., et al. The nitric oxide pathway modulat es hemangioblast activity of adult hematopoietic stem cells. Blood 105, 1916-1922 (2005). 171. Schachinger, V., Britten, M.B. & Zeiher, A. M. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outco me of coronary heart disease. Circulation 101 1899-1906 (2000). 172. Zeiher, A.M. Endothelial vasodilator dys function: pathogenetic link to myocardial ischaemia or epiphenomenon? Lancet 348 Suppl 1 s10-12 (1996). 173. Pannirselvam, M., Verma, S., Anderson, T. J. & Triggle, C.R. Cellular basis of endothelial dysfunction in small mesenteric arte ries from spontaneously diabetic (db/db /-) mice: role of decreased te trahydrobiopterin bioavailability. British jo urnal of pharmacology 136, 255-263 (2002). 174. Craven, P.A., Studer, R.K. & DeRubertis, F.R. Impaired nitric oxide-dependent cyclic guanosine monophosphate generation in glomer uli from diabetic rats. Evidence for protein kinase C-mediated suppres sion of the cholinergic response. The Journal of clinical investigation 93, 311-320 (1994).

PAGE 115

115 175. Rosselli, M., Imthurn, B., Keller, P.J., Jack son, E.K. & Dubey, R.K. Circulating nitric oxide (nitrite/nitrate) levels in postmenopausal women substitut ed with 17 beta-estradiol and norethisterone acetate. A two-year follow-up study. Hypertension 25, 848-853 (1995). 176. Bak, I. et al. Heme oxygenase-1-related carbon monoxide production and ventricular fibrillation in isolated ischem ic/reperfused mouse myocardium. Faseb J 17, 2133-2135 (2003). 177. Yoshida, T. & Kikuchi, G. Purification a nd properties of heme oxygenase from pig spleen microsomes. The Journal of biological chemistry 253 4224-4229 (1978). 178. Maines, M.D., Ibrahim, N.G. & Kappas, A. Solubilization and partial purification of heme oxygenase from rat liver. The Journal of biological chemistry 252, 5900-5903 (1977). 179. Yoshida, T. & Kikuchi, G. Features of the reaction of heme degrad ation catalyzed by the reconstituted microsomal heme oxygenase system. The Journal of biological chemistry 253, 4230-4236 (1978). 180. Yoshida, T. & Kikuchi, G. Reaction of th e microsomal heme oxygenase with cobaltic protoporphyrin IX, and extremely poor substrate. The Journal of biological chemistry 253, 8479-8482 (1978). 181. Carter, E.P. et al. Regulation of heme oxygenase -1 by nitric oxide during hepatopulmonary syndrome. Am J Physiol Lung Cell Mol Physiol 283, L346-353 (2002). 182. Miyazono, M., Garat, C., Morris, K.G., Jr & Carter, E.P. Decreased renal heme oxygenase-1 expression contri butes to decreased renal function during cirrhosis. Am J Physiol Renal Physiol 283 F1123-1131 (2002). 183. Ozawa, N. et al. Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress. The Journal of clinical investigation 109, 457-467 (2002). 184. Johnson, F.K., Durante, W., Peyton, K.J. & Johnson, R.A. Heme oxygenase inhibitor restores arteriolar nitric oxide function in dahl rats. Hypertension 41, 149-155 (2003). 185. Brann, D.W., Bhat, G.K., Lamar, C.A. & Mahesh, V.B. Gaseous transmitters and neuroendocrine regulation. Neuroendocrinology 65 385-395 (1997). 186. Carvajal, J.A., Germain, A.M., HuidobroToro, J.P. & Weiner, C.P. Molecular mechanism of cGMP-mediated smooth muscle relaxation. Journal of cellular physiology 184, 409-420 (2000).

PAGE 116

116 187. Furchgott, R.F. & Jothianandan, D. Endothelium-dependent and -independent vasodilation involving cyclic GMP: relaxation induced by ni tric oxide, carbon monoxide and light. Blood vessels 28, 52-61 (1991). 188. Kajimura, M., Goda, N. & Suematsu, M. Organ design for generati on and reception of CO: lessons from the liver. Antioxidants & redox signaling 4, 633-637 (2002). 189. Stone, J.R. & Marletta, M.A. Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33, 5636-5640 (1994). 190. Bussolati, B. et al. Bifunctional role for VEGF-indu ced heme oxygenase-1 in vivo: induction of angiogenesis and inhibition of leukocy tic infiltration. Blood 103, 761-766 (2004). 191. Cisowski, J., et al. Role of heme oxygenase-1 in hydrogen peroxide-induced VEGF synthesis: effect of HO-1 knockout. Biochemical and biophysical research communications 326, 670-676 (2005). 192. Kimura, H. & Esumi, H. Reciprocal regul ation between nitric oxide and vascular endothelial growth fact or in angiogenesis. Acta biochimica Polonica 50, 49-59 (2003). 193. Deshane, J., et al. Stromal cell-derived factor 1 pr omotes angiogenesis via a heme oxygenase 1-dependent mechanism. The Journal of experimental medicine 204, 605-618 (2007). 194. Foresti, R. et al. Vasoactive properties of CORM -3, a novel water-soluble carbon monoxide-releasing molecule. British journal of pharmacology 142, 453-460 (2004). 195. Motterlini, R. et al. CORM-A1: a new pharmacologically active carbon monoxidereleasing molecule. Faseb J 19, 284-286 (2005). 196. Clark, J.E., et al. Cardioprotective acti ons by a water-soluble carbon monoxide-releasing molecule. Circulation research 93, e2-8 (2003). 197. Foresti, R., Hoque, M., Bains, S., Green, C. J. & Motterlini, R. Ha em and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway. The Biochemical journal 372 381-390 (2003). 198. Guo, Y. et al. Administration of a CO-releasing mol ecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol 286, H1649-1653 (2004). 199. Gertler, F.B., Niebuhr, K., Reinhard, M., We hland, J. & Soriano, P. Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-239 (1996).

PAGE 117

117 200. Bachmann, C., Fischer, L., Walter, U. & Reinhard, M. The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation. The Journal of biological chemistry 274 23549-23557 (1999). 201. Niebuhr, K. et al. A novel proline-rich mo tif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. The EMBO journal 16, 5433-5444 (1997). 202. Ahern-Djamali, S.M. et al. Mutations in Drosophila en abled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains. Molecular biology of the cell 9, 2157-2171 (1998). 203. Lambrechts, A. et al. cAMP-dependent protein kina se phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains. The Journal of biological chemistry 275 36143-36151 (2000). 204. Massberg, S., et al. Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice. Blood 103 136-142 (2004). 205. Krause, M., Dent, E.W., Bear, J.E., Loureir o, J.J. & Gertler, F.B. Ena/VASP proteins: regulators of the actin cyto skeleton and cell migration. Annual review of cell and developmental biology 19 541-564 (2003). 206. Aszodi, A. et al. The vasodilator-stimulated phos phoprotein (VASP) is involved in cGMPand cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. The EMBO journal 18, 37-48 (1999). 207. Anderson, S.I., Behrendt, B., Machesky, L. M., Insall, R.H. & Nash, G.B. Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelli ng of the cytoskeleton. Cell motility and the cytoskeleton 54, 135-146 (2003). 208. Bear, J.E. et al. Negative regulation of fibroblas t motility by Ena/VASP proteins. Cell 101, 717-728 (2000). 209. Bear, J.E. et al. Antagonism between Ena/VASP prot eins and actin filament capping regulates fibroblast motility. Cell 109 509-521 (2002). 210. Coppolino, M.G. et al. Evidence for a molecular complex consisting of Fyb/SLAP, SLP76, Nck, VASP and WASP that links the ac tin cytoskeleton to Fcgamma receptor signalling during phagocytosis. Journal of cell science 114, 4307-4318 (2001). 211. Goh, K.L., Cai, L., Cepko, C.L. & Gertler, F.B. Ena/VASP proteins regulate cortical neuronal positioning. Curr Biol 12, 565-569 (2002).

PAGE 118

118 212. Krause, M., et al. Fyn-binding protein (Fyb)/SLP76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton. The Journal of cell biology 149 181-194 (2000). 213. Price, C.J. & Brindle, N.P. Vasodilator-s timulated phosphoprotein is involved in stressfiber and membrane ruffle formation in endothelial cells. Arteriosclerosis, thrombosis, and vascular biology 20 2051-2056 (2000). 214. Rosenberger, P. et al. Identification of vasodilator-stimulated phosphoprotein (VASP) as an HIF-regulated tissue permeability factor during hypoxia. Faseb J 21, 2613-2621 (2007). 215. Comerford, K.M., Lawrence, D.W., Synnestvedt K., Levi, B.P. & Colgan, S.P. Role of vasodilator-stimulated phosphoprotein in PKAinduced changes in e ndothelial junctional permeability. Faseb J 16, 583-585 (2002). 216. Halbrugge, M. & Walter, U. Purification of a vasodilator-regula ted phosphoprotein from human platelets. European journal of biochemistry / FEBS 185, 41-50 (1989). 217. Oelze, M. et al. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxid e/cGMP signaling and e ndothelial dysfunction. Circulation research 87, 999-1005 (2000). 218. Lawrence, D.W. & Pryzwansky, K.B. The vasodilator-stimulated phosphoprotein is regulated by cyclic GMPdependent protein kinase during neutrophil spreading. J Immunol 166, 5550-5556 (2001). 219. Howe, A.K., Hogan, B.P. & Juliano, R.L. Regulation of vasodilator-stimulated phosphoprotein phosphorylation and interaction with Abl by protein kinase A and cell adhesion. The Journal of biological chemistry 277 38121-38126 (2002). 220. Shaw, L.C. et al. Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retin al neovascularization. Gene therapy 13 752-760 (2006). 221. Szymanski, P., Anwer, K. & Sullivan, S.M. Development and characterization of a synthetic promoter for selective expre ssion in proliferating endothelial cells. The journal of gene medicine 8, 514-523 (2006). 222. Segal, M.S. et al. Nitric oxide cytoskel etal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes. Diabetes 55, 102-109 (2006). 223. Hiratsuka, S., et al. MMP9 induction by vascul ar endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer cell 2, 289-300 (2002).

PAGE 119

119 224. Cursiefen, C. et al. Roles of thrombospondin-1 and -2 in regulating corneal and iris angiogenesis. Investigative ophthalmology & visual science 45 1117-1124 (2004). 225. Luttun, A. et al. Revascularization of ischemic tissu es by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nature medicine 8, 831840 (2002). 226. Li, H. et al. Hypoxia-inducible factor-1alpha (HIF-1alpha) gene polymorphisms, circulating insulin-like growth factor bindi ng protein (IGFBP)-3 le vels and prostate cancer. The Prostate 67, 1354-1361 (2007). 227. Le Jan, S. et al. Characterization of the expres sion of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer. FEBS letters 580, 3395-3400 (2006). 228. Liu, L.Q. et al. Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoti ng proliferation of primitive CD34+CD38hematopoietic cells in vitro. Oncology research 13 359-371 (2003). 229. Kee, T.H., Vit, P. & Melendez, A.J. S phingosine kinase signalling in immune cells. Clinical and experimenta l pharmacology & physiology 32, 153-161 (2005). 230. Lee, O.H. et al. Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells. Biochemical and biophysical research communications 264, 743-750 (1999). 231. Roviezzo, F. et al. Essential requirement for sphingosine kinase activity in eNOSdependent NO release and vasorelaxation. Faseb J 20, 340-342 (2006). 232. Igarashi, J. & Michel, T. Sphingosine 1phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptorregulated endothelial nitric-oxide synthase signaling pathways. The Journal of biological chemistry 276 36281-36288 (2001). 233. Loureiro, J.J. et al. Critical roles of phosphor ylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration. Molecular biology of the cell 13 2533-2546 (2002). 234. Hauser, W., et al. Megakaryocyte hyperplasia and e nhanced agonist-induced platelet activation in vasodilator-stim ul ated phosphoprotein knockout mice. Proceedings of the National Academy of Sciences of the United States of America 96, 8120-8125 (1999). 235. Li, Z., Ajdic, J., Eigentha ler, M. & Du, X. A predomin ant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans. Blood 101, 4423-4429 (2003).

PAGE 120

120 BIOGRAPHICAL SKETCH Kyung Hee Chang was born in Seoul, South Kore a. She received her Bachelor of Science degree in anim al science from Seoul National Un iversity in 2001. She c ontinued her graduate education in Seoul National University and obt ained her Master degree majoring embryology in March 2003. In 2003, she joined the Interdisciplinary Program in Biomedical Sciences at College of Medicine, University of Florida, where in May 2008 she received the degree of Doctor of Philosophy.


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101109_AAAAAJ INGEST_TIME 2010-11-09T10:23:55Z PACKAGE UFE0022015_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2770 DFID F20101109_AAAJKL ORIGIN DEPOSITOR PATH chang_k_Page_117.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
dd0a71d3a1c5ec0cb004883be7c60508
SHA-1
d649dcbadcdee1a998a282ac2110e665b644b012
2162 F20101109_AAAKNN chang_k_Page_043.txt
710ddc06ca5e1056081038231e2b3b86
8ddf2e77eb2939fc93e0abbe458d737e2b681800
65391 F20101109_AAAKMY chang_k_Page_112.pro
2924664e669ad37fbfc97ca333dd489e
838d7f6a4a39d7200f8817c4525941e91816ece8
2039 F20101109_AAAKOB chang_k_Page_095.txt
1e824d3b1f48ff57a36807e96879d33e
1f632533a9adf1ae1b8a47cb7d36817dcb72a569
33618 F20101109_AAAJKM chang_k_Page_051.pro
bcc20b4700a6ce0b74d289ce54249ddd
ef616e15c4ef82a6e96742b27dc5469d474323c2
2127 F20101109_AAAKNO chang_k_Page_047.txt
664b91d8805330cc8205dbe4dd095565
b6bcebe6155ce7edf68148f86606763fef7e444e
62158 F20101109_AAAKMZ chang_k_Page_113.pro
3aef7978ace4c2490f401190eb2375c0
2d79d517ca27793a59edb74ac566840a31ff3169
2119 F20101109_AAAJLA chang_k_Page_032.txt
34670c59801726b33187754c4d5f29a6
29374eabdb5c0876bd91b2dfaa5854b36a267f03
2439 F20101109_AAAKOC chang_k_Page_106.txt
9019f459c42f764201faccf86eb2bedd
9569bad45eeb344ced3b45b7c8b5aa4f73fcdd70
1942 F20101109_AAAJKN chang_k_Page_062.txt
bfd946917a7ba007f4176fba70209223
8bedfd00f189b0796226dc0ca77e2fc27fedb095
1966 F20101109_AAAKNP chang_k_Page_055.txt
9e52bb27d45262eae8b0e181d99a6b9f
4704d3e155eab83e7673fd39d464053ea714b6c0
340205 F20101109_AAAJLB chang_k_Page_009.jp2
0d42f0313df1570c3df45af9d0dddfc3
8d0504d2d199b8927a71152ca76d00d76ede034b
2664 F20101109_AAAKOD chang_k_Page_112.txt
7c360115ef3aa5fa5290594abb52951d
6a18473093d3d7199afe40011f27067778ee8fb4
1051927 F20101109_AAAJLC chang_k_Page_019.jp2
c5071ecf6e1313a77115a2195e3edb63
1a2e44e59585a9bf5bd04725efa297b355c544e2
2399 F20101109_AAAKOE chang_k_Page_001thm.jpg
be40a7db02b120d916feadf4d87eb9e6
9c1e174ce8c682775372038961472e5eed7529f4
132909 F20101109_AAAJKO chang_k_Page_108.jpg
386461d9efd4958770c4040ca702c5af
7346642838c4b2432b6dae4937463ef078ac4a68
2154 F20101109_AAAKNQ chang_k_Page_060.txt
f6313d26f7d8ea08e7bf45d8992324f0
6c7e713c822488b0eabe6d42776469ceab830623
8568 F20101109_AAAJLD chang_k_Page_095thm.jpg
2791c4183b63290b87e83188e10864f6
277a68f4b53b5c4a07321820e18c91de63f78468
4075390 F20101109_AAAKOF chang_k.pdf
20ff444dea509f36987f4bf950ca559d
08f2fc67b4caacfa11254a52859b2a8294b93ccc
99238 F20101109_AAAJKP chang_k_Page_034.jpg
7c88045d22dfea321247f07c6977bef0
c536f9dbec33cbe60a4896626c9d4a3b653d9676
1964 F20101109_AAAKNR chang_k_Page_064.txt
1e50486b99c9825b3c2b9f2d0195e65d
6a31c7c96bdf7befc413db06be7f5d2a22da2334
1051979 F20101109_AAAJLE chang_k_Page_008.jp2
8456da2453cdf969ef1b87dfc26cc674
21137c5d0c499ff497abdd6b80bb512470dca026
8531 F20101109_AAAKOG chang_k_Page_069thm.jpg
0e49ff7af54f44266c34675e8ddd5826
d70d93b415c4b7835c17cf0dee6ceb3d2bd269ac
55553 F20101109_AAAJKQ chang_k_Page_027.pro
dd7d847f69f66002ab078156b374207f
e04a56a1d45bf3ee85cefaf1b0c9da573eb28e3d
2065 F20101109_AAAKNS chang_k_Page_068.txt
26ca10ed49c41fe6b6893a1bde75a6b2
0e1ecd1f1b29f924c87bfdfa96a8537e5e1a8b75
9033 F20101109_AAAJLF chang_k_Page_108thm.jpg
e0a3b83959f07dca5f1d12744c5e4f52
7e6fd3f0c0588d38e62ac4acfc1b96f7410a6901
8716 F20101109_AAAKOH chang_k_Page_029thm.jpg
2323b8afedd0d9ae0bc646ed102c15de
9f04d9e81be3ed54d173fa2a39a9ecdba4621416
32238 F20101109_AAAKOI chang_k_Page_008.QC.jpg
a0e471fa249789c02a6bbe6dd7a7ba57
3d24be097c741db6289c3e29d9b2d79ccb3570ca
329 F20101109_AAAJKR chang_k_Page_009.txt
46c6371acf139d61bd3a96e34d8996a0
b354f1d3c2e4d6331252cac40c41a3c40730e66a
1986 F20101109_AAAKNT chang_k_Page_069.txt
d277343f43882c8394c6b0141db4af39
b70810c634f6e2825d50603c1cf3364e2cc36359
2543 F20101109_AAAJLG chang_k_Page_113.txt
5d4163fada5c36d43b6d83d805bb9fed
0e7b23755689cfe197015c4e622aa91cadfa9b99
6074 F20101109_AAAKOJ chang_k_Page_051thm.jpg
8fa3eab30d6a66aa0821dc7a68073cc0
d2f4edc0bea436ef59babde96d979877f7d3ce24
25271604 F20101109_AAAJKS chang_k_Page_079.tif
05b847b190ab6d73fd7556b2544dda68
47f4d889ddb7daa97ab4406808e18f8daf70d237
1844 F20101109_AAAKNU chang_k_Page_070.txt
91895802fcc61eb0eefcb8c8d8ffa166
7a2901497574b73cda8570c50b7badc735054662
8849 F20101109_AAAJLH chang_k_Page_097thm.jpg
3ac215952ea683eaaf719e459ebaebb6
fe5ac0afca24778ed5723927cf35268dcd6d2c7e
8981 F20101109_AAAKOK chang_k_Page_057thm.jpg
932d4d82e0477b08c2d3b6381c3fab5d
d4bb37c3197b958592feec4a4bf9dba8a2bf9447
37088 F20101109_AAAJKT chang_k_Page_117.QC.jpg
1764253aa0bd20efa09b3a4a8f9b5967
f4e7694339441a7a5ded20a9edbe68384399359a
1702 F20101109_AAAKNV chang_k_Page_071.txt
60ed5786366f18bf69c3243525224d94
e8f862bd6379faf4960161f6d8e927964ab8753e
F20101109_AAAJLI chang_k_Page_118.tif
76b01d77fda052e0be153a3fef177074
c65eb410a9fd8a14eca5319945bba7666bcc08b0
33257 F20101109_AAAKOL chang_k_Page_114.QC.jpg
6df6f0ee89d0b91af55a34acae9c3d7a
d9fb0eb986396239419376aadb3a5f629d1cc1c9
2034 F20101109_AAAJKU chang_k_Page_058.txt
42b3291621299c8fa6b694908a1b8c78
95534a1cc0e6cf6373d63da0ecebf0b89bfeeffc
805 F20101109_AAAKNW chang_k_Page_073.txt
78fa31862e7b1052b0d392e336709dc0
b966c75b25a82f796547f6098a5198adce51ba86
787990 F20101109_AAAJLJ chang_k_Page_051.jp2
07ce34b22de1641b0ab76cece96c9092
59fd1f7e91bf15da7b15162e974775242196257e
179687 F20101109_AAAKPA UFE0022015_00001.xml FULL
b125862b4f14b960f7d5cf8a96bbc0e0
36cc459936522e5ceb4b8e5930b4bc0db9512818
2562 F20101109_AAAKOM chang_k_Page_120thm.jpg
69dc2dd2ab81ca7bcf2c179b4e896923
d7d8dd5f99b847574f781d97139a89614e392255
2058 F20101109_AAAJKV chang_k_Page_067.txt
5dfef98d3e6a12b3e2fe2904d53d65c0
2077593319fbbe96d94a676840d7368bc79f946f
366 F20101109_AAAKNX chang_k_Page_077.txt
58e6109ce2fe4a1b0cb5a4d2b627c6d1
bb1fb5c3103af868f4c6290799e0446f5f7cb995
F20101109_AAAJLK chang_k_Page_042.tif
7f1fe3fd72f09ff3d43d6dc1d045bb72
9aab1c2867933114d424ae18dbad582ddf7a0b9d
563 F20101109_AAAKPB chang_k_Page_002thm.jpg
191424e1cba18b9358e2c2c60c61fec2
0e03421b08dd147f3cce3cdbd1db02786e9047f3
12649 F20101109_AAAKON chang_k_Page_075.QC.jpg
c41fb2b68aef7440afe1bc55b7c02be2
ffbac54de783b8a260adf479ca6b0bf4b2547a17
999738 F20101109_AAAJKW chang_k_Page_041.jp2
b3e535d218917e186c4c69e8c68729d9
24aab81c3f0af7c7dc067d59e8f8af6a2245368b
F20101109_AAAKNY chang_k_Page_081.txt
6816af0c833b4c01a665c152c04eacf1
51fa391a19b9b2bc911c8245bb9f8ec5f9c2e68e
54336 F20101109_AAAJLL chang_k_Page_048.pro
fb4beb651dfda148c3cd18c92fb5cf3e
4dfde80939864033612b6f141239d2b4a4b0dd51
34034 F20101109_AAAKPC chang_k_Page_004.QC.jpg
98ca776f34735c07239b934b132018f0
387dfadefb62422de61625fa7ecab8ed0f7db0cc
9171 F20101109_AAAKOO chang_k_Page_098thm.jpg
c7a60414dfb246e7014f00c9cc2ded54
8c01a420cfe7785e766e467085dd85f5984430b8
8214 F20101109_AAAJKX chang_k_Page_033thm.jpg
4d3f473d20c53dc172e6d1ebb3542d4f
d5f6bfb12b3f2230667d28290eb05b69e715abe8
649 F20101109_AAAKNZ chang_k_Page_084.txt
14c4fcf378d01fc43ed6a24d59cdb398
f0ae1faddbcb7f74ce091ec7f99a6af4e79e0764
8824 F20101109_AAAJMA chang_k_Page_110thm.jpg
df4df2f9cf0721af336cacee542bac11
1184a30d3aac5064ead48823d42b3a61333d2dfa
2370 F20101109_AAAJLM chang_k_Page_008.txt
30d682a546046347b8a620d8826b0ab2
b42b19f0422dbb5b6ff2d776dd104485b60dc86a
8453 F20101109_AAAKPD chang_k_Page_004thm.jpg
acd6bd1f9c96b87a31d4abb846450d88
6605bf9fbf8b3f4587b1828b066638688cfa5e0a
36077 F20101109_AAAKOP chang_k_Page_031.QC.jpg
ed64a444fe1e40f710fd91e9c37a37fb
7e06db8943157893137927b2a4cf3e57f0e5b49e
F20101109_AAAJMB chang_k_Page_107.jp2
da820216ecf4ad64910785b460804867
3febaa2be59ebe96a3c3a54176982fafbcdc6f7e
1233 F20101109_AAAJLN chang_k_Page_100.txt
ae7c46bb489a335fb3d4c12582bc8fa8
4b1567e9d9a37b933c0dfd55fbbe6cf98139e581
116626 F20101109_AAAJKY chang_k_Page_066.jpg
31fa954d132fab95b3a18b1095460f9b
89d60571ba7ce3478ccb8839bee13c05444f48b1
6505 F20101109_AAAKPE chang_k_Page_006thm.jpg
29560d7eb50090e12fe7384a85adcade
c1ff0b160a003826f425c66089f73c4972c67a13
9338 F20101109_AAAKOQ chang_k_Page_017thm.jpg
7435379b99d50c72dcf64b8e11656955
8f0a3def9383462fb3c5847e33b9eb86d02a0cca
2155 F20101109_AAAJMC chang_k_Page_096.txt
b5e01048b93879206f10a6bef9341247
a1362ced33f3094d1cef737129f097da1746133e
2183 F20101109_AAAJLO chang_k_Page_039.txt
9d09dcd6a197c9046d0b1b6a63825e08
e0825f9bc9693cf6c6dd3f82ce86cc8e73fd72c4
2120 F20101109_AAAJKZ chang_k_Page_029.txt
7e7426879e7f318cf4be8cbf94e095d4
cb3c2a1de26c66a694ad20d94d93e4bdee7eb40c
4749 F20101109_AAAKPF chang_k_Page_012thm.jpg
e0c537981c5c1128a9d7e080eb8305fa
fc41d2207e6d077155a0647666fba51a1517834f
F20101109_AAAJMD chang_k_Page_013.tif
6f1c7902f89e571a5f8e10b85f7de1f5
beb64715c7664d5b21ac7cfa848d96871ef73b7d
32508 F20101109_AAAKPG chang_k_Page_016.QC.jpg
4076c19ab83122c3491aa54b68ae1b9c
81f5e8053def9469b9c65653d179c7ddf47eab5f
34417 F20101109_AAAKOR chang_k_Page_115.QC.jpg
a4fa2092f8163a178c563ecb74ff3baf
21447854d9b33ff9d859d037a5bdae92b4e117c8
4981 F20101109_AAAJME chang_k_Page_086thm.jpg
02949b850a0ca2f9d795976193472c11
f385f4902cfbe9161721504ccd759f310d1bd331
F20101109_AAAJLP chang_k_Page_086.tif
47997125c07a88c82b0cc7532f9b2eb8
60e2d9f3646cc6ad3c6c80cc040774018bff7165
37583 F20101109_AAAKPH chang_k_Page_017.QC.jpg
2169efe41f22b0dd06e3b7099733399e
f302f1cd8fb98f2d063bbc740c43ad9651964e33
3616 F20101109_AAAKOS chang_k_Page_076thm.jpg
a9f94f0dfe5de8bc676f6b899a69fe8e
724985f8f151330cc5def3fe8e5fdfebca095cb8
F20101109_AAAJMF chang_k_Page_112.tif
94c27fc04ca0d5a87a9bde18406fe253
e58fa0cb88a97d48b512d014499f14a1f2bbc9f5
34577 F20101109_AAAJLQ chang_k_Page_101.QC.jpg
f7d80d6d8863f4044e1f3827bf3af9dc
afb2b62deae4588a089efac89e00f4ce7a5ee51c
9024 F20101109_AAAKPI chang_k_Page_020thm.jpg
2d71777fae413bbbbff1d9c46120f950
dc3fd22acad50f729888cd46e2042e800a23fe37
37600 F20101109_AAAKOT chang_k_Page_035.QC.jpg
ee9be0ad727c404e516d4fa39f39a4cc
999a5683f80d3e73310c9c7fb438c1bcf92ee5d2
8970 F20101109_AAAJMG chang_k_Page_059thm.jpg
b3af3ac2e5c8a509922b71bfc58e4f67
806c9a91e509add964bdc195ee673b377d577c23
1051974 F20101109_AAAJLR chang_k_Page_111.jp2
7c63e1abc5463ba4187cfeead8aba5ec
af77390e8a1228b0a8651e0f3f72233e3cf7b19d
35243 F20101109_AAAKPJ chang_k_Page_024.QC.jpg
635ae6bd0cd05023bbaa9a23478db6c6
a4f7f5f8ddb813ce0f0f6dc5cabcb57109f4a3dd
8533 F20101109_AAAKOU chang_k_Page_103thm.jpg
12c25a968ee2c8b45b6ad5a96afdd274
d70901be6aebfc6de6c660b4177aac48546e1405
2446 F20101109_AAAJMH chang_k_Page_105.txt
b5ec3154d6b7db01bd9cf0cb5d866d3f
7ab111e8cca651e831d522569b2ffea83e286f95
F20101109_AAAJLS chang_k_Page_015.tif
3f96c4efbd197ab1e037683c84d46773
9cfd5894fe5b5bb7139cbcefeba40b9d080902bc
8665 F20101109_AAAKPK chang_k_Page_024thm.jpg
140af946d6d37b117d68e2a30591286c
16618fb2a1d3ab0c6879cc820c83bca4ce13718d
7948 F20101109_AAAKOV chang_k_Page_070thm.jpg
67d00d00c7d1d1943167e2ef4535d0e9
dae0c552e98e91c0e6bd5b2c87f2ed33f1c128ae
114995 F20101109_AAAJMI chang_k_Page_098.jpg
48c990eaab77a091bef813870aeddd07
887e5672583fa0e7b8007da12b233893cd502b42
F20101109_AAAJLT chang_k_Page_047.tif
f701c5ffe5c31b3305a37e1ea6a12566
35936396ab7bfb64d91e269b661f944ac0d2b1b5
38029 F20101109_AAAKPL chang_k_Page_048.QC.jpg
8bfc875bda56b8b1ff629804dbf1e9e8
64a3ec71f68b6671c6a73f0a5c00ab9e9d790504
7895 F20101109_AAAKOW chang_k_Page_014thm.jpg
3e951492605f47fbf62fb1126af6f81d
33fd5da07631f5087264e633f3a66d27fca03f4e
331403 F20101109_AAAJMJ chang_k_Page_090.jp2
dce4e6974441d2de9b7194554e4eb0ba
f5ead9ad899d562680c9af7213237ddc671ec9d1
107488 F20101109_AAAJLU chang_k_Page_065.jpg
6f9ca3ccc9893fa78f2e62ae24881d68
09199343c6cccb1c700cee064b4256ae80855064
8752 F20101109_AAAKQA chang_k_Page_078thm.jpg
111b10f48a0671dbd03aa07bfd4ec30a
b8c041c78e773e00e896fd167129b7a61e3c107e
32810 F20101109_AAAKPM chang_k_Page_049.QC.jpg
4f4ddbe6341ca64fde2d18cea43cd0ba
e76d06a0e4877fcee4678212cc660302fd84da3a
8769 F20101109_AAAKOX chang_k_Page_107thm.jpg
c758fb458216a17dabaa0bda8cd6b4c2
0d659cec92bc93343066103a556d5073fc0a7ebc
51101 F20101109_AAAJMK chang_k_Page_010.jpg
5f439ff4038e3708d7ae0175c4df012b
11876ef677c1c206ad7410020eba828af9d36fbf
1051954 F20101109_AAAJLV chang_k_Page_032.jp2
257982539b7075d89cc829faf7e735d5
1f884e7ff2532f334a0972e77dc7b4792a609857
12739 F20101109_AAAKQB chang_k_Page_079.QC.jpg
91cb50cfb30cb33cc3c70e864a87aa0b
225510fa60311aa20bd7fe2b69a4aa2a22f9f35f
23279 F20101109_AAAKPN chang_k_Page_051.QC.jpg
a6f0d307ddf68f069e8d951a8ee47a0a
23f1699d88f6bf4557b83d5e91f9f279073021ad
32727 F20101109_AAAKOY chang_k_Page_105.QC.jpg
3da32f1f3b508abb895e167b3578d9a1
afd169e26a53e7d4460eca0617c63cb7641baa69
33052 F20101109_AAAJML chang_k_Page_055.QC.jpg
70a42b2316379b65a44e3dc85d7bfe3f
cdf544666f804dec4a221b923a9fb33415c5fc9d
2208 F20101109_AAAJLW chang_k_Page_056.txt
7f4984633a80c894e662fb1bfdf0a7f9
a2566ee3ecc1acae06ec92e1ae4ff46e5a5df019
11873 F20101109_AAAKQC chang_k_Page_080.QC.jpg
244414053750b9bf12d510a2791fc2b7
b90657f216061fcf37810623b29f7d4cf62e35c1
8323 F20101109_AAAKPO chang_k_Page_052thm.jpg
1d48412edc1b87298666f22b30a01282
41846684a5ccf44b26dacede5862d9d31711df8a
8118 F20101109_AAAKOZ chang_k_Page_045thm.jpg
03125ddc2138764b07708d24cf4e7bee
9a51cdc7509adb15e88458ef94390669678a2c4d
3759 F20101109_AAAJMM chang_k_Page_090thm.jpg
88bc230d9aed0139d6cdc25c8ca58f33
0aa72f064d4f5fad04f1a5381eb65f6b6ae7fd4e
4384 F20101109_AAAJLX chang_k_Page_084thm.jpg
ddc51ab3be54efb2fda297df4e4e3381
8ab65d7c0222074a2753b0f9cdea1f411b545ec7
100350 F20101109_AAAJNA chang_k_Page_021.jpg
79f83764b2410007c0245db0ef05d3dc
dda89a17f41d426c92bce4bfb842c6ef98c11cfa
6215 F20101109_AAAKQD chang_k_Page_081thm.jpg
dc5bfb389c37f5cdf85796d68a094b65
4ed22e2f3dfb21dad9d0db1ddb343d926f21f8f7
8743 F20101109_AAAKPP chang_k_Page_053thm.jpg
eaf56a06747ddd1fa453626be71aa4b6
51d6603ff4e855ed48a0b87e7957989ac9dc50f5
F20101109_AAAJMN chang_k_Page_045.tif
995e308675bee6ae3047ec00039ead0a
8108c815af8f1c37ba94bc56597bb60c8a0cfd8a
1051935 F20101109_AAAJLY chang_k_Page_046.jp2
9264ff2236e67fb94e1eae35eb150475
6de5d795f3fb9161e46d0cd40a93480f241eba8d
31873 F20101109_AAAJNB chang_k_Page_080.jpg
9e6cf1dafef4ca712af23738f0c97c29
6cb3e68b06754693b87dcf92f3c360527c88d4b6
18295 F20101109_AAAKQE chang_k_Page_082.QC.jpg
b977ec931116862a86d809f54747e4bb
ff5cb6912de06085a988dc500a38cfe33dbc877e
38286 F20101109_AAAKPQ chang_k_Page_057.QC.jpg
c43d325bd7b0bec16e2834e905bf10be
43c862b48952dcae94f72ef2afcee5aa009467ba
55260 F20101109_AAAJMO chang_k_Page_086.jpg
d25e67f9999b0cd8ac441da811f9db00
6582762a212c505da1c67fe31de5a0fa713f0963
38886 F20101109_AAAJLZ chang_k_Page_066.QC.jpg
1fa2520014ccb01f42515fda82a58d20
57d2cbb4e31f358bb1c03244890d596b1c7846af
1051931 F20101109_AAAJNC chang_k_Page_053.jp2
30b31b79c9f212785ff787e6ab05e6fc
433ea07584ea5198430e0c8b6d3b35cfee2d833e
12611 F20101109_AAAKQF chang_k_Page_083.QC.jpg
96bf2124596b37e6700de1aade948d36
4009c6b50585a5e737deb3cfc0e00c71ff8e7cca
38492 F20101109_AAAKPR chang_k_Page_059.QC.jpg
1928b1aa096fff7d50b70d7c77299690
76e2d9958fd76362fe5b59c85776364183ee6e1e
126155 F20101109_AAAJMP chang_k_Page_112.jpg
3175c237962878d7bf7d6e61fddd00df
8e11a4d714dc0dc2d5c22f705c7f75506bc3b756
F20101109_AAAJND chang_k_Page_030.tif
d6aefbe5b00d9aaf8eb75bc2aab45b46
746a151df585e988b082282939bee38a7289586b
3858 F20101109_AAAKQG chang_k_Page_083thm.jpg
8dcb32c7ee92a5c27c57aaa736d09b36
1e5931220b8013d8c5e21ef70caf3ea309eee9a7
36343 F20101109_AAAJNE chang_k_Page_111.QC.jpg
6f78271544b758e9ce51c330346f2047
33e335cd71a0c90c9d9f96857c152aa4adf4d25b
8822 F20101109_AAAKQH chang_k_Page_092thm.jpg
5acc4982b14086c370dd3f51abf552bc
feb54899ebb7f13acad43071885fcbc84db595c0
9000 F20101109_AAAKPS chang_k_Page_060thm.jpg
d80ffe3d704751346596d0fb66ce20cd
6eb5951a17bd61840ea43d3fb42887c1c9388e49
48326 F20101109_AAAJMQ chang_k_Page_034.pro
375e808c516eae9b72f45c51aa667ec1
24729b938793f038abb86b466158a12a0d927738
9059 F20101109_AAAJNF chang_k_Page_056thm.jpg
925df6722faffc0fb0623a2fd5f0f1c3
0bb9b323eb3c3a4f3572b6d48f1e69ea83bde771
33628 F20101109_AAAKQI chang_k_Page_094.QC.jpg
90d56d4009fa8c8115f5b2b21658ad91
deeb777f30ad4c2f6f0300f24cd3f0ac54bb01d3
33383 F20101109_AAAKPT chang_k_Page_062.QC.jpg
2ba0a03c6a076434a4da051cada69238
131b84c069c039f6ee10fc1d08a56134463a4ddc
609 F20101109_AAAJMR chang_k_Page_090.txt
9c174df39e44b8b57f6ef531543a2689
82e09986838c2870ac9938f2388b788476c27081
F20101109_AAAJNG chang_k_Page_091.tif
6a1f4b5fdd13f8c7ad653b8f40be9899
9c9f09a91d5d0e3bb95fcb912f41ed1d64f596f1
37836 F20101109_AAAKQJ chang_k_Page_097.QC.jpg
7412746e4a97ae1147618c81dfc46c46
f815b6ed619a68e41b9da17bfff5b90fa6579556
34351 F20101109_AAAKPU chang_k_Page_067.QC.jpg
13f93e34ad67fb40f4985b4f64aef9b9
ffdf7b0d24a13727d81706c7c4a4a8af5a53494c
F20101109_AAAJMS chang_k_Page_027.tif
4ff7cc2f0e62ac092b190f4a3ef72574
4bb9f38c9e06f8cf08562355e858bc832e6bf011
102866 F20101109_AAAJNH chang_k_Page_004.jpg
c921fa9824fbbe325c142206dd17c434
37756ae17e40ae1e226bba2a51cfb8461fcd53b7
33761 F20101109_AAAKQK chang_k_Page_099.QC.jpg
e65ce4d089158e2d70b9be35e694fe01
06919fff80e3088a1e987a6ab375ff3ffbe31270
35658 F20101109_AAAKPV chang_k_Page_068.QC.jpg
2190f6b7ede019c7f7b453b2f5d7b579
d3e7c56bc31a3a4fc850909d76727348c0e44a4c
2759 F20101109_AAAJMT chang_k_Page_108.txt
b9c596b6f2d71e167ae7d7496c8800da
5563911f86b63e23d4cb69864a3642ff8deffbeb
6692 F20101109_AAAJNI chang_k_Page_041thm.jpg
9c1589772dcbeff3da8f6087654558a3
58a59e591d1af970673a5a4568a96619991d9c8a
8957 F20101109_AAAKQL chang_k_Page_101thm.jpg
70fe4b5b29038b23784246724a0e95c0
7972bd5298f11b0b8bf0fd28fd8447757a792830
8698 F20101109_AAAKPW chang_k_Page_068thm.jpg
7945225662c95056f169c443a9312111
d81246da1a38203153441ada4b433cf09526859e
47113 F20101109_AAAJMU chang_k_Page_050.pro
9fac2def3d7cdba042f335c0d0350680
fdf30d780dd2084d8fe786ee8faa930930ce14f5
132703 F20101109_AAAJNJ chang_k_Page_119.jpg
034a47eb00f475250e1d1d4f30fb648c
4e0439925649cfb38700a1ec45afb6e7e97d7340
9004 F20101109_AAAKQM chang_k_Page_102thm.jpg
660c7cd7f543ba72354fd4a2ff38debe
ba5e2674cb76042d68707ce7adbb2e760e301dd0
32572 F20101109_AAAKPX chang_k_Page_070.QC.jpg
99d16bef2550af73e723543e544645bc
af00ba1a9c2810325b63054466499a081d1279b9
36756 F20101109_AAAJMV chang_k_Page_046.QC.jpg
7db900b2e842384b91e98f8fabd4bfa3
4747297d5177d4b5c6c4c086a8f5e0acf192b1a3
2481 F20101109_AAAJNK chang_k_Page_103.txt
afa50f55327aee731bde711adfb26032
e2103d01e384aa5df8a7f068f505ab8ca1ea182a
F20101109_AAAKQN chang_k_Page_106thm.jpg
92df5f30387c6266c242b15f73204c20
8dee18e1fd1ce85cde6398ce547124f7d6d6e86f
7117 F20101109_AAAKPY chang_k_Page_073thm.jpg
57d8f79afbb2a36f41413a1c123bf720
f20e90eb5d6a649115740b2969032d558ff3820a
50257 F20101109_AAAJMW chang_k_Page_099.pro
2900481cb72723f2b493017662a4feb3
a9bf26c3c89533fa33193b003cf135375981c325
F20101109_AAAJNL chang_k_Page_052.tif
4f2210838772343aac1f98b468f34cb3
effe5411193478642870837e213fe50ec2194698
37139 F20101109_AAAKQO chang_k_Page_119.QC.jpg
d967c3240feda3ec4163b19bb2deb6c7
4411fc4af034b6795dabc12afe826548ed095fe4
5678 F20101109_AAAKPZ chang_k_Page_074thm.jpg
bcf82e61c4f25dec4a6fdc85aaea832f
6b484b736670ea06028cd8ca1782d439bf973b78
67562 F20101109_AAAJMX chang_k_Page_108.pro
73f7f33b23c29ebe2a069fd1b8768b0d
7633d13a62972ffc66379633f2a41850b628520e
1051952 F20101109_AAAJOA chang_k_Page_031.jp2
fe0cb295403b5e74272a5e6c0ee9c342
9990daa4d1549394f31dc9e3224242e5dba7ae8f
2048 F20101109_AAAJNM chang_k_Page_019.txt
0ce30e138161f0201e67f2b260a09dd8
251bf40731ff3f9dfdaa0d49af9a4874405de601
2063 F20101109_AAAJMY chang_k_Page_045.txt
db6df52c5969c7be955480d09b8c578c
0751dfd90a5afac1fc9926964581bdc8e75542f4
1051959 F20101109_AAAJOB chang_k_Page_118.jp2
08636a7e8ab7f59be887201dfd6647d2
84835da96b82037f0141a206244dde36387c4fd9
8211 F20101109_AAAJNN chang_k_Page_114thm.jpg
8a27ffd9f678dde2c2e41c59cd338d45
f16faa660c86f0c5b350e1a35930e8c6c736fe10
855 F20101109_AAAJMZ chang_k_Page_002.pro
59ab11c4c4c076605ed5d166f7ff3b29
d7f83f53f82cc6844f5f42300eb1b31dc0c72cdd
F20101109_AAAJOC chang_k_Page_120.tif
98f480967681697480ac33caaa4b29ea
87728f2580a31415f909365b6815c6ea17c68b0c
5002 F20101109_AAAJNO chang_k_Page_011thm.jpg
817a924dca378cef6544c9154b3d2b30
5e4c389b51f9f3a3e8e4b29dd7a8eab94d350c0c
111713 F20101109_AAAJOD chang_k_Page_026.jpg
c8eb91b75a47e6f40460001a8d5d102d
fb0d8efb34368c9f5484253775881b8ec11cbf61
F20101109_AAAJNP chang_k_Page_101.tif
708acea9e2937293982209ab7a135882
4f0ff32d31e9005ae109e2fa26bb0fed0d53bbd1
7930 F20101109_AAAJOE chang_k_Page_050thm.jpg
c0d4b1c9d467d4d5deb426509b860d95
cbe2385d021c03190fda915dcf2f76fbf3d9750c
32960 F20101109_AAAJNQ chang_k_Page_050.QC.jpg
b3e384fcdf96906f94d8dd1a7f10ad2a
58caf073c80dde5d4b7a63bcad5e2e7f9c921b89
F20101109_AAAJOF chang_k_Page_027.txt
a0e0f2c3b4f37e68dd20c129b285c51f
99ec7b79143206519bab8025ad1a56f06aba57ba
20820 F20101109_AAAJOG chang_k_Page_089.QC.jpg
26dad0f6d3341c30f32ccda58d7966b8
31998793f302c0d38090a50e31b67a6d2786f068
115853 F20101109_AAAJNR chang_k_Page_102.jpg
e0976f2aae9a65df60a364719ff66ee7
911a40c880844a0ae698f9f7e7caa55f4b471fc5
35760 F20101109_AAAJOH chang_k_Page_065.QC.jpg
941f6f081734196d5c2ae8f6e444a889
3cd6441d4bf8e528e4c1a480cf7dcc1cc96c4c7c
108050 F20101109_AAAJNS chang_k_Page_068.jpg
dd2e8384da07546ad63dc74d320407ca
a498004ccd7be59c3a0d31a1368bd3c086ec544d
132974 F20101109_AAAJOI chang_k_Page_117.jpg
449b262f2856542ae3e864dc593e9354
21b6024654e9b083a34e41fe2ed6e8868b4b0cdc
2389 F20101109_AAAJNT chang_k_Page_101.txt
ea0c913b38b0f574f8ce8bf523cabd9e
340517cd4ddb2b313da248ecf1594d38872fac63
16768 F20101109_AAAJOJ chang_k_Page_086.QC.jpg
929be9aa4c485ee162f1c87565bdc4a4
3b1209cc6e9c6c32f8093da98c00e4bc279e851f
5990 F20101109_AAAJNU chang_k_Page_040thm.jpg
771323b45c429d06d7b15bd1277b6b15
7d53ae9a18a0166d60a0bb7a7d2a545b1cfe425c
26100 F20101109_AAAJOK chang_k_Page_063.QC.jpg
9b3ea11ab069c7eb22b43e058405047f
a49bc41f9770359c2154b69142aa1170a2dbacc3
18960 F20101109_AAAJNV chang_k_Page_011.QC.jpg
00544e3929be04e94e87b912be5e2ce2
92448a7b25c05c11934a5c6760974581add03393
32481 F20101109_AAAJOL chang_k_Page_078.QC.jpg
0a66eb1d53b4281b3c8a4c888c085174
04cb067fe20e70b217147fad451ad4f6b7c76d2c
2795 F20101109_AAAJNW chang_k_Page_111.txt
698b12d67e4565a3fb780ff85e710d97
7fb14f04f42fd158805320d595539b6418ecb0e6
64791 F20101109_AAAJPA chang_k_Page_073.jpg
0ed9e5f322cbc245143cefbb05554661
815ad118bd323155a69e65aa3d81cac49f6ca430
8423998 F20101109_AAAJOM chang_k_Page_083.tif
ab0b270c833af4ccf77b61d9b91d7f87
f265f8b839c8873d54b729f93a9d40678bb2d574
2827 F20101109_AAAJNX chang_k_Page_119.txt
dc8de69c336c3075d931af955a821db2
392f1b4a199f9eb658314888789d986ea1bf4f87
6055 F20101109_AAAJPB chang_k_Page_082thm.jpg
cc6dc5487130027a2bc5ca039ca33b9f
5db3d3ee033d38cbf39c834ec3e0fd150407371e
108881 F20101109_AAAJON chang_k_Page_058.jpg
ec448aec5dc7a7e9faf686883c321cc2
2bfc949780232951d3fc9365b2486aafaaa9be8e
866960 F20101109_AAAJNY chang_k_Page_063.jp2
2b617e16e253a401b6648ab40335f0f5
1b7326a5412fb605c3d343909d9f784b243c863d
F20101109_AAAJPC chang_k_Page_018.jp2
d970b7e57aa35a2768120d8bb172048d
1f64edf6b9ff5bd6d07ce45cfa88decbaf7d7560
F20101109_AAAJOO chang_k_Page_072.tif
4a9281f845b0cb673efb26f7336de242
903b0c296c33838e0c405bf6fa36bf798bd166ad
2069 F20101109_AAAJNZ chang_k_Page_061.txt
b1a62bd3948d861cea80c4ae5f166b75
213f11b511012e4eae74043a3b543b09ab855f60
12240 F20101109_AAAJPD chang_k_Page_086.pro
76bc57f670ae78774e4498f020508d91
e402504260a1b096cb178caa6a521411012b1db3
F20101109_AAAJOP chang_k_Page_095.tif
f5af7fbe0e3e324fa37934171efa2b55
bc80621ae685d2f050739d2c83b00a8f56f6eec2
101635 F20101109_AAAJPE chang_k_Page_028.jpg
7a128be7b160b18ef75a98821fc2ff24
5cdbe8b9b4749bbc6cb0741b11fe16a29dbefbbb
302954 F20101109_AAAJOQ chang_k_Page_001.jp2
43cd866007dab28f56ba071d8808aecc
f866241ed0c911674848d4ff0de03e0dc759ebb7
2100 F20101109_AAAJPF chang_k_Page_093.txt
2e97c10045499b998a44bd86e648cfcf
6dd506037d7c479ec7a9092b0c80c0687fadc9c5
4272 F20101109_AAAJOR chang_k_Page_038thm.jpg
74dbc89ebeb706c9acdab56742d42969
4589070d8658743f94dac1248dc5f0f2494ca2f6
55073 F20101109_AAAJPG chang_k_Page_026.pro
392b6624a5b8766d436f7df251ff5514
72bc467817e504ee7d041579c827ee94fb7e9c55
1051968 F20101109_AAAJPH chang_k_Page_069.jp2
f57db8b7892f7cb05da097317fc82311
a7335be9aafd1dff497a19d3f0d267a86ef13c07
440 F20101109_AAAJOS chang_k_Page_040.txt
d207d422a926fb63e601861e8c667f6c
a5cafaf785b3940fa28071fd87010cffbcc37b53
8973 F20101109_AAAJPI chang_k_Page_104thm.jpg
7f46df471c3b9cc56eb299a39f985468
4996e7fb88d4f086cc6629349123b5150b583925
95447 F20101109_AAAJOT chang_k_Page_044.jpg
0d1e15f800638f5a37fa10bc0779aef3
3e700663a6abef9188986b1bcb91f50ad7a47912
8257 F20101109_AAAJPJ chang_k_Page_049thm.jpg
ba8f8fd5a230e0bbd2167cd496011aa9
ab66335aa2380e316c8530ab125f4b27f912aeac
8996 F20101109_AAAJOU chang_k_Page_058thm.jpg
d38600557fb369765766611b2bbadfcc
f48d16859761e4744a835c399cc30e64b81472a6
34571 F20101109_AAAJPK chang_k_Page_069.QC.jpg
4efee0a7551447847116ac83624a9965
2a26a68faf7e668d6084f236aa511e1a466c502c
F20101109_AAAJOV chang_k_Page_114.tif
126f8c1aa5da862c02f4fb5ac7ad1ce4
4d78d76e19cced1c67340803f951bb50d342f26d
2618 F20101109_AAAJPL chang_k_Page_107.txt
54eb41e1652d4bb5a816dfb25557c48f
a19a6dfe93b632553fb1d63a1b6d8fc06ca7426e
6579 F20101109_AAAJOW chang_k_Page_089thm.jpg
9c6dbf03ecbdce723b3b57abbb532a39
c068bd4a2cb980f735f6c974d24795566c10e651
38188 F20101109_AAAJPM chang_k_Page_060.QC.jpg
50ce0e8d4d1462d7d95e3bcff431b91c
6668abd6f93e63e9a40bdb47668ae2f14178ab3c
F20101109_AAAJOX chang_k_Page_068.tif
092c574f82354efa0a6f8159d06278ee
b6c690aae53d2fa597a50744912065a852f0f868
2104 F20101109_AAAJQA chang_k_Page_059.txt
050942e4d64c2dcbfd7d3ae7e997c542
b63ca527027bb2fa0d373f1493dbe3435f4b10e6
112193 F20101109_AAAJPN chang_k_Page_047.jpg
a7e77cd9607e047e407c9a4b597a95ce
9872028489318d71848eb94b61cc9f7b692271c3
32158 F20101109_AAAJOY chang_k_Page_001.jpg
06075f0e0340adcfb6846cb223ba3f3d
f4efe2c1d5cebfe66c5e311f4287c2fdd8744620
F20101109_AAAJQB chang_k_Page_035.txt
2149b1b0a840768ff27d0b9b0c6c2b65
fac2c8430056e29a1e83fc8b857ea35425c00e38
2151 F20101109_AAAJPO chang_k_Page_098.txt
9a1d593dca2d4e63904dbef741b6e075
a6fa90a060d8901acab1968fa1f0e7d46ec0cb5c
13113 F20101109_AAAJOZ chang_k_Page_120.pro
fc037b5dd27b530c5c18f5c64cb129d9
19e7228b4b0d7b4ff1860d9fa0a495fe24ad7bf6
75517 F20101109_AAAJQC chang_k_Page_006.pro
d59311e09adcc47b76ef6b042f15c233
a48ade92986efa70b77485384901a8419ec9fb74
36438 F20101109_AAAJPP chang_k_Page_058.QC.jpg
71e00145a9a5c6edc55e8a2a04d1d1ea
0d1a1d2f6a554136aa956e6fd266c92c2fb7f117
2025 F20101109_AAAJQD chang_k_Page_054.txt
c1c76641bd01dbb512f38390296a9cec
6e40e27387c19a00489f348e0b08f9b29b6ed33c
123678 F20101109_AAAJPQ chang_k_Page_116.jpg
c74f4a843af3863d935af9bc3ab1085c
aefb2d95af891c3296901f6a1d71fe87c81952c5
115417 F20101109_AAAJQE chang_k_Page_105.jpg
c9fa7b0bcaed1f66eb0ac1cf9776020c
e4ea4e437f0c699237b4d2c41d35f1444bfb4178
1051981 F20101109_AAAJPR chang_k_Page_092.jp2
5463cf5cbb1125779e7a95844c653697
963ba23e1e6acdabee82903f3b9df16072bdee2f
F20101109_AAAJQF chang_k_Page_087.tif
43fa7e7e96116c393756a9651f35f334
f9bd772b72082e1fe173578762f4d0a090e9c0e9
1051965 F20101109_AAAJPS chang_k_Page_106.jp2
bd7699d83206c854a0088995467006f1
474f565b0feb40f0beb2d5760895c4b8cc68c0a8
F20101109_AAAJQG chang_k_Page_061.tif
68a386cd420cc66f68a0d62b60881496
0150554f377072ef47588448bed409d824dac8a1
F20101109_AAAJQH chang_k_Page_069.tif
a7cb8cfa88516d391661561796d16ca5
8eac1e07406a54d5a3dfee0ceedfd75e46abb904
98223 F20101109_AAAJPT chang_k_Page_016.jpg
3e4a8e1f502ce013167e363d92b164ac
bb089dc7fe8255c1afa8424ac15fbeb658774d5b
F20101109_AAAJQI chang_k_Page_048.tif
907fbd57bfd90f4743c08c5f9e715d19
29cf9458059b7d613cec6f18b97dacd1ec7c1e8d
1051967 F20101109_AAAJPU chang_k_Page_047.jp2
37d31313f342f6713f54ff39ab5eb340
69622ba8252edc82864de6f9bddb0af0b3d11362
49873 F20101109_AAAJQJ chang_k_Page_054.pro
5867e1a4ac0e1b40ecce1107775be533
aef4c1a23fdc938332647a856d2c0fd4fbf16617
4796 F20101109_AAAJQK chang_k_Page_005.pro
3c36da194309cfe088346e0f024f2384
b39e6551da01182b31d6b009eb5fab8996c2a360
8298 F20101109_AAAJPV chang_k_Page_064thm.jpg
368db4d52c7414e27fab936683757d6c
9b6350b95ce7c3f9290dcd9f11563b73f7d20d00
33328 F20101109_AAAJQL chang_k_Page_019.QC.jpg
3ed970d98537b63b21850ef1b6b348f8
0858414e6d0836f492344096667ae5350dc8d6e3
55512 F20101109_AAAJPW chang_k_Page_056.pro
61f73af89efde64b5d0c9172955682a1
6e425211107d0c1253f6b4d340aa330057442e8a
32892 F20101109_AAAJRA chang_k_Page_091.QC.jpg
6ec699e73a947069f3a3d73a6f583b37
f65a3318c7cadae88e9160950f82c15acdbaf444
F20101109_AAAJQM chang_k_Page_012.tif
dccc1fb780b6dd029e3fb8c5d479b3d5
326700b73e9be9c0352ddf0c58799fa77e165811
F20101109_AAAJPX chang_k_Page_109.tif
b0de194397a7062e9ec2df10e4addc19
5d101b8294acadca6693b2f7210089757981e9eb
1051980 F20101109_AAAJRB chang_k_Page_112.jp2
b778aebcb061977166cc5d577244d1a0
2cc1187123647a213cbb4f2a11a37703f361d7b7
1841 F20101109_AAAJQN chang_k_Page_044.txt
defb9dd0e6455c288c61b06826b2625b
948262dcef2c59541f21938c5d431234b6034276
353813 F20101109_AAAJPY chang_k_Page_076.jp2
9cfb87dba4e831c8977aa686d7dd5290
8a2e09d06fc7bd2271fcbec3baac180032cee9f1
2138 F20101109_AAAJRC chang_k_Page_057.txt
3363ee1e1721f7c5a21527b98e26033a
035b6b0882c8c16062969716374449467c9b472a
1051984 F20101109_AAAJQO chang_k_Page_024.jp2
783b56b5354a9ae7ebee87198f5860ab
497c5be463e898935bf2cee1e44f0a2ab638f86a
36280 F20101109_AAAJPZ chang_k_Page_029.QC.jpg
64b1861b1c07ef500c8527f5c98a48e4
c5fe7fb0b8eb5e511d7317567db694c5d8b3db8f
7923 F20101109_AAAJRD chang_k_Page_007thm.jpg
53f4322d49c742564cc7aea8bc189d46
367866197a871641f587af1c6d91b9dd0d732cbd
F20101109_AAAJQP chang_k_Page_058.tif
9d4bdeff5dd1e62ff1583be192003035
2f10d206878c983edbe1d82fa71c5495a0bef835
35559 F20101109_AAAJRE chang_k_Page_030.QC.jpg
9fa14e2d112cef11d4442a4320b7412e
6ebc0736c0837668804fb96e3bedbf4d7f953b4b
87 F20101109_AAAJQQ chang_k_Page_002.txt
21f2faf90d570adb534e09fe8bda86f4
4850c18ee5748840637e5beb6037b99496274eac
18245 F20101109_AAAJRF chang_k_Page_087.QC.jpg
35f6e9386ac08aabd8cbc4a23b912570
3a770d4397a2a3fb70220531daf6aebf582bc032
32631 F20101109_AAAJQR chang_k_Page_076.jpg
2a74ff5e87d399be85adc58c288dbdde
7467bfe2677ca6bb6b59dca7f4e9a69e27414dbe
8940 F20101109_AAAJRG chang_k_Page_048thm.jpg
c99706a801807024d8ae2eb650fba996
22e36af96974b50df6caf32472f8aa5449489ab4
8855 F20101109_AAAJQS chang_k_Page_027thm.jpg
7e43f66fcff945f028631a7c2eb2bad6
c7f16f6818a71e25a01846972b344c83cb8d772a
1017314 F20101109_AAAJRH chang_k_Page_040.jp2
b608cee70bce933e987904956726632f
1e0f3c5e02542be8caf5b21337e2b421ee8dab9f
1051957 F20101109_AAAJQT chang_k_Page_030.jp2
a6460636eaad9e7712e87b9fb2ac6d4d
641100ebe65add8897071946d912dbbbeccf7545
8880 F20101109_AAAJRI chang_k_Page_096thm.jpg
5cebeb1baac889ee239759872c693415
489588ff98ce5615bee4c6eac518f45d87ee65de
33467 F20101109_AAAJRJ chang_k_Page_028.QC.jpg
3fecf3d1b2166cac823b5c48a046db83
ab3f2a4f8f04169e1443b4511ff87b8b0b6f4278
7170 F20101109_AAAJQU chang_k_Page_071thm.jpg
14c1f18afe13d9d983bc629ede64901a
d55342163dc45a3488f2fcfb5ec5a7a642af6b49
56185 F20101109_AAAJRK chang_k_Page_020.pro
32321a63b062d2eda61fddfec343c5b0
dc708e06e115a4d4f292c997b39ac0b79545155b
123709 F20101109_AAAJQV chang_k_Page_104.jpg
c915e490f39c46b5ab0fe5cc5898bf31
aa9c8fdc1f9f11273a2b1c007721eccf36418e20
420638 F20101109_AAAJRL chang_k_Page_084.jp2
1e7007b3509c9f10b665c9b3009e2e9d
b096d800a8f85204d08d6d68e845a701b3ac1ac3
67502 F20101109_AAAJQW chang_k_Page_110.pro
142a9763805b306fb2b3a88ddfaffa5b
996cf112885bbacdb4c061d1148b6107656093ef
36233 F20101109_AAAJRM chang_k_Page_112.QC.jpg
9cf93e60cb08d15e7910ed81d79f210a
2814be7cecd3f1a2747f912714dfc325a1c8deb7
847 F20101109_AAAJQX chang_k_Page_072.txt
510824daa74caeeaed0062238c3acfa2
46ab5aea7c60fb42796eb803a6a24bb5aba717c2
4820 F20101109_AAAJSA chang_k_Page_005.QC.jpg
62e6068d90d217205b91c0cd9f4f7fec
e54a7af32b5891e6495717590d9dab824c0cab6d
76037 F20101109_AAAJRN chang_k_Page_041.jpg
b7c4fd626277abe9979d40739b2ada23
1ab7a0bbcca8c5f3ed1df885156583881da7e108
F20101109_AAAJQY chang_k_Page_107.tif
1b49a088778080dffb685ac6fdb7f49b
b2a4e1a0f8b02d333ee47ef84cd56aae840b08ae
F20101109_AAAJSB chang_k_Page_082.tif
8fba022a21e97530637d59d4845e1e8e
a59e4ac6b414476aedd3cfaae7ee2c25e54e9a57
34019 F20101109_AAAJRO chang_k_Page_042.QC.jpg
9f290ca8b79457d9cfddcf39e3d1045e
a19775e175f71d2f27fdeaf0f28b840a4e0a7b29
366850 F20101109_AAAJQZ chang_k_Page_083.jp2
920bd10b167090ac3f3e9c8fef6c01b1
b030ed0b38649e6c93da19f2115f1e1150df61cc
9387 F20101109_AAAJSC chang_k_Page_076.pro
8eb0cda861869682c39a6475586b53dc
bbfc7945597ca6627570deb72e818401a7a0a39b
36366 F20101109_AAAJRP chang_k_Page_037.QC.jpg
189e83e6faeefb9edea801c6a7832858
a764bb2917c9d6e8dac8504c4c9b604852650d2a
36106 F20101109_AAAJSD chang_k_Page_118.QC.jpg
874c20b5339a24c5c1d154c42da7a4a9
3fb13da5393b4bb50b3fa6b6fdea950b15ae4f32
2197 F20101109_AAAJRQ chang_k_Page_020.txt
3cd91505a9c355c2f4cb75a23de7b9a5
1d0997e5a02cd50d01e87abc36f8cbee2d000f1a
2222 F20101109_AAAJSE chang_k_Page_066.txt
cef1a6abd64ed8fb2c8b445e83d48ef9
b983025d4cf948b4fcaecd42d977d7e350dfa659
2087 F20101109_AAAJRR chang_k_Page_014.txt
58dfa475af7957b1d3a33e50701960e9
f4e908c3624cea9fa9461be11a3f49eeb77c434a
103274 F20101109_AAAJSF chang_k_Page_014.jpg
42b010609d0ecc64024c1aa13f46bf08
ec4c38a328e73b2eff47c86122e32f1c0e902680
1051969 F20101109_AAAJRS chang_k_Page_103.jp2
c8c7b6dfe479389870104f052823b4a8
a9808ad7e25e33a0661712cf5eeb8c7668b630aa
F20101109_AAAJSG chang_k_Page_003.txt
e18258d0c3a0db83adfeb21af00a7944
c24bda12f8adffb70a3acbc32a7233d049bb65df
3594 F20101109_AAAJRT chang_k_Page_007.txt
5ad32e680203432bfe274067ad9ee148
3c0300af5f659d55557a613bc6ff4ce53836ccb7
10668 F20101109_AAAJSH chang_k_Page_120.QC.jpg
1e436f47ae212f84838d317b932943f4
5aada44423631d1a5ebf81b0a66161b7d83dac3c
27864 F20101109_AAAJRU chang_k_Page_002.jp2
c8483ace221cc86b61b83cf928255eb6
d072d45a3b2cb978d2215b1c557d42d1bb43417c
574 F20101109_AAAJSI chang_k_Page_087.txt
d97d2fc9cf3f65b46a80f09269fc1ae3
9b12ef9ca824331f363894f5771eeeb87c16e468
3392 F20101109_AAAJSJ chang_k_Page_006.txt
15f67260a63ef3b46f66be723871c4c3
4eff7fce96cd394778a2b968fdee508e0f62160a
90938 F20101109_AAAJRV chang_k_Page_071.jpg
4100aea57f04cd977fc37006e7bd16cc
7cd87b639e0690f6d01b95a595d912974e5d1bd7
1912 F20101109_AAAJSK chang_k_Page_034.txt
e9023a5c186c9172d383853b7bf10082
402398854582cfac535eb0eb60dba772c3f1a7ad
F20101109_AAAJRW chang_k_Page_007.jp2
c9252f9c7a635b9ad2cfa67f829b81a4
70f95821219bcb969b375568fd1c0e338aa9e981
16789 F20101109_AAAJSL chang_k_Page_072.QC.jpg
a4e35f3ee68759e695d98532d81b2221
011c6baeefe336c13853f7d4463db0610400213a
9485 F20101109_AAAJRX chang_k_Page_001.QC.jpg
0cc647c064e1311ccef385d9d2e3f8c7
8828cccf817686f9f7bc14e1bc71b52c7205f463
2062 F20101109_AAAJTA chang_k_Page_065.txt
0922d5e05fb74740870ef7d8688ccec1
218bf870ee556f307ea6943ba98959e8f0d73d08
1545 F20101109_AAAJSM chang_k_Page_063.txt
1b784a92ba4cd282aa20348e455b8639
cb1d7b7befce2e97f9f7307abc6e35db3c427f3b
2679 F20101109_AAAJRY chang_k_Page_118.txt
43f794c23688e9a8fd23260d5995ba1c
4e3a3622448dcf1f2c68a6fd33d10e4f2e5866eb
37425 F20101109_AAAJTB chang_k_Page_108.QC.jpg
57008df12b6ac182451b496b11c728ba
b11f320585ae88bb0038397821814c203d622270
6133 F20101109_AAAJSN chang_k_Page_087thm.jpg
066249ab7a18cf7bf44878a2b97ff8d0
a7bee756fc36bf90a8f82353cd2ff80dce2bf3bb
38295 F20101109_AAAJRZ chang_k_Page_063.pro
62974ee6020866812417232bcf5ef334
e273277143604e91e65230c3d370e399300316ab
32317 F20101109_AAAJTC chang_k_Page_120.jpg
6b8ab1354b435411dfe9d548dc0e02b6
dd767954a6bcca1dd44087a60cb31a32673c0d39
100239 F20101109_AAAJSO chang_k_Page_064.jpg
9cfaf38541050f3ae1ef2038129b4e19
b9507174baff7dbbe44d574b26c133ec51f84694
19898 F20101109_AAAJTD chang_k_Page_040.QC.jpg
eed9a04f989953318c71bcf901c4290b
d4cec26cad486e9d5d20262b14c159c3a984b037
F20101109_AAAJSP chang_k_Page_050.tif
5150f52d422ffdb0b7ccfa4b3a2d3be9
089f1fac3a23a87cc42f98310b5a53e0c288efa5
8412 F20101109_AAAJTE chang_k_Page_094thm.jpg
85380316ba1a08e8b443124d0daf0aa7
2a462e7255ed1c559f32f3eb64125ef240e5c4ab
F20101109_AAAJSQ chang_k_Page_064.tif
d810e50369878e4c317652ec0a555483
c3dd2f01ab28f93c95891a91515719308aa257a0
35410 F20101109_AAAJTF chang_k_Page_116.QC.jpg
501a551cf795fa0da459faf73106e54c
b207533ff88b4ef0ee011b6ad53489fb3d88c998
F20101109_AAAJSR chang_k_Page_105.tif
71c4cb3a00013288fc203bc36b6d8183
54aaca05556b7e059bf06d92371c71c826cb3747
1051937 F20101109_AAAJTG chang_k_Page_097.jp2
075cadfe966758eabb556561e6b4c8f0
db36c9fb5cd69f90b97f05ffd40d1d4eb576848d
8400 F20101109_AAAJSS chang_k_Page_019thm.jpg
2aab935339eb4d8d401759c58f11d5c8
4079a84f3468743a9c4b7cca7963b3fd50c0c490
F20101109_AAAJTH chang_k_Page_056.tif
16ff66a53b1157060066a49858728333
7f23897b5b22a80d8a4bbeb3ab4383139b754b61
8043 F20101109_AAAJST chang_k_Page_044thm.jpg
85e2b643871de4b36a2dd03500ff30e1
a9e63949744dbdcd8c6ac8261019cce5081a9018
F20101109_AAAJTI chang_k_Page_048.jp2
094e518362d045bb1e8c3bad47fad544
4fe6ae6ef1b1dcc841caad8c4073fa11e4cc95da
36903 F20101109_AAAJSU chang_k_Page_088.jpg
b0a145a1d0a899b8cadbe71310015d7e
eecb6f6287218c03ed182895b7e97281571a9cf1
112044 F20101109_AAAJTJ chang_k_Page_020.jpg
24f52912ab62df88ecbce43fbc092dd9
12ba4789e28fbb4b1f71c66050b0d5355b455a71
F20101109_AAAJSV chang_k_Page_111.tif
53f450cc01463f9434df5f2aa0198b50
74c430cfd989b07f686742de6f899ee5065d4bfe
36433 F20101109_AAAJTK chang_k_Page_077.QC.jpg
8b7bee8d0fd45f0acc14d111b694c493
1d00049eb2bf3dbc974e354f42a566bd00b3d4f4
703603 F20101109_AAAJTL chang_k_Page_036.jp2
9444c8a09a37126a9a6317de49e641ac
9c6abb233cf1fd2f85f8605a9a59d8de2831d54a
8985 F20101109_AAAJSW chang_k_Page_118thm.jpg
40635baec19d01e14509deedf22ec12b
b7cf3384ef71cbadf7426d37cd81dd472b745811
9012 F20101109_AAAJUA chang_k_Page_025thm.jpg
453655db74de22af472dc781e2f27669
57b845177c3832c3837e23310deb3deab53032ec
495 F20101109_AAAJTM chang_k_Page_003thm.jpg
08745de87644c9224b1920e5c6914d65
82e7cc2769cdb78c74d6030be298b702057f3946
F20101109_AAAJSX chang_k_Page_022.txt
7a1a8e1cb95536e8a8221cbd29e3f646
bc6436275994487dbeb1be408509165fb9d59755
F20101109_AAAJUB chang_k_Page_070.tif
71eb1b30593314e5cb1ec2b20ee5d63c
46a2719808d31f18c1a03cc25e26551400c0b36f
F20101109_AAAJTN chang_k_Page_099.tif
680b270cb21f1fe99db49b11ea2a93d5
d80bf0e352877d61b983fa988d047549101dc40a
11568 F20101109_AAAJSY chang_k_Page_076.QC.jpg
910efbfd504f43ad1826151d907f3d3d
23353379c755d4127ffbd482c4ce873936be8cb1
103529 F20101109_AAAJUC chang_k_Page_042.jpg
e28c6f8ef07e6bb07891e6432506b7b6
9078b3be4825422985d9f335a2ad0ac41d948057
111052 F20101109_AAAJTO chang_k_Page_078.jpg
07cc4c7ff2aa981a01c2faeff1233ed6
c294665fec30d400a787c47ce2ad65b2cc925642
F20101109_AAAJSZ chang_k_Page_047.QC.jpg
0071dec5eda2e8be93668180ba202b0f
e887a24ebf70a443ef238eb472aac96cd45c2f45
31014 F20101109_AAAJUD chang_k_Page_044.QC.jpg
524892929abff991d43f9e6511cbabb9
49c12adb1d907c4062ab45a733f079a9babb8792
735965 F20101109_AAAJTP chang_k_Page_100.jp2
7a5aa9fcaf6a55405b6a6b1b1dd12498
ff2a74848c7e47be21dca869d1f20d2360576151
17308 F20101109_AAAJUE chang_k_Page_012.QC.jpg
747ad387ff581b940d90f09b79a4c7aa
0b4258ffe7a8bfb8546a5e69905cd569a05f705c
51794 F20101109_AAAJTQ chang_k_Page_061.pro
70049076d629e3725713fc8944114e66
284e522269b1eba56c65b1384051a486a977d7b9
1026 F20101109_AAAJUF chang_k_Page_015.txt
af2b93ca6c3b3bb51f15ae62adfaf588
7fe8680541e7a1f8dbda6755c040553a75de921b
8775 F20101109_AAAJTR chang_k_Page_116thm.jpg
d31847c36c1cf4a61fe41d88a61548d0
571b2bb655e4b9addff9b3de9e889841fd2b5bfc
585 F20101109_AAAKAA chang_k_Page_082.txt
dd924b3b0024d4bdb291d737b43729dc
ae6e6bbeefdf689055b76a263e406412f8da46d3
F20101109_AAAJUG chang_k_Page_005.tif
78fbebb0c70c8bbe06aa93bce9a5e2f3
e727dd4b7138da941e88a3cbdf146eeeead3fe32
64357 F20101109_AAAJTS chang_k_Page_074.jpg
1b209135fadbc88a3b9911f7ce9a3ed8
421d20650e359860f93af3d8585c4397ae7541a3
F20101109_AAAKAB chang_k_Page_090.tif
71b0f728e215c7482a87c8212bc285ae
ce084c20e2456b3fc073d1ffc15a66f09f685dc3
F20101109_AAAJUH chang_k_Page_115.tif
c28356b5e6b2eac759d2709208f0df5c
4419fa1e96c9de21482a3e1fedc1e2feaddca124
49326 F20101109_AAAJTT chang_k_Page_049.pro
e7340535c89ad002dc55b31d6cf516ce
e3ea45d0c6ae96a0426c19967ba333e770cf3f36
2137 F20101109_AAAKAC chang_k_Page_048.txt
6cf06a16028295d9567522978bcebfcb
0b12f635f2a032bf296ab35ceed02ba03455d0fc
34706 F20101109_AAAJUI chang_k_Page_092.QC.jpg
2cb3b22a536808832eed98f0ef8de605
1b6e963b0abe2e151454a24d75ac4368a8708975
112389 F20101109_AAAJTU chang_k_Page_060.jpg
0a43d60eb22742838d9a0c81425525a1
e12f1f8c2c34fc744cceb258d8529cc23133ecd5
8598 F20101109_AAAJUJ chang_k_Page_115thm.jpg
22268bd86aa568e8bc164c6381afd069
0b4d190d73c9b924e07ee6837827d166a1ec9da5
116638 F20101109_AAAJTV chang_k_Page_056.jpg
ae21988894fb0647ac2682055538ceb5
a78dcd5dcec3496b5f52423fdd8517fc7fd9367b
1051922 F20101109_AAAKAD chang_k_Page_039.jp2
481ec4db1f28dae90755ef7d14556c0d
bc22fcead8faf4c93a90c44dda71e46473b6c044
2475 F20101109_AAAJUK chang_k_Page_114.txt
e224e8e89f678b49cde70ab7d1822175
947c2ba721300bd3dfcbefdf691e946468063f76
F20101109_AAAJTW chang_k_Page_098.jp2
130893a75466513cb91c9de63e2be127
a9721f308c922f9ff4e1796cd11e9fbacb783f0e
1051951 F20101109_AAAKAE chang_k_Page_102.jp2
a629307f481895daefd9340db769be65
a229f718cd87b0131b2a73ac446559fe3974bf1c
2041 F20101109_AAAJUL chang_k_Page_004.txt
bca25b442768d059a9aec642fef8e886
41cabadc0bb8d9ca86e79cb1d3760299116f389a
5282 F20101109_AAAKAF chang_k_Page_077.pro
3594d4b4a7c8b39df4dd33e393aa02f5
252e948a87ce2a356fc5a03a038a67f7f7f93088
60336 F20101109_AAAJUM chang_k_Page_115.pro
0b09535ddec1dbb531a4e302513659de
85eadd870e35114a1a1046622f3b5a11a64dcad3
1194 F20101109_AAAJTX chang_k_Page_002.QC.jpg
11ff5df4781f7726a2e85a2e438f3844
47ee7270d2e61efa5d5d410468ad7940ad9db303
F20101109_AAAKAG chang_k_Page_023.txt
ec4c41de37b52e691f5ce1baa07f44b0
c82140db944b71c5c38684ef6d46f205f1b66a91
1051976 F20101109_AAAJVA chang_k_Page_057.jp2
b82fe49df05b6016a697725f39e88464
508c1634ddbbde99a552f065ecd20a32e1ced343
F20101109_AAAJUN chang_k_Page_084.tif
51b8f3bb1b8787698618eeaadcea7b21
4c5aa02d6d4fc8c74b51220cb59e2e9544a4e91a
37400 F20101109_AAAJTY chang_k_Page_020.QC.jpg
1c4e0a4b8d6200c2c3bae63a29d03caa
261c05e42f727ff46ccbf203fc6b8c7866921db1
47942 F20101109_AAAKAH chang_k_Page_014.pro
59e5da1c2dddd3530827336f9957ca14
41b60634e66c99cd2e9e8c2a87ed40778954e7db
4077 F20101109_AAAJVB chang_k_Page_002.jpg
2a4abc1c286ea0c13ddcd46442ca91de
992496e1325c9f64ed25661d65ca1507c19fc5b4
F20101109_AAAJUO chang_k_Page_017.tif
6361a0c5eafe620d32c4061fe1868a14
d069f1732c0ac143820d1d18db64fb19b999b9ce
53968 F20101109_AAAJTZ chang_k_Page_037.pro
cb5c241538e14fefebe3671b53d70c5e
056ef651839319f8d418bff0ef4cb7e50cf618ce
34044 F20101109_AAAKAI chang_k_Page_103.QC.jpg
c32b1c77308a259fbb3f9c5815222ab9
557960831725e08e1bfa89b5a65570cb658529fe
54395 F20101109_AAAJVC chang_k_Page_031.pro
3deae4f07c9db20e740e6c74f94285aa
3410187ae41f7cc6ce574f6e729b42aac9f1ce45
31358 F20101109_AAAJUP chang_k_Page_014.QC.jpg
1073be99ff5923187c9916e668e12bfd
257b60ff3111a00f244fcdfc00af53a0f4beaa28
22571 F20101109_AAAKAJ chang_k_Page_073.QC.jpg
997e06e0a347bcacd0ab4dbd22efa820
5e1e68588a5e0ed489b95fc6553acb9d30841e0f
2763 F20101109_AAAJVD chang_k_Page_110.txt
09015abfcbfbc8923039b3097292a737
020b42b52efb57908c0d98057bbce6d8bc81057d
F20101109_AAAJUQ chang_k_Page_039.tif
c22450040c7d87c966985117848ca370
270f92570569dc8ba745d73c76b8f3f5478c89bf
1937 F20101109_AAAKAK chang_k_Page_042.txt
0d7e272ceb3470d91cac32d7dcada0bf
58846f083e34ce9473ff895a530097139f8bfcfa
569 F20101109_AAAJVE chang_k_Page_083.txt
238c8827ef772243396d94749aa2d326
f6243458f07f6b98933aa983831762908068fbcf
33861 F20101109_AAAJUR chang_k_Page_007.QC.jpg
a12d3fb84cd2a1a151bfb2f3f867d697
df55132c3f0168fca1864adf35ec5b6259c96b0a
2009 F20101109_AAAKBA chang_k_Page_053.txt
c15b316ac1a227fbd986ee5d5082945b
7254e0d7859ff40f8c8b15d6a3b7eaf0a9de0b46
60439 F20101109_AAAKAL chang_k_Page_103.pro
8010e16dee734a3d5ee165ebd9b2a8dc
c89c81201cf1815570253b56d643b61e5083c819
8669 F20101109_AAAJVF chang_k_Page_065thm.jpg
79a580fcec7ad3452ec34d22bba9ed98
6c2fad36ad87aa44b64f791921ccce48d20aba8d
2128 F20101109_AAAJUS chang_k_Page_097.txt
bf74cedb94509c8b12a73a86d6fbd1fe
8c6b42e686414f4c851af9249b007f4cc66a0b08
F20101109_AAAKBB chang_k_Page_007.tif
e5439d4bc37519abfc98c959793f7303
156a3c76a450a468339dcdd2b5737f2f1526ff7a
12140 F20101109_AAAKAM chang_k_Page_090.QC.jpg
97de86da2b0c14503979831a166fa520
558b2aa33fe2a0a46a4ee3341f91b885816ac433
F20101109_AAAJVG chang_k_Page_110.tif
be3c634f2fbbaf77c144cf6f280aee5e
5fe4cfb741f3d81047ac046182e1634ee83141f6
F20101109_AAAKBC chang_k_Page_001.tif
284727d78fe61abb9d5976a08fe71a15
6312ece06ec7059d6c2d742b068f36484a7ff04a
63600 F20101109_AAAKAN chang_k_Page_104.pro
2ccce61bb99fc5fb7f6872682fafbb7d
2f571253ee3d424e677716b8079058d84f7a8fb3
9704 F20101109_AAAJVH chang_k_Page_077thm.jpg
7c9c9d2d479c16fe9216c27202694622
145e3c70339ca1c2d191cfe1930f0a97b92987d2
61462 F20101109_AAAJUT chang_k_Page_040.jpg
20a98e581f3b5fe85eca660f6925f3b7
cd3d8eb7bb6203a6b3fcfaf39aaaa84abdf9b7f8
9968 F20101109_AAAKBD chang_k_Page_088.pro
ad9bb1835aca5113f4a24c0e3050afc4
b8d0d63090f6e199edc776786bad15ee526c5f5f
F20101109_AAAKAO chang_k_Page_117.jp2
b4293d25800320097bf8281e5ded2eb2
3f5f78ae2abe2d9358baafd64d3621a5a7ff8cc4
8937 F20101109_AAAJVI chang_k_Page_031thm.jpg
20b6dedc94176c9430b6bd02f37c65de
a3f46ee389949e4acf39d17803fde4defd35d3f8
F20101109_AAAJUU chang_k_Page_044.tif
5648a987d37a403ac0b3822cb479b789
52d858c070fc6af50afda16ec26aaf0483aba7dd
118128 F20101109_AAAKAP chang_k_Page_103.jpg
d2428e708ed4f57b0d41579d9a6309f5
288a0d2c9e284edcf55c2fbf62d6f962a11977a3
80175 F20101109_AAAJVJ chang_k_Page_063.jpg
e378346671f3f2c0800ac29a49df0214
d4f1c03bdd16c34f94cdd0680c6bc79e36f44117
111302 F20101109_AAAJUV chang_k_Page_029.jpg
718b42c41461f35c22bd71a3f6277d64
c473d68a915818e35661a9a9ea116a4a63b5460c
3894 F20101109_AAAKBE chang_k_Page_013thm.jpg
a0121b89d26fc3fc3d516d6f861e1091
15d89c7a337fe23aa2ed20506d2da51b41625a90
35738 F20101109_AAAKAQ chang_k_Page_053.QC.jpg
f32d54eaec348306d5e73b04e17e677a
8396af2cbc6eb5626f726e5e6292d5813d578b89
9120 F20101109_AAAJVK chang_k_Page_022thm.jpg
427695cf71bb10163a00eab30d92c153
e4e48579b995db7962b11bb64f2b9021bc621175
35561 F20101109_AAAJUW chang_k_Page_032.QC.jpg
41c3075a3d76c530ae0a87137e9619d5
5c8939f6e878be6db623fd2ac363ac88541a12b5
27521 F20101109_AAAKBF chang_k_Page_080.jp2
9862d45661e1fddc80e1bad846fe405e
13c8c38862a86c7348b61c9289029d48ec8c59f0
F20101109_AAAKAR chang_k_Page_008.tif
1c92ccc9c327929345da84c0224eb9f6
a70c7b63e882fac7d2f34498a8d3590138a3275c
1051936 F20101109_AAAJVL chang_k_Page_114.jp2
e35cf645f1409ccfd766fbd622f29322
a637fb744fb81663ccca6eeb9ca1410852514862
F20101109_AAAJUX chang_k_Page_024.tif
8eef78a66356c96e11b92121764f9ca7
65b9b6b2ff7c53f2a02c5b6405f42bf5dedcd047
32519 F20101109_AAAKBG chang_k_Page_034.QC.jpg
7b26e2ac050776c538827cd8286bb675
1c5076e98bf8af82bf1296f67d8c902eb2da9813
110899 F20101109_AAAJWA chang_k_Page_022.jpg
e1da681cb70af0c0fde5a4505e125839
ea360337b479e68012993996dfc7fb4509ce849c
2060 F20101109_AAAKAS chang_k_Page_030.txt
827954e69750c632663984b8396d5f69
117a72dac530f96f06e8fa1d9f06b4d3b8e3a895
5520 F20101109_AAAJVM chang_k_Page_036thm.jpg
d49fabe0ef5785a4ba8793b22841168a
700b132fc3f6af761e07a8175d61209abf8b3bc7
F20101109_AAAKBH chang_k_Page_067.tif
0e73dcf799ce577f2e1273143f18e4c4
7547aad468ec5875d8a0fc76d41a7d450644b80d
39095 F20101109_AAAJWB chang_k_Page_079.jpg
b459bdcb5cdf78425078aac0925f5dc4
1b532725282b47df8f4e6814697b12865e75678b
1051929 F20101109_AAAKAT chang_k_Page_027.jp2
a345cdc74713b27bec9587bbe70da49b
640a4345d96683891a2d08695e58de4d824d22d5
2589 F20101109_AAAJVN chang_k_Page_116.txt
4789a7e9407fbc293686639e9646bf08
c4b7609b9a9dd093cc9f896720adb50243831802
8871 F20101109_AAAJUY chang_k_Page_080.pro
e886fd6a2b4fec331cf78b999a2fdd0a
9f61cdb43058bf36ba674602cbcef6937382ca94
9104 F20101109_AAAKBI chang_k_Page_112thm.jpg
8ac7aca56b4165331ce4f572c55aa409
f6885661f4a606b85275c6726d2b3bf90cd95dfb
35417 F20101109_AAAJWC chang_k_Page_109.QC.jpg
67e38563a83c1d95defb02c363198ef2
342006d06b6a5e5383517a7305ee75f5206fc25e
53847 F20101109_AAAKAU chang_k_Page_029.pro
54d695fc474995d71290bb03cf358e50
969d6c5bd89059d31c664208b2a3ac4f102e3728
2135 F20101109_AAAJVO chang_k_Page_031.txt
2de508888a3fe0cc7ef0f5d47889118c
bf3ba8a66f28dbb2e33e41d4019ef8550c846043
319393 F20101109_AAAJUZ chang_k_Page_120.jp2
eefed0b68e821f2bf282d9b9e86a30f7
153b29abf08d562119a2348f563451a61b68c2c4
551025 F20101109_AAAKBJ chang_k_Page_073.jp2
ff1b85ca1916ff4796243229406f2c50
1926c3c8ba0abb3cc2f07dccac1be8b2c4b6db36
14505 F20101109_AAAJWD chang_k_Page_085.QC.jpg
a5b62bdc770852f000e6fa81edfa2727
c03c37d07da17a651d26b9ff0e14f5663e1f0123
1051938 F20101109_AAAKAV chang_k_Page_066.jp2
ad3f735c6684b8475aef2ffe3a68d2f8
5689525abb636f43ad180315669683042fcff268
25643 F20101109_AAAJVP chang_k_Page_018.QC.jpg
82c1385a23d4239c4057d866316b49f9
afee2f2a14d3af7552e8373304b5a79fe00f2634
F20101109_AAAKBK chang_k_Page_100.tif
20edb71c2a410b8e93d4c283730681ea
6a9f6ac090b3b136cff7d92979750393894cda49
112306 F20101109_AAAJWE chang_k_Page_025.jpg
05fb9023192b8e0e3f5c6448602365cd
2bb305d5c26409a070d1f620b0b8894d5c49ec99
1870 F20101109_AAAKAW chang_k_Page_050.txt
bdd2f893e6bb0cd87c7f9688776ea1dd
795deb07b51917aabcc2fb7efda48aa618e126fe
86327 F20101109_AAAJVQ chang_k_Page_007.pro
26e9229aa447640f6cae9c74e259670d
76748c638c467371be92d93526041bac41f5e33c
743819 F20101109_AAAKBL chang_k_Page_038.jp2
d01af5e80ee89c606f5690ec35879d40
a72e38369bc5898c1683c41b667b972da9eea9a5
37361 F20101109_AAAJWF chang_k_Page_022.QC.jpg
e14b1d584a53ba7c744ad6cdd4b6af32
11ad69b63bd8a73babed669baf82701d39042409
48608 F20101109_AAAKAX chang_k_Page_016.pro
dddde636e7c3557b42b32003e34e8e66
4d7f56b734c6c4ba09f165ac83ac6c5534a3a14e
833617 F20101109_AAAJVR chang_k_Page_074.jp2
e350c24985b208f6e879861961d9d9b7
f54e842ea90cf7e14e3777ded36de33d45ae62df
15241 F20101109_AAAKCA chang_k_Page_089.pro
991ebdfaf76e7e682f9f7e79f85ac865
d8169bd626cabaef46e7ef9c1e8f6ebe50cdc6b5
8333 F20101109_AAAKBM chang_k_Page_021thm.jpg
256af7d5ef4cf98a4290b463885eddc4
76c021427dd39fe624577a4e7b0fd5bf1b25c7cf
2592 F20101109_AAAJWG chang_k_Page_104.txt
95d0959ee6fb90645dc0ddb8dd37176f
236dc473daf98728d0c9bc01604c829b11e10a80
103291 F20101109_AAAKAY chang_k_Page_054.jpg
95b21cd1d584f5ad6efd4b64c43baf02
e0a70959d78a0c8fdae8f29043a9106fde3778d1
53116 F20101109_AAAJVS chang_k_Page_059.pro
9e8d8956452dba0d16265d3fb0191262
c29759788c786c90f78cf1ef36734a74e565c106
7481 F20101109_AAAKCB chang_k_Page_018thm.jpg
3c12ad9c76e3300e1161ed82cab13b25
91d4678ed8b2f5a4877e89a07d863c5114b70c98
59172 F20101109_AAAKBN chang_k_Page_105.pro
01365e0b6cf4247025dd8b37788ed8d2
f3a8a113cd79a71ec1c72830c1a0ab78c373a193
104875 F20101109_AAAJWH chang_k_Page_030.jpg
30fadd538ecd123781db75b03ac42bed
d9aafea5cd6a3032220c53437a7709037b61dabd
566 F20101109_AAAKAZ chang_k_Page_079.txt
49280685caea5cb47121147d5306e8a3
cc817879c9de96d92b507d83601b45e8b10a077f
468867 F20101109_AAAJVT chang_k_Page_082.jp2
370f1c2a184dde1a23d41c0d27e08a3c
95888ed88351555934ff45ba5e898aa8c43f94d1
1051983 F20101109_AAAKCC chang_k_Page_064.jp2
31a264e5d36406c9e16dbd8a36f141f5
c3f7ce3e16f581f4987db3e9a204428fd7cf4ea4
95226 F20101109_AAAKBO chang_k_Page_070.jpg
200f4d38a40f73ff1eb3e88aa8d3a03b
b8eb886288c313f6cc0e90d12746a5169f726e14
33669 F20101109_AAAJWI chang_k_Page_102.QC.jpg
d74a1ea6563292065af8b2499124674d
7a96e3894a0780e9c4eea5d848c9045a428ec170
684 F20101109_AAAJVU chang_k_Page_085.txt
44178097bb2f300de040302b6d69c302
44101cf5e7cefe9d1f835c5856d93a3ae54cc2ce
1069 F20101109_AAAKCD chang_k_Page_011.txt
02574fd6285ba75e0d6a454b158dfa9c
e47da968439d47aec55a4d8980568a0eba6fff5e
12937 F20101109_AAAKBP chang_k_Page_088.QC.jpg
edb961d57a77403f0c893cb720b33656
2cbfd17d39483fbfbc92275fc39f973d1df17225
68498 F20101109_AAAJWJ chang_k_Page_100.jpg
dec8f757685506424e737f75c18ef8c5
e0d4c01a97a823d65a4ee78dc7f2ca34d97981e9
7809 F20101109_AAAJVV chang_k_Page_034thm.jpg
17dce2b14751bb3ce88622900acd87f3
53ff339b6a7fefea1e1069c8bec9842fe3ecd155
63267 F20101109_AAAKCE chang_k_Page_116.pro
b764a87f483cae73723b25694ad5d8d1
b834d20edb8265560f45ef055c9b66d6cad129a5
9150 F20101109_AAAJWK chang_k_Page_117thm.jpg
05b1a6220d9ad453bf75689856dd8752
188a60d6bb3a50721cf05b04f828dd32eb34a1ea
43729 F20101109_AAAJVW chang_k_Page_084.jpg
aca59486859641ebe8c9bd316852af73
db3ce3b58c07c968e9f0fa2bdbad826ec98384e8
35199 F20101109_AAAKBQ chang_k_Page_107.QC.jpg
53172c0fa6d6b9a83eb8cf3845065caf
e4af443b4e8586b505e6a0b5f1869c5ed6a51627
105426 F20101109_AAAJWL chang_k_Page_069.jpg
219a9375039b5f93ce472cd7473b70dc
4903bd00034cfb25596cbf52b6e9b0a7cfd869d2
105279 F20101109_AAAJVX chang_k_Page_052.jpg
e4df0a26045cb66156a1386b42d02503
18139bcdc83f850263e1ed2228bc59e2b18e7c1d
51038 F20101109_AAAKCF chang_k_Page_065.pro
bc860f2056e97dd06cd5549e5f490df2
a3da7d9017eac808121f15bceabdcff455572608
23842 F20101109_AAAKBR chang_k_Page_041.QC.jpg
55f115880e788c7bfb297b42861316e8
a95bd6efa2557f96be39e1a948bdf071e288971e
106717 F20101109_AAAJWM chang_k_Page_024.jpg
6d3e0204373e81f0d9cf1e2cc0391ac3
43aa002128dc1cd54ab6e67d3b2799b33d7ab7c6
8335 F20101109_AAAJVY chang_k_Page_055thm.jpg
148a222806f68d18395761408086744b
493b2a1cb6fc8a9586443b4251a6c7cd8bf1d3d8
1051960 F20101109_AAAKCG chang_k_Page_016.jp2
ceba7d3676fff39bef7d73571e5e4ab8
8a1ab9543f1aeaf017a78c76e9b0d65ce201f817
117817 F20101109_AAAJXA chang_k_Page_106.jpg
1c4284d1f92adca252268e1b9cab21ae
be2f9b01352e8c0f02abeb0634783305b77301d7
F20101109_AAAKBS chang_k_Page_050.jp2
7356a9fd550dedf5b81669128ced7f82
b6655d7d5655c068fa367c566b9b0c934c173a45
8803 F20101109_AAAJWN chang_k_Page_035thm.jpg
36dd096181aba258aa8e5fced020fd2e
09f8b34e3a95bdc5db9b1f0e74fb3582176e33b8
8138 F20101109_AAAKCH chang_k_Page_016thm.jpg
8b05e08addca2d2324ea5e37d9312083
0d40d624c1d3d49e0e1778b7ef06d5782118f99d
101453 F20101109_AAAJXB chang_k_Page_049.jpg
67fe946cc9bd708fcb1213363fb13d72
c1b1293e49e7935888b3893edfef50f42c818389
34613 F20101109_AAAKBT chang_k_Page_052.QC.jpg
4f56838c2ac775c7872f57eeae1a24b2
862b364e052a84fad2c3123b71d0fbfc6a626682
1120 F20101109_AAAJWO chang_k_Page_003.QC.jpg
360a4163de0a34438e0cd38c90e12065
27eeb994e5cac09201f2ab486a495c80cae51c79
33131 F20101109_AAAJVZ chang_k_Page_106.QC.jpg
762b2cdf30963981400586f7bf246ead
4833d756cb81880c73e7ccf20dc4e2532f40b41b
22610 F20101109_AAAKCI chang_k_Page_012.pro
77780312add0aab3e03bf9b88116a2a6
a88baf27bd5046cf40a12dff6123c903939b24ba
49838 F20101109_AAAJXC chang_k_Page_053.pro
e0e4cd866b3cb9c14ebdce83f53f80d4
27011fa23f39d17f0ddcf980b1fe17bee213d6d3
34531 F20101109_AAAKBU chang_k_Page_095.QC.jpg
0a65d30be3ec92078911f654036423ce
edfed3b3c7c35f2a5f433557857847e1fa77c645
F20101109_AAAJWP chang_k_Page_042.jp2
4d26e057da1d83267f88dc54328a6297
e356428aa58575b798a8e93c1a9561ec070ac7fa
F20101109_AAAKCJ chang_k_Page_053.tif
f6b6c1ad1c8065d8e594677d80ab9eeb
bdca94687e4d74a9a37635ff5acd262b5014dbbe
16669 F20101109_AAAJXD chang_k_Page_036.QC.jpg
9aca4325613182892a0f5ddc78f98b2b
4ddbf875d61fe81fe13a656fd065f828a10a9635
112922 F20101109_AAAKBV chang_k_Page_097.jpg
2197dd071b45194869cd4b179987dfa8
570fef471ef5a70ab67489582eca8aae54103875
3413 F20101109_AAAJWQ chang_k_Page_080thm.jpg
304e20721b22e47734056c9eeb871761
ad9d20b652a3b2dd01e37e5f624744daa301dacd
8310 F20101109_AAAKCK chang_k_Page_054thm.jpg
e0e9f126054fe1b88008b0b652912660
1494398c7dbcdf69a23c37b4114f0a5f40584cea
F20101109_AAAJXE chang_k_Page_052.jp2
b2b680164f2933adfea4e6695af29862
0469e6c3fe0b839c755a55e75fc4c5cde36f5c22
F20101109_AAAKBW chang_k_Page_022.tif
d9e13c00f47fc524932ce4ece5c2c4f2
5b8056ab057a27ded4cd43e1f2ba64c4244cf895
20157 F20101109_AAAJWR chang_k_Page_074.QC.jpg
b7048997722a4605d7c8ba894a641ca2
88c982af72420e8e3582627c7c7f12cc16ee830b
9118 F20101109_AAAKDA chang_k_Page_043thm.jpg
278146dfcf318ed75a9085016668d822
eb910739cd9cc3de491f8ac25ba648b4fb6c0b3f
8646 F20101109_AAAKCL chang_k_Page_099thm.jpg
4c517289cdfd6365d4c9a625cb8f6b97
1285327aace8a95395981d4f79a8d22cbfeeee93
1393 F20101109_AAAJXF chang_k_Page_051.txt
f2027ac5ba5a0b558b2bded242e1c857
1391786ec24a31cf56f027f223e89bab6479960a
48904 F20101109_AAAKBX chang_k_Page_036.jpg
66e0568ba228012dd61a3ba5361f781f
9e6638f0a7a24b34f5532275c1f35d8460416226
F20101109_AAAJWS chang_k_Page_045.jp2
7103a401ee09945a5db927284da72396
437c9f85d635c35904134826d75b33baf15d08f3
17632 F20101109_AAAKDB chang_k_Page_010.QC.jpg
7e58343674d2e6a3eddcc9e9e9cf2119
133ac852c802541576741d5acd9148dc06484232
1051977 F20101109_AAAKCM chang_k_Page_096.jp2
e1875a1ec9eeb14e5465d3eb039b8965
1eb2bf538271316af832fc83a94faf118a612f87
591 F20101109_AAAJXG chang_k_Page_086.txt
394937872a187ee9226d768c32fe2420
da8026444e587145638096214849d3f2f3bec805
35351 F20101109_AAAKBY chang_k_Page_023.QC.jpg
4eb6de4c385f5aff98c6b27f233255c6
fc740070516fbb9dea8d4eb4d3e6865c76636590
818 F20101109_AAAJWT chang_k_Page_013.txt
0d31e0cd9efa93315236e24d742a07c2
f2110e86bfc59975a6db7a1c833cb12b402f928e
F20101109_AAAKDC chang_k_Page_071.tif
ea8889ca0d1edd7bdeac0c8ea9675a14
10cc721ef4199f6d4367c571078461abfde42174
F20101109_AAAKCN chang_k_Page_078.tif
c276b09e4caae6d7631300ef3789af74
c317ec248f057fc215dd288f134a00eb167cc98e
13540 F20101109_AAAJXH chang_k_Page_085.pro
c8c516324089e7fc771a725b809a3e3b
5e362584a6f12e34f6f3a4a267687b8e78864e3b
107317 F20101109_AAAKBZ chang_k_Page_093.jpg
7b02ed2f658a380afcab5889052c9aa0
9622bdcf14e6ada96b1a4cb64ebe0b2ea0143030
36440 F20101109_AAAJWU chang_k_Page_025.QC.jpg
b8f3c4ffc3ead4387600fb3502d70d04
745a1717f8bfc93d16d599e5f5854c2b231c48b1
9295 F20101109_AAAKDD chang_k_Page_066thm.jpg
bdfdfd7d4fc1153414b420b9146e5230
8f7bfe14198cc4ba95f2bb0b5ad1f02321fcc6ce
F20101109_AAAKCO chang_k_Page_026.tif
1d9200a4930e003b95ea79102803ad63
a8ee4068a203ac81a96a721a4565a1ddf470152f
F20101109_AAAJXI chang_k_Page_088.tif
de36a5a237b94d7e42482bbe075dc8f7
d70d37adbbfd24c25ab5c1528d33a6601ce625be
F20101109_AAAJWV chang_k_Page_075.tif
071ec56614915bcdf34ce271517cb802
0b82c5e0de8aee210a8871589ae7b63573611c8c
1952 F20101109_AAAKDE chang_k_Page_049.txt
357e5213aee38bce2d59c4f6c61090de
312a88e24c4596a7703b43ff8bc48ca627c8f3d2
19410 F20101109_AAAKCP chang_k_Page_015.QC.jpg
f916503d3e7e8eaa9be763a9325b0ca5
b07fa39ec0b50f30bc67fb1f982aa32aa88acc51
1053954 F20101109_AAAJXJ chang_k_Page_080.tif
b043c7342b146b1a693465819cf459ef
af269d396f27ba8afb2395e180657727b0007cdf
F20101109_AAAJWW chang_k_Page_037.tif
5efbc8285cbb5db85a4b9e90bd7d32d1
c768d054556d03671fd3049b45ed0eedbc9d7cd2
114288 F20101109_AAAKDF chang_k_Page_059.jpg
df3ff666028e4f26cc1eb3173c3bf534
787e541e5ba53fee2bb9afd4f08df12cd5ce49f0
F20101109_AAAKCQ chang_k_Page_108.jp2
11d7d4f703153b3823851a49306bbc5e
5790c99d274f2d978e01d2e443ee1d04ab96bca5
F20101109_AAAJXK chang_k_Page_041.tif
a34542027537693f7d98d63d82236b07
93fe1ad1097b82818325ed3445c99de3b1a6ffe8
20784 F20101109_AAAJWX chang_k_Page_009.jpg
ebb059627b3b82dd05bcc327b5987174
2df42072932259a5fb5e9696ec24af5447d73a3f
F20101109_AAAKCR chang_k_Page_035.tif
6ad94473f9fe8c5380606ab1f2f4d867
95e368205857e7a79d73fff9dd55d06ded3496d8
1998 F20101109_AAAJXL chang_k_Page_052.txt
41d8a3d547c3dce00906ce0a182c75af
8edf7f018520ee170246fc94558cce1a1fb2ed8e
F20101109_AAAJWY chang_k_Page_055.tif
58da30380364a23346607f1a988c87a3
dd1972e51a54606e02fa97b8a19703c3caad5420
F20101109_AAAKDG chang_k_Page_078.jp2
75c0a8801659e65df297ef9b5885dca5
a279d1dc334f627d78ac7e9a1996cc5c17691781
7979 F20101109_AAAJYA chang_k_Page_008thm.jpg
b5ac948cf7147b6653a9a2456f7673fe
20017eebd45da983ef98f0414d94acf507ac6608
1051940 F20101109_AAAKCS chang_k_Page_119.jp2
b73e995e007790ff9ac04ca4c2378d18
4c4a3fccb963af4b91dd018867fc067dbd1e334d
106100 F20101109_AAAJXM chang_k_Page_023.jpg
84102b9990008c4d7fe099e77c5619e8
1142f6873bbb71975cd8e8d6bdf47b586356ac39
8083 F20101109_AAAJWZ chang_k_Page_009.pro
670b0d2caa02f2e44873809c044d1f58
7b22c4e253d3b57ebd19c34736e6b31f916d1961
36431 F20101109_AAAKDH chang_k_Page_096.QC.jpg
59364633c8bd7dbda78a03bdd6fc5cfb
4656ad284dfa9ea545f4c87a4a9a8665f9e686fa
111868 F20101109_AAAJYB chang_k_Page_037.jpg
00b07cc9bd04f0f9a0def7af2fc9e972
5dc66494ece459960c1f6f6b535b6f45e9c75d05
F20101109_AAAKCT chang_k_Page_016.tif
8bec9288dde40138d30b8a175a417db5
a57d49d1dcbf7d0fe4ffa0bb9d0e9935a284d88b
8251 F20101109_AAAJXN chang_k_Page_062thm.jpg
14cba43a2949e50ae77f395b8df740b6
01aa6c9558928b943f6e9f7985d45f8b43c0d725
37168 F20101109_AAAKDI chang_k_Page_039.QC.jpg
e7514eb95443d20beb1b4cb59eecb30d
c2c8783be03cafe5e3cf879f46c0570cccb9b870
509 F20101109_AAAJYC chang_k_Page_080.txt
5df4a3893c63acc55d57b9b47dd3cc27
5a3013ecf84d6eb9ee0e1f3e043a62242f59741a
100857 F20101109_AAAKCU chang_k_Page_055.jpg
4a3f2705106cdb75886e53c477f58b78
2d49c26791b4452901c76dec46723a8ff7a634da
64942 F20101109_AAAJXO chang_k_Page_109.pro
a2ad9b22ad9996dd1bd0fe3eb2abc717
e8e921177022e5092a155502fc5c203ed49ab90c
63505 F20101109_AAAKDJ chang_k_Page_107.pro
b913707f2c563c0901b4b2ff4e5cfe26
2a7072c1e06a28323e226aad2e51eae771b9fc13
1051956 F20101109_AAAJYD chang_k_Page_109.jp2
063747cec4f60375b06100c74a397241
6c3cf12ca6a1e0d439c32cc7660531fc5bc84bae
1051978 F20101109_AAAKCV chang_k_Page_056.jp2
17cba491d955f05cbce4fed7d87fc8bf
704213b47a80cc010289164ce58049f3af576280
1921 F20101109_AAAJXP chang_k_Page_009thm.jpg
9980cac540d883fb133337005198a07d
fc7a73ad3fade1b6d8c77543ce85f350f5d8ba67
8599 F20101109_AAAKDK chang_k_Page_030thm.jpg
e8c44198a38ecd537b7292f139d1d08e
a91d24a5d08fc739e250ef9a91dd8344357e62be
F20101109_AAAJYE chang_k_Page_117.tif
2c7ee91bb1af6f8797e8e33cc68733aa
744a072364c657abc80de40e76a3d706ece79b7d
17035 F20101109_AAAKCW chang_k_Page_073.pro
fc92a20840fbdfc3acff9702b7e0334c
0e3f077f7c39ce90a0547e71414da7f5e9e1696c
F20101109_AAAJXQ chang_k_Page_081.tif
21b524c1d7289a3037539e6f9074e7b9
f91f4bc251a13344b590806c3694983e308e11f3
104747 F20101109_AAAKDL chang_k_Page_094.jpg
cacd4bd181ea455ff095d9a6e0da6297
73886a71b5924f6acda309f1be8ef36131f58a10
4009 F20101109_AAAJYF chang_k_Page_075thm.jpg
78e7b77cd91d03f8e66c71e06f48bb4d
be24050d12f6f4a152167b2336ce1cb917395029
6380 F20101109_AAAKCX chang_k_Page_009.QC.jpg
f04f36e52f6787a37d2255abd2b23cdf
b218e6e6a615838c311f68a2b3df3d4afc3b8c0b
401463 F20101109_AAAJXR chang_k_Page_085.jp2
ffd654b4ca46ecbacb01152dff48d160
2b509e5c6aea921370d75c13071b84d42dc8d085
37689 F20101109_AAAKEA chang_k_Page_098.QC.jpg
9286e3499a84a51fcacb1a085a0ec418
e5b6554c897b5a3869b6af4a7e46f9be1363592e
37550 F20101109_AAAKDM chang_k_Page_027.QC.jpg
919be405c46adedffcb94f103cd43d18
8fc27d2f8ea8e2cb272643ab3142339d35c61564
33854 F20101109_AAAJYG chang_k_Page_054.QC.jpg
c886ac38d73fd02b0094aa3c9f18e55e
ae8dedca2b237be3e2aafaed775c35231238768d
F20101109_AAAKCY chang_k_Page_031.tif
b29d4c6ffbaabd8d25315976e9894857
a6279e9ed4a3547c507caaaedc38b2ae34554e13
F20101109_AAAJXS chang_k_Page_093.jp2
ea518c5546cca6e60f31df014ae8fa6f
e2744e78e7bbabfb3303b16c67a994750da693b5
68000 F20101109_AAAKEB chang_k_Page_111.pro
c23015b5fbdaa25d2fc3a3d931865375
1d84feec4fbdfee615548e488ee6fe500a2d3f5b
F20101109_AAAKDN chang_k_Page_098.tif
b8f460ecb54457f31f9059b7821c1364
73a2b0fb71896309b997743c03a2d1474faf4871
42786 F20101109_AAAJYH chang_k_Page_013.jpg
9932dd13834092655fe0cb42debf6190
24dd040f89ce033b95746e05df78ff01dc1dfb76
51699 F20101109_AAAKCZ chang_k_Page_095.pro
763e133c60bd42e87a7edfdfa80295a2
ca0be578d2ba3eddc01bbb9f5e231b4bba013771
2670 F20101109_AAAJXT chang_k_Page_109.txt
22d825a6a6b94f75297951d5eed7be47
486e56a79e3f114354c365586d27f155b15e4cad
1051955 F20101109_AAAKEC chang_k_Page_029.jp2
4ddc15f7cb58090de2ed7f5d1c061e8e
b56f4aecddea0d360d0562b33a2091241248b693
F20101109_AAAKDO chang_k_Page_093.tif
125c0efbf131a8df7103bc4f226af578
47ab9c0d2c32b2416886f43bd4c7458c766e6e5e
53801 F20101109_AAAJYI chang_k_Page_047.pro
80b156900a1bb65862cea3b618917b45
8b0b8e097a0a64fd9cf584f797a96fe8592e1773
2293 F20101109_AAAJXU chang_k_Page_017.txt
f788b0a342724b7c447ef030eec63b27
2d172f0512c0ab7c0e25c8d41fbba91f1d589fee
2024 F20101109_AAAKED chang_k_Page_099.txt
a84735ca62d3f8a6f6305b9d6daceb18
9a1ccb7f00f29ce224216fe7c526dd897a09116f
44748 F20101109_AAAKDP chang_k_Page_085.jpg
39eece5aeb303b92704ba288e43f2819
38710c8971fa6e0783ddb0f038451750f82d9466
127231 F20101109_AAAJYJ chang_k_Page_107.jpg
54e6023b844fc40f8647170fbc18ce2d
779d6e75897ce5c7bb217ad78317c9ad1ddbf1c2
F20101109_AAAJXV chang_k_Page_073.tif
05bc3c75f701f631716493402e2fe6ee
a72358c6f1eea4c2108ae11f5c01f1e08a537a62
446735 F20101109_AAAKEE chang_k_Page_013.jp2
b68e65b81f1e1059e71de6968c4ef79b
9e17caeb35e9361e5c870ac5a953fc8ea643738e
33750 F20101109_AAAKDQ chang_k_Page_045.QC.jpg
d9eb453a82bc389c85b3d61d00ebe99f
f0b003dde1827fec4106fb92c731aed21f9a87c9
F20101109_AAAJYK chang_k_Page_022.jp2
0c6ad82139ea0eccbb7fefef1d21d0ed
87cfbf38096bb4c91f6e628a8ddd114e20867b7e
8861 F20101109_AAAJXW chang_k_Page_047thm.jpg
fbc9b9042ee87736cb1e1337534acf73
5a385e3b04dd8255f1a34d7cac013d3df3a3317f
5880 F20101109_AAAKEF chang_k_Page_038.pro
72b6f2a8b9b7a5232eb11d14d1bb1efc
3dd21e05cf60cdc15e4ebbc144f86e4f2cfcfeff
2074 F20101109_AAAKDR chang_k_Page_091.txt
8bc15d4eafbe2cfd10a34a791da940ad
ca1a730e02514a63bc519a0019ada032a8c7631a
2487 F20101109_AAAJYL chang_k_Page_115.txt
fae340bc112df8f670378cd25687d5d2
88f0f7ff48650bd94243b64e69c32ba9d2a3ad2d
8485 F20101109_AAAJXX chang_k_Page_028thm.jpg
d926b9789b36a1c6289ff8685a8d444f
990e55fbca39c98f0c3239247dab824c17d9530c
51266 F20101109_AAAKEG chang_k_Page_087.jpg
26b6bcadf9e9c476436aafe5b0c7e922
e66307fb8b10cdbb2c03625a0e9fb26a01f3aa53
2167 F20101109_AAAJZA chang_k_Page_046.txt
ba888a1c4fd16611b854a05a54367061
05265640334180b846833532da9637cdf8e6c115
1051961 F20101109_AAAKDS chang_k_Page_043.jp2
360dfe51cefcbb5bf10f508d11218419
decf2534c9bc4ec269d4a0d625f8156fe6949985
38094 F20101109_AAAJYM chang_k_Page_043.QC.jpg
0300669ae806c70b46671bf45cf00121
e09bb8cc5c59b890776779d99638deb88093515c
130359 F20101109_AAAJXY chang_k_Page_118.jpg
afe6cc1937cb1233230660ddfd0aa885
d8870822d92c38073c4eb1c337705ff4d3c9bc5e
2110 F20101109_AAAJZB chang_k_Page_092.txt
5fc1d7395ccc73ba581459c3e9cb4649
7f69575423eded8599c409633750dcdc8e405725
12223 F20101109_AAAKDT chang_k_Page_090.pro
55f1dfb12b77ac6810d7fe844887de94
73eeeaf15db974b19fcc24bc6c7c57f89ccb0386
53289 F20101109_AAAJYN chang_k_Page_024.pro
1ee7be0f2c79de44321f9203f9aebfdf
8f2b21f269f064e3b9dd034667eadbfb8a930a4c
114819 F20101109_AAAJXZ chang_k_Page_035.jpg
dc5f6bc44d0c4c7c515c7ed45aba8bba
f70fa2e93804ad0dba62632cdf5d8771388d223b
40901 F20101109_AAAKEH chang_k_Page_071.pro
4ca30eab42c8ba2942d4d762a0631fda
a0729ef18bd06b26fe7a96d926158f92a8fc1aef
110462 F20101109_AAAJZC chang_k_Page_005.jp2
84cf32582bcce4bb68d9ee74bfe377f8
74b95117a5da7a70e8f6d765a369a53d0aa54bd6
1045750 F20101109_AAAKDU chang_k_Page_070.jp2
3fa7dce3ac598b4cafc4aec478332b1b
6dfab4e3e6f21d792971eccf46c507705b0a7eb1
F20101109_AAAJYO chang_k_Page_016.txt
337c6202c258a44ac730f7eb3e281658
95c6e6cd5425bc019e108416bdc1981a34983c6c
10342 F20101109_AAAKEI chang_k_Page_079.pro
2448b717eba9753a6802a344ef93832e
7b6dea4520f90a97a245e056d399c08d1c2387d3
18488 F20101109_AAAJZD chang_k_Page_021.pro
1886ab8cd0030c7467e675dfb085560a
9a83bcfdbf1d2645b3df183af8b69615f06e62e7
35211 F20101109_AAAKDV chang_k_Page_061.QC.jpg
b545026fc8f6708fe675d09161f9280e
486386e8e5039399848dc76e8349560575d24e5a
8684 F20101109_AAAJYP chang_k_Page_067thm.jpg
6a0ae8da0b915e3a410c2eabfa96a0fa
601488bf5167e33ecbfff8bd793ea1c9812c6e40
8840 F20101109_AAAKEJ chang_k_Page_119thm.jpg
f0f7e4d57f554bb829c0a554e1e974b1
3f67fbc5b5752bcd98baa7fe0dbfd98ee459df10
F20101109_AAAJZE chang_k_Page_065.tif
13e6ff1f8fa6e8660d601aeae7298910
51750fb955138aa9f21f8fb14d1a451f72d84561
1160 F20101109_AAAKDW chang_k_Page_041.txt
608dc26d052c3f7fbb391014f7764a84
f338ca877222b04519613d917ad390f8133cafc4
4233 F20101109_AAAJYQ chang_k_Page_085thm.jpg
124fbb848dc5962d50b3c8da7c34d1de
bc6b28e9c123c46513d4e4cb9515060c168b1600
4004 F20101109_AAAKEK chang_k_Page_079thm.jpg
e58050af02cdc4a7bdf6b2fb15831717
0d096d3dbe3eb10739901c8fe19f3a7b91a37675
527404 F20101109_AAAJZF chang_k_Page_010.jp2
9e30b963f81fc71315e62c04a598b0ed
767371515c5dadde52c3838bef657a3dc8570cb8
F20101109_AAAKDX chang_k_Page_089.tif
2b885d10df5b989e5af89280bfccf536
909b222c265539ee5354528ee2c33434804fa302
2004 F20101109_AAAJYR chang_k_Page_094.txt
75d2030dba890f38d0c9ad0054865117
2629fd8cd3626fbeeba4d999e81d5d7cc60a2d4f
14218 F20101109_AAAKFA chang_k_Page_084.QC.jpg
936fe6cac81a4763ad4b0536f7a5af12
d161ecd66dad15c39a6fe826915a8f9a137aab80
53689 F20101109_AAAKEL chang_k_Page_032.pro
20ac4eaecba362de3f2081b9cce29c2b
edf4f12bcc0508f0ef2da1541fcb59cdef5fcc83
53346 F20101109_AAAJZG chang_k_Page_060.pro
a6839346b2d85a52a888732253941971
0fe262c397eeb8ab8cbfab19bd8c831a1f25a70d
9089 F20101109_AAAKDY chang_k_Page_111thm.jpg
203c8f74eff6ebf54dc8557cbfb8a3f9
084d6e9b513239b2edf05a557128607d625bd0ab
527333 F20101109_AAAJYS chang_k_Page_089.jp2
5af755eef03427472b4eeab9b61a2ff2
0be0eeb9fb1f568bb87f2b365e02ee1adcd2d93a
24830 F20101109_AAAKFB chang_k_Page_041.pro
4d4481ac1cab716ec0b6209042fccecc
8184622145637375d8eb9a257b9b99f83667eb9b
8605 F20101109_AAAKEM chang_k_Page_093thm.jpg
99fd77f4bcba6c7400c27274b634e47a
185aed1359a1ca51361f76169546aae5260ac2c8
121503 F20101109_AAAJZH chang_k_Page_115.jpg
57f925f75b84c513abc1799d435d0c2b
668e3ae9cab6f703f9c174d34b65ed595bace17a
36324 F20101109_AAAKDZ chang_k_Page_026.QC.jpg
d9188a3a2570a98b8a67867ac7ec87cd
02aa09800ac5ee28f2dd75c51a9a768a4dec1b5f
50910 F20101109_AAAJYT chang_k_Page_068.pro
71a786e047cf356e3d06d974bb54530d
9ec62adea6149eb1d64d2a028dd2f13b0f086177
106064 F20101109_AAAKFC chang_k_Page_061.jpg
99d1db4d50f07c06183a6940283d2b96
c0e24786872b0a8db487dd71ff197ba23e21ce84
F20101109_AAAKEN chang_k_Page_021.txt
5058d6f7e4907429df04b7c9106b9ee8
036c1a7620d7529a997d8d2ee9822e824751e069
F20101109_AAAJZI chang_k_Page_119.tif
b1ec0a6dcc23f43ae7314a0736eb13b9
c1ec07afa741bd73546624cde717fd40d46b9e6a
F20101109_AAAJYU chang_k_Page_019.tif
f17b91db16bd3563e40d4cb597d7c20a
c1b882f56c48d2fe04015603fd2c9a7da210206b
47965 F20101109_AAAKFD chang_k_Page_062.pro
273f20dbf348093404b1be24ba41d7c8
9af1d32e63a19a3f6cf7a9d4bdb7f64841164321
14930 F20101109_AAAKEO chang_k_Page_013.QC.jpg
88e0044e33ba737f44466dcd9b140e4e
34763144c73425a0dad7e962bfd389715694a8fe
36949 F20101109_AAAJZJ chang_k_Page_110.QC.jpg
5201860aee71b4c03a24b157afa7dc94
a38126f2651ca6c17a4ed1dfdcaebdfd4558473d
124045 F20101109_AAAJYV chang_k_Page_006.jpg
6b7fdfcfd212fa6331f6def7d581a836
f5371269a470bc53148dad743c19d2a11b2b2025
4223 F20101109_AAAKFE chang_k_Page_088thm.jpg
229ec04e8bc1d0722b0456ab611be6bc
f2f5495e7e0a9910665346fb01be5f5ac2c09a86
F20101109_AAAKEP chang_k_Page_105thm.jpg
5a4bdf143c3c12d3976a4defabcf60de
839f2f2b0998ca22ef94a3cb5cf5811908bc0d31
F20101109_AAAJZK chang_k_Page_094.jp2
24223421a00ac1a58acab92fa209b172
8f442aaf64528cdba75c8a4b2e995fd56a96a30b
50743 F20101109_AAAJYW chang_k_Page_094.pro
7a0eaeec40eba5ed753cb78f838d16ba
889f5224c0fa742c819854e39ba0e897efea23d6
23016 F20101109_AAAKFF chang_k_Page_100.QC.jpg
033abfd3a27c2dd73e347b6aa620046b
ade81b027f22fff235acfb1691f7d4930b6febf0
8502 F20101109_AAAKEQ chang_k_Page_042thm.jpg
e443cc5ed6e90790dbbf3f6ac8abf592
a50c6aeabb5966869fa20c63174ca610dcd32a24
35701 F20101109_AAAJZL chang_k_Page_093.QC.jpg
bec2273927a7ef7fa003351f9eff40b7
7f9362cbac1cc54c2fb5617095d7b8e335249d48
113055 F20101109_AAAJYX chang_k_Page_043.jpg
4720647f7ba65dd1ee2b24df63ae647d
f2c9685abe9aa49b84dba198cce35515e9cfa1ce
22786 F20101109_AAAKFG chang_k_Page_033.pro
e10cc88461e8a2a3fe8eeef1d2543269
50a3600d1a7c293b74f6a8daae56b6ca58677915
14546 F20101109_AAAKER chang_k_Page_072.pro
d0bedc3e05e7640fab5dc8f0ea459ddf
147c30c2512b15f0f28d2bfe0c526f12985a020d
53988 F20101109_AAAJZM chang_k_Page_096.pro
3b0e320f98d9932903e4601cd98fd443
93f9837d55435da80ce6ff6f988616a4cd7987d4
1051886 F20101109_AAAJYY chang_k_Page_062.jp2
2aae6c3013ec60e3fbce82fa10c0f3e0
52eb581a8646ff38441c3b7b5b8aeb73b9bc4c22
8873 F20101109_AAAKFH chang_k_Page_113thm.jpg
d6e1dc8d6ad5c0d87888e3fe1c00c0ed
ca430b65a0b3f21262dfa7c0e129180b09224dda
4656 F20101109_AAAKES chang_k_Page_010thm.jpg
c720381a5a52ed672f392b160273b568
6f04b7af8c14651a41805203cb086743fa2bb0c9
F20101109_AAAJZN chang_k_Page_023.tif
e1f07b37693231d23bd913cb0d1113af
5317f0f3f6e5d39006edb60a269c4dbc92eefb98
F20101109_AAAJYZ chang_k_Page_038.tif
098ebca19575e654f656a50749d14b96
a9bb3c144846e04e959077bd2330215ae1c99859
F20101109_AAAKET chang_k_Page_094.tif
69246bdb1a0437b131561c7498b0ce60
0c912244648b5cc22f17f683f0002f947955f843
F20101109_AAAJZO chang_k_Page_046.tif
450cc5d30981fa7029750e091534be76
b66288813f356a6e4b9a159bce3a317076a1bcf2
33245 F20101109_AAAKFI chang_k_Page_064.QC.jpg
0c828b04344c1173b8dc1944f81d1686
adf055328e614384683fbbca21e6a1b100fd9a51
5423 F20101109_AAAKEU chang_k_Page_100thm.jpg
507d57792299c17d6447c0b45c427ea2
bbc773e7238e91120c943e43ad25f23f858aa132
8710 F20101109_AAAJZP chang_k_Page_039thm.jpg
7873a7c6cb1857a16eeb6ea7b3e8efcd
614210593038b512eb2f9db354966a43f82f643f
113447 F20101109_AAAKFJ chang_k_Page_027.jpg
075deebd651ec855287785a9116b88ec
dccb085dfd38d3cb50ec6ae94536cea3de0bc1a6
1051958 F20101109_AAAKEV chang_k_Page_037.jp2
31650e3b0a64045dc51fe7196cebfe1c
a0b5e58568677d26662bd1fd71b30982d584ff97
560 F20101109_AAAKFK chang_k_Page_120.txt
60b8cf8d5f7101b5fe20d5a5c83194b6
29c2b54ecaad2e4d639842e214588641c562ad7f
8584 F20101109_AAAKEW chang_k_Page_109thm.jpg
bf33d8804e8a87dceabfa54c6ae7bc43
b4bc601d9887d1141f916f1859170dab4fdf7453
29855 F20101109_AAAJZQ chang_k_Page_021.QC.jpg
526748aafac32611cec6361173c447c3
77c93c3d7143753161f0ce43b0e953ce983d43f9
34905 F20101109_AAAKGA chang_k_Page_113.QC.jpg
967c1188e1c1165cd726f540382ab72b
46b0c08a7cd5b1cbcb004cb29119cdb47ea14969
46980 F20101109_AAAKFL chang_k_Page_064.pro
d01ada8541115f4a05940ebc683272b5
1d3da273285e945fd0faa7be48ec5e7ee6e84ac3
F20101109_AAAKEX chang_k_Page_062.tif
8643a689d48d26ea7c605c8c598519eb
59d493e710de45b431ecec2e92915e9019a16e82
3789 F20101109_AAAJZR chang_k_Page_003.jpg
8081b6e4ef6722f851c567a9907a0a81
244c632795c39e6951a40573bc4947cc44fb5d1c
635 F20101109_AAAKGB chang_k_Page_078.txt
7c82726f770a3a3a56679c4d94709d9e
f7b169a754bd3c86c0f11e6fd31db61b63dd0039
491193 F20101109_AAAKFM chang_k_Page_072.jp2
f4be45efd3de5340a1b086fa4562d43a
4364f72f67553023efa0b751b94b81d90deb1b74
50396 F20101109_AAAKEY chang_k_Page_004.pro
538480f711ab0c808d6fa636f19cb660
49d35e5c510fb84ebcc2284d60da1a81f599b68f
27061 F20101109_AAAJZS chang_k_Page_006.QC.jpg
75e6ecd25dc8fb2ad6dcc1face9f19fd
94c0abfb3ecd0e3fce9f6c1ca3d7b4453c66a189
6385 F20101109_AAAKGC chang_k_Page_063thm.jpg
fc2940f4c9221edd90c84959ab2a8b25
45792427110b27d4cb22d5c865aaf3c35f9c1ac1
5710 F20101109_AAAKFN chang_k_Page_018.pro
ea3392645bda31fc8f097b4e0cf83b83
b13c2345db7f4401c53adc7f78ff9f8b2660152c
2413 F20101109_AAAKEZ chang_k_Page_102.txt
fe5a49ca1cbc7f502012b7c920bed06e
37a3f59cd96499a6fe0335034adb1b4c35e623b1
967696 F20101109_AAAJZT chang_k_Page_071.jp2
6eeef79ef44590953c3bc1e2d12990ca
5bc17e67507ee00d44951c32d395a35bc7603d37
F20101109_AAAKGD chang_k_Page_018.tif
74652305b4eb7fa80e01a68dc9c1eea2
63407b8d8d038eb633cfc7837a53ced6966a25a7
27639 F20101109_AAAKFO chang_k_Page_033.QC.jpg
5d042dc2d124ed44e849b25a99ea13d7
e7788f1b5581d38ba8f63151a2695cfb74b3785b
1051964 F20101109_AAAJZU chang_k_Page_095.jp2
c023021067420fb94483291563a3d7ab
c6d50518459732da0dabab7a403fa15c45f12186
F20101109_AAAKGE chang_k_Page_074.tif
702f7801c18fcffb37cc34b545732d4b
8805bef3f421a139167f7dada956278bbd5b2e5f
56556 F20101109_AAAKFP chang_k_Page_066.pro
e04afca287c7e758a3a514bf7810046c
cf2bd03da0820097ea40af732a99d370e217065c
60383 F20101109_AAAJZV chang_k_Page_114.pro
948b23108ab7893be2f60eb27917ebe1
6f94731904e20eb206f9d3e67d73e7f13c30971c
F20101109_AAAKGF chang_k_Page_108.tif
216893f2fc34dd426053ff372dd16c02
186f025218d0a3ff197d5dc5614bbe59c54833e6
8514 F20101109_AAAKFQ chang_k_Page_061thm.jpg
7449656f2822dc96e9abad22dc6124c6
bde284ad9cc6038257e3314061b76fcdf0af2752
F20101109_AAAJZW chang_k_Page_051.tif
a3927fc8163163117c2af33dab6e60b5
b6cf4c768c6dda82e4a50cfdf6fedf950b24ae82
F20101109_AAAKGG chang_k_Page_043.tif
1c0c53f468727f47f0217014160f334a
0615fd29c75fd377dd7ef14aeff351b48ed835d2
30949 F20101109_AAAKFR chang_k_Page_100.pro
ca7b83b33c0a40911150f43403a16713
e9f70ad060f3479f392959f6a4a4042d6279796a
35748 F20101109_AAAJZX chang_k_Page_104.QC.jpg
89fa6e9a57028664d310743ebcdcd6cc
4c26af735751761426e1fa67b8b0c7ee953624b3
1027 F20101109_AAAKGH chang_k_Page_033.txt
6c59e9aa68d1ede42a97edfa9d8207b0
fd3424ae6557f5e9afb9d68810930f55a3324596
108182 F20101109_AAAKFS chang_k_Page_032.jpg
0be3077fbf430b13732db027f4939e18
9797b019ec5694e4debc7d877e00c82801c479a8
8742 F20101109_AAAJZY chang_k_Page_032thm.jpg
e53114ccc989460b8360e41a396567b0
de7134ecacbf977034989317a300e419bc7fdc09
99782 F20101109_AAAKGI chang_k_Page_050.jpg
42f92d808e7b3aae86dfcc915addd87a
4498a98a4d16f6cd5364ddec06a214f928570cc8
526 F20101109_AAAKFT chang_k_Page_076.txt
fde9664515b4010d44108c6b3e6858cc
7121ef03635c5954077f84ae1f45fefadc05b296
F20101109_AAAJZZ chang_k_Page_009.tif
029d264fb74942c63e9583c57597ac09
625fc174a3e6068b1fb70a331486c068fe6c39f7
114833 F20101109_AAAKFU chang_k_Page_008.jpg
341091ddb84ca5e9b196038bc219edcb
a429f37e53eb5825e17257bad4529259a06f9115
1051944 F20101109_AAAKGJ chang_k_Page_026.jp2
825ed51ff06bed5f22fff7ad40379192
8649fe3d3f559be95582710321f851903a6b5359
F20101109_AAAKFV chang_k_Page_006.tif
84dac0d59cb93e5127c21f992e3353a1
518f4ea5aa809a8beb0f494deed992ff8530ae20
48804 F20101109_AAAKGK chang_k_Page_069.pro
9b8f4db0eee75ef80829024d2a008736
f04ff1e31b81fc507e3cb873bec303877cd5cef5
F20101109_AAAKFW chang_k_Page_021.tif
5a720e3975c89d908075275df86eed24
5a4c4d54ca3116438e4ae3ae3fe0f5fb027b57fd
104601 F20101109_AAAKGL chang_k_Page_053.jpg
03d1dccaa850c9f1dea08ad8426b5039
fb6c9c3e07fb09d311d099cf25b1d245d69d4cb8
655 F20101109_AAAKFX chang_k_Page_074.txt
55b6862e1d7dfb9ffd2a8f5998aaf34c
097ac72df153264ccdc547058bf05baa8ae405b4
952120 F20101109_AAAKHA chang_k_Page_081.jp2
db2cb6f88799f7b7896e51381094f93e
a43ae1e98429de9bf8b386dcc8fe46201b8ee2da
F20101109_AAAKGM chang_k_Page_036.tif
b9f7be6bdcf7277816cee28ca7025670
73e891f03c18cf188e9aaf4fa4c2120ae79d9776
45434 F20101109_AAAKFY chang_k_Page_070.pro
ee02fbb9b47050b3b40219aba5d2e575
244ba663a202c58d2be28b07391440ffbe0a9352
52132 F20101109_AAAKHB chang_k_Page_045.pro
760b2d7477d982666333ef8dc15831c2
1e1fcdaef3062d074f1ff608baaa62a0ac934169
F20101109_AAAKFZ chang_k_Page_014.jp2
b5da9f6aae515b75b30c1c678deb89da
74997f1624c9ae58239b7639f6546531074f66f6
F20101109_AAAKHC chang_k_Page_046thm.jpg
e6a5460f83ddc387d6ee6371ba4dd787
3ecfdce2ef190820bc0c3d44aa962bb0e02bf3a9
F20101109_AAAKGN chang_k_Page_104.jp2
4810e9bfbd0c88f8c6119d8e5a662462
dbc8e8eebb80edddf2b4f5b341bcfb7e26002845
1978 F20101109_AAAKHD chang_k_Page_028.txt
7f6f92822cffa3b8a05243c5eb99df0b
48463d3c6950698be0828c5d42e57cea0604d9a0
1283 F20101109_AAAKGO chang_k_Page_005thm.jpg
25498edbeafad0ae81abe20de64761ff
25d077cbb1f4dfad263f26a775eae51f2c0923a0
9019 F20101109_AAAKHE chang_k_Page_026thm.jpg
bd932d6d06476123410660568296207f
c4322c073e9eeaec4ce1c235a2abcace68d8623c
550 F20101109_AAAKGP chang_k_Page_088.txt
34f18a0696cffe5f42f5ef622a31c360
b051af72e7900441c5e3954c1625d24c58e801b8
39481 F20101109_AAAKHF chang_k_Page_056.QC.jpg
04550d68e3e5a8081107f66729374e7e
8e99f911c1e5adb8a9a4bc84d533720c64375044
F20101109_AAAKGQ chang_k_Page_025.tif
5139cb188b605fb97b013c670e3442f8
eb1d23b7703a0ec48f619593926ffd1e4274ab58
103382 F20101109_AAAKHG chang_k_Page_019.jpg
dab8564b85470c3835c8f0e9eb6fda44
b8ee76fe797e8ab9714c8d09e1161f5232513bc8
111269 F20101109_AAAKGR chang_k_Page_096.jpg
841fa4071d5ba3db361c81a2fa754976
5d3f61f2873210c3945f31387cf93b1ab741ab43
4697 F20101109_AAAKHH chang_k_Page_015thm.jpg
aa0192afc202f8e55d990d2e8cc6b11d
a011365fe8e92e9078fe179868f0f8f90df93ba9
497971 F20101109_AAAKGS chang_k_Page_087.jp2
3ecb9bca66e7ba57922a7637e4de5bc0
5a5b34c4a00be3456fbc7f985e935a842bb15f9d
2164 F20101109_AAAKHI chang_k_Page_025.txt
d1f42f651cbb7ece4228ea7660633fbd
873f3f98f15b3a981fe186749910299f15d72956
F20101109_AAAKGT chang_k_Page_085.tif
3ad454a160e0b25efe95aec19b0924c5
b69670957e529ccb862ec5a3b9f52a37c2d8baa9
8186 F20101109_AAAKHJ chang_k_Page_091thm.jpg
dfb996034a7ccf3dd1d870eedc714e3d
0cf43bde5b058e1810408036fe83f0b7d799ae19
5189 F20101109_AAAKGU chang_k_Page_072thm.jpg
64e760a56839839ce4ffea8c105e8c17
0bdca200309881e6a53db65f30e70c70391f8f26
132061 F20101109_AAAKGV chang_k_Page_110.jpg
0bddc450d3185783bf8babf7255fa696
b65c43692c5f6d9435bc5274b12d0b768aeb4601
58596 F20101109_AAAKHK chang_k_Page_017.pro
2fcafc0a2962da17e496ce752eef13c0
152b8ef374936cb3ec7bf672dca9dffee9665321
55835 F20101109_AAAKGW chang_k_Page_011.jpg
fe7d24cbe69f5332565055869b78d33d
f0dc37201449383f7943918cd53c93432b43a155
107378 F20101109_AAAKIA chang_k_Page_045.jpg
76c61694f6ea82b55a2a7e4d180f41c8
309a1bc58fb6e64c7a52c18816df57798b2339b1
8771 F20101109_AAAKHL chang_k_Page_037thm.jpg
e54455e10a88a7b44fc7ddf8235836b6
62fe427c5d5ad0f4b639754cfc1d0d1d4cc56634
54870 F20101109_AAAKGX chang_k_Page_098.pro
340682ed61fe5369db582f319ea8c239
2dac9b12887d7170a80c4eedf7f0ba4f40ca517b
113157 F20101109_AAAKIB chang_k_Page_046.jpg
a8d2b9ce1f3d7acb53058fd6896e16f8
c987cf246426e8c89441ff939e50f06b5191feae
67100 F20101109_AAAKHM chang_k_Page_081.jpg
05b3f210e64eeefd95c58b150f95f849
aefd9eb9a4ee356cd1c9848d9bd83eafffda2728
23136 F20101109_AAAKGY chang_k_Page_003.jp2
4ade144121778e4119a2ea4a95c12934
eef24a8beabd45751538b46405e0bab18df842dd
113254 F20101109_AAAKIC chang_k_Page_048.jpg
816e1c9c6cfb62d678d1a19d8d208e6a
8b90b11bcffd7ec7a3da4aa1aa2cac9368ebd6d5
58803 F20101109_AAAKHN chang_k_Page_015.jpg
bbcd81ac2a8af169302fff0a40fad1b8
f5af7b708dafca6d686468c8b7e4c00ee0f216fa
F20101109_AAAKGZ chang_k_Page_077.jp2
037b4939ab38706218f05e5a48cdbf84
d1f35ba2196d2703160beed7821389b56116a116
72791 F20101109_AAAKID chang_k_Page_051.jpg
e22a852d3530a10b99d4436a583752e7
0cd21984022b34d1f269a03c69252a77fa8b869a
138960 F20101109_AAAKHO UFE0022015_00001.mets
ee6c13491a1399bf66cedc663f024b81
096b71a26cc3e8463b8301663d6726c1a9f96db6
113739 F20101109_AAAKIE chang_k_Page_057.jpg
ac8e29fba2178c1e17211df78eb44d14
504d3e5e8a801d12da9f6820b3fb444653bab86b
102463 F20101109_AAAKIF chang_k_Page_062.jpg
26f331466ef6eea4e67e104187650f29
814166a0c9853d99b8ef2ea7f2950db9120e6126
104198 F20101109_AAAKIG chang_k_Page_067.jpg
a590e055eed917a2b61128dd572cf04e
3bf39d0fcfed9dc0c1a207b84956eb31a241f38a
12684 F20101109_AAAKHR chang_k_Page_005.jpg
9690471039fc02da2c2de62eaaf1e14b
2e645ce79ce4df939152dd3b24f70e5c8902f99d
50118 F20101109_AAAKIH chang_k_Page_072.jpg
d66965d08855cb342054d3777692bb28
c6b898b5cf8f743f40aab7753764e75ec0a822f0
152157 F20101109_AAAKHS chang_k_Page_007.jpg
9c98f3d932a87c2650720f9fe43386b6
7122c0d1d7e6202c041245fb1f9b7fe166d1486b
37571 F20101109_AAAKII chang_k_Page_075.jpg
903aa0ad31e61e6783f851f0fc2e653c
fe481974aeb140e51d25d5d0c85969e170e401c9
50575 F20101109_AAAKHT chang_k_Page_012.jpg
05bf466aa3b45f016beb7f5d46407674
e36fd18dce67e48e3eef1c6b406caae75dbdb863
122509 F20101109_AAAKIJ chang_k_Page_077.jpg
6453ff9e688aa996c47c3e8ba0c831fc
1d6f00903adae73a723d7bdaab81b51576218140
114701 F20101109_AAAKHU chang_k_Page_017.jpg
f2a81f6a242824d6742bd0a6cc24592d
a33a94e6a35b896d5d857cec92f0d6ea65989e71
52485 F20101109_AAAKIK chang_k_Page_082.jpg
be950d65630aedd22ccfda2235ecf6c3
c2e1b474b1a1588113bf1b5da7bd871e94a7a0b8
80509 F20101109_AAAKHV chang_k_Page_018.jpg
977e466e4e4bc4328aff82fe73ce4f7c
59a003c8a50e9afc4a5bca6e44273f16145ba0c2
110149 F20101109_AAAKHW chang_k_Page_031.jpg
f7e68ee2bce3192e458ddcdee8f470ca
4dd440307f866f082e40c68789bea0df781faefc
38923 F20101109_AAAKIL chang_k_Page_083.jpg
7b6b69b95c45b68d270cb177088b00c6
360174f9aa3057b8480a0f486bd50942113af04c
88207 F20101109_AAAKHX chang_k_Page_033.jpg
9521b7477150fec3d9ac67f37aec135d
992e47e6ae8685f53b3e606a553e9bae2fb98552
534075 F20101109_AAAKJA chang_k_Page_012.jp2
aca3798d87ab460e88dff1e0d831e04a
23d20d9efe961642df877a630a36883032f881c4
60020 F20101109_AAAKIM chang_k_Page_089.jpg
2a2024287f9a184c64bbbfd810679e49
70c93233379572fb54c1912a1711785d63c284df
45740 F20101109_AAAKHY chang_k_Page_038.jpg
9b55a5b7bc34940bf844fa6e1ae6a42d
454f977b5aa784dcd20d390b3e40b8df71b0b2c2
620780 F20101109_AAAKJB chang_k_Page_015.jp2
b7d4927f21e170f073e5bea6e083bb36
a9fc4149be97044cec7917377ec3cdc3854cecc3
36398 F20101109_AAAKIN chang_k_Page_090.jpg
06caa9e1b2a5446780994ba014623bf2
94c9f3d31ae868cc41cf6d84bee03d7cb5266272
113703 F20101109_AAAKHZ chang_k_Page_039.jpg
01f57cedff5491d0ea47b418d67d5f0e
4373af9c1b007e4da25eb0bd3eb0c5e4c82db20f
1051919 F20101109_AAAKJC chang_k_Page_017.jp2
76b3a9071d14c565e4543de7e98c7dd2
c229b9a762d734128bb9b0f071e5d6ec00a5699d
102274 F20101109_AAAKIO chang_k_Page_091.jpg
0d9926bd60d924a4aa4ffbb85f1755b3
25b7a1c428d7214f16624095848e030d89cf2c8d
F20101109_AAAKJD chang_k_Page_020.jp2
076efc34e138be77de09f409538e70d9
c119889b33506cb6f9d0a0e739bd43c13772cb90
107728 F20101109_AAAKIP chang_k_Page_092.jpg
229a30b1a9c25756ab19cb7452a4866a
d79ecd78a265c736202523e4c93d1b6780a7c764
F20101109_AAAKJE chang_k_Page_021.jp2
b3d78a5590688df641884e25b0b30cbb
02bb0e1834669825054d1a6ed448120da29b807f
107208 F20101109_AAAKIQ chang_k_Page_095.jpg
89ff843d8d6d674a24644c9ff6613c0b
02a86755961d78996c9b330befbe7ffdf40db6bb
1051946 F20101109_AAAKJF chang_k_Page_023.jp2
f44a4bcf0425593c33742e353ba9dd83
5177a1351ce2be96ac2df51a7539b037ed24eb6f
104373 F20101109_AAAKIR chang_k_Page_099.jpg
cd602a676648cb6f41f281430256a871
b85f2ef0957b602a48df305d63415e5991f01321
F20101109_AAAKJG chang_k_Page_025.jp2
d13f6736a83fcb2eac7c4720a97c219e
35eb9a1db5a19dfa61bfd7135bb17584e29f0345
117632 F20101109_AAAKIS chang_k_Page_101.jpg
d1317be1e28a02c7dd594d8b15ee4cb7
28a9f06e1dbdb1bff629131852b75f342127d5e6
F20101109_AAAKJH chang_k_Page_028.jp2
b1359742b78dd73676b31346b30c9fe0
139731efa0761cef25f9a911cce9ac666ba67c52
128125 F20101109_AAAKIT chang_k_Page_109.jpg
2e222e3ae71f313d05cc1f8b1da6ac01
1816bc918068153aabf32d7bc481d0a081f04431
1051982 F20101109_AAAKJI chang_k_Page_033.jp2
5972d469ebd437c672aac373a0d58499
32acf9a1a99ac49c79249aede48f66cadf9cdea1
129123 F20101109_AAAKIU chang_k_Page_111.jpg
3ea38772ada6adc5f796764ccc6c6051
a53c3267afdf83a1999365eb068d71d075db8427
F20101109_AAAKJJ chang_k_Page_034.jp2
0b99125e2dadafbb25f281085824354c
4c20a5debe4c496456800eff0d311d47d8bfd3e5
121866 F20101109_AAAKIV chang_k_Page_113.jpg
1eda1b0cd44d4c1fe6ac13a9a265fe88
bd98b8df56775581079a10da399cd02cd16a7426
1051900 F20101109_AAAKJK chang_k_Page_035.jp2
5dcb54ae2544fb7b4679e51116fcb6c2
7ccc4e566aca206c2c658b95160cff068e9d846a
117126 F20101109_AAAKIW chang_k_Page_114.jpg
1296690dae8138f756b5b2f7fffd307f
99e424b0a952dc2a5e9e99eb1bdf50c3ff2b0f87
1034391 F20101109_AAAKJL chang_k_Page_044.jp2
2f82ac35cf344c8ff09c293445bb9e7d
25ca37fbc17828bba76139ce17081e29695991b1
F20101109_AAAKIX chang_k_Page_004.jp2
9c4036e6d49b9f9ae9546cc70a1126ea
4d5a1cb1d0538b3b149fb3cca6c398c755d3fa4c
1051966 F20101109_AAAKKA chang_k_Page_091.jp2
2b7897070cfc1938bcef57e017e62f97
7f1f330bf97228b7190827c763c0ff3925464305
F20101109_AAAKIY chang_k_Page_006.jp2
d75b8b3c9047e2cc5fb7da4028963979
c2f7488508dadc48d5885ba2efdd758e54757bd6
F20101109_AAAKKB chang_k_Page_099.jp2
320c0021bcb6d1292d2766a5b9c0cb76
da8b822afaebdd1a8600bce75bcea23bb2b5f8ee
F20101109_AAAKJM chang_k_Page_049.jp2
a9e7db19f33e16b617a1df1f4d02ce4c
09197f5bafa284610456f0ebcd8f53ef180e1b78
593610 F20101109_AAAKIZ chang_k_Page_011.jp2
7edb228ea08a3ea513f0df148d6c071d
cddb397b42062c6ae5dab8dc45c8d73b299de747
F20101109_AAAKKC chang_k_Page_101.jp2
2ed163b4f746f22b1ad54f83c4ccf7e7
f5ddd164032a07f19ac44dbce7b115e6515630ee
F20101109_AAAKJN chang_k_Page_054.jp2
fdbe5d76786682a8a1ce657d9910e694
138bf627c2ff45148502de64adf5aea3b669784d
1051985 F20101109_AAAKKD chang_k_Page_105.jp2
d6866de67a01bd726d5f68e488264b5a
36e0a4672666816c598fc61d32d241d688a9d131
F20101109_AAAKJO chang_k_Page_055.jp2
bc71622619682f64a63969e38bb02f55
0f5c6c336445d49321c6b40c1cead15eecef9b00
F20101109_AAAKKE chang_k_Page_110.jp2
7e3c6b44911904ccae92f1e7889cb3f6
53187e810534244169b95cb8a5f0543ab80c0ffd
F20101109_AAAKJP chang_k_Page_058.jp2
7d62ce83961518b8425a59fbaf2dfdf6
b9434035d4828bd2bf6f714f1085aca35b37682a
1051804 F20101109_AAAKKF chang_k_Page_113.jp2
437d32c1cc4a883296bac92b1e161ca8
59d331fc8ccc89e856dd8714629b67a48505a0d0
1051972 F20101109_AAAKJQ chang_k_Page_059.jp2
b34204baf5a27511dfabce4dffae1825
3c3eb78c5115d47e8f6c56743f3ce9d679c03ede
1051832 F20101109_AAAKKG chang_k_Page_115.jp2
c02594b66d37ce6f149ad22158ace7c4
20865bec87790bbb7658b358d1429f5892bd5f78
1051925 F20101109_AAAKJR chang_k_Page_060.jp2
5e8912b201544e3cf583acfe5c92b16b
88208fa54fb51062e5bf36642a9249fa069fe328
F20101109_AAAKKH chang_k_Page_116.jp2
e1abffeecce9d2ab5a7eba341eaa0f57
0d04bae4a277c7161cd5accb7f50f846841d2dad
F20101109_AAAKJS chang_k_Page_061.jp2
ca5359803bad51a2602879031d8b21fa
9ef6410efef5df6aa91487d7137d9f56a00a170c
F20101109_AAAKKI chang_k_Page_002.tif
0be884a44a76e8b4b0825c1f80d850c1
9424bb239a19ec484eb3a29a730f7355c5605eda
F20101109_AAAKJT chang_k_Page_065.jp2
c653cba46a70ffb8fc54af83a523ab60
9197acb147eed8ade17a8ebfb2034183bf9bb409
F20101109_AAAKKJ chang_k_Page_003.tif
ba695aa09dca4b65b3b970b837a32121
978a64517ba9f9064ca3d6505b46c56ba16e6e77
1051971 F20101109_AAAKJU chang_k_Page_067.jp2
782b4133eb0ccdd6fad9da9f02deeb47
bd6103f8b0cee353a9c9d1e925f23fef4a39b5d5
F20101109_AAAKKK chang_k_Page_004.tif
2425c04d950a6faba3f52ca73c6c7f3e
4c8932f5ed60015f70e3a2573107f0f2c77b79ee
F20101109_AAAKJV chang_k_Page_068.jp2
7610f6916f1e80cdbc19a6abb32b81a0
a590150f383a2649dc5ce9a13c42bdb643807c94
F20101109_AAAKKL chang_k_Page_010.tif
5a355955632f259a4512dff4090913c4
7e9472704ef2888f8a74447d6933d8f438094c5f
336417 F20101109_AAAKJW chang_k_Page_075.jp2
4591311edb53d09f1d67c58c40a1733c
eec7eb1acd473477e9b6a5bef46172a92b4ceb04
F20101109_AAAKLA chang_k_Page_077.tif
8eefdb69eba304beae27bdf0fed2e810
ff1f4d0e1ba817db72b3eca6b85884ad201f1ef7
F20101109_AAAKKM chang_k_Page_011.tif
6103ec7ea77a10dd8d0dc32246c48e54
4a399720f53a8cc1a09be7188027439a14896db9
386404 F20101109_AAAKJX chang_k_Page_079.jp2
82d7ca1d54d508b01b1b0fb540b38612
74dcb0760ae5a6c030261c79229d9e5b583d4eb1
F20101109_AAAKLB chang_k_Page_092.tif
00452014370b10e7bbf97df06dfd5372
19ff7b033b69bd3c7685e69568dc1e04979da2fc
873255 F20101109_AAAKJY chang_k_Page_086.jp2
3c54b103587688f90cd98568d4ac44b2
3c646feba04e21dd72b696ebeba251fba1f4250e
F20101109_AAAKLC chang_k_Page_096.tif
aa92eb99b7fcb6ecc9ef395ff6b65e48
b331588ceecbe2dbd5f1a041af965027917ce513
F20101109_AAAKKN chang_k_Page_014.tif
db5e7d82ca8c414ca1bd57ac3e1158f7
3f1910058a42423d0d6c2e577685da3e9b1634fb
308207 F20101109_AAAKJZ chang_k_Page_088.jp2
e7e629bd00fcf8429ae317f6db5e9fac
1ef203af1d6ac7d6bb9fdd70e00bd91c36c541a6
F20101109_AAAKLD chang_k_Page_097.tif
54dd2299279620c1646273b1d407a6f5
0ee2293799bf3db6418398943816aab2fe2b7f4f
F20101109_AAAKKO chang_k_Page_020.tif
77bc6163e63d5ab9142d36006eaaeda6
daae24fa971e3c5d05a0bb52ae358edb7c7dc252
F20101109_AAAKLE chang_k_Page_102.tif
7f02b492c99533d1cd050a4b3e833165
4b0061fe859afb449e8935681493d2074d53a0e8
F20101109_AAAKKP chang_k_Page_028.tif
60dbb8ac7ff972d46e9c0790ca23d22b
8fadb9d4caae031fb8aff68a29e0b1fd66d8c894
F20101109_AAAKLF chang_k_Page_103.tif
d0b146272ba47872fa9e9cbcc3e187e4
5505ec1923898af41dc00ae2b02b5ac4045bf0c6
F20101109_AAAKKQ chang_k_Page_029.tif
1f0bed1b47a20b0621bdeb6b2ef7c052
d10a8c2d79431fbccf91d3b83b6b6c93617eb22b
F20101109_AAAKLG chang_k_Page_104.tif
1b0409273a5ca8c558b56ee84164f2a3
b0db11b45eb4a8129b76c3e268717dba79a9c10b
F20101109_AAAKKR chang_k_Page_032.tif
2df10702348f2eb09ec47d91607f7fb6
5e65e4cd95dc3868bbfada79f11f65606f3146c2
F20101109_AAAKLH chang_k_Page_106.tif
a2fb4e98c40c7615e886502174cf5c7c
cdb317d09520e72467677d308e03b1efa054beb0
F20101109_AAAKKS chang_k_Page_033.tif
93f5a1f28c2ba2542baa26d0614a6c43
214640266855f5841e2127c75332a9fd6fa57bd9
F20101109_AAAKLI chang_k_Page_113.tif
de08303996acdf7cdc7d584f24c3b107
cf84ba72801a4a223bf74a0021b064ac7434c317
F20101109_AAAKKT chang_k_Page_034.tif
3852b648efe47265a03a731248a75688
0d49e42d82b7ce096994e6bb5a911f42636ea406
F20101109_AAAKLJ chang_k_Page_116.tif
8e38dd367bedca2338129903314b0a6c
954ff8f80b0979045369d4c999564e7d55154a52
F20101109_AAAKKU chang_k_Page_040.tif
feff3113a68994c72967a91303d5362d
2a8fb94eb27dc2e9051ac60ed991292c2bcd8d58
10020 F20101109_AAAKLK chang_k_Page_001.pro
84355648bb4d2f77208606ca628d8bcf
f4e5ed0f25a37260d99e2f5ecfd37b83a20b657b
F20101109_AAAKKV chang_k_Page_049.tif
4fdf934ee9f506269c3c851ebf73c744
78d36061daeaeadf36917b4ac86a4acd681aa4ea
F20101109_AAAKKW chang_k_Page_059.tif
eb364733c76a659ccf5b3746ad0d399f
d8cad8d5e6375f6023c9a3d7813d9096aa4b48e0
803 F20101109_AAAKLL chang_k_Page_003.pro
ac5888e3801ee626439fb2aef6a6722c
f9c896f8622e4b86563fef4bd4bea44e28fa4684
F20101109_AAAKKX chang_k_Page_060.tif
8810b08ee60431aa3ea7bf637ad0e45a
c04cd2cc5f449b07577cadc725f6c10634a529d3
48976 F20101109_AAAKMA chang_k_Page_042.pro
46b85215edf03117ee4fd6716efac604
faef8c916abd45af91f3f0060b06968e30aafaa3
58519 F20101109_AAAKLM chang_k_Page_008.pro
d42123bd1c3b88404571af8809bafcae
45fa35a71911e6067ea28a1bbb0fde4bfe651f8a
F20101109_AAAKKY chang_k_Page_063.tif
303edc99d7047da4b0ba927668551521
7034fa51d7d8824a06abc24ca0c6db34e0dbbd0e
55147 F20101109_AAAKMB chang_k_Page_043.pro
a8216a5402c5a87b883245b2009dd158
5d9ed5092f8ba9e881b366bd01296a48edc3c5e2
21745 F20101109_AAAKLN chang_k_Page_010.pro
582f23e8334fec6b3776d5c5b8e1c51e
e1b1d0dd0b373fdf4b828e3d97bb18cadc0c908f
F20101109_AAAKKZ chang_k_Page_076.tif
96bb6d147aee4c812bf1e214e3cb6bda
ae02acfecc060993bbb2956cd2e34b2a5db7d36e
46066 F20101109_AAAKMC chang_k_Page_044.pro
989f0d44e99d0bad131bcaeec642b0a4
a3b2c0bfa636b9934725e6f20e372a964eb48e76
55182 F20101109_AAAKMD chang_k_Page_046.pro
79da84bc39fa9550071d19502890311c
74d38ba404b04d3a1a4bb7a7ddeb138503aa688f
24808 F20101109_AAAKLO chang_k_Page_011.pro
eee8d1826377e0a5d70201e13119507e
4cada4ef769f12a6d61223a0c99544cb5e02d2c4
48152 F20101109_AAAKME chang_k_Page_052.pro
0adba97e190e399e275bf4e67b4e9e58
98e74a5b29fdbc857380f3380d3caa22d3456cb3
18539 F20101109_AAAKLP chang_k_Page_013.pro
77daff3f2a0c5546ae00691e78887e6b
dc78db5b5ada21aa8327d5ac971f7250adfe30ec
48314 F20101109_AAAKMF chang_k_Page_055.pro
16a463b06d75a1d896844fae51272595
8e6fe201adfb3b789d4c7024a356f0a9fd07be2b
25887 F20101109_AAAKLQ chang_k_Page_015.pro
fbe10935ce01b093e8095dd12f20e7ed
fccdd663696a10f49f49815cda6b8a58ce5c95d9
53692 F20101109_AAAKMG chang_k_Page_057.pro
c94b6ee25f99034b56fecf3f7f8ae338
9db407550bcb7b0fb5d5a5ca0a1a03d410421389
51659 F20101109_AAAKLR chang_k_Page_019.pro
d2dcbee61f50753f1cfa45109da83e1a
5cc508b768d6437a3d68d381cf5a88717e905f1e
51110 F20101109_AAAKMH chang_k_Page_058.pro
328ec9eb4b29d18bb908f2d7c6382bb4
efc5c4e8cc8d3a195f61e707c17b4cedfa26655a
56062 F20101109_AAAKLS chang_k_Page_022.pro
16ae081a9d0654779f2ea85237e714b1
f9501fe22ea9445ff738118a2ab7e09dab998c2c
50429 F20101109_AAAKMI chang_k_Page_067.pro
0af851ed4d3e997e8c5cc053d2d967e8
ad7874782dcf640cb93084298ee645e718f5da1e
52244 F20101109_AAAKLT chang_k_Page_023.pro
665b41ec2366fa05a00d1b16c99d2234
fc069f19f6f6eccccdc89d0dbccb01913435ffd0
13253 F20101109_AAAKMJ chang_k_Page_074.pro
a5ce3bd130e5317fcbefa9ec113dfd9f
2afff8b049bb1c53fdf79bd95e2d140a8ac2b761
55110 F20101109_AAAKLU chang_k_Page_025.pro
d9c3d5a2c6e76352c9d0252493b9af05
4bcf88dff4142c49667a095a87f150a0b58fddac
11434 F20101109_AAAKMK chang_k_Page_075.pro
c5cd1d3d3d89085ff401ef3e91a51ad6
974e0cb87b0322af24427d7b1d29489266dff7f3
50059 F20101109_AAAKLV chang_k_Page_028.pro
473d065d1fa910f7df8f331e3f406f38
77d59d9549e317534f22e17c715d95da0ec8d85e
13238 F20101109_AAAKML chang_k_Page_078.pro
34702278bd80a2e7816827a2ea7538ab
66908f55da6f82c924635c666a26889bf6b898ac
51711 F20101109_AAAKLW chang_k_Page_030.pro
858d59010ceaf019d9d57f6003f62e3a
60cad7434c1b8025f16f0569528ecf3410e0b940
67705 F20101109_AAAKNA chang_k_Page_117.pro
cdc88d323c689c770199bfc0dfd76f12
00606a0fd084ef72f9db8252d9eabac9b7562718
7265 F20101109_AAAKMM chang_k_Page_081.pro
7e4d8b0739603f9c5c6eb479fd7dbc59
29bb3feb329c7af345f978cb99aeb4ab0aef7bda
53907 F20101109_AAAKLX chang_k_Page_035.pro
c51347132265dd7c1455354962bc752b
6025e2eb1536c367271192fe52604484a5b3e79d
65433 F20101109_AAAKNB chang_k_Page_118.pro
7028c918d2fa32cbf4f419d7f00f4c69
0e17cb79195baaf97c1ffc30359862b05119c8da
13203 F20101109_AAAKMN chang_k_Page_082.pro
5d7b65126ba06e2593b12af3f72a8916
699697eec690e28bc157db36f699b9e4a5a151b5
11049 F20101109_AAAKLY chang_k_Page_036.pro
8394853406712b12a070184e0e0694f9
5505a9118f53d7e7599afe8078dd7d195ea2398b
69345 F20101109_AAAKNC chang_k_Page_119.pro
552234debd4f4bc8b7ed3b3059cdeff7
8b93fa6779d0cb55132b7071d617ea9ca5498d04
10532 F20101109_AAAKMO chang_k_Page_083.pro
6ef3eeea78cb675ec1b898fc1baf8138
8021ada72597b0859ee4b5650474417905108eab
55603 F20101109_AAAKLZ chang_k_Page_039.pro
7ca6d74c09c79860019d067b5fe38352
2f9dfcb0fc91d8da792731ab7ef203ebe58965a3
F20101109_AAAKND chang_k_Page_001.txt
afb8d11edbe713e3694172ff749c3f41
86387a21f0b510a26244757a28b6effcff46ff01
F20101109_AAAJKC chang_k_Page_057.tif
3129b7b14f33a36589e67906a03072c6
7553bd0fa58c4a83c0702d52ac5bcef3731edc5d
193 F20101109_AAAKNE chang_k_Page_005.txt
144e798c1c9be1e57124518043bfd1f9
19f0a37f3b5659833322dd26c10cf94d17e1e1c1
13450 F20101109_AAAKMP chang_k_Page_084.pro
3d97f431e53695616259b0fb472d12fa
63a3ea257ec99896d4fb989723850f1dc1242dfc
29217 F20101109_AAAJKD chang_k_Page_071.QC.jpg
a6a974efbfc3a59b7ca740195deb7da4
29e960a30ca858f81b897b5ec37d9304c2d39182
957 F20101109_AAAKNF chang_k_Page_010.txt
f89ec52672096d9b6cc5d0efe2fcb533
e9ec18cdf932f280cd247a11544b24717711486e
10973 F20101109_AAAKMQ chang_k_Page_087.pro
8f0fe0525d19949404d806ec2514a943
13b70e04729da5ceaae5e50ab09566cbfe69be75
14450 F20101109_AAAJKE chang_k_Page_038.QC.jpg
79e6d6da84ba121d507a709c28940ece
d3eba9e498fd8652aac37d3c7954a8ad3b4712c6
922 F20101109_AAAKNG chang_k_Page_012.txt
641fc20cb8df596983a9540837f73dcd
2cdb8dd7bc303e55319b2dd8f2d1a6038abfd1a3
49673 F20101109_AAAKMR chang_k_Page_091.pro
0a2d23b40d03881427497849d8e78f66
b664041e8b874d5eb66c93aafe83510f6b36c4f9
8565 F20101109_AAAJKF chang_k_Page_023thm.jpg
fe51007cc5bfd2c778f3453ac5f09ac7
fa53e919d1a5bc4608c8878cfa85ae7cce4989e7
315 F20101109_AAAKNH chang_k_Page_018.txt
e250797c454dfcfcbf57860415e50b2f
ae87d579257897e7c501600593dfdcc74bb293d7
53668 F20101109_AAAKMS chang_k_Page_092.pro
69279957278b19d7f7ec8eaeb3e90550
4781884629fdc762fef9d33f896c4e74279c1c26
20878 F20101109_AAAJKG chang_k_Page_081.QC.jpg
3627759afe89dfc41b91d9e143726932
8c5667cf655fa76c4a96a3815dda12d19d4e6fb3
2149 F20101109_AAAKNI chang_k_Page_024.txt
a2334fd6d98cf0779ed0f9dc3e15c1bb
4c2d4e1193e49d0a3dc3dfbca7373a2edd09e26b
52568 F20101109_AAAKMT chang_k_Page_093.pro
8c7712fa2053e31b1e78f30a5667fecb
2373cb498652ea33634e4593ba158168032acd8e
10179 F20101109_AAAJKH chang_k_Page_040.pro
a4465f0ceacabbe1529f8fdbf3d5dced
7dc6a07de576648d40ba1a0ff6e8adb3fbf164f4
2171 F20101109_AAAKNJ chang_k_Page_026.txt
cb2651644aa3683955dc387f1e89a150
40df6bb6d5742e2801ff3364fcb1ff0a9467d037
53422 F20101109_AAAKMU chang_k_Page_097.pro
f745cc082f99a45ea6703bb72d55fa62
ff551ab640ec516eed8c701c48862fdbdda5187a
F20101109_AAAJKI chang_k_Page_066.tif
95ed2a7c076c5d2e3cdb5df311a4b4b5
a6e30ec0383f9368243a1def242643cf31f0f2b9
722 F20101109_AAAKNK chang_k_Page_036.txt
f0bbdfd1d59c805499ad6ade258a587c
d2d99cbd68e605544eb704ef80c2de525e4b82e6
58484 F20101109_AAAKMV chang_k_Page_101.pro
dac3e38fedf379aafda8376b88a20bcc
a148428eb590e819e819e77a16534bb772da07a8
F20101109_AAAJKJ chang_k_Page_054.tif
1bd0cf5fecd308c5ca28c7be1b022a4b
a8ac8eb70beda64cd387208f04bf3e7057b8f540
2131 F20101109_AAAKNL chang_k_Page_037.txt
fb15ba8a469e42965ee770da4890e7cb
b63453030fd658ff4a3c0ac734e5079965ff94d5
59196 F20101109_AAAKMW chang_k_Page_102.pro
c2910ad6911d7133cc9ba58a78257439
f084fa007fd216d943cba14d9292ac5f684dc1c0
510 F20101109_AAAJKK chang_k_Page_075.txt
1712580cad388b76fed5c539c4aedc47
5898f41442aefea526323e547984623a5b6df12d
319 F20101109_AAAKNM chang_k_Page_038.txt
2b01685882dc8f88ef467105c692a378
7bb724cceb6eb028b2513514fb3f19e788091c3e
59598 F20101109_AAAKMX chang_k_Page_106.pro
3bb872493ba047189c282513ac6824d2
4ea3c2b902ff8616fd3c7da2ff5ff4f432ec0257
761 F20101109_AAAKOA chang_k_Page_089.txt
cf457cde987e202035bc4c72b0147352
67afd4d8c4e36d71f0461e826e1e1571aeadf024