Mechanisms of Action of Insulin-like Growth Factor Binding Protein-3 in Promoting Repair in Ischemic Retinal Vascular Mo...

Permanent Link: http://ufdc.ufl.edu/UFE0041478/00001

Material Information

Title: Mechanisms of Action of Insulin-like Growth Factor Binding Protein-3 in Promoting Repair in Ischemic Retinal Vascular Mouse Injury Models
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Kielczewski, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010


Subjects / Keywords: enos, epc, eye, homing, hsc, igfbp3, ischemia, migration, no, recruitment, repair, retina, retinopathy, s1p, srb1, vasp, vessels
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


Abstract: Endothelial progenitor cells (EPCs) are bone-marrow derived cells, which give rise to mature endothelial cells. IGFBP-3 can promote EPC repair and maintainence of blood vessel integrity. This study was conducted to gain insight into IGFBP-3?s cellular mechanism of action on EPC-mediated repair in damaged retinal vasculature. To unveil how IGFBP-3 modulates EPC functional repair, studies were designed to focus on how IGFBP-3 promotes cell migration. For in vivo studies, 2 complementary vascular injury models were utilized: laser occlusion of retinal vessels in adult green fluroscent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3 expressing plasmid into lasered adult gfp+ chimeric mouse retinas stimulated homing of EPCs into retinal blood vessels. The gfp+ EPCs also differentiated into various vascular cell types such as pericytes, astrocytes, and endothelial cells. In the OIR model, IGFBP-3 injection prevented cell death of resident vascular endothelial cells, while simulateneously increasing astrocytic ensheathment of retinal blood vessels. For EPCs to orchestrate these cytoprotective and homing effects, they must migrate into ischemic or damaged tissue. Experimental in vitro studies showed their migratory ability is mediated, in part, by endogenous nitric oxide (NO) generation. IGFBP-3 treated EPCs significantly increased NO generation compared to untreated EPCs. IGFBP-3 can signal through the high-density lipoprotein receptor, Scavenger Receptor class B, type 1 (SR-B1), to increase NO production and activity in mature endothelial cells. In EPCs and mature endothelial cells SR-B1 blockade with a neutralizing antibody resulted in a decrease in NO production. Furthermore, when EPCs and mature endothelial cells underwent IGFBP-3 treatment, there was an increase in phospo-endothelial nitric oxide synthase (peNOS) protein expression. IGFBP-3 exposure led to the redistribution of vasodilator stimulated phosphoprotein (VASP), a NO regulated protein, critical for cell migration of endothelial cells. Lastly, IGFBP-3 effects on vascular permeability were examined. Distinct differences in permeability were found depending on whether the IGFBP-3 was adminstered acutely or there was chronic exposure. IGFBP-3 acutely increased pemeability,while upon longer exposure IGFBP-3 reduced retinal vascular permeability, supportive of its vascular stabilizing ability. In summay, identification of a new signaling receptor that IGFBP-3 can activate was uncovered. IGFBP-3 can activate the SR-B1 receptor to increase exogenous NO production in EPCs, which leads to increased cell migration of these cells. Due to the positive stimulatory effects of IGFBP-3 on cell migration, it can act as an in vivo homing, resulting in vascular repair and stabilization of injured mouse retinal vasculature. IGFBP-3 has the potential to be used as a therapeutic agent to treat ischemic vascular eye conditions.
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.
Statement of Responsibility: by Jennifer Kielczewski.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Grant, Maria A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041478:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041478/00001

Material Information

Title: Mechanisms of Action of Insulin-like Growth Factor Binding Protein-3 in Promoting Repair in Ischemic Retinal Vascular Mouse Injury Models
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Kielczewski, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010


Subjects / Keywords: enos, epc, eye, homing, hsc, igfbp3, ischemia, migration, no, recruitment, repair, retina, retinopathy, s1p, srb1, vasp, vessels
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


Abstract: Endothelial progenitor cells (EPCs) are bone-marrow derived cells, which give rise to mature endothelial cells. IGFBP-3 can promote EPC repair and maintainence of blood vessel integrity. This study was conducted to gain insight into IGFBP-3?s cellular mechanism of action on EPC-mediated repair in damaged retinal vasculature. To unveil how IGFBP-3 modulates EPC functional repair, studies were designed to focus on how IGFBP-3 promotes cell migration. For in vivo studies, 2 complementary vascular injury models were utilized: laser occlusion of retinal vessels in adult green fluroscent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3 expressing plasmid into lasered adult gfp+ chimeric mouse retinas stimulated homing of EPCs into retinal blood vessels. The gfp+ EPCs also differentiated into various vascular cell types such as pericytes, astrocytes, and endothelial cells. In the OIR model, IGFBP-3 injection prevented cell death of resident vascular endothelial cells, while simulateneously increasing astrocytic ensheathment of retinal blood vessels. For EPCs to orchestrate these cytoprotective and homing effects, they must migrate into ischemic or damaged tissue. Experimental in vitro studies showed their migratory ability is mediated, in part, by endogenous nitric oxide (NO) generation. IGFBP-3 treated EPCs significantly increased NO generation compared to untreated EPCs. IGFBP-3 can signal through the high-density lipoprotein receptor, Scavenger Receptor class B, type 1 (SR-B1), to increase NO production and activity in mature endothelial cells. In EPCs and mature endothelial cells SR-B1 blockade with a neutralizing antibody resulted in a decrease in NO production. Furthermore, when EPCs and mature endothelial cells underwent IGFBP-3 treatment, there was an increase in phospo-endothelial nitric oxide synthase (peNOS) protein expression. IGFBP-3 exposure led to the redistribution of vasodilator stimulated phosphoprotein (VASP), a NO regulated protein, critical for cell migration of endothelial cells. Lastly, IGFBP-3 effects on vascular permeability were examined. Distinct differences in permeability were found depending on whether the IGFBP-3 was adminstered acutely or there was chronic exposure. IGFBP-3 acutely increased pemeability,while upon longer exposure IGFBP-3 reduced retinal vascular permeability, supportive of its vascular stabilizing ability. In summay, identification of a new signaling receptor that IGFBP-3 can activate was uncovered. IGFBP-3 can activate the SR-B1 receptor to increase exogenous NO production in EPCs, which leads to increased cell migration of these cells. Due to the positive stimulatory effects of IGFBP-3 on cell migration, it can act as an in vivo homing, resulting in vascular repair and stabilization of injured mouse retinal vasculature. IGFBP-3 has the potential to be used as a therapeutic agent to treat ischemic vascular eye conditions.
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.
Statement of Responsibility: by Jennifer Kielczewski.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Grant, Maria A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041478:00001

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2010 Jennifer Lee Kielczewski 2


To my mother 3


ACKNOWLEDGMENTS It is my pleasure to ackn owledge all individuals who aided me in completing my dissertation. I would like to firs t thank my mentor, Dr. Maria Grant, who has guided me for the past 3 years. Her creative and enthusiastic guidance has been a source of constant motivation during my graduate studies. I would like to thank my committee members, Dr. Stephen Baker, Dr. Paul Oh, Dr. Bryon Petersen, and Dr. Daniel Purich. Undoubtedly, their insightful comm ents and constructive criticism proved to be a great asset to my studies. Each of them provided engaging and challenging questions that helped shape my scientific thinking. My committee successfully established a solid foundation upon which I can continue to build my scientific knowledge and critical thinking skills. I would also like to acknowledge all the me mbers of the Grant Laboratory. Whenever I needed help, I could count on my lab members. I especially thank Dr. Lynn Shaw who was helpful in constructing diagrams and figures for my project and dissertati on. Also, special thanks goes to Dr. Aqeela Afzal. She was instrumental in training me during the firs t 2 years in the lab. I am very grateful for all of her time, effort, and patience. She was trul y a great role model. In addition, I would like to e xpress gratitude to Dr. Robert Mames and Dr. Guoqin Niu for all their help, which enabled me to complete my in vivo studies for my project. I also express my gratitude to Dr. Michael Boult on and his lab for all their help and support. Amy Davis, the graduate secretary in my department was also of great assistance during my graduate studies. Most important, I would like to express my indebted gratitude to my mother who has always supported my educational endeavors. Sh e has always provided me with encouragement and motivation to complete my goals. She raised me to be an independent thinker and is responsible for shaping the person I am today. Her dedication, generosity, and warmth have 4


made my educational journey a much smoothe r experience. Therefore, I dedicate my dissertation to her. I would also like to acknowle dge the pre-doctoral fellowships I received during from my graduate studies. They include a National Ey e Institute training fellowship from 2007-2009 in vision science and a Clinical and Translational Science Institute (CTSI) training fellowship from the National Institute of Health from 2009-2010. I am very grateful for these training grants and the opportunities they provided to me during my graduate studies. 5


TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF FIGURES .........................................................................................................................9LIST OF ABBREVIATIONS ........................................................................................................1 1ABSTRACT ...................................................................................................................... .............15 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ..............................................................................17The Eye ...................................................................................................................................18Eye Structure and Function .............................................................................................19The Retina .......................................................................................................................20Anatomy of the Retina .....................................................................................................21Retinal Blood Supply ......................................................................................................22Retinopathies ..........................................................................................................................24Retinopathy of Prematurity .............................................................................................24Diabetic Retinopathy .......................................................................................................27Neovascularization ............................................................................................................ .....29Endothelial Progenitor Cells ............................................................................................30Hypoxia ....................................................................................................................... ....32Vascular Endothelial Gr owth Factor (VEGF) .................................................................34Stromal Derived Factor-1 (SDF-1) ..................................................................................36Insulin-like Growth Factor Binding Proteins (IGFBPs) ..................................................38Insulin Like Growth Factor Binding Protein-3 (IGFBP-3) .............................................39Scavenger Receptor Class B type 1 (SR-B1) ..................................................................41EPC Homing and Cell Migration ...........................................................................................42Nitric Oxide (NO) ............................................................................................................43Sphingosine-1-Phosphate (S1P) ......................................................................................45Vasodilator Stimulated Phosphoprotein (VASP) ............................................................47Significance .................................................................................................................. ..........49Specific Aims and Hypotheses ........................................................................................492 METHODS AND MATERIALS ...........................................................................................60In Vivo Studies ...................................................................................................................... ..60Oxygen Induced Retinopathy (OIR) Model ....................................................................60Statistical Analysis of Cell Death in OIR Model ............................................................62Quantification of Astrocyte Ensheathment in OIR Model ..............................................62Electron Microscopy in the OIR Model ..........................................................................63Generation of Adult Chimeric gfp+ Mice ........................................................................63Bone Marrow Isolation & Transplant ..............................................................................63 6


Chimeric Engraftment Confirmation ...............................................................................65Retinal Laser Vessel Occlusion Model ...........................................................................65Immunostaining & Microscopy Analysis ........................................................................66Mass Spectrophotometry Analysis ..................................................................................67Vascular Pe rmeability .....................................................................................................68In Vitro Analysis .....................................................................................................................69EPC Isolation ...................................................................................................................69Cell Culture of Endothelial Cells ....................................................................................70Nitric Oxide Measurement ..............................................................................................70DAF-FM NO Production Assay ......................................................................................70eNOS Activity Assay ......................................................................................................71Western Blot Analysis .....................................................................................................72Real Time Polymerase Chain Reaction (RT-PCR) .........................................................72Immunocytochemistry .....................................................................................................73Cell Preparation and Fixation ..........................................................................................73Immunostaining ...............................................................................................................7 3Quantification of Immunocytochemistry ........................................................................743 RESULTS ..................................................................................................................... ..........79IGFBP-3 Prevents Endothelial Ce ll Death in the OIR Model ................................................79IGFBP-3 Increases Astrocytic Ensheathment of OIR Blood Vessels ....................................79IGFBP-3 Protects Retinal Neurons fr om Apoptosis in the OIR Model .................................80IGFBP-3 Increases Incorporation of EPCs in Adult Retinal Blood Vessels ..........................81IGFBP-3 Expressing Plasmid is Upregul ated in the Adult Mouse Retina .............................81IGFBP-3 Causes Differentiation of EPCs into Pericytes, Astrocytes, and Endothelial Cells ......................................................................................................................... ...........81IGFBP-3 Decreases the Ceramide/Sphingomyelin Ratio in Lasered Mice ............................82IGFBP-3 Increases NO Production in CD 34+ Cells and Endothelial Cells ...........................82IGFBP-3 increases eNOS phosphoryl ation at Serine 1177 in CD34+ Cells ...........................83Blockade of SR-B1 Leads to Decrease d NO Production in Endothelial Cells .......................83Blockade of SR-B1 Leads to Decrease d NO Activity in Endothelial Cells ...........................84Blockade of PI3K/Akt Reduces NO Generation and Activity ...............................................84Blockade of Sphingosine Kinase Decreases NO Release in CD34+ Cells .............................84IGFBP-3 Stimulates VASP Re-distr ibution in Endothelial Cells ...........................................85IGFBP-3 Decreases Vascular Permeabil ity in Laser Inju red Adult Mice ..............................85IGFBP-3 Increases Vascular Permea bility in Unlasered Adult Mice ....................................86IGFBP-3 Reduces Sphingomyelinase mRNA Expression .....................................................864 DISCUSSION .................................................................................................................. .....104IGFBP-3 Acts as an EPC/HSC Homing Factor and Provides Cytoprotection .....................104IGFBP-3 Increases NO and Activates SR-B 1 to Mediate its Protective Effects ..................106IGFBP-3: New Perspectives and Remaining Questions .......................................................108IGFBP-3 as a Therapeu tic in the Future ...............................................................................109Conclusions ...........................................................................................................................110 7


LIST OF REFERENCES .............................................................................................................112BIOGRAPHICAL SKETCH .......................................................................................................130 8


LIST OF FIGURES Figure page 1-1 Anatomy of the human eye.. ..............................................................................................511-2 Human retinal layers.. .................................................................................................... ....521-3 Retinopathies.............................................................................................................. ........531-4 Blood vessel development.. ...............................................................................................5 41-5 Hypoxia-regulated growth factor s and bone-marrow derived cells.. .................................551-6 Recruitment of EPCs in ischemic tissue.. ..........................................................................561-7 IGF system ................................................................................................................ .........571-8 IGFBP-3 signaling and cross-talk ......................................................................................581-9 IGFBP-3 signaling to increase NO leading to cell migration.. ..........................................592-1 Oxygen-Induced Retinopathy (OIR) Mouse Model.. ........................................................752-2 In vivo studies with IGFBP-3 plasmid in OIR Model. .......................................................762-3 In vivo studies with IGFBP-3 plasmid in Laser Vessel Occlusion Model. ........................772-4 In vitro studies with IGFBP-3 recombinant protein. .........................................................783-1 IGFBP-3 prevents endothelia l cell death in the OIR model. .............................................873-2 IGFBP-3 increases astrocytic en sheathment in OIR blood vessels ...................................883-3 IGFBP-3 protects retinal neurons fr om apoptosis in the OIR model. ................................893-4 IGFBP-3 increases incorporation of EPCs in adult retinal blood vessels ..........................903-5 IGFBP-3 expressing plasmid is upr egulated in the adult retina ........................................913-6 IGFBP-3 causes differentiation of EPCs into endothelial cells in the adult retina. ...........923-7 IGFBP-3 stimulates differentiation of EP Cs into astrocytes and pericytes. ......................933-8 IGFBP-3 decreases the ceramide/sph ingomyelin ratio in lasered retina. ..........................943-9 IGFBP-3 increases NO production in CD34+ cells.. ..........................................................95 9


3-10 IGFBP-3 increases phosphorylation of eNOS at Serine 1177 in CD34+ cells. ..................963-11 IGFBP-3 activates the SR-B1 receptor leading to NO generation in endothelial cells. ....973-12 Blockade of PI3K/Akt reduces NO generation in e ndothelial cells. .................................983-13 IGFBP-3 phosphorylates eNOS at Serine 1177 in HUVECs. ...........................................993-14 IGFBP-3 induces VASP re-distribution in HMVEC-L. ..................................................1003-15 IGFBP-3 decreases vascular perm eability in laser injured mice .....................................1013-16 IGFBP-3 acutely increases vascul ar permeability in unlasered mice ..............................1023-17 IGFBP-3 reduces sphingomyelinase mRNA expression levels .......................................1034-1 Mechanisms of action of IGFBP-3. .................................................................................111 10


LIST OF ABBREVIATIONS AKT Protein kinase B ALS Acid-labile subuit AMD Age-related macular degeneration BBB Blood-brain barrier BM Bone marrow BMD Bone marrow derived BMDC Bone marrow derived cell BRB Blood-retinal barrier BSA Bovine serum albumin Cdc6 Cell division cycle 6 cDNA chromosomal deoxyribonucleic acid cGMP Guanosine 3,5-cyclic monophosphate CNV Choroidal neovascularization DAF-FM 4-amino-5-methylamino-2,7 -difluorofluorescein diacetate DM Diabetes mellitus DMEM Dulbeccos modified eagle medium DMS Dimethylsphingosine DR Diabetic retinopathy EBM-2 Endothelial cell basal medium-2 EC Endothelial cell ECM Extracellular matrix EDG Endothelial differentiation gene EDTA Ethylenediamine tetraacetic acid Ena Drosophila melanogaster protein enabled 11


eNOS Endothelial nitr ic oxide synthase ENDO Endothelium derived nitric oxide EOMs Extraocular muscles EPC Endothelial progenitor cell EPO Erythropoietin EVH Ena/VASP homology FACS Fluorescence-activated cell sorting FITC Fluorescein isothiocyanate Flk Fetal liver kinase Flt-1 Vascular endothelial growth factor receptor-1 Flt-2 Vascular endothelial growth factor receptor-2 GCL Ganglion cell layer GFP Green fluorescent protein GM-SCF Granulocyte/macrophage colony-stimulating factor GPCR G protein-coupled receptor GS isolectin Griffonia simplicifolia isolectin HIF-1 Hypoxia inducible factor-1 HMVEC-L Human lung microvasc ular endothelial cells HREC Human retinal endothelial cells HSC Hematopoietic stem cell HUVEC Human umbilical vein endothelial cells IACUC Institutional Animal Care and Use Committee IGF-1 Insulin-like growth factor-1 IGF-2 Insulin-like growth factor-2 IGFBP Insulin-like growth factor binding protein 12


IGFBP-3 Insulin-like growth factor binding protein-3 ILM Inner limiting membrane INL Inner nuclear layer IOP Intraocular pressure KDR Kinase insert domain-c ontaining tyrosine kinase LGN Lateral geniculate nucleus mRNA messenger ribonucleic acid NIH National Institutes of Health NO Nitric oxide NOD Non-obese diabetic 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 peNOS phospo-endothelial nitric oxide synthase PFA Paraformaldehyde PIGF Placenta growth factor PI3K Phosphatidylinositol-3-kinase RGC-5 Rat retinal ganglion cells ROP Retinopathy of prematurity ROS Retro-orbital sinus 13


RPE Retinal pigment epithelium RT-PCR Real time polymerase chain reaction S1P Sphingosine-1-phosphate S1PR Sphingosine-1-phosphate receptor SCF Stem cell factor SCID Severe combined immunodeficiency SDF-1 Stromal cell derived factor-1 SD Standard deviation Sphk Sphingosine kinase SR-B1 Scavenger receptor class B type 1 TBS Tris buffered saline TGFTransforming growth factor VASP Vasodilator stimulated phosphoprotein VCAM vascular cell adhesion molecule VEGFR-1 Vascular endothelial growth factor receptor-1 VEGFR-2 Vascular endothelial growth factor receptor-2 VEGF Vascular endothe lial growth factor VPF Vascular permeability factor VSMC Vascular smooth muscle cell vWF von Willebrand factor 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 MECHANISMS OF ACTION OF INSULIN-LI KE GROWTH FACTOR BINDING PROTEIN3 IN PROMOTING REPAIR IN ISCHEMIC RETINAL VASCULAR MOUSE INJURY MODELS By Jennifer Lee Kielczewski May 2010 Chair: Maria Grant Major: Medical SciencesPhysiology and Pharmacology Endothelial progenitor cells (EPCs) are bone -marrow derived cells, which give rise to mature endothelial cells. IGFBP-3 can prom ote EPC repair and maintainence of blood vessel integrity. This study was conducted to gain insi ght into IGFBP-3s cellu lar mechanism of action on EPC-mediated repair in da maged retinal vasculature. To unveil how IGFBP-3 modulates EPC functional repair, studies were designed to focus on how IGFBP-3 promotes cell migration. For in vivo studies, 2 complementary vascular injury models were utilized: laser occl usion of retinal vessels in adul t green fluroscent protein (GFP) chimeric mice and oxygen-induced retinopathy in m ouse pups. Intravitreal injection of IGFBP-3 expressing plasmid into lasered adult gfp+ chimeric mouse retinas stimulated homing of EPCs into retinal blood vessels. The gfp+ EPCs also differentiated into various vascular cell types such as pericytes, astrocytes, and endothelial cells. In the OIR mode l, IGFBP-3 injection prevented cell death of resident vascular endothelial cells, while simulate neously increasing astrocytic ensheathment of retinal blood vessels. For EPCs to orchestrate these cy toprotective and homing effects, they must migrate into isch emic or damaged tissue. Experimental in vitro studies showed their migratory ability is mediated, in part, by endogenous nitric oxide (NO) generation. IGFBP15


16 3 treated EPCs significantly in creased NO generation compared to untreated EPCs. IGFBP-3 can signal through the high-density lipoprotein recep tor, Scavenger Receptor class B, type 1 (SRB1), to increase NO production and activity in mature endothelial cells. In EPCs and mature endothelial cells SR-B1 blockade with a neutralizing antibody resulted in a decrease in NO production. Furthermore, when EPCs and mature endothelial cells underwent IGFBP-3 treatment, there was an increase in phospo-endot helial nitric oxide synt hase (peNOS) protein expression. IGFBP-3 exposure led to the re-distributi on of vasodilator stimulated phosphoprotein (VASP), a NO regulated protein, critical for cell migration of endothelial cells. Lastly, IGFBP-3 effects on vascular permeability were examined. Distinct differences in permeability were found depending on whether the IGFBP-3 was adminste red acutely or there was chronic exposure. IGFBP-3 acutely increased pemeability, while upon longer exposure IGFBP-3 reduced retinal vascular permeability, supportive of its vascular stabilizing ability. In summay, identification of a new signali ng receptor that IGFBP-3 can activate was uncovered. IGFBP-3 can activate the SR-B1 recep tor to increase exogenous NO production in EPCs, which leads to increased cell migration of these cells. Due to the positive stimulatory effects of IGFBP-3 on cell mi gration, it can act as an in vivo homing, resulting in vascular repair and stabilization of injured mouse retinal vasculature. IGFBP-3 has the potential to be used as a therapeutic agent to treat isch emic vascular eye conditions.


CHAPTER 1 BACKGROUND AND SIGNIFICANCE Sight is one of our most precious senses. When it is compromised or lost, it can significantly impair quality of lif e. As life expectancy continue s to increase, blindness is an imminent threat for the aging population.1-4 In fact, more than 2 million Americans age 50 and older have age-related macular degeneration (AMD) and more than 4.4 million Americans age 40 and older suffer from diabetic retinopathy, according to the Nationa l Eye Institute in 2008. Over the next 20 years the number of people with compromised vision and blindness will continue to climb, not only in the United States, but worldwide.4,5 This will undoubtedly place a strain on not only the visually impaired, but th eir families, as well as society as a whole. Therefore, therapeutic strategies to combat b lindness are necessary, such as stem cell based therapies. Stem cell therapy in the eye has become an a ttractive therapeutic strategy to correct lost vision.6-11 This is particularly true of ocular di seases with vascular complications, such as diabetic retinopathy.12 Diabetic retinopathy results from uncontrolled hyperglycemia, which ultimately leads to neovascularization and aberrant vessel formation in the eye. The vessels that form in the eye are leaky, fragile, and unstable, which leads to obscured vision.13 In order to overcome vision impairment, adult hematopoietic stem cells (HSCs) derived from the bone marrow, specifically endothelial progenitor cells (E PCs), can be potentially used to repair and reendothelialize damaged retinal vessels in diabetic retinopathy patients.14-17 Previously, EPCs have yielded improved blood perfusion in ischemic vascular injury animal models involving the hind limb18-24 and heart.25-31 Also, EPCs incorporate into damaged retinal blood vessels in ocular injury mouse models with promising results.15-17,32-34 However, the extent of EPC repair and therapeutic benefit still requires improve ment with regard to increased homing and 17


regenerative efficiency. When EP Cs are provided the right cue a nd/or stimulus, such as a potent growth factor, their migratory efficiency and re parative capability can be greatly enhanced. But the question remains open as to what growth f actor stimulus will best improve EPC mediated repair, particularly in th e retinal vasculature. Insulin-like Growth Factor Binding Protein-3 (I GFBP-3) has garnered much interest within the past few years as a potent ial therapeutic to treat ischem ic vascular eye complications, especially in pre-mature infant s with retinopathy of prematurity (ROP). This is based on studies using a mouse model of ROP, called oxygen induced retinopathy (OIR). By increasing exogenous levels of IGFBP-3 in the mouse pup ey e reduced pathological neovascularization was observed.35,36 Thus, IGFBP-3 has vascular protective eff ects. It is believed IGFBP-3s vascular protective nature may be due to its in fluence on EPC behavior. Exogenous IGFBP-3 administration has been shown to have a prof ound effect on EPC migration, tube formation, and differentiation, all of which al low the progenitor cells to hom e, stabilize and promote normal vessel development in the mouse OIR model.36 IGFBP-3s cellular mechanism of action on EPC mediated vascular repair is not yet understood. Hence, this study was undertaken to illuminate how IGFBP-3 influences EPC driven repair in re tinal vascular mouse injury models at both the cellular and molecular levels to better understand its therapeutic po tential. The Eye The vertebrate eye is a well designed and highly efficient or gan. All of the various cell types and structures contained in the eye work in harmony to crea te a clear image which is sent to the brain for visual processing. If any part of the eye is damaged, it can disrupt the intricate process of visual phototransduction. The eye, although an important organ, can be under appreciated for its intricacies. Nevertheless, the ey e is truly spectacular in its anatomical shape, form, and function. 18


Eye Structure and Function The eye is a complex organ composed of many structures. The ability to see is dependent on all the structures working t ogether to creat e a clear image.33 Figure 1-1 depicts the essential components of the eye. Light ente rs the eye through the cornea, which is considered the window of the eye due to its transparency and avascular nature.37 After light rays pass through the cornea, they travel through the aqueous humor which provides nourishment for the surrounding lens and cornea, as well as main tains intraocular pressure (IOP).38 The aqueous humor is produced by the ciliary body. Th e ciliary body changes the shape of the lens for focusing. The iris is the pigmented part of th e eye. It separates the anterior chamber from the posterior chamber and regulates the amount of li ght entering through the pupil.39 The size of the pupil is regulated by the dilator and sphincter muscle s of the iris and controls the amount of li ght that enters the eye. After light travels through th e pupil, it passes through the lens. The lens is surrounded by ligaments (zonule fibers) that are attached to the anterior portion of the ciliary body. The lens changes shape, contracts or relaxes, by th e ciliary muscles and attached ligaments.40 Light then passes through a clear, jelly-like substance called the vitreous hu mor before it finally reaches the retina. The retina is a thin, multi-layered transparent, neuro-sensory tissue lining the back of the eye. It allows light rays to be converted into electrical impulses, which are transmitted to the optic nerve leading to the brain.38 The optic nerve is a bundle of nerve fibers that carries visual information from the eye to the brain. It consists of approximately 1 million axons arising from the ganglion cells of the retina. The optic nerv e runs from the optic disc through the optic foramen to the optic chiasma where it becomes the optic tract. The visual fibers synapse in the lateral geniculate nucleus (LGN). The cell bodies of this structure give rise to the neurons that comprise the visual pathway. The eye is made up of three distinct layers: th e external layer, the intermediate layer and 19


the internal layer. The external layer is formed by the sclera and cornea. The cornea, as described earlier, is quite refractive, thus provides the eye with exquisite focusing power.38 The sclera is composed of a tough, fibrous tissue that serves to protect the eye. Extraocular muscles surround the eye and are attached to the sc lera. There are a total of six ex traocular muscles, four rectus muscles and two oblique muscles, which work to gether in accord to keep both eyes properly aligned.41 The intermediate layer is divided into tw o parts: the anterior (iris and ciliary body) and the posterior part called the choroid. The chor oid contains a layer of blood vessels and lies between the retina and sclera. The choroid supplies oxygen and nutri ents to the outer layers of the retina. The choroid connects th e ciliary body with the front part of the eye and is attached to the edges of the optic nerve.42 The internal layer is the sensory part of the eye d eemed the retina. The Retina The retina is one of the most important stru ctures in the eye and is where a large amount of the eyes blood supply is located. Most eye diseases result in some degree of retinal damage.43-45 The retinal blood vessels are frequently subjected to damage in diseases such as retinopathy of prematurity (ROP) and diabetic retinopathy (DR).33,43,46 In these disease processes, neovascularization occurs, which is the formation of new blood vessels. The new blood vessels that form are abnormal in nature thus unable to support normal blood flow and result in retinal ischemia.46-48 The abnormal blood vessels that form in the retina, ultimately contribute to blindness due to the formati on of edema, exudate build up, scar tissue accumulation, and/or retinal detachment.13,33 It is these abnormal bl ood vessels that are a target for repair and stabilization. Fortunately, th e retinal vasculature is easy to visualize in vivo. This facilitates studying pathological ne ovascularization and potential cel l based therapies, such as with EPCs, to target this process from obliterating the retina. 20


Anatomy of the Retina The retina is a multi-layered structure that is essential for visual processing. The human retina is approximately 0.2 mm th ick and has an area of roughly 1100 mm.38 The human retina contains well over 200 million neurons.49 The retina captures light and converts it into electrical impulses by way of photoreceptor cells called ro ds and cones. There are close to 125 million rods in the human retina.50 They are situated throughout the peripheral retina and function best in dim light. Hence, rods are responsible for peri pheral and night vision. In contrast, the human retina contains 6 million cones, which functi on best in bright light and color perception.51 The highest density of cones can be found in the macu la. The center part of the macula, called the fovea, is densely packed with cones. The f ovea permits greater light absorption by the dense array of photoreceptors, thus is the site of our most acute vi sion. The fovea contains no blood vessels, permitting increased visual acuity in the macular region. Nevertheless, the vascularization of the remainder of the macula is very dense and increases the likelihood of several vascular related diseases. The retina is loosely attached to the retinal pigment epithelium (RPE). These cells contain an abundant amount of pigment that is necessary for light absorption and transportation of oxygen, nutrients, and wa stes between the photoreceptors and the choroid.42 Bruchs membrane is tightly associated with the RPE, stabilizing the RPE layer by separating it from the blood vessels of th e choroid. Oxygen diffuses across the Bruchs membrane and this membrane thickens with age.42 Breaks in Bruchs membrane are the hallmark for choroidal neovascularization (CNV) in the retina. Beneath the Bruchs membrane is the choroid, which contains a network of blood vessels, nerves, and provides all of the nutritional needs of the RPE and the outer part of the sensory retina. The human retina consists of ten layers (F igure 1-2). Among them, three layers of nerve cell bodies and two layers of synapses are respon sible for converting a light signal into a neural 21


signal. The photoreceptor cell bodies fo rm the outer nuclear layer (ONL).52 While the inner nuclear layer (INL) contains th e cell bodies of the bi polar, horizontal, and amacrine cells, the ganglion cell layer (GCL) contains the cell bodies of ga nglion cells and displaced amacrine cells. The outer plexiform layer (OPL) is located be tween the outer nuclear layer (ONL) and inner nuclear layer (INL).53 In the OPL, photoreceptors relay thei r information to the bipolar cells, as well as the horizontal cells. The bipolar cells th en transfer information to the inner plexiform layer (IPL), which separates the INL and GCL. Bipolar cells are connected to the retinal ganglion cells in addition to amacrine cells in the IPL.53 The ganglion cells are the output neurons of the retina that transmit information from the eye to the brain. Retinal Blood Supply The retina is a metabolically active tissu e and requires among the highest blood flow rates of any tissue.45,54 The blood supply to the retina orig inates from the ophthalamic artery.37 There are two sources of blood supply to the mamma lian retina: the central retinal artery and the choroidal blood vessels. The outer retina is supplied by the chor iocapillaris, which is an extensive network of fenestrate d capillaries. The choroid rece ives the greatest blood flow between 65-85%, which is critical for the maintena nce of the outer retina specifically the inner and outer segments of the photoreceptors.42 The central retinal artery supplies the remaining 2030% blood flow from the optic nerve head to nourish the inner retinal layers. All blood vessels share a number of common features. For exam ple, the insides of blood vessels are lined with endothelium, a thin layer of endothelial cells (ECs), which separates the blood from tissues.55 Blood vessels are also covered with a specialized layer of connective tissue called the basement membrane followed by a la yer of mural cells know n as pericytes and vascular smooth muscle cells. Astrocytes, which are characteristically star-shaped glial cells, are also a component of blood vessels. They form a layer around blood vessels and provide 22


biochemical, as well as structural support to endothelia l cells. Astrocytes help maintain the blood brain barrier (BBB) in cerebr al blood vessels in the brain.56,57 Retinal blood vessels are remarkably simila r to cerebral blood vessels in that they maintain the blood-retinal barrier (BRB), which is similar to the BBB.58 The BRB consists of two distinct monolayers of cells: the retinal pigment epithelium ( outer barrier) and the retinal capillary endothelial cells (inn er barrier). Both monolayers fo rm tight junctions, which are responsible for maintenance of the barrier. The inner BRB is cove red with pericytes and glial cells. Glial Muller cells predominately support retinal endothelia l cells and glial astrocytes are partly responsible for supporting endothelial functions at the inner BRB.56 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. The outer BRB consists of specialized nonfenestrated capil laries and tight junctions within the RPE.57 The outer BRB forms a transport barrier betw een the retinal capillaries and choroidal capillaries. Also, it prevents the passage of large molecules fr om the choriocapillaris into the retina.48 Transmembrane proteins such as occludin, claudi n, junctional adhesion molecule (JAM), as well as adherens junctions he lp maintain BRB integrity.59 The eyes blood supply is crucial for its prop er function and any disruption in it or the BRB can have dire consequences on visual pe rception. The BRB maintains the ocular milieu by protecting the neural retina fr om circulating inflammatory cel ls and their cytotoxic products. This allows the retina to regulat e its own extracellular chemical co mposition for proper neuronal function.59 The retina essentially co ntrols its own blood flow and BRB by a variety of cellular and chemical interactions. Therefore, any breakdown of the BRB, such as ischemic injury or inflammation, can lead to detrimental altera tions in blood flow and increased vascular 23


permeability.33,47 This in turn can lead to serious va scular eye diseases called retinopathies (Figure 1-3). Retinopathies Loss of vision is a difficult health problem to overcome. When vision is compromised it leads to disability, suffering, loss of productivity, and a lower quality of life. In the United States alone, more than 25 million people suffer from vision loss according to the 2008 National Health Interview Survey. Approximately 1.3 million people in the United States ar e legally blind. Many of these cases of blindness are attributed to re tinopathies, which are ocul ar diseases in which deterioration of the retina is caused by a bnormal neovascularization, resulting in vision impairment (Figure 1-3). Vascular retinopathies are the leading causes of visual disability and blindness worldwide.46 Pathological growth of new blood vessels is the hallmark of retinopathies. Retinopathies affect all age gr oups. Retinopathy of prem aturity (ROP) affects premature infants, while diabetic retinopa thy (DR) strikes the working age population. Retinopathies are debilitating eye diseases, wh ich are increasing in number and frequency. Retinopathy of Prematurity ROP is the leading cause of blindness in children in both deve loped and undeveloped countries.60 Two major risks factors of ROP are the use of oxygen and a decreased gestational period.61 ROP mainly affects premature infants weighing approximately 2.75 pounds or less that are born before 31 weeks of gestation.62 In general, the more premature the baby and the lower the birth weight, the greater the risk for ROP.63 Growth of the human fetal eye occurs within the last 12 weeks of full term deliver y, 28-40 weeks gestation. Vessels reach the anterior edge of the retina and then regress at about 40 weeks of gestation. Therefore, infants born pre-maturely (before 31 weeks) have incompletely vasculari zed retinas with a peri pheral avascular zone. 60,64 In premature infants, vascular growth that normally occurs in utero slows and is accompanied by 24


regression of developed retinal vessels. ROP was first described by Terry in 1942. At this time, administration of high oxygen was considered the standard of care for prem ature infants to supplement their underdeveloped lungs and maintain adequate respiration.65 Although the high oxygen a ssisted premature infants with their breathing, it was discovered upon removal from the high oxygen environment, the return to normal levels of atmospheric oxygen was often seen as a hypoxic environment in the eye.66 As a result, many infants suffered pathol ogical neovascularization within the retina. Despite adjustment of oxygen de livery and other medical advances the total number of infants with ROP has not decreased over th e years because of increased surv ival rates in very low birth weight infants.64 There are approximately 4 million babies bor n in the United States annually. According to the National Eye Institute, about 28,000 pr emature infants are born each year. Between 14,000 and 16,000 of these premature infants develop some degree of ROP. There ar e 5 stages of ROP, from a mild stage 1 to the most severe stage 5.61 Most infants, close to 90%, have mild ROP and do not require extensive treatment. ROP in most cases regresses spontaneously.67 Therefore, only a small number between 1,100 and 1,500 of the premature babies develop severe ROP and require significant medical care. 61 As a consequence, 400 to 600 infants each year in the United States become legally blind from ROP. ROP progresses in two phases.60,64,66 The first phase includes the hyperoxia extrauterine environment surrounding the baby and the suppl emental oxygen administered to the baby. The growth inhibition of retinal vasc ular growth after birth and part ial regression of existing retinal vessels is the first phase, followed by a s econd phase of ROP i nvolving hypoxia-induced uncontrolled proliferative vessel growth. The pat hological growth of vesse ls produces a fibrous 25


scar that extends from the reti na to the vitreous and lens. Re traction of this scar tissue can separate the retina from the RPE, resulting in retinal detachment, bleeding, and ultimately blindness in neonates.64 The biphasic disease process of ROP is associated with unbalanced levels of growth factors.64 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.68 IGF-1 plays a critical role in ROP in infants. Reducing IGF-1 levels inhibits vessel growth even in the presence of VEGF.69 Low levels of IGF-1 re duces vascular density, which subsequently causes early vessel degenerati on in phase I. The mean serum levels of IGF-1 in age-matched premature babies are directly correlated with the se verity of ROP disease stages.70 Likewise, infants with ROP have lower IGFBP3 levels than those of healthy infants. In the second phase of ROP, which is driven by hypoxia, VEGF expression is increased in the retina. This results in pathol ogical neovascularization because blood vessels grow toward the concentrated VEGF areas in the retina.71 Inhibition of VEGF can prevent a certain degree of hypoxia-induced retinal neovascularization in the second phase of ROP. However, VEGF inhibition does not completely prevent neovasc ularization in the second phase of ROP, suggesting ROP is a multi-factoria l process involving an interpla y between a number of factors related to growth and development.64 There are several therapies, such as cryot herapy and laser photocoagulation, that can be used to reduce visual loss in ROP infants.62 However, these therapies can reduce peripheral vision and include risks from anesthesia. Therefor e, preventive and less invasive therapies for ROP are warranted. Also, understanding how IGF-1 and VEGF contribute to ROP and the use of these growth factors as potential therapeutic targets is crucial. The two phases of ROP are quite 26


distinct and require different therapeutic interventions. In ph ase 1, the hypoxia-induced vessel loss can be partially prevented by administering VEGF or PIGF-1.60,72,73 While an injection of anti-VEGF aptamer, as well as anti-VEGF antibody can be used to treat neovascularization in phase II, pharmacological interv ention related to the prevention of vessel loss may be a more effective therapeutic strategy.64 This is true becaus e the extent of the second destructive phase of ROP is determined by the amount of ve ssel loss in the first phase. In 1994, Smith and colleagues developed a m ouse model of ROP to study the molecular mechanisms involved in the disease.71 This model was developed on the premise that retinal vessel development in mice is incomplete at birth.74 Therefore, this model is intended to mimic the first and second phases of ROP in human premature infants. Neonatal mice are exposed to 75% oxygen from postnatal day 7 until day postn atal 12. When neonatal mice are exposed to hyperoxia, vessel regression and the cessation of normal radial vessel growth occurs mirroring the first phase of ROP. The hypero xia primarily targets capillaries adjacent to arteries in the center of the retina and does not affect larger, more mature veins and arteries.75 Upon return to room air, the non-perfused portions of re tina become hypoxic, resulting in retinal neovascularization. This neovascul ar phase in the OIR model is similar to the second phase of ROP in humans. The mouse ROP model has been used extensively to st udy neovascularization in the early developing vasculat ure and to unravel molecular changes in both phases of the disease. It has proven to be a reproducible and quantifiable model and is used in studies described in subsequent chapters. Diabetic Retinopathy Diabetes mellitus (DM) affects 100 million people worldwide.5 A common complication of DM is diabetic retinopathy, which can have detrimental effects on vision. Nearly 4.1 million Americans are affected by diabetic retinopathy (DR).76 Of these individuals, close to 900,000 27


are affected by retinopathy that is vision threatening. Vascular di seases are the principal cause for death or disability in peopl e with diabetes. The metabolic dysfunctions that characterize diabetes include elevated blood glucose levels, in creased levels of free fatty acids, and insulin resistance, all of which contribu te to vascular complications.77 According the National Eye Institute, the microvascular complic ations of DR is one of the mo st common ailments of diabetes and frequently results in visual loss. DR is known to affect diab etic patients who have had the disease for longer than 20 year s and have poor glucose control.78 Although the best way to prevent vision loss is to initia te treatment before symptoms develop, many diabetic patients do not experience visual complications until significa nt vascular damage has already occurred. DR is classified into two stages: nonproliferative diabetic retinopathy (NPD R) and proliferative diabetic retinopathy (PDR). PDR typically devel ops in patients with type 1 diabetes, while NPDR is more common in patients wi th type 2 diabetes (Figure 1-3).76 The progression of DR begins with increased reactive oxygen species (ROS) and changes in nitric oxide synthase (NOS) isoforms follo wed by apoptosis of pericytes and adhesion of leukocytes to the vessel wall that result in microvascular occlusion, basement membrane thickening, and increased vascular permeability.13,77 As a result of these pathological changes, the blood vessels become leaky, allowing blood and va scular fluids to accumulate in the retinal tissue and form exudative deposits.59,79 This then results in macular edema, which is commonly seen in patients with NPDR.59 NPDR is classified according to three stages: mild, moderate, and severe. NPDR is associated with areas of capillary non-perfusion, which lead s to hypoxia in the retina in the severe stages of the disease. To compensate for the decreased oxygen supply, angiogenic factors such as VEGF are released from the hypoxic retinal tissues and stimulate the growth of new blood vessels on the surface of the retina.13,80 This late stage is called PDR. The 28


walls of the newly formed vessels are fragile and can easily break, allowing blood to leak out. This can cloud the vitreous and obscure vision. In the advanced stage of PDR, newly formed fibrous vascular tissue grows from the retinal su rface into the vitreous cavity. This can cause retinal detachment leading to blindness.59 The current therapy for DR is laser photocoagulation.76,81 However, this treatment often causes unwanted side effects such as neural tissu e loss, peripheral visi on loss, impairment of night vision and change in color perception. Additi onally, in some patients, their retinopathy still continues to progress after laser treatment.46 Therefore, new therapeutic treatments are greatly needed to treat diabetic retinal vascular disease. Pharmacological agents that directly inhibit angiogenesis have been developed to treat DR. Particular emphasi s has been placed on inhibiting pro-angiogenic growth factors such as VEGF. The overexpression of VEGF plays a key role in the pathogenesis of diabetes and induction of retinal vascular dysfunction.13 The development of agents that directly target VEGF and its rece ptors have been vigorously pursued in clinical research trials. The success of these agents has been limited in patients. However, the use of EPCs to repair damaged retinal vessels is a nove l and exciting strategy to treat DR. Neovascularization Blood and lymphatic vessels are either developed by vasculogenesis or angiogenesis.26,47,82 During de novo vasculogenesis, endothelial lineage committed angioblasts assemble to form new vessels during embryogene sis. During angiogenesis, sprouts form from pre-existing blood vessels and migrate into the surrounding tissue in the a dult. This involves sprouting, pruning and intussuscep tion of pre-existing vessels.83 This process relies on proliferation, migration, and remodeling of fully differentiated endothelial cells. Most organs are vascularized by vascuologenes is, like the brain and kidney.84 Neovascularization during adult life has long been attributed to angiogenesis only.85 However, this dogma has recently been 29


challenged.20,86-89 Studies have shown that EPCs also circ ulate postnatally in the peripheral blood and can be recruited and incorporated into site s of active neovascularization in ischemic hind limbs, ischemic myocardium, injured corneas and retina, cutaneous wounds, and even the tumor vasculature.90 Therefore, retinal vascular development occurs by a combination of both vasculogenesis and angiogenesis, which is called neovasculariz ation (Figure 1-4).47 A variety of factors are known to contribute to neovascularization such as cyt okines, chemotactic factors, and angiogenic factors. Essentially, EPCs can functionally revasculari ze ischemic tissues, participate in neovessel formation, and maintain overall vascular homeostasis (Figure1-5).26,43,55,82,87,90-92 Endothelial Progenitor Cells The bone marrow (BM) is the major reservoir of adult stem cells.93,94 The bone marrow microenvironment, commonly known as the bone marro w niche, remains relatively quiescent. It is comprised of stromal cells and extracellular matrix (ECM) co mponents. A special subtype of BM-derived stem cells, known as EPCs, are able to differentiate into mature endothelial cells and incorporate into sites of neovascularization under physiological as we ll as pathological conditions, such as wound healing, or gan regeneration, and tumor growth.87,95-97 EPCs were first isolated by Asahara et al. in 1997.89 Over the past ten years EPCs have been extensively studied.17-20,27,34,43,91,98-114 EPCs are a rare population in the periphera l blood and bone marrow. They represent between 0.01 and 0.001% of the total peripheral blood mononuclear cell fraction from a normal blood sample.43,95 EPCs can be isolated from not only peripheral blood, but fetal liver or umbilical cord blood.87,89 EPCs are characterized by specific antigens, such as CD34+ in humans and c-kit+/Sca-1+ in mice, expressed on the surface of the cells.27,93,95,115-118 Stem cells maintain immature, primitive markers so that they can diffe rentiate or transdifferentiate into a wide spectrum of cell types. This proce ss is known as stem cell plasticity.94 30


The identification of true EPCs has been highly debated in terms of their stem cell plasticity ability. Defining EPCs has been contro versial to say the least because several studies have demonstrated overlapping antigens among ot her types of bone marrow-derived cells, such as monocytes.116,119-121 Moreover, many have suggested size, cell cycle, cell a dhesion, and other functional characteristics, rather than cell su rface markers alone, may be more useful when isolating and characterizing EPCs.112 Little effort has been e xpended to carefully characterize the definitive EPC. However, even with disc repancies in the EPC fi eld regarding phenotypic characteristics that define EPCs, it is widely accepted that CD34, vascular endothelial growth factor receptor-2 (VEGFR-2), and CD133 (AC133) are the common antigens used in the sorting and isolation of human EPCs.27,87,115,118 It has also been suggested that endothelial nitric oxide synthase (eNOS) is an additional marker used to define EPCs. EPCs have other characteristics of endothelial cells including acetylated low densit y lipoprotein uptake and endothelial specific lectin binding in vitro Additionally, EPCs can produce nitr ic oxide (NO). With maturation, EPCs begin to lose expression of CD34 and CD 133, and start to express CD31, also known as PECAM-1 (platelet /endothelial cell adhesion molecule), vascul ar endothelial cadherin, and von Willebrand factor.122 The differentiation of EPCs occurs wh en circulating EPCs home to sites of injured vessels or integrate into mature endothe lium, based on molecular stimuli that govern their rapid differentiation.87 EPC function and number can vary am ong individuals due to pathological, pharmacological, and physiological factors. In fact, the number of circulating EPCs and colony forming ability of EPCs are directly correlated with certain disease states.43,86,104,113,123-130 Fewer CD34+ EPCs are circulating in patients with diabetes131-134 and peripheral artery disease.135-137 Also, increased levels of oxidative stress and inflammatory cyt okines have negative 31


consequences on EPC function and mobilization. On the other hand, increasing numbers of EPCs were found in patients with limb ischemia or ve ssel wall damage after coronary thrombosis, burn injury, or coronary bypass surger y to rescue damaged vessels.113 Exercise and physical training have also been found to increase circulating EPCs.126 Clearly EPC numbers vary among individuals. But it is clear reduced EPC numbers can have a negative impact on vascular homeostasis and consequen tly vascular repair. EPC recruitment, as well as release from the bone marrow, is influenced by various exogenous factors (Figure 1-5). Pr o-angiogenic growth factors su ch as granulocyte/macrophage colony-stimulating factor (GM-C SF) and erythropoietin (EPO) have been shown to modulate EPC functions that play a critic al role in embryonic development, as well as in homeostasis in the adult.14,82,87,138 However, three well studied stim uli, hypoxia, VEGF, and SDF-1, have a major impact on not only EPC mobilization, but their overall f unction and reparative capacity.14,20,90 Each of these stimuli are discussed separately. However, hypoxia, VEGF, and SDF-1, frequently work together in accord through cross-talk signa ling to influence EPC behavior and homing. Hypoxia Hypoxia occurs when there is an imbalance between oxygen supply and demand in cancerous or ischemic tissues. Cons equently, hypoxia is a critical stimulus for expansion of the vascular bed.139 In wounds, capillary injury genera tes a hypoxic environment, and altered oxygenation induces a reconstr uctive angiogenic response.99 Hypoxia triggers vessel growth by signaling through hypoxia-inducible transcription factors (HIFs) to stimulate SDF-1, VEGF, EPO and other factors. It has been suggested HIF-1 may be ultimately responsible for initiating progenitor mobilization and targeting to sites of neovascularization. 32


Hypoxia serves as a critical cue for both physiological and pathol ogical angiogenesis in the brain, heart, kidneys, lungs, and muscles. Hypoxia is also a potent stimulus for mobilization of bone marrow derived cells, such as EPCs.14,20,32,140 When a gradient of hypoxia was created in skin wounds in mice, bone-marrow derived (BMD) cells followed the gradient, and the greatest number of cells homed to and integrated into vessels of ischemic tissue.141 Kalka et al transplanted human EPCs into athym ic nude mice with hindlimb ischemia.19 They found blood flow recovery and capillary density in the ischem ic hindlimb was significantly improved in mice receiving human transplanted EPCs. Also, Annabi et al. report hypoxia promotes murine BMD stromal cell migration and tube formation.140 They suggest hypoxia-driven angiogenesis may be a critical condition for remodeling by bone marrow-derived stem cells. More or less, the prolifera tion, patterning, and assembly of recruited progenitors into functional blood vessels are influenced by tissue tension and hypoxia.99 Hypoxia may be regarded as fundamental requirement for progeni tor cell trafficking and function. Since the bone marrow environment itself is hypoxic, where EPCs or iginally reside prior to release into the circulation, ischemic tissue may represent a conditional stem cell niche, which may attract circulating EPCs.99 This premise appears to hold true for the eye, in which EPCs are recruited to sites of ischemic ocular injury (Figure 16).16,17,34,92,102,138 Takahashi et al. showed in 1999 that EPCs contribute to enhanced corneal neovascularization, which were mobilized from the bone marrow in response to ischemia and GM-CSF.14 Two years later, Grant et al. showed that recruitment of endothelial progenitors to sites of retinal ischemic venous occlusion injury had a significant role in neovascularization in retina.17 They concluded HSCs are major contribut ors to the functional vessel formation that occurs during neovascularization in the retina. Furthermore in 2006, Ritter et al. showed a 33


population of adult BM-derived myeloid progenitor cells migrated to av ascular regions in the retina.32 They found myeloid progenito rs differentiate into microg lia and promoted vascular repair in a model of ischemic oxygen induced retinopathy. Essentially, circulating EPCs and HSCs are mobilized endogenously in response to tissue ischemia. 25,28,30,31,33,36,43,104,140,142 Vascular Endothelial Growth Factor (VEGF) VEGF, also known as vascular permeability fact or (VPF), plays an important role in both normal physiological angiogenesis a nd pathological angiogenesis asso ciated with disease states such as diabetic retinopathy, rheumatoid arthritis, and solid tumor formation.143,144 It modulates vascular growth and morphogenesis, vascular tone, as well as ch emotaxis of endothelial cells.145 VEGF is especially well known as a pro-angiogenic growth factor and is implicated in inducing microvascular hyperpermeability, which can both precede and accompany angiogenesis.144 VEGF is a highly specific mitogen for endothelia l cells and is a potent cell survival factor.143 Signal transduction involves binding to tyrosine kinase receptors, resu lting in endothelial cell proliferation, migration, and new blood vessel formation.83 The VEGF family consists of seven struct urally related homodimeric glycoproteins: VEGF-A, placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, orf virus-encoded VEGF-like proteins (VEGF-E), and a series of snake venoms (VEGF-F).83,143 Although structurally similar, the VEGF homologs have distinct functions and roles and bind to specific subtypes of VEGF receptors (VEGFRs). VEGF exer ts its effects by binding to one of its three receptors that belong to the superfam ily of receptor tyrosine kinases. VEGFR-1 is a 180 kDa glycoprot ein expressed on many hemat opoietic cells. It regulates blood vessel morphogenesis. 83 This receptor is also required for normal blood vessel development during embryogenesis. Homozygous deletion of VEGF R-1 is lethal in mice at embryonic day E8.5 due to severe malformation of the vasculature.83 VEGFR-2, on the other 34


hand, regulates a diverse range of cellular func tions such as angioge nesis, mitogenesis, cytoskeletal organization, cell migration, vasc ular permeability and mediates cell survival.143 It is a 200 kDa glycoprotein expressed on hematopoietic, neural, and retinal cells. VEGFR-2 is considered the most important mediator in angiogenesis.83 VEGFR-3 is synthesized as a 195 kDa precursor protein consisting of seven extrace llular Ig-like domain, a transmembrane and an intracellular kinase domain. It re gulates lymphangiogenesis and angiogenesis. Expression of this receptor starts at E8.5 of mouse development in all embryonic endothelial cells. After E8.5, VEGFR-3 expression is only seen on developing veins and lymphatics.83 Later in development, the expression gradually becomes restricted to lymphatic vessels. VEGF-A binds to either VEGFR1 (Flt-1) or VEGFR2 (KDR/Flk-1). PI GF and VEGF-B binds only to VEGFR-1. VEGFC and VEGF-D are specific ligands for VE GFR-2 and VEGFR-3, regulating both blood and lymphatic vessel development. Viral VEGF-E and some of the snake venom VEGF-F variants exclusively activate VEGFR-2.83,143 VEGF-A, commonly referred to as simply VEGF, has been extensively studied and is regarded at the most poten t mediator of angiogenesis.13 It is a 34-42 kDa, dimeric, disulfidebound glycoprotein highly expressed in the eye, l ung, kidney, heart, and adrenal gland. There are several splice variants of VEGF including VEGF-121, -145, -165, -189, and -206. VEGF and its receptors are naturally present in the retina. They are important for maintaining angiogenesis and homeostasis in the vascular bed and retinal tissue. VEGF, howeve r, plays a role in not only normal angiogenesis, but also pathological neovascul arization in the eye. Specifically, VEGF is involved in intraocular neovascular syndromes in diseases, such as diabetic retinopathy and retinopathy of prematurity. VEGF expression is induced when cel ls are subjected to hypoxia or hypoglycemia precipitating these disease states. In all these ocular diseas e states, VEGF not only 35


causes uncontrolled neovascular growth that dama ges the retina, but also promotes vascular leakage leading to blood barrier breakdown, which can lead to vision loss. The primary sources of VEGF in the eye include RPE and neural cells such as ganglion cells and Muller cells. VEGF is both necessary and sufficient for the occurren ce of pathological ocular neovascularization. VEGF clearly regulates several key endot helial cell functions in both normal and pathological states. Yet even more noteworthy, VEGF also plays a role in HSC and EPC cell function. VEGF is a potent chemoattractant and plays a role in EPC recruitment. 146-148 It promotes monocyte chemotaxis.149 Gill et al demonstrated that in bu rn patients and those who undergo coronary artery bypass grafting, there is a rapid elevation of VEGF levels followed by immediate mobilization, within 6 to 12 hours of vascular trauma, of VEGFR2+ AC133 cells into the peripheral circulation.27 Recruitment of circulating EPCs to sites of active angiogenesis is mediated through VEGFR2. Galiano and coworkers show topical VEGF is able to improve wound healing by systemically mobilizing BM derived cells to wound injury where they accelerate repair.150 Also, VEGF controls HSC and EP C survival. VEGF-deficient HSCs and bone marrow mononuclear cells show an inability to repopulate lethally irra diated hosts, despite co-administration of wild-type cells.151 More interestingly, VEGF is induced by HIF-1 which then upregulates SDF-1, another po tent EPC modulator and stimulus.99 SDF-1 and VEGF can work in concert such that VEGF and SDF-1 i nduce each other to influence EPC behavior. SDF-1 can mobilize and sequester HSCs, while VEGF recr uits HSCs and promotes their differentiation and proliferation. 152 VEGF clearly has a powerful influence on EPC and HSC recruitment, homing, as well as differentiation of these cells. Stromal Derived Factor-1 (SDF-1) Stromal cell derived factor-1 (SDF-1) bel ongs to a group of chemokine CXC subfamily, 36


originally isolated from murine bone marrow stromal cells. It is produced by multiple bone marrow stromal cell types, as well as epithelial cells. CXCR4, a 7-transmembrane spanning G protein coupled receptor and is the only known receptor for SDF-1.153 Both hematopoietic and endothelial progenitor cells express CXCR4.154 SDF-1 is a potent EPC cytokine and chemoattractant.155-157 SDF-1 mediates homing of stem cells to the bone marrow by binding to its receptor CXCR4 on circulating cells. CXCR4 is also required for maintaining quiescence of primitive hematopoietic cells.153 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.158 Inhibition of the SDF1/CXCR4 axis partially blocks the homing of progenitor stem cells to the ischemic myocardium.159 Also, inhibition of CXCR4 by neutraliz ing anti-CXCR4 antibodies significantly reduces SDF-1 induced migration of EPCs in vitro and reduces in vivo homing of myeloid EPCs to the ischemic limb.160 Overexpression of CXCR4 on stem and progenitor cells promotes their proliferation, migration, and in vivo engraftment of NOD/SCID mice.103 SDF-1 gene expression is regulated by the tran scriptional factor, HIF.99 Progenitor cell mobilization is activated by hypoxia gradients through HIF-1 induction of SD F-1. HIF-1 induced secretion of SDF-1 in ischemic tissue has a direct correlation with reduced oxygen tension. It is proposed SDF-1 induces increased expression of metalloproteina se-9 (MMP-9) activity, which causes cleavage of membrane bound Kit ligand into so luble Kit ligand, known as stem cell factor (SCF). Hence, SDF-1 promotes stem cell mobilization into the circulation. SDF-1 likely acts to sequester HSCs at sites of injury since studies have shown it is required for adhesion of HSCs at sites of injury.146,147 37


SDF-1 has also been implicated in neovascul arization. SDF-1 promotes revascularization of ischemic hind limbs through recruitment of CXCR4+ hemangiocytes. 161 De Falco et al. report that SDF-1 expression fo llowing hind-limb ischemia was up-regulated in plasma and down-regulated in bone marrow, thus mobilizing c-kit+ cells into the peripheral blood. Additionally, SDF-1 and CXCR4 c ontribute to the involvement of bone marrow derived cells and collaborates with VEGF in the development of several types of oc ular neovascularization.138 SDF-1 alone is not sufficient to recruit BMD ce lls to tissues. SDF-1 works in conjunction with others signals such as VEGF to promote BMD mobilization. SDF-1 plays an important role in regulating BM derived cell engraftment a nd function in vascular remodeling and neovascularization. However, SDF-1 is not likely to be the only cytokine that can profoundly impact stem cells. There are likely to be more f actors identified that mobilize and influence stem cells in coming years. One such group of fact ors is the Insulin-like growth factor binding proteins (IGFBPs). Insulin-like Growth Factor Binding Proteins (IGFBPs) Insulin-like growth factor-1 (IGF-1) and II (IGF-II) modulate a diverse range of biological activities including growth, differe ntiation, survival, and regulation of cell metabolism.162-164 In serum and extracellular fluid, circul ating IGFs are sequestered into 150 kDa ternary complexes with IGF bindi ng proteins (IGFBPs) and the liv er derived glycoprotein (acid labile subunit, ALS).163 This complex prolongs the half lif e of IGFs in the circulation and prevents them from crossing the capillary barrier. IGFBPs consist of six homologous secreted proteins, which specifically bind to IGF-1 with high affinity (Figure 1-7). There has been increasing research on IGFBPs in recent years rega rding their role in angiogenesis. Of the six IGFBPs, IGFBP-3 or Insulin-like growth factor binding protein3, has attracted considerable interest as a pro-angiogenic factor and EPC modulator.35,36,165-167 38


Insulin Like Growth Factor Binding Protein-3 (IGFBP-3) IGFBP-3 was first described in 1989 as a tran sporter of 70-90% of all circulating IGF.162 Today, IGFBP-3 is known as the most abundant binding protein in serum and milk. IGFBP-3 can either transport IGF-1 to its receptor, enhan ce IGF-1 actions, or sequester IGF-1 from its receptor.164 It circulates in the seru m, binding IGF-I or IGF-II in conjunction with ALS, to form a 150 kDa circulating complex at a serum concen tration of 100 nM. Human IGFBP-3 is present in various glycosylated forms between 40 and 44 kDa and contains a tota l of 264 amino acids. It contains 3 functional domains: a nonconserved central domain and highl y conserved cysteinerich carboxyl and amino domains. Interesti ngly, IGFBP-3 contains a nuclear localization sequence (NLS), which is responsible for transl ocation into the nucleus via nuclear transport factor importin(Figure 1-8) where it can impact transcription.163 IGFBP-3 is produced and released by Kupffer hepatic cel ls and endothelial cells. The liver and kidney are the main sources of IGFBP-3.163 The level of IGFBP-3 in the serum is regulated by not only its rate of synthesis, but also its post-tr anslational modification and proteolysis. IGFBP-3 proteases include pl asmin, matrix metalloproteases, kallikreins, prostate-specific antigen, and cathepsin. Norm al individuals have mi nimal IGFBP-3 protease activity; however, protease activity is increased in individu als who are pregnant, diabetic or have acute catabolic illnesses.163 Post-translational modifi cations of IGFBP-3 include phosphorylation, methylation, glycosylation, and ubiquitination.163,168 IGFBP-3 concentration in serum is also regulated by other factors such as IGF-1, HIF-1, VEGF, NO, and TGF. IGF-1 affects HIF-1, which increases VEGF and IGFBP-3 expression.162,163 More importantly, IGFBP-3 is hypoxi a-regulated. Hypoxia can induce IGFBP3 mRNA through p53 dependent and independent mechanisms. Likewise, induction of IGFBP-3 39


mRNA was observed in response to hypoxia in wild -type embryonic stem cells. Furthermore, it is upregulated in ischemic tissues.169,170 For example, IGFBP-3 mRNA is upregulated in ischemic brain, specifically in ce rebral vascular endothelial cells.171 The activation of IGFBP-3 is a likely mechanism by which endothelial ce lls respond to hypoxic insult and increase cell survival. IGFBP-3 is known to bind many factors involved in wound hea ling such as heparin, fibrinogen, humanin, plasminogen, plasmin, dermatan sulfate and fibronectin.163 IGFBP-3 can also activate several receptor signaling pathways like TGF, the integrins, and proteoglycans (Figure 1-8). However, IGFBP-3 has no official cell surface receptor. There are two putative receptors: one that was cloned vi a a yeast two-hybrid system us ing the midregion of IGFBP-3 and another termed the low-density lipoprotein-related protein-1 (LRP-1)/ 2M receptor.164 However, these putative IGFBP-3 receptors have not been confirmed or validated. The exact cell surface receptor structure and signaling mechanism of IGFBP-3 remains unresolved. IGFBP-3 has been recognized to have IGF-1 independent effects.172,173 In fact, more research is being focused on IGFBP-3 IGF i ndependent effects thr ough the use of IGFBP-3 mutants that do not bind IGF-1 or IGF-1 analogs with reduced affinity for IGFBP-3. Also, available are IGFBP-3 fragments with total and partial loss of IGF affinity, as well as the existence of IGF-1 negative cell lines (breast cancer and chondrocytes).164 IGFBP-3 depending on the cell type and environment, can be either proor anti-angiogenic.163 IGFBP-3 has been widely studied in cancer regardi ng its anti-angiogenic effects. In vitro IGFBP-3 can inhibit cell proliferation in human breast cancer cells devoid of IGF-1. Also, IGFBP-3, when co-treated with VEGF, inhibits VEGF induced vessel formation in human endothelial cellular vessel formation in matrigel. Furthermore, IGFBP-3 can inte ract with the retinoic acid receptor (RXR), to reduce prostate tumor growth and prostate specific antigen in vivo Likewise, in vivo CD31 40


staining in microvessels and endothelial cells was re duced by half in tumors treated with IGFBP3 versus control, indicating IGFBP-3 s uppresses intra-tumoral angiogenesis. Regarding IGFBP-3s pro-angi ogenic effects, Granata et al. found IGFBP-3 can stimulate neovessel formation in human endothelial cells, but at a very high concentration, 1000 ng/ml, which is well above the physiolo gically relevant concentration.174 Also, IGFBP-3 has been shown to have pro-angiogenic effects on EPCs with regard to enhancing their cell migration, proliferation, and tube formation in vitro, but at physiologically low concentrations.36 Additionally, IGFBP-3 prevents oxygen-induced vessel loss and pr omotes vascular re-growth after ischemic insult in vivo thus reduces patholog ical neovascularization.35,36 IGFBP-3 is now considered more than just a bind ing protein, but an eff ector molecule with clear IGF independent effects on cell growth and proliferation. Even though IGFBP-3 has been widely studied, it still remains an el usive growth factor with no known cell surface receptor. We propose it can signal through another receptor system, the scavenger type receptor (SR-B1) to activ ate downstream cell survival pathways, which promote its pro-angiogenic effect s on EPCs and vascular reparative ability by increasing nitric oxide production (Figur e 1-9). Scavenger Receptor Class B type 1 (SR-B1) In 1996, Krieger and co-workers identified th e Scavenger Receptor class B type 1 (SRB1).175 SR-B1 is a cell surface multiligand r eceptor that can bind high density lipoproteins (HDL) and mediate exchange of lipids with cells. It is a member of the CD36 family of proteins and contains two transmembrane domains, short N and C-terminal cytoplasmic regions. It is 509 amino acids in length. The SR-B1 has a horses hoe-like membrane topology and is heavily N-glycosylated, like IGFBP-3.176 It is also highly expre ssed in the liver and cells of the vascular wall, similar to that of IGFBP3. Also, IGFBP-3 and HDL have been found to be 41


complexed together in the same lipid fraction wh en isolated. Therefore, it is reasonable to postulate SR-B1 may indeed uptake HDL comp lexed with IGFBP-3. Both IGFBP-3 and HDL are sticky molecules. Conse quently, IGFBP-3 may activate SR -B1 and lead to subsequent downstream signaling that may be responsible for IGFBP-3s vascular prot ective nature. SR-B1 activation is well known to gene rate nitric oxide, which play s an important role in the cardiovascular system and plays an important role in endothelial cell migration. IGFBP-3 has been reported to increase nitric oxide produc tion in EPCs, thus we hypothesize IGFBP-3 may signal through the SR-B1 pathway to increase NO production, leading to enhanced EPC cell migration. SR-B1, along with HDL has spar ked the interest of many re searchers regarding their possible role on endothelial cell and EPC function. Recently, Van Eck et al. found SR-B1 in bone marrow-derived cells is eith er pro-atherogenic or anti-atherogenic, i ndicating a unique dual role in pathogenesis of atherosclerosis.177 Furthermore, Mineo et al. report that HDL-SR-B1 promotes endothelial repair.178 Likewise, Seetharam et al. demonstrated impaired reendothelialization was obs erved in SR-B1 knockout mice.179 Even more interesting, HDL by itself has been found to play a role in progeni tor mobilization for endothelium repair. Increased levels of HDL can improve EPC availability in patients with Type 2 Diabetes. Tso and coworkers report injection of HDL into mice increases Sca1+ cells in damaged aortic endothelium.180 Similarly, Sumi et al. found HDL directly stimulates EPC differentiation via PI3K/Akt pathway and enhances ischemia-induced angiogenesis.181 Overall, SR-B1 signaling can influence EPC differentia tion and migration. EPC Homing and Cell Migration Cell migration is an essentia l biological proce ss involved in development, cell growth, wound healing, vascular remodeling, and angiogenesis. If cells fail to migrate, then biological 42


processes can be easily disputed in an organi sm, leading to alterati ons in homeostasis and development of pathological states In order to ensure cells migrate, particularly EPCs, there are a number of stimuli that activate cell migration. EP Cs require potent stimuli to attract, recruit, and direct them to areas in need of vascular re pair or required vascular homeostasis. Some of these include nitric oxide (NO) and sphingosine-1-phosphate (S1P). Also, various cytoskeletal components are involved in cell migration, such as vasodilator associated stimulated protein (VASP). Each of these are disc ussed in detail in the context of their role in EPC homing. Nitric Oxide (NO) Vascular endothelial cells pr oduce numerous vasoactive substa nces that play important roles in the regulation of vascular tone, inflam matory responses, and growth and migration of vascular smooth muscle cells (VSMCs).182-185 Nitric oxide (NO) is one of the most important nonpeptide endothelium-deriv ed vasoactive factors.186 Endothelium-derived NO (ENDO) was initially identified as a main molecule representing the endothelium-derived relaxing factors. It was originally identified in 1980 by Furchgott and Zawadzki. NO is structurally one of the simplest biological molecules. NO is a small, diffusible, lipophilic free reactive radical that participates in a variety of signaling activities in nearly ev ery organ system in the body.186 NO has been shown to inhibit platelet aggregation, leukocyte-endo thelium interaction, and VSMC proliferation and differentiation.187 NO has multiple important regulatory roles in the maintenance of vascular ho meostasis and angiogenesis. 188,189 190,191 NO is generated by NO synthases (NOS). NOS is a heme-containing enzyme that is linked to NADPH-derived electron transport. NOS catalyzes the oxidation of L-arginine to Lcitrulline and NO, with tetrahydrobiot erin and NADPH as essential cofactors.192 Three NOS isoforms have been identified and named after the cell type or condition in which they were first discovered. They include: endothelial NOS (e NOS), neuronal NOS (nNOS) and inducible or 43


inflammatory (iNOS).189 NO has a very short half life, roughly 5 seconds, therefore many studies on NO have reli ed on measuring NOS. All three isoforms of NOS are found in different cell types in the eye.193 nNOS is responsible for producing NO in phot oreceptors and bipolar cells, whereas eNOS is present in vascular endothelial cells. iNOS is found in Muller cells and in th e retinal pigment epithelium. It is involved in inflammatory processes and pha gocytosis of the photorec eptor outer segment. iNOS is also thought to be responsible fo r the pathogenesis of diabetic retinopathy.194,195 The retinal endothelium produces significant NO and as a result plays an important role in eye function and homeostasis. 187,195 NO defects have been implicated in a wide ra nge of diseases. It is well established that endothelial NO bioavailability is systemically reduced in patients with coronary artery disease and heart failure. In diabetic patients redu ced NO bioavailability may result from altered NO metabolism.123,131 In diabetic mice, vascular endothelial dysfunction is associated with uncoupling of eNOS within the endo thelium that is caused by oxidation of its essential cofactor tetrahydrobiopterin, resulting in a specific loss of NO bioavailability.13 The 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 reduced. NO modul ates various physiological pro cesses and has a major role on endothelium health and maintenance. NO also has an important function in the stem cell microenvironment as a molecular mediator in controlling the stem cell niche.196 eNOS deficient mice have an impaired capacity to mobilize stem cells from the bone marrow as reported by Aicher and associates. Guthrie et al. found that NO/NOS pathway is a significant re gulator of neovascular ization. NO can modulate 44


hemangioblast and EPC activity by dictating th e size and branching characteristics of blood vessels that are formed in res ponse to ischemic retinal injury.102 Moreover, Kaminski et al. show the presence of eNOS appears to be a crucial and specific factor for firm c-kit+ cell adhesion to the vascular endothelium.197 Michurina et al. report NO is a regulator of HSC activity and Grant et al. show NO modulates HSC behavior and vascular phenotype in the retina. Essentially, NO is a multifactorial signaling molecule and plays a key role in EPC migration. Sphingosine-1-Phosphate (S1P) Sphingosine 1-Phosphate is a bioreactive sphing olipid metabolite that is an extracellular mediator.198-201 It has been implicated in angiogene sis, wound healing, embryonic development, cytoskeletal organization, adherens junction assembly, vascular permeability, and morphogenesis. 198,202,203 Sphingosine Kinase (Sphk) catalyz es the formation of S1P from sphingosine, an abundant lipid in most cell me mbranes, while sphingosine lyase breaks down S1P in most cells. Basal levels of S1P in cells is low, but can rapidly increase when cells are exposed to mitogenic and/or angiongenic grow th factors such as EGF, IGF-1, or VEGF.200,204 These signals activate Sphk, the enzyme that phosphorylates sphingosine to produce S1P, which is ultimately responsible for incr eased levels of S1P. S1P levels inside cells are tightly regulated. The plasma concentration of S1P is approximate ly 200 nM and more than doubles in the serum to 500 nM.198,199,202,205 Both red blood cells and endothelial cells are reported to be the major sources of S1P. However, activated platelet s also store and produce large amounts of S1P.198,205 Sphk is an evolutionarily conserved lipid kinase, which consists of five conserved domains. 198,205 There are two isoforms of mammalian Sphk, Sphingosine Kinase type 1 (Sphk1) and Sphingosine Kinase type 2 (Sphk2) that have been characterized. Sphk1 is mainly expressed in the cytosol, whereas Sphk2 is localized to the nucleus.206 Sphk1 and Sphk2 have contrasting biological roles. Sphk1 is consid ered anti-apoptotic, thus promo ting cell growth and survival, 45


while Sphk2 enhances apoptosis. The biological roles of Sphk have been studied using N,N dimethylsphingosine (DMS), a pharmacological inhibitor, which blocks both Sphk1 and 2. This inhibitor was used in in vitro studies, which are described in subsequent chapters. S1P acts as a ligand for plasma membrane localized G protein coupled receptors (GPCRs) known as endothelial differentiation gene receptors (EDGR) or more commonly S1PRs.207 S1P binds to the five members of this receptor family: S1PR1, S1PR2, S1PR3, S1PR4, or S1PR5 (previously referred to as EDG 1,5,3,6 and 8 respectively). The S1P receptors are expressed in wide range of organs and systems such as the cardiovascular, immune, reproductive, respiratory, and nervous systems.200 They also have wide range of biological effects such as regulating ce ll migration, differentiation, proliferation, and survival. S1P receptors are particularly important in vascular maturation and angiogenesis.208 S1PR1 null mice are embryonic lethal due to massive hemorrhag ing that is caused by incomplete vascular maturation of arteries and capillaries.208,209 Endothelial cells form a vascular network, but smooth muscle cells and pericytes fail to be recr uited to stabil ize the vessels in S1PR1 knockout mice. Also, in vitro, reduction of S1PR1 expression by antis ense oligos inhib its endothelial cell proliferation, migration, and tube formation. S1PR 1 plays an important in endothelial cells and consequently is the most highly expressed on endothelial cells. S1P has been shown to have profound e ffects on cell migration, specifically on endothelial cells. S1P has an impact on the cytosk eletal actions such as aggregation, contraction, shape change, and adhesion. S1P activates the small GTPases Rac and Rho, thus acting as a chemoattractant for endothelial cells. S1P indu ces Rho-dependent integr in clustering in focal contact sites, which modulate cel l adhesion, spreading, and migration.210 S1P can also phosphorylate protein kinase Akt in endothelial cells, leading to cell migration. At low levels 46


S1P can even act as a chemoattractant for CD4 and CD8 T immune cells. S1P can also trigger invasion of primitive hematopoietic cells into stromal layers.211 Yet even more striking, S1P has also been shown to have a substantial role in the mobilization and homing of stem ce ll and progenitor populations. Whetton et al. was the first to show that murine HSCs that are Lin-Sca+Kit+, express S1P receptors, specifically S1PRs1-4.212 Additionally, it was shown S1P substantially enha nced the chemotactic migratory response of mouse HSCs to SDF-1.213 With regard to human CD34+ cells, Seitz et al. found that S1P can act as a direct chemoattractant for these progenitors in vitro .214 Furthermore, they showed sustained S1P1R activation in vivo via FTY720, a specific S1PR1 agon ist, increased engraftment of human CD34+ cells in NOD/SCID mice. Similarly, Walter et al. found that human EPCs treated with S1P or FTY720, increased flow in ischemic murine limbs via S1PR3.213 Bonder et al. recently showed sphingosine kinase regulates the rate of EPC differentiation.215 When Sphk1 levels are reduced, EPC mobilizatio n of the bone marrow is increased, and the rate of endothelial cell differentiation is hastened. Together, these studies demonstrate an emerging picture for the importance of S1P in regulation HSCs and EPCs, sp ecifically regarding thei r migratory potential. Thus, we propose IGFBP-3 may activate S1PRs in order to enhance EPC migration (Figure 19). Vasodilator Stimulated Phosphoprotein (VASP) Nitric oxide-dependent, vasodilator stimul ated phosphoprotein (VASP) plays a pivotal role in cytoskeletal actin regul ation. VASP belongs to a family of proline-rich proteins that include the Drosophilia melangaster protein Enabled (Ena), it s mammalian ortholog Mena, and the Ena-VASP-like protein Evl.216 All Ena/VASP family memb ers share a highly conserved amino-terminal Ena/VASP homology 1 (EVH1) doma in followed by a proline rich central region and a carboxy-terminal Ena/VASP homology 2 (ENVH2) domain.217 The structure of ENV1 47


domain of Ena/VASP family has been identifi ed by using X-ray crys tallography and nuclear magnetic resonance spectroscopy. The EVH1 doma in serves as an Ena/VASP protein-binding site for the focal adhesion proteins includi ng vinculin, zyxin, and axon guidance proteins roundabout (Robo). EVH1 domain-protein inter actions are necessary for localization of Ena/VASP family to focal adhesions, as well as to the periphery of protruding lamellipodia.216,218 The central proline rich region has binding si tes for several SH3 and EE 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 importa nt for both actin filament bundling and stabilization.216,217,219 VASP is a cytoskeletal actin filament protein, which is involved in platelet activation, cell adhesion, and migration.220,221 VASP mutant mice exhibit defects in actin-dependent process of platelet aggregation.222 The results from site-directed mutagenesis and overexpression studies, suggest the importance of Ena/VASP pr oteins in the developmental and physiological processes in various cell type s. For example, VASP modulates T cell activation, phagocytosis, and epithelial morphogenesis. It also induces migration of neutrophils, fibroblasts, and neurons.223,224 In mammalian cells, Ena/VASP proteins localiz e within cells to areas of dynamic actin organization such as the leading edge of lamelli podia and at the tips of filopodia and other actindependent intracellular structures like cell-cell contact s, focal adhesions, and in periodic puncta along stress fibers. In endotheli al cells, VASP functi ons in membrane ruffling, aggregation, as well as tethering of actin filaments during the form ation of endothelial cell -substrate and cell-cell contacts. VASP plays a distinct role in endothelial cell migration and function. Oelze et al. 48


reported the levels of vessel phospho-VASP lead s closely follows changes in endothelial function and vascular oxidative stress.225 Li Calzi et al. showed NO mediates cytoskeletal changes through siteand cell-type specific VASP phosphorylation in endothelial cells.127 Hence, we postulate exogenous IGFBP-3 ge nerated nitric oxide can promote VASP redistribution in endothelial cells which can ultimately enhance e ndothelial cell migration (Figure 1-9). Significance IGFBP-3 has been identified as an impor tant binding and carrier protein for IGF. However, it physiological and syst emic roles extend much further than a simple binding protein. In fact, considerable research has been conducte d on IGFBP-3s IGF independe nt effects, as well as its proand antiangiogenic effects. IGFB P-3 has direct IGF inde pendent effects on cell migration, proliferation, differentiation, and a poptosis. However, IGFBP-3s mechanism of action still baffles many researchers, especia lly since no known cell surf ace receptor has been identified. IGFBP-3s pro-angiogenic role on EP C-mediated repair is especially confounding. The goal of this study was to highlight IGFB P-3s role in enhancing cell migration of EPCs, and its vascular reparative effects. Here we demonstrate IGFBP-3 increases EPC migration through NO generation via act ivation of the SR-B1 receptor. Specific Aims and Hypotheses Three specific aims were set forth for the st udies of IGFBP-3 in hum an EPCs and in two mouse retinal vascular injury models. The firs t aim was to examine the effects of IGFBP-3 on cell migration in both EPCs and endothelial cells. The second aim was to determine the mechanism of action of IGFBP-3 on promoting cell migration. The third aim was to determine whether IGFBP-3 is cytoprotectiv e in two different types of mouse retinal vascular injury models. 49


The underlying hypotheses are: (1) IGFBP3 can act as an EPC homing factor in vivo in an adult retinal ischemiainduced injury mode l by stimulating EPCs to migrate to sites of vascular injury. (2) IGFBP-3s mechanism of action on cell migration in both EPCs and resident vascular endothelial cells is generation of exogenous NO via SR-B1. (3) IGFBP-3 can aid in trafficking EPCs to unstable, damaged vessels for needed repair by influencing vascular permeability, which leads to vascular stabiliza tion and cytoprotection of retinal blood vessels. Overall, it is hypothesized IGFBP-3 has vascular protective e ffects by influencing EPC and endothelial cell migration via NO generati on by SR-B1, which leads to the homing and reparative effects of these cells in injured retinal vasculature. 50


Figure 1-1. Anatomy of the human eye. The eye is made up of many structures, which are essential for clear vi sual perception. 51


Figure 1-2. Human retinal layers. This diagram is a depiction of a retinal cross section of the human retina. From the most anterior layers of the retina, the retinal layers consist of the sclera, choroid, re tinal pigment epithelium, rod and cone photoreceptors, outer nuclear layer, outer plexiform layer, inner nuclear layer, inne r plexiform layer, ganglion cell layer, and the inner liming membrane. 52


Figure 1-3. Retinopathies. A) S hows stages of ROP. Stage 1 i nvolves a demarcation line where normal and abnormal vessels meet. Stage 2 is characterized by an intraretinal ridge that rises up from the retina as a result of abnormal vessel growth. Stage 3 contains a ridge which grows from the spread of abnor mal vessels and extends into the vitreous. Stage 4 and 5 (plus) is characterized by re tinal detachment with Stage 5 being total retinal detachment and blindne ss. B) Contains representati ve images of the two forms of Diabetic Retinopathy, Proliferativ e Diabetic Retinopathy (PDR) and NonProliferative Diabetic Retinopathy (NPDR). NPDR, also known as background retinopathy, involves the accumulation of bl ood and/or exudative deposits in the retina from tiny blood vessels that leak (m icroaneurysums). In advanced stages of NPDR fibrous vascular tissue accumulates in the retina leading to visual impairment. PDR is characterized by the formation of abnormal blood vessels (neovascularization) growing on the surface of the retina or optic disc. These blood vessels are typically fragile and easily ruptured. 53


Figure 1-4. Blood vessel development. Blood vessels are either developed by vasculogenesis angiogenesis. During vasculogenesis, endothelial progenitor cells (CD34+ cells) can assemble to form new vessels during embryogenesis. During angi ogenesis in the adult, sprouts form from pre-existing blood vessels and migrat e into the surrounding tissue in th e adult. This process re on proliferation, migration, and remodeling of fully differentia ted endothelial cells. Neovascularization can also occur with EPCs, wh ich circulate postnatally in the peripheral blood. They can be recruited and in corporated into sites of active neovascularization in ischemic tissue by or lies growth factors such as IGFBP-3, which ar e released by ischemic tissue in response to injury. 54


Figure 1-5. Hypoxia-regulated gr owth factors and bone-marrow de rived cells. HSCs and EPCs are derived from a common precursor called the hemangioblast. These cells maintain primitive characteristics and can differentiate into a wide range of cells types as shown. CD34+ cells are EPCs. These cells are mobilized by many factors such as SDF-1 and VEGF, which govern their mi gratory and homing ability into blood vessels undergoing ischemic insult. EPCs ar e recruited to areas of ischemia where they can participate in vascular repa ir and functional revascularization. 55


Figure 1-6. Recruitment of EPCs in ischemic tissue. Circulating EPCs, specifically CD34+ cells, are recruited to areas of hypoxic vascular inju ry such as in the retina where they can home, integrate, stabilize, and promote proper va scular repair. 56


Figure 1-7. IGF system. Ligands (IGF-1, IGF-2 and insulin) IGFBPs (1-6), as well as receptors (IR, IGF-1R, hybrid IR/IGF-1R, IGF-2R, I RR, and IGFBP-R) are depicted. IGF-I interacts with IGF-1R, IR, hybrid IR/IGF-1R, and IGFBPs; IGF-2 interacts with IR, IGF-1R hybrid IR/IGF-1R, IGF-2R, and IGFBPs; insulin interacts with IR, IGF-1R, and hybrid IR/IGF-1R. Some IGFBPs ar e cleaved by IGFBP proteases releasing proteolysed fragments, which have low a ffinity for IGFs. IGFBP-3 and IGFBP-5 may act through their own receptor, which to da te has not been identified. 57


Figure 1-8. IGFBP-3 signaling a nd cross-talk. IGFBP-3 has no know n receptor. However, it can signal through other receptors such as TGFreceptor. IGFBP-3 can also interact with proteoglycans and integrins to cont rol cell adhesion and/or growth. IGFBP-3 contains a NLS sequence which allows it to enter the nucleus via importin. In the nucleus IGFBP-3 can interact with the RX R receptor to influence transcription. 58


59 Figure 1-9. IGFBP-3 signaling to increase NO leading to cell migration. IGFBP-3 can activate the SR-B1 or S1PR to increase NO production and release via the PI3K/Akt cell survival pathway. IGFBP-3 also induces VASP re-distribution, which promotes cell migration. IGFBP-3 increases NO producti on in both EPCs and endothelial cells, which allows them to migrate to areas of needed repair or vascular instability. Likewise, NO production and release by these cells can also attract other cells to promote vascular remodeling in ar eas of vascular damage.


CHAPTER 2 METHODS AND MATERIALS In Vivo Studies All animal procedures conducted in this study were in agreemen t with the National Institute of Health (NIH) Guide for the Care a nd Use of Laboratory An imals (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, adult female C57BL/6 mice, and C57BL/6-tg (UBC) transgenic gfp+ mice were obtained from Jack son Laboratories (Bar Harbor, ME) and housed in a temperat ure controlled room with a 12 hour light/dark cycle in the University of Florida Health Science Center Animal Care Resource facilities. Approximately 50 mouse pups, 50 adult female C57BL/6 mice and 50 adult female chimeric gfp+ mice were used in various studies. We performed a series of in vivo assays to assess IGFBP-3s effect on EPC migration, incorporation, and differentiation in th e mouse retinal vasculature. We also evaluated IGFBP-3s anti-apoptotic effects in the mouse vasculature via TUNEL analysis and mass spectrophotometry. Lastly, we assessed IGFBP-3 s effects on vascular permeability. Figures 2-1 through 2-3 summarize the in vivo studies conducted. Oxygen Induced Retinopathy (OIR) Model We used the OIR model developed by Smith et al. which was described in chapter 1. It is also depicted in Figure 2-1. The OIR mode l induces the formation of pre-retinal neovascularization in mouse pups. On postnatal day 7 (P7), the pups and the nursing dam were placed into an oxygen (O2) chamber and exposed to hyperoxic levels of O2 (~75%) for 5 days. On P12 the mice were returned to room air. The return to normoxic conditions (21% O2) after being held at high O2 stimulates a hypoxic stimulus that ini tiates the formation of the pre-retinal 60


vasculature. The mice were sacrificed at either P12 or P17 and their retinas were harvested for various studies. One cohort of mice (n=24) underwent the OIR model and were euthanized at P17 to examine TUNEL apoptosis analysis of IGFBP-3 injected and vector in jected control mouse pups. We used the OIR model to assess whether IGFBP-3 has anti-apoptotic effects on specific vascular cell types such as endothelial cells, astrocytes, and pe ricytes. (Figure 2-2) The OIR model was ideal for this since the developing neonate vasculat ure is actively undergoing rapid cell turnover and remodeling, compared to the adult vasculature, wh ich is relatively quiescent. The mice were intravitr eally injected with 0.5 l in the right eye on P1 with either plasmid expressing mouse IGFBP-3 (2 g/ml) under control of a proliferating endothelial cellspecific promoter (Cdc6) (n=12) or the cloning vector as an injection control (n=12). By utilizing a proliferating endothelial cell-specific promoter IGFBP-3 expression was targeted to endothelial cells, thus to areas of neovascularization. For intravitreal injection in to P1 mouse pups, iceinduced anaesthesia was performe d by placing the neonate on a pl astic shield over a layer of crushed ice for 1-2 minutes. The mice were euthanized at either P12 or P17 for simultaneous TUNEL analysis of retinal flatmounts (n=12) and for immunohistoc hemical staining of endothelial cells, pericytes, and astrocytes (n=12). The eyes were fixed with 4% paraformaldehyde for 1 hour at room temperature. The retinas were dissected from the posterior eye cup and incubated with antibodies for astr ocytes (S-100, Sigma), endothelial cells (GS isolectin B4, Sigma) and pericytes (NG2, Chem icon) followed by staining with a commercial TUNEL detection kit (Roche, Switzerland) to detect cells undergoing DNA fragmentation. The eyes from the neonate mice injected with IGFBP3 plasmid were compared with eyes from mice 61


injected with empty plasmid vector or the uninjected eye of the same animal (contralateral eye). Three different regions of the re tinal flatmounts were studied: the central, mid-peripheral, and peripheral regions. Statistical Analysis of Cell Death in OIR Model In order to quantify cell death in the OIR model, representati ve fields of view from the central, mid-peripheral, and periphe ral retinas were counted using a 20x objective as the field of view for analysis as modified by Hughes et al .226 In each field of view TUNEL+/GS Lectin+ vascular endothelial cells, TUNEL+/NG2+ pericytes, and TUNEL+/S-100+ for astrocytes were counted. The data is collected as mean standard deviation (SD) N=6 per experimental group, and the statistical significance of differences among mean values was determined by one-way ANOVA. ANOVA statistical analysis was performed with SPSS 13.0 software (SPSS, Chicago, IL). A p-value of less than 0.05 was considered statistically significant. Quantification of Astrocyte Ensheathment in OIR Model We also assessed the frequency of S-100 ensheathment of retinal vessels in the OIR model. We studied the astrocytes in OIR model, since they serv e as a template for developing vessels. Astrocytes also help to stabilize bl ood vessels and we propose IGFBP-3 may have an influence on these pa rticular cells. We determined the astrocyte ensheathmen t using a modified method previously described. Representative fields of view from the mid-peripheral retina were counted using a 20x objective as the field of view for analysis. Ea ch confocal image was overlaid with a 10 x 10 equally spaced grid using Adobe Photoshop V 5.0. The grid was superimposed onto each image. The occurrence of S-100 labeling relative to lectin labeling at the 100 intersection points yielded the percentage of astrocyte ensh eathment. The data was collected as mean standard deviation (SD) where n=6 per experimental group and th e statistical significance of differences among 62


mean values was determined by t-Test. A p-valu e of less than 0.05 was considered statistically significant. Electron Microscopy in the OIR Model In order to assess apop tosis in the neural retina, we us ed electron microscopy to assess retinal cross sections for their morphology and cell structure. Electron microscopys fine detail allowed us to clearly delineate whether neuron al cells in the retina were undergoing active apoptosis in IGFBP-3 treated versus untreated neonatal mouse retinas. A second cohort of mice (n=12) was injected as previously describe d with a total of 6 mice injected with IGFBP-3 plasmid and 6 mice inj ected with the empty vector plasmid control. (Figure 2-2) The mice were euthanized at P17, whole eyes globes were harvested, and processed for electron microscopy analysis. The eyes were fixed with 4% paraformaldehyde/2.5% glutaraldehyde for 4 hours. The eyes were embedded in epoxy resin and stained for electron microscopy as previously described. 227 Generation of Adult Chimeric gfp+ Mice Chimeric mice have been used extensively for in vivo studies to assess homing and the reparative capacity of EPCs, especially in ocul ar injury models. Green fluorescent protein (gfp+) chimeric mice are easy to produce, have high engr aftment efficiency, and are relatively stable over time. They serve as an excellent tool to study and track EPC recr uitment, differentiation, and incorporation into the retinal vasculature. Bone Marrow Isolation & Transplant C57BL/6 Tg mice (Jackson Laboratories, Bar Harbor, ME) are homozygous for gfp expression under control of the ubiquitin promot er. These mice were bred and housed at the University of Florida. For the bone marrow isol ation, male C57BL/6 mice were anesthetized by intraperiotoneal injection of a mixture of xylazine (30 mg/ml) and ketamine (14 mg/ml) at a dose 63


of 5 l per 20 grams of body weight. The animals we re sacrificed by cervical dislocation. The tibias and fibulas were removed and placed on ic e. Both ends of all bones were removed and bones were flushed with ice-cold PBS using a 27 gauge needle. The bone marrow mixture was collected in a 15 ml conical tube, centrifuged at 19,000 g for 10 minutes at 4 degrees. The supernatant was discarded and the pellet was re suspended in 2 ml of PBS. Controls were prepared in 5 ml polypropylene tubes. The fi rst control contained 1 ml of PBS with 30 l of the resuspended bone marrow. The second control had 1 ml of PBS, 30 l of bone marrow, and 1 l of Sca-1-phycoerythrin antibody (S ca-1-PE, BD Pharmingen, San Diego, CA). The third control consisted of 1 ml of PBS, 30 l of bone marrow, and 1 l of c-kit-allophycocyanate antibody (ckit-APC, BD Pharmingen). The sample c ontained the remaining bone marrow and 120 l each of the Sca-1-PE antibody and the c-kit-APC antibody in a 15 ml conical tube. These were all incubated at 4 degrees for 15 minutes to allow th e antibodies to attach to the antigens. PBS was added to the controls and sample. All tubes we re centrifuged. The supernatant was discarded to remove unbound antibodies. The controls were resuspended in 500 l of PBS, while the sample was resuspended in 10 ml of PBS. The total volume was then filtered using polystyrene tubes with cell strainer caps (Becton Dickinson, Frank lin Lakes, NJ). The bone marrow cell population was then enriched for HSCs by fluorescenceactivated cell sorting (FACS Calibur flow cytometer, BD Biosciences, San Jose, CA). Only cells that were c-kit+/Sca-1+/ gfp+ were selected and placed in PBS supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) on ice until needed. The cells were then centrif uged and resuspended in a volume adequate to yield 5,000 cells per 100 l. Adult female C57BL/6 mice were anesthetized and lethally irradiated (950 rads) using a cesium source. The recipient mice (6-8 weeks age of age) were 64


injected in the retro-orbital sinus (ROS) with 100 l of c-kit+/sca-1+/gfp+ cells. The transplanted mice were followed and allowed to engraft for three months. Chimeric Engraftment Confirmation After three months, successful engraftment of the mice was confirmed by flow cytometry analysis of peripheral blood. Approximately 500 l of blood was obtained from the transplanted mice by tail venipuncture and added to the same volume of PBS with 10 mM EDTA (ethylenediaminetetraacetic acid) to prevent coagulation. Two ml of Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ) was added to each sample and the samples were centrifuged at 125 g at room temperature for 30 minutes. After centrifugation, glass pipettes were used to collect the buffy coat (comprised of ly mphocytes), which was then placed in a clean tube. A total of 2 ml of PBS was added and th e samples were centrifuged at 55 g at 4 for 5 minutes to remove any residual Ficoll. Afte r discarding the supernatant, 500 l of PBS was added and the number of gfp+ cells was determined by flow cytometry. Mouse samples in which the concentration of gfp+ cells was higher than 80% were consid ered adequately reconstituted. Only these highly engrafted mice were used for experimentation. Retinal Laser Vessel Occlusion Model In order to assess whether IGFBP-3 enhan ces EPC homing and recruitment to retinal vessels, we had to induce ischemic injury to th e adult retinal vessels. Th e mice were lasered by delivering ~50 spots at 150 milliwatts for 0.2 seconds with an Argon green laser in a transpupillary manner to the right eye to indu ce retinal vessel occlusi on. Two groups of adult C57BL/6gfp+ chimeric mice: laser only (n=15) and la ser with IGFBP-3 injection (n=18) were subjected to laser injury in th eir right eye. A third group of ch imeric mice received only IGFBP3 injection into their right eye (n=16). The IGFB P-3 plasmid under the endothelial cell specific 65


promoter (Cdc6), was packaged into liposomes Immediately following laser treatment to the right eye a total of 2 l of IGFBP-3 plasmid (1 g/ml) packaged into liposomes was delivered intravitreally into the right eye of the de signated groups with a 39 gauge Hamilton syringe (Reno, NV). All mice were sacrificed 3 weeks post laser treatment and thei r eyes were harvested for microscopy analysis. The mouse eyes were fi xed for 1 hour at room temperature with 4% PFA. The neural retinas were dissected from the eye cup and washed with PBS prior to immunostaining. Immunostaining & Microscopy Analysis In order to visualize the retinal vasculatur e in the adult chimeric mice, retinas were stained with rhodamine-agglutinin (Vector Labs, Burlingame, CA), a blood vessel specific stain, which clearly labels both large and small vesse ls in the mouse retina. The retinas were permeabilized with 0.2% triton-X-100 and 0.2% BSA in 10mM HEPES buffer overnight at 4 degrees followed by incubation with rhodamine-agglutinin at 1:1000 overnight. The retinas were then stained with an anti-GFP antibody (Chemi con, Temecula, CA) at 1:500 to confirm gfp+ cell incorporation into the retinal vasculature. Also, the adult re tinas were stained with three additional markers to determine the fate of the gfp+ cells. (Figure 2-3) These markers include: Griffonia Simplicifolia isolectin B4 (Sigma) for detection of endothelial cells, NG2 (Chemicon) for detection of pericytes and smooth muscle cells and S100 (Sigma) for detection of astrocytes. After immunostaining, the mouse retinas were fl atmounted by placing radial cuts in the tissue and mounting on a glass coverslip with VectaShiel d (Vector Labs). Retinas were examined using a Leica-argon krypton laser mounted on a Le ica DMRBE epifluorescence photomicroscope (Leica, Wetzlar, Germany) using OpenLab Imag ing software. Images were taken at 20x and 40x to examine fine detail of the retinal blood vessels. 66


Mass Spectrophotometry Analysis Adult non-chimeric C57BL/6 mice were subjec ted to retinal vessel occlusion (n=6) and either injected (n=6) or uninjected (n=6) with IGFBP-3 plasmid as described earlier for mass spectrophotometry analysis. The mi ce were sacrificed th ree weeks later and their retinas were harvested to assess sphingosine1-phosphate, ceramide, and sphingom yelin lipid ratios. Retinal homogenates were extracted with 200 l/mg tissue of chloroform:methanol (2:1 v/v) by vortexing for 1 minute. After centrifugation at 3000g for 10 minutes, the lower organic phase was recovered and the aqueous upper phase was re-extracted with 200 l/mg tissue of chloroform. The pooled organic phases were evaporated under nitrogen, resuspended in chloroform:methanol (2:1 v/v) and washed w ith HPLC-grade water. The solvent was again evaporated under nitrogen, and lip ids were further dried overnight under vacuum. Lipid extracts were resuspended in 50 l/mg tissue of isopropanol:methanol:chl oroform (4:2:1v/v/v) and stored under nitrogen in glass vials in the dark at -80 C until further use. Prior to mass spectrophotometry analysis, lipid extracts were diluted 1:20 in isopropanol/methanol/chloroform (4:2:1 v/v/ v) containing 20 mM ammonium hydroxide. The final concentrations of internal standards added to the lipid ex tracts were 300 nM for GPCho and 12.5 nM for sphingolipids. All samples were centrifuged, loaded into Whatman Multichem 96 well plates (Fisher Scientific) and sealed with Teflon Ultra-Thin Sealing Tape (Analytical Sales and Services, Pompton Plains, NJ). Lipids we re introduced to a Thermo model TSQ Quantum Ultra triple quadruple mass spectr ometer (San Jose, CA) at a flow rate of ~250 nL/min via a chip-based nanoelectrospray ionization (nESI) source (Advion NanoMate, Ithaca, NY) operating in infusion mode using an ESI HD-A chip, a sp ray voltage of 1.4kV, a gas pressure of 0.3 psi and an air gap of 2 L. The ion transfer tube of the mass spectrometer was maintained at 150 C. All MS/MS spectra were acquired automatically by methods created using Xcalibur software 67


(Thermo, San Jose, CA) with a scan rate of 500 m/z second1, and Q2 collision gas pressure of 0.5 mtorr. GPCho and SM species were analyzed as [M+H]+ ions by parent ion scanning of m/z 184 in positive ion mode, and as [M+Cl-H]ions by neutral loss scanni ng of 50 Da in negative ion mode. Ceramide species were analyzed as [M+H]+ions by parent ion scanning of m/z 264.4 in positive ion mode. GPIns species were analyzed as [M-H]ions by parent ion scanning of m/z 241 in negative ion mode. Fatty acid constituents of GPCho species were confirmed in negative ion mode by product ion scanning of [M+Cl-H]ions of interest. Collision energies for each MS/MS scan were optimized using commercially available synthetic lipi d standards. MS/MS spectra were averaged over a pe riod of 5 minutes, and a five po int Gaussian smooth was applied to all spectra prior to data analysis. Vascular Permeability Non-chimeric C57Bl/6 mice were used for in vivo permeability experiments. In the first set of permeability experiments, there were a to tal of five experimental groups. These included IGFBP-3 alone (n=12), VEGF alone (n=12), VE GF followed by IGFBP-3 injected 6 hours later (n=12), VEGF followed by IGFBP-3 injected 24 hours later (n=12), and IGFBP-3 vehicle consisting of acetic acid (n=12). The mice were intravitreally injected with recombinant VEGF (100 ng/ml) and/or recombinant non-IGF binder mutant IGFBP-3 (100 ng/ml). Recombinant VEGF was administered to induce permeability fo llowed by IGFBP-3 injection at 6 hours or 24 post VEGF injection. After 48 hours following the initial VEGF injection, the mice received a tail injection of FITC albumi n. Two hours later the mice were sacrificed by cardiac perfusion with 4% PFA. The retinas were immediately harves ted and processed for fluorescence analysis. In the second set of permeability experiments, non-chimeric C57BL/6 mice underwent laser injury and IGFBP-3 intravit real injection as previously de scribed earlier in the chapter. There were a total of three experimental gr oups: laser only (n=12), IGFBP-3 injected only 68


(n=12), and laser and IGFBP-3 injected (n=12). After 4 days post laser treatment, the mice received a tail injection of FI TC albumin. Two hours later the mice were sacrificed by cardiac perfusion with 4% PFA. The retinas were imme diately harvested and processed for fluorescence analysis. In Vitro Analysis Various in vitro assays were conducted to assess IGFBP-3s effects on nitric oxide production, eNOS expression, and VASP re-distri bution in endothelial cells. Figure 2-4 summarizes the in vitro experiments conducted. Four type s of cells were used in the in vitro experiments. These include human CD34+ cells (EPCs) from normal patients, human microvascular endothelial cells from the lung (HMVEC-L), human umbilical vein endothelial cells (HUVECs), and rat retinal ganglion ce ll line (RGC-5). Human recombinant IGFBP-3 protein (Upstate Cell Signaling) was used at a concentrati on of 100ng/ml in all in vitro experiments. This concentration is reflectiv e of physiological leve ls found in human blood serum. We have previously shown this concen tration has the desired maximum effect on EPCs.36 Also, various inhibitors were us ed to block nitric oxide such as L-NAME, a blocking antibody to SR-B1, and inhibitors for Akt and PI3K. These inhib itors were used at con centrations previously reported in the literature for e ndothelial cells. Also, human HD L isolated from normal patient blood, was used at a concentration of 1mg/ml, within the physiological range found in human serum. Basically, the in vitro studies were conducted to seek a mechanism of action of IGFBP-3 in promoting vascular repair. Our in vitro studies were designed w ith emphasis on how IGFBP-3 influences cell migration, both EPC and mature endothelial cell migration. EPC Isolation Endothelial progenitor cells (EPCs) were isolated from peripheral blood from healthy patients. The blood was collected into cell prepar ation tubes (CPT; BD Biosciences, San Jose, 69


CA) and spun to obtain mononuclear cells. EPCs were separated from the mononuclear fraction using a CD34+ isolation kit (StemCell Technologies, Vancouver, CA). Mononuclear cells (2 x107) were incubated with a CD34+ selection cocktail for 15 minutes. A total of 50 l of nanoparticles were added to the cells and incuba ted for an additional 10 minutes. The suspension volume was increased to 2.5 ml a nd 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 (StemSpan, Vancouver, BC, Canada). The isolated EPCs were used for western blot and nitric oxide studies. Cell Culture of Endothelial Cells Mature endothelial cells, HMVEC-L and HUVECs were maintained in culture with EBM-2 media enriched with ali quot growth factors (Lonza, Walk ersville, MD). The cells were used at passage 2-4 with distinct cobble stone morphology, which is indicative of their endothelial phenotype. The RGC-5 cells were grown in DMEM supplemented with 20% FBS. All three cell types were gr own at 37 degrees under 5% CO2. Nitric Oxide Measurement In order to assess whether IGFBP-3 increa ses nitric oxide, a DAF-FM nitric oxide production assay and radioactive nitric oxide activity assay were performed. Also, eNOS expression and peNOS expression was evaluated vi a western blotting, as well as evaluation of expression levels of the S1PRs1-5 and SR-B1 in the mature endothelial cells and EPCs. DAF-FM NO Production Assay NO production was quantified in HUVECs, HMVE C-Ls, rat retinal ganglion cells (RGC5) and human CD34+ cells using NO sensitive cell permeant fluorescent dye DAF-FM. HUVECs, HMVEC-L, and RGC-5s were cultured on cover-slipped bottomed dishes (MatTek, Ashland, MA). CD34+ cells in suspension were loaded with DAF-FM diacetate (Invitrogen) (10 70


M for HUVECs, HMVEC-Ls, and RGC-5s; 20 M for the CD34+ cells) for 30 minutes in Dulbeccoss phosphate buffered saline with calciu m and magnesium (Mediatech, Inc., Manasses, VA) supplemented with glucose (1 mg/ml) and L-arginine (1mM). DAF -FM loaded cells in dishes were placed on the stage of Axiovert inve rted microscope with 20x fluar objective (Zeiss) for fluorescence imaging. In the case of CD34+ cells, approximately 75 l of cell suspension was placed in the coverslipped dish to perform imaging. Fluorescent images were obtained using a computer-controlled monochromator excitati on light source (TILL Polychrome II, TILLPhotonics, Martinsried, Germany) and a cooled CCD camera with exposure control. Images were analyzed and fluorescence was measured in arbitrary units using Till vision. To evaluate the effect of IGFBP-3 and/or HDL on NO production, cells were treated with these agents for 30 minutes. Some dishes were incubated with Scav enger Receptor (SR-B1, Novus Biologicals, Littleton, CO) blocking antibo dy for 30 minutes (1:100) before loading cells with DAF-FM. Changes in DAF fluorescence wi th different treatments were expressed as percent change. Results were evaluated for statistical significance by one-way ANOVA. eNOS Activity Assay Activation of eNOS by IGFBP-3 was evaluate d by measuring L-citrulline synthesis in HUVECs using radioactive L-argini ne as substrate. Briefly, th e cell suspension was incubated with L-(14C) arginine at 37 de grees with constant agitation in the present or absence of 500 M L-NAME, a nitric oxide synthase inhibitor. Following incubation, cells were lysed by sonication for 10 seconds and the sample suspension was run through 1 ml columns of Dowex AG50WX-8. Radioactivity corresponding to (14C) citrulline within the eluate was quantified by liquid scintillation counting. Enzyme activity was expre ssed as L-NAME inhibitable radioactivity/mg of cell protein. In order to evalua te the effects of different bloc kers on IGFBP-3 stimulated eNOS 71


activity, cell suspension was incubated with vari ous blockers (SR-B1 antibody, PI3K inhibitor, or Akt inhibitor) for 30 minutes before the addition of IGFBP-3. Western Blot Analysis Since CD34+ cells are nonadherent suspension cells, CD34+ cells (106) were treated in eppendorf tubes with 100ng/ml of recombinant IGFBP-3 (UpState Biotechnology) 0, 10, 30 and 60 minutes. The cells were harvested at each time point by lysing them with a mixture of lysis buffer (Cell Signaling Technology, Danvers, MA) and proteinase inhibitor (Sigma). The homogenates were vortexed and then centrif uged at 1000 G for 1 minute to collect the supernatant. The protein concentration of th e samples was assayed using a protein assay to assure equal protein loading (Pie rce, Rockford, IL). Proteins were separated by 10-20% sodium dodecylsulfate-polyacrylamide gel electrophoresis, with 15 g of protein loaded in each lane. Proteins were transferred to nitrocellulose membrane with 0.2 m pore size (Bio-Rad Laboratories, Inc, Hercules, CA). Effective prot ein transfer was verified by Coomassie Blue protein staining. Membranes were blocked for 1 hour with Odyssey Buffer (LI-COR, Lincoln, Nebraska) and probed overnight in Odyssey blocking buffer at 4oC with the rabbit polyclonal antibody for peNOS (Abcam used at 1:4000 d ilution. A fluorescent conjugated donkey anti rabbit secondary antibody (Rockland Immunochemical s, Inc., Gilbertsville, PA) was applied for 1 hour at room temperature at a dilution of 1:5000. All samples were normalized to cofilin, rabbit polyclonal, (Santa Cruz Biotec hnology, Inc., Santa Cruz, CA) at a 1:1000 dilution. Immunoblots were visualized using an Odyssey LI-COR infrar ed imaging scanner. Image analysis software (LI-COR) was used to quantify the intensity of the specific bands. Real Time Polymerase Ch ain Reaction (RT-PCR) Total mRNA from human CD34+ cells, RGC-5, HMVEC-L, a nd HUVECs or retina from adult mice was isolated using the total RNA Mini Kit (BioRad Laboratories, Inc, Hercules, CA). 72


The mRNA was transcribed usi ng iScript cDNA synthesis kit (BioRad Laboratories, Inc, Hercules, CA) and real-time PCR was performed using ABI Mastermix (ABI Biosystems, Foster City, CA). Primers for the PCR were purchased from ABI BioSystems. All samples were normalized to -actin or TATABP (mouse retina). Real time PCR wa s performed on an ABI 7500 Fast PCR machine for 60 cycles. All reac tions were performed in triplicate. Immunocytochemistry In order to assess whether IGFBP-3 influe nces VASP expression in mature endothelial cells, immunocytochemistry was performed. Imm unocytochemistry was used because if IGFBP3 has an effect on VASP re-distribution and rear rangement, it can be clearly visualized using fluorescence microscopy vers us assessing protein cha nges by western blotting. Cell Preparation and Fixation HMVEC-L (Lonza, Walkersville, MD) were cultured on fibronectin coated coverslips (BD Biosciences) with EBM-2 media (Lonza, Walkersville, MD). Cell passage 3 was used for experiments. Cells were either untreated (cont rol), treated for 15 minutes with recombinant 100 ng/ml of IGFBP-3 (Upstate Biotechnology, Lake Placid, NY) or with IGFBP-3 preceded by 1 hour pretreatment with 100 mM L-NAME, a selectiv e non-inhibitor of nitric oxide synthase. At the end of the treatment, the media was removed a nd cells were fixed at room temperature in 4% PFA in PBS, supplemented with calcium and magnesium ions, and adjusted to pH 7.4. Immunostaining After fixation, the cells were washed in PB S with 0.1% Triton-X 100 for 5 minutes at room temperature. After three additional PBS wa shes, cells were blocked with 10% normal goat serum with 1% BSA in PBS for 1 hour at room temperature to block nonspecific antigens. Cells were incubated with 5 mg/ml mous e anti-VASP antibody (BD Biosciences) in blocking solution overnight at 4 degrees and then with FITC labe led goat anti-mouse IgG 73


(Abcam, Cambridge, MA) at 1:200 dilution in blocking solution for 1 hour at room temperature. Samples were mounted with Vectashield (V ector Labs) mounting medium. Samples were examined by fluorescence microscopy (Zeiss Axiopl an 2), using a Zeiss Plan-neofluar 40x oil objective. Micrographs were captured using a QImaging 12 bit cooled CDC digital camera (QImaging, Surrey, BC, Canada) and processed using Openlab imaging software (Improvision, Waltham, MA) for Macintosh. Quantification of Immunocytochemistry The micrographs were analyzed as TIFF imag es. Analysis of the images was performed using Image J software from NIH. For each cell, three measurements were made by drawing a line from the outer edge of the nuclear membrane to the outer edge of the plasma membrane. A fluorescence profile was determined by plotting the profile of each line. This yielded a set of distance coordinates (x-values) and fluorescence values (Y-values) for each line. This was done for a set of five cells for each condition and result ed in 15 sets of X-Y values for each condition. The sets of distance values (Xs) were normalized by setting the distance from the nucleus to the outer plasma membrane of each set to one and then adjusting the remaining Y-values relative to this value so that all Y-values are within the range of zero an d 100. Each set of 15 XY coordinates were graphed using Excel (Microso ft) and a graph with fifteen plots for each condition was obtained. The area under the curve was determined for each condition at the relative distance from the nucleus of 0.2-0.25, 0.5-0.55, and 0.95-1.0 using Scion image (based on NIH Image for MacIntosh by W. Rasband at NIH and modified for Windows by Scion Corporation). These areas were averaged and th e standard error of the mean was determined. 74


Figure 2-1. Oxygen-Induced Retinopathy (OIR) M ouse Model. Mouse pups are injected with IGFBP-3 expressing plasmid on P1 and ma intained at normal room oxygen levels (21% O2). On P7 the mice are transferred to ~75% oxygen until P12 when they are returned back to normal oxygen levels (21% O2). The mouse pups are sacrificed at P17. Phase 1 (P7-P12) of the OIR model includes vaso-obliteration in which the retinal blood vessels regress follo wed by Phase 2 (P12-17) in which neovascularization or new retinal bl ood vessel formation occurs. 75


Figure 2-2. In vivo studies with IGFBP-3 plasmid in OIR Model. 76


Figure 2-3. In vivo studies with IGFBP-3 plasmid in Laser Vessel Occlusion Model. 77


78 Figure 2-4. In vitro studies with IGFBP-3 recombinant protein.


CHAPTER 3 RESULTS IGFBP-3 Prevents Endothelial Cell Death in the OIR Model IGFBP-3 is known to have anti-apoptotic eff ects. We used the OIR model to assess cell death of endothelial cells in IGFBP-3 injected mo use retinas. IGFBP-3 plas mid was injected into the vitreous of mouse pups on P1 and then th e mice were subjected to hyperoxia from P7-P12 (phase 1) and returned to room air P13-P17 (phase 2). The mouse pups were sacrificed at both P12 and P17 and their retinas harvested for TUN EL analysis. The OIR model is ideal to assess apoptosis because the early de veloping mouse vasculature un dergoes constant remodeling through combined cell death and proliferation. Mo reover, in the OIR model endothelial cells are the most sensitive to cell death. Therefore, if IG FBP-3 has an anti-apoptotic effect in ischemic mouse pup retinas, we would be able to readily detect this through TUNEL analysis of OIR retinal flatmounts. We found IGFBP-3 injected mouse pups subjected to OIR had significantly reduced cell death of endothelial cells in the re tina compared to control plasmid injected OIR control mice in both phase 1 and phase 2 of th e OIR model (Figure 3-1 D-F IGFBP-3 injected versus A-C control). This reducti on in endothelial cell death was evident in the mid-peripehral (p<0.05) and peripheral regions (p<0.05) of the IGFBP-3 injected retinas. In contrast, IGFBP-3 injected OIR mice did not show a significant reducti on in cell of pericytes or astrocytes in the retina (data not shown). IGFBP-3 Increases Astrocytic Ensheath ment of OIR Blood Vessels Although IGFBP-3 injected OIR mice showed no decrease in the number of apoptotic astrocytic cells in the retina, we found that IGFBP-3 still had an influence on astrocyte morphology. IGFBP-3 injected OIR mice had mu ch thicker and elaborate astrocytic 79


ensheathment of retinal blood vessels compared to uninjected control OIR mice as shown in Figure 3-2 A-H. Also, the IGFBP-3 in jected eyes had far greater S-100+/gfp+ immunoreactivity (red stain E-H) compared to uninjected mice (A-D). Quantification of the frequency of astrocytic ensheathment of retinal vasculature (Figure 3-2M) was significan tly increased at both P12 and P17 stages of development in IGFBP-3 injected OIR mice compared to control injected OIR mice (p<0.05). Retinal astrocytes critically supp ort the development of the retinal vasculature and can modulate angiogenesis during OIR. IGFBP-3 has a protective effect on retinal astrocytes in mice subjected to OIR by likely enhancing thei r stability, which lends support to the overall retinal vasculature. IGFBP-3 Protects Retinal Neurons from Apoptosis in the OIR Model In addition to evaluating cell death of endothelial cells, pericytes and astrocytes in OIR mouse retinas, we also assessed neuronal cell death via microscopy analysis. We found that IGFBP-3 injected OIR mice had reduced neuronal cel l death in retinal cross sections compared to uninjected control OIR mice (P<0.01). Figure 3-3 A&B contains representative images and quantification. The black arrows denote cells undergoing apoptosis, which are stained darkly denoting their breakdown and condensation of geneti c material. There are significantly fewer of these cells present in the IGFBP-3 injected OIR mice (p<0.001) compared to the control uninjected OIR mice (Figure 3-3G). The neuronal cells that appear to have the least cell death in the IGFBP-3 injected mice are ganglion cells lo cated in the ganglion cell layer. Most cells undergoing cell death are located in the inner retinal layers, pre dominately in the inner nuclear and inner plexiform layers. Electron microscopy analysis further confirmed the presence of apoptotic cells in the retina of vector control injected mice (Fi gure 3-3 C&E) versus the IGFBP-3 injected mice (Figure 3-3 D&F), which showed few apoptotic cells in the retina. 80


IGFBP-3 Increases Incorporation of EP Cs in Adult Retinal Blood Vessels Besides assessing the cellular pr otective effects of IGFBP-3 in the OIR model, we also assessed the cytoprotective effects of IGFBP-3 in the adult vasculature of chimeric mice. The adult vasculature is relatively quiescent. Therefore, we questi oned whether IGFBP-3 would still actively recruit EPCs into the retinal vasculature. We evaluated IGFBP-3s effect on EPCs in the adult vasculature both in the abse nce of laser injury, as well as in the presence of laser induced injury. Greater gfp+ cell incorporation was obs erved in IGFBP-3 injected adult chimeric mice (Figure 3-4 D-F) and IGFBP-3 injected chimeric mice subjected to retinal vessel occlusion injury (Figure 3-4 G-I) compared to uninjected eyes at 3 weeks post laser injury (Figure 3-4 A-C). There is clear incorporation of gfp+ HSCs/EPCs into the retinal va sculature of IGFBP-3 injected mice both lasered and unlasered. We al so see some incorporation of gfp+ HSCs/EPCs in the lasered only mice (Figure 3-4 J-L) However, most of the gfp+ cells are outside the retinal vessels and do not appear to be directly incorpor ated into the retinal blood vessels. IGFBP-3 Expressing Plasmid is Upregulated in the Adult Mouse Retina In order to confirm the IGFBP-3 plasmid expressed IGFBP-3 following injection, we performed mRNA analysis of retinas injected w ith this plasmid. The IGFBP-3 plasmid begins to express IGFBP-3 at 48 hours and remains high at 72 hours post injection (p<0.05). The IGFBP-3 expressing plasmid remains significantly elevated until 1 week post injection in the mouse retina, after which time the expression levels begin to decline (Figure 3-5). Thus, the IGFBP-3 plasmid transiently upregulates IGFBP-3 ex pression in the retina. IGFBP-3 Causes Differentiation of EPCs into Pericytes, Astrocytes, and Endothelial Cells IGFBP-3 increased in corporation of gfp+ HSCs/EPCs into retinal blood vessels. However, we questioned what the gfp+ cells were in terms of their cell type. We stained wholemount retinas for mature vascular cells ty pes such as endothelial cells, pericytes, and 81


astrocytes. In Figures 3-3.6 and 3-3.7 it is shown IGFBP-3 results in the differentiation of gfp+ HSCs/EPCs into endothe lial cells (Figure 3-3.6), pericytes (Figure 3-3.7), and astrocytes (Figure 3-3.7). There appears to be the greatest increase in gfp+ cells differentiating into endothelial cells, pericytes, and astrocytes in the IGFBP-3 inje cted and lasered mice compared to the IGFBP-3 injected alone mice. However, this was not quantified. IGFBP-3 Decreases the Ceramide/Sphingom yelin Ratio in Lasered Mice In the OIR model, IGFBP-3 has anti-apoptoti c effects on endothelial cells. We reasoned IGFBP-3 may also have anti-apoptotic effect s in the adult vasculature. The ceramide/ sphingomyelin ratio is an indicator of a pro-infla mmatory and pro-apoptotic state in a tissue. The graph in Figure 3-3.8 shows lasered mice have a significantly higher ceramide/sphingomyelin ratio in their retinas (p<0.05), at 3 weeks post lase r treatment compared to untreated control. This is in contrast to laser and IGFBP-3 injected mice. This group has a ceramide/sphingomyelin ratio similar to that of untreated mice. Hence, IGFBP-3 can lower the pro-inflammatory and proapoptotic state in lasered retinas to that of control levels. IGFBP-3 Increases NO Production in CD34+ Cells and Endothelial Cells IGFBP-3 stimulates in corporation of gfp+ HSCs/EPCs in the retinal vasculature. These cells have to migrate in order to home to re tinal blood vessels. IGFBP-3 may act as a potent migratory stimulus by recruiting EPCs into the retinal vasculature by st imulating NO generation. In Figure 3-3.9, IGFBP-3 at 100 ng/ml in creases exogenous NO production in CD34+ cells. The increase in DAF-FM fluorescence, which is an indicator of NO produc tion, is statistically significant (p<0.01). Also, IGFBP-3 at a concentr ation of 100 ng/ml increases NO generation in endothelial cells as shown in Figure 3.3-11. There is a significant increase in NO generation by IGFBP-3 treatment in both cultures of HUVEC s, a well known macrova scular endothelial cell line, and HMVEC-L, a microvascular type of endothelial cell. IGFBP3 clearly increases NO 82


generation in both human CD34+ cells and human endothelial cells. IGFBP-3 increases eNOS phosphoryla tion at Serine 1177 in CD34+ Cells NO production in CD34+ EPCs must occur through NOS activation. We tested whether IGFBP-3 increases eNOS phosphorylation at Serine 1177. IGFBP-3 increased eNOS phosphorylation protein levels at Serine 1177 in EPCs. As depi cted in Figure 3.3-10. IGFBP-3 increases Serine 1177 phosphorylation in a time-dependent manner. eNOS Serine 1177 phosphorylation significantly incr eases by 30 minutes (p<0.05) and significantly peaks at 60 minutes (p<0.01). Likewise, IGFBP-3 leads to in creased eNOS phosphoryl ation at Serine 1177 in HUVECs at 60 minutes (p<0.05) as shown in Figure 3.3-13. Blockade of SR-B1 Leads to Decreased NO Production in Endothelial Cells IGFBP-3 clearly increases nitric oxide production. There are a number of ways nitric oxide can be generated exogenously in the cell. We postulated IGFBP-3 can signal through the HDL receptor called SR-B1 to increase intracellula r levels of NO in endothelial cells. In Figure 3.3-11, we demonstrate blocking the SR-B1 with a blocking antibody leads to decreased NO production in mature endothelial cells treated with a physiologica lly relevant concentration of IGFBP-3 (100 ng/ml). We evaluated two types of endothelial cells, microvascular (HUVECs) and macrovascular (HMVEC-L) human endothelial cells. The decrease in NO generation when the SR-B1 was blocked was equivalent in both cell lines (p<0.02). The RGC-5 cell line is rat ganglion cell line that does not express SR-B1, hence serves as a negative control. There was no observed increase in NO upon addition of IG FBP-3 or HDL, nor was NO production altered upon blocking SR-B1 in this cell type. Interestingly, addition of exogenous HDL combined with IGFBP-3 led to increased NO production in both HMVEC-L (p<0.001) and HUVECs (p<0.05), but was most pronounced in the HMVEC-L. Addition of HDL alone produced NO levels similar to that of IGFBP-3 alone. Also, blocking th e SR-B1 in HDL treated cells decreased NO 83


production significantly (p<0.05). However, blocking the SR-B1 in IGFBP-3 treated and HDL treated HMVEC-L did significantly lower NO production, but in the HUVECs the reverse is true (p<0.01). (Figure 3-11) Blockade of SR-B1 Leads to Decreased NO Activity in Endothelial Cells In addition to evaluating NO generation, NO activity was also assessed. In Figure 3.312C, NO activity was measured in HUVECs bl ocked for SR-B1 receptor and treated with IGFBP-3. There was a significant reduction in eNOS activity as measured by the conversion of L-arginine to L-citrulline in HUVECs treate d with SR-B1 blocking antibody and IGFBP-3 (p<0.05). Blockade of PI3K/Akt Reduces NO Generation and Activity IGFBP-3 increases NO through activation of the SR-B1 receptor. To further understand the signaling cascade we examined the PI3K/Akt cell survival pathway, since IGFBP-3 promotes cell survival. Pharmacological inhi bitors for PI3K (LY294002) or Ak t (Triciribine) were used to block IGFBP-3 treated HUVECs. We found IGFBP-3 tr eated cells both inhibitors resulted in decreased NO production as shown in Figure 3.3-12B (p<0.01). Also, L-NAME treated HUVECs had decreased NO genera tion (p<0.01). We further confirmed these observations by evaluating eNOS activity in Figure 3.3-12C. IGFBP-3 stimulated eNOS activity was significantly reduced by pre-tr eatment with SR-B1 or LY294002 or triciribine (p<0.05). Additionally, in Figure 3.3-13, IG FBP-3 decreases eNOS phospor ylation at Serine 1177 in HUVECs upon exposure to SR-B1 or LY294002 or triciribine either individually or in combination (p<0.05). Blockade of Sphingosine Kinase Decreases NO Release in CD34+ Cells IGFBP-3 has previously been shown to in crease the activity of sphingosine kinase (Sphk), the enzyme responsible for the genera tion of the potent pro-angiogenic factor 84


sphingosine-1-phosphate, S1P. Addition of the S phk inhibitor, N,N-dimethylsphingosine as shown in Figure 3.3-9, resulted in a significan t reduction in NO generation in response to IGFBP-3, as well as HDL, supporting that S1P gene ration can contribute to the effects of both HDL and IGFBP-3 on NO generation (p<0.01). CD34+ cells and endothelial cells express S1PR, predominately S1PR1 and S1PR3, as well as Sphingos ine Kinase 1 and 2 (data not shown). IGFBP-3 Stimulates VASP Re-distribution in Endothelial Cells Nitric oxide is a critical regulator of cell migration. VASP, vasodilator-stimulated phosphoprotein (VASP), plays a pivo tal role in promoting actin filament elongation at the leading edge of the cell by forming an active molecular motor complex that propels motility. Migration of BMDCs into areas of ischemia is paramount to their ability to initiate and orchestrate repair. HUVECs treated with IGFB P-3 underwent significant VASP re-distribution (p<0.05) compared to untreated cells as depict ed in Figure 3.3-14 A&C. IGFBP-3 results in lamellipodia formation at the periphery (Figur e 3.3-14C). Pre-treatment with L-NAME, a NO scavenger, resulted in significant decreases in VASP re-distribution and lamellipodia formation as shown in Figure 3.3-14E (p< 0.05). VASP biodistribution wa s quantified in Figure 3.3-14 H in endothelial cells. Due to CD34+ cells being nonadherent, VASP re-distribution could not be studied in this cell type. IGFBP-3 Decreases Vascular Permeability in Laser Injured Adult Mice IGFBP-3 clearly has dramatic effects on the ce ll migration of endothe lial cells and EPCs. IGFBP-3 facilitates their recruitment and incorp oration into retinal blood vessels. We assessed IGFBP-3 on vascular permeability in the retinal vessel occlusion injury model. We found IGFBP-3 injected animals subjected to laser injury have decreased vascular permeability compared to laser alone treated mice (Figure 3-15 ). This effect was observed 4 days after laser injury when the IGFBP-3 expressing plasmid was at its optimal peak expression. 85


IGFBP-3 Increases Vascular Permeab ility in Unlasered Adult Mice Besides evaluating vascular permeability in the laser injury model, we also decided to look at VEGF induced vascular permeability in normal unlasered mouse retina. VEGF, as mentioned previously, is one of the most, if not the most, potent vascular permeability factor. We injected VEGF intravitreally in normal mice and as expected there was an increase in vascular permeability in vivo IGFBP-3 by itself, increased vascular permeability more so than VEGF. IGFBP-3 and VEGF injected together in combination increased vascular permeability at 12 hours, but fell at 24 hours to control levels. IGFB P-3 coupled with VEGF can increase vascular permeability transiently in normal mice (Figure 3-16). IGFBP-3 by itself significantly increases vascular permeability, suggesting IGFBP-3 has sim ilar effects to that of IGFBP-3 in normal mouse retina. These results were further confirmed in vitro IGFBP-3 by itself acutely increases vascular permeability, however, by 24 hours it dec lines to control levels (data not shown). IGFBP-3 and VEGF combination increases vascul ar permeability at 5 minutes, 1 hour, and 5 hours, but declined by 24 hours (data not shown). IGFBP-3 depending on the type of injury in the vasculature, laser injured versus VEGF inj ected, can impact vascular permeability in two distinct ways. Also, the exposure time to IGFBP3 can influence vascular permeability. IGFBP-3 Reduces Sphingomye linase mRNA Expression IGFBP-3 has an impact on vascular permeab ility in retinal blood vessels. We suspect IGFBP-3 influences sphingomyelinase, which in turn can affect vascular permeability. We found IGFBP-3 and laser mice have decreased acid and neutral sphingomyelinase levels in mouse retina at 4 days post laser treatment comp ared to laser only treat ed mice (Figure 3-17). Likewise, we confirmed these results in a sphi ngomyelinase activ ity assay. We f ound that laser treated and IGFBP-3 treated mice have lower s phingomyelinase activity co mpared to laser only treated mice (data not shown). 86


Figure 3-1. IGFBP-3 prevents endothelial cell death in the OIR model. Panels A-C) are uninjected control mice. A) shows an abundance of TUNEL+ green cells B) represents TUNEL+ cells (white arrows), and GS lectin staining of endothelial cells (blue stain). C) depicts a higher magnificat ion shown in white box in B of numerous colocalized TUNEL+/GS lectin+ staining endothelial cells. DF) are IGFBP-3 injected mice. D) shows fewer TUNEL+ green cells. E) shows a small number of TUNEL+ cells (white arrows), and GS lectin staining of endothelial cells. F) represents a higher magnification of white box shown in E of colocalized TUNEL+/GS lectin+ staining of endothelial cells. 87


Figure 3-2. IGFBP-3 increases astrocytic ensheathment in OIR blood vessels. In comparison to control A-D), the S-100/GFAP ensheathment of underlying va sculature in the IGFBP-3 injected eyes E-H) was much more completed and astrocytes showed larger, thicker processes. I-L) show representative fields of view from the mid-peripheral retina using a 20x objective during normal development and during exposure to the OIR model. The ensheathment of GS lectin+ vascular endothelia l cells (blue) by S100+ astrocytes (red) and GFAP (green) were determined. S-100 ensheathment of underlying vasculature in the IG FBP-3 injected L) was much more complete than in control injected eyes K) at P17. M) Show s quantification of astrocytic ensheathment of retinal blood vessels. Astrocyte ensheat hment was found to be more significant (p<0.05) in both P12 and P17 injected eyes in comparison to controls. 88


Figure 3-3. IGFBP-3 protects retin al neurons from apoptosis in the OIR model. Panels A-B) show representative light micrographs stained with Toluidine Blue to clearly visualize the retinal layers in OIR mice at P17. The vector control OIR mice A) have increased numbers of apoptotic cells in the inner re tina (black arrows) compared to the IGFBP3 plasmid injected mice B). Electron microscopy analysis revealed retinal cells undergoing active apoptosis as shown in C-F). C&E show obvious cells undergoing active apoptosis in the vector injected mice versus D&F which show little signs of retinal apoptosis in the IGFBP-3 injected OIR mice. Yellow arrows denote apoptotic cells. The graph in G) shows quantification of the number of apoptotic cells counted in the retinas of vector injected mice (unt reated control) and the IGFBP-3 injected mice. 89


Figure 3-4. IGFBP-3 increases in corporation of EPCs in adult retinal blood vessels.Uninjected control mice A-C) show very little incorpor ation of gfp+ EPCs/HSCs directly into the retinal blood vessels. IGFBP-3 inject ed mice D-F) show increased gfp+ EPCs/HSCs incorporation and homing as well as mice la sered and injected with IGFBP-3 G-I). Laser only mice J-L) show the presence of gfp+ EPCs/HSCs, however, these cells are present outside the blood ve ssels and do not incorporate directly into retinal blood vessels. The red stain represents rhodamine aggl utinin (far left side panels) which is a blood vessel specific stain and the gr een stain is indicative of gfp+ cells (middle panels). The merged images of the rhodamine stain and gfp+ cells are shown in the far right panels. 90


Figure 3-5. IGFBP-3 expressing pl asmid is upregulated in the a dult retina.RT-PCR analysis of mouse retina evaluating IG FBP-3 mRNA expression over a 3 week time course. IGFBP-3 mRNA remains highly expressed in the treated eye (OD) of mice subjected to laser injury followed by intravitreal in jection of IGFBP-3 up to 1 week compared to the contralateral control eye (OS). By 3 weeks, IGFBP-3 expression levels in the treated eye (OD) returned to normal control levels. *p<0.05 91


Figure 3-6. IGFBP-3 causes differen tiation of EPCs into endothelial cells in the adult retina. The green stain is gfp+ cells and the blue repr esents GS isolectin B4 for detection of endothelial cells and activated microglia /macrophages. A-C) represents laser only retina with 2 laser burn sites indicated by pigment changes and tissue damage (dashed circles) and surrounding vascular remodeling. There are few gfp+ cells colocalized with lectin indicating li ttle differentiation of gfp+ cells into endothelial cells. D-F) illustrates IGFBP-3 injected plus laser retina showing large incorporation and differentiation of gfp+ cells, at and around the site of laser injury, participating in wound healing response. gfp+ cells differentiate into GS Lectin labeled endothelial cells (white arrows). G-I) illustrates IG FBP-3 injected eyes showing significant gfp+ BMDC incorporation and differentiation in to GS Lectin labeled vessels (white arrows). 92


Figure 3-7. IGFBP-3 stimulates di fferentiation of EPCs into astrocytes and pericytes. A-I) The green stain is gfp+ cells and the purple represents NG2 for detection of pericytes. AC) Represents Laser only retina showing very little gfp+ cells giving rise to NG2+ pericytes. D-F) Illustrates IGFBP-3 inj ected laser retina showing increased gfp+ cell differentiation into NG2+ pericytes (white arrows). G-I) Illustrates IGFBP-3 injected eyes showing more gfp+ cell differentiation into NG2+ pericytes (white arrows). J-R) The green stain is gfp+ cells and the red represents S 100 for detection of astrocytes. J-L) Represents Laser only retin a showing markedly increased S-100 immunoreactivity. gfp+ HSC differentiated S100+ astrocytes (white arrows) were evident though in very low numbers in the laser only eyes. M-O) Illustrates IGFBP-3 injected plus laser injured retina showing markedly increased S-100 immunoreactivity. gfp+ HSC gave rise to S100+ astrocytes (white arrows) which were evident in greater numbers in the laser plus IGFBP-3 inject ed eyes compared to laser only eyes. P-R) Illustrates IGFBP-3 in jected eyes showing the most gfp+ cell differentiation into S100+ astrocytes (white arrows). 93


Figure 3-8. IGFBP-3 decreases the ceramide/sph ingomyelin ratio in lasered retina. Retinal Lipids extracts from control, laser, or la ser plus IGFBP-3 treated mice were analyzed for ceramide and sphingomyelin molecular species as their [M+H]+ ions by precursor ion scanning of m/z 264.4 m/z 184, respectively, after alkaline hydrolysis of glycerophospholipids. The data was normalized to internal standards and ratios of total retinal ceramide/sphingomyelin were cal culated. The results are the means SD of 3 independent experiments. *p<0.05 vs. c ontrol is statistica lly significant. 94


Figure 3-9. IGFBP-3 incr eases NO production in CD34+ cells. Determination of NOrelease by DAF-FM fluorescence imaging in human CD 34+ cells. A) Showsrepresentative images of cells treated with DAF-FM. Images obtained were cells that were either untreated (control) or treate d as labeled. B) Graph disp layschanges in fluorescence with different treatments expressed as pe rcentageincrease over the control. *p<0.01 compared with IGFBP-3 or HDL alone ;**p<0.001 compared with IGFBP-3; ***p<0.0001 compared with HDL.Representat ive results from 3 independent experiments are shown. 95


Figure 3-10. IGFBP-3 increases phosphorylation of eNOS at Serine 1177 in CD34+ cells. A) Western Blot analysis depicting eNOS phosphorylation in human CD34+ cells treated with 100 ng/ml of IGFBP-3 at 10,30, and 60 minutes. Protein expression levels were normalized to cofilin. B) Quantification of western blot in panel A. There is a time dependent increase in eNOS phosphor ylation at Ser 1177 in human CD34+ cells (p<0.05 and p<0.01) at 30 a nd 60 minutes, respectively. 96


Figure 3-11. IGFBP-3 activates the SR-B1 recep tor leading to NO generation in endothelial cells. Determination of NO release in response to IGFBP-3 and HDL by DAF-FM fluorescence imaging. NO release in res ponse to IGFBP-3 and/or HDL in HUVECs, HMVEC-L, and RGC-5 cells. Treatment w ith SR-B1 blocking antibody significantly decreased NO release by either IGFBP-3 or HDL or the combination of IGFBP-3 and HDL. (A-C) show respective scales for fluores cence in 3 different cell types. Images obtained were cells that were either treated or untreated as labeled. (D) Changes in fluorescence with different treatments were expressed as percentage increase over the control. *p<0.05 and **p<0.02 compared with IGFBP-3; ***p<0.01 compared with HDL+IGFBP-3; ****p<0.0001 compared with HDL; #p<0.05 compared with IGFBP-3 or HDL alone; ##p<0.01 compared with HDL and IGFBP-3; ###p<0.01 compared with HDL or IGFBP-3. Repres entative results from 3 independent experiments are shown. 97


Figure 3-12. Blockade of PI3K/Akt reduces NO generation in endot helial cells. A) NO production in response to IGFBP-3 was measured by DAF-FM fluorescence in HUVECs and effects of different pharmacol ogical blockers were evaluated (color scale for fluorescence). Images obtained were cells that were either untreated or treated as labeled. B) Changes in fluorescence with different treatments were expressed as percentage increase over the time control. NO release by IGFBP-3 was significantly decreased by pretreatment with SR-B1 blocking antibody (SR-B1-Ab), LY294002, triciribine, or L-NAME. *p<0.01 and ***p<0.0001 compared with IGFBP-3. C) eNOS activity expressed as L-NAME-inhibitable c onversion of [14C]Larginine to [14C]L-citrulline was stimulated by 100 ng/mL IGFBP-3. Pre-treatment with SR-B1-Ab, 30 mol/L LY294002 or 30 mol/L triciribine significantly decreased IGFBP-3-induced eNOS activation (p<0.05). Representa tive results from 3 independent experiments are shown. 98


Figure 3-13. IGFBP-3 phosphorylates eNOS at Serine 1177 in HUVECs. HUVECs were treated with 100 ng/ml of IGFBP-3 for 60 minutes Protein expression levels were normalized to pan-caderin. Phosphorylation was significantly incr eased in IGFBP-3 compared to untreated cells (p<0.05). In the presence of blockers SR-B1 or triciribine, the phosphorylation wa s significantly decreased. p<0.05 99

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Figure 3-14. IGFBP-3 induces VASP re-distrib ution in HMVEC-L. HMVEC-L were treated with IGFBP-3 and VASP biodistribution was detected by immunofluorescence A,C,E,G) and quantified. A untreated control cell showing uniform VASP localization (green) al ong the actin filaments throughout the cytoplasm. C) IGFBP-3 induced VASP redistribution to llamellipodi a at the leading edge of HUVECs. E) Pretreatment with an inhibitor of nitric oxi de synthase (L-NAME) abolishes the effect of IGFBP-3 on VASP redistribution. G) Control in which VASP primary antibody was omitted. B,D,F) Quantification of VASP biodistribution in A,C, and E respectively. H) Area under the curve calculated from the th ree areas of interest in B,D, and F (region shaded blue) Gr een: VASP; Blue: DAPI (nuclei). 100

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Figure 3-15. IGFBP-3 decreases vascular permeab ility in laser injured mice. Four days after laser treatment, lasered mice displayed a significant increase in vascular permeability. In mice lasered and injected with IGFBP-3 expressing plasmid, vascular permeability declined at four days post laser treatm ent compared to laser treated only mice. *p<0.05 101

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Figure 3-16. IGFBP-3 acutely in creases vascular permeability in unlasered mice. IGFBP-3 and VEGF given in combination at 6 hours increases vascular permeability. At 24 hours, VEGF and IGFBP-3 combination leads to reduced vascular permeability. IGFBP-3 by itself increases vascular permeability greater than VEGF alone. *p<0.05 102

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Figure 3-17. IGFBP-3 reduces sphingomyelinase mR NA expression levels. Four days after laser treatment, laser alone mice displayed a significant increase sphingomyelinase mRNA levels, both SMase 1 (acid form) as shown in A) and SMase 2 (neutral form) as shown in B). In laser treated mice inject ed with IGFBP-3 there is significant reduction in sphingomyelinase mRNA levels at 4 days post treatment. By 7 days the sphingomylinase mRNA levels reversed. OS (left eye) in the c ontrol untreated eye and the OD (right eye) is the treated. *p<0.05 103

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CHAPTER 4 DISCUSSION The main purpose of this study was to determine how IGFBP-3 influences EPC driven repair in mouse retinal vascular injury models at the cellular and molecular levels to better understand its therapeutic potential as a robust EPC migratory stim ulus. Particular emphasis was placed on understanding how IGFBP-3 impacts bot h EPC and EC migration, which is key to vascular repair. Directing, homing, and trafficking cells to the proper areas in need of repair is crucial for vascular stabilization, remodeli ng, and revascularizati on. IGFBP-3 mediates NO generation, which allows for functional revascul arization of ischemic injured retinal blood vessels by enhancing both EPC and endothelial cell migration (Figure 4-1). IGFBP-3 Acts as an EPC/HSC Homing Factor and Provides Cytoprotection In this study, the cellula r and signaling mechanisms responsible for the vascular protective effects of IGFBP-3 were identified. The effects of IG FBP-3 on two distinct types of vasculature, a stable vascular bed (adult chimeric mice undergoing laser vessel occlusion) and an immature unstable vascular bed undergoing activ e endothelial cell prolif eration and migration (hyperoxia-induced re tina injury model) were examined. In the adult model, IGFBP-3 enhanced repair by recruiting BMDCs to sites of laser occlusion within the ischemic retina. Even in the absence of injury, overexpression of IGFBP-3 by the resident retinal endothelium promoted extr avasation of BMDCs from the circulation into perivascular regions and their incorporation into areas of vascular remo deling. The incorporation of EPCs/HSCs into the endothelium appears to be a result of direct integration and not due to cell fusion. Cell fusion can take place with adult stem cells, but usually occurs with embryonic stem cells. There is little evidence for significant fusion of BM C cells in the endothelium and in situations where BMD cells are recruited to repa ir acute damage in otherwise healthy tissue, 104

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fusion rarely occurs.112 As reported by Harris et al., cell fusion was unlikely in their studies involving HSC donor-derived differentiati on of RPE regenerating injured RPE.92 Essentially, there are no clear reports of ce ll fusion of BMDCs in the retina and the observed results described here are unlikely due to cell fusion. The reparative effects of IGFBP-3 are not limited to simply promoting BMDC homing, because IGFBP-3 reduced both endot helial and neuronal cell death, in the OIR model, as well as decreased inflammatory lipids in the adult retina. IGFBP-3 is know n to have both proand antiapoptotic effects. The fact that IGFBP-3 is anti-apoptotic gives cr edence to its vascular stabilizing and reparative nature, which are supported by the studies described, as well as those previously published by both Chang et al. and Lofquist et al. in 2007. During repair of the retina, IGFBP-3 increased astrocyte-endot helial cell interactions likely leading to enhanced barrier properties of the neovasculature. Moreover, IGFBP-3 decreased vascular permeability in vivo in mice subjected to laser retinal vessel occlusion and is likely mediated through decreased proinflammatory sphingomyelinase levels in the reti na. IGFBP-3 also stimulates the differentiation of EPCs/HSCs into not only endothe lial cells, but also astrocytes a nd pericytes, which likely aids in decreasing vascular permeability and reducing retinal inflammation. Astrocytes specifically serve as a template for both developmental and injury associated angiogenesis. The fact that IGFBP-3 increases astrocytic ensheathment a nd stimulates differentiation of EPCs into astrocytes suggests that IGFBP3 has a specific effect on astroc ytes, which likely stabilizes the resident vasculature allowing for proper repair to occur. Interestingly, however, in normal mice subj ected to VEGF induced retinal vascular permeability, IGFBP-3 increases permeability sugge sting IGFBP-3 can be pro-angiogenic or anti-angiogenic. Depending upon the in jury state of the tissue, IGFB P-3 can act very differently. 105

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Thus, IGFBP-3 may act to augment VEGF effect s to increase vascular permeability in normal retina, while providing vascular st abilization in injured retina. In vitro data in bovine retinal endothelial cells also supports the in vivo findings that IGFBP-3 a nd VEGF administered in combination increase permeability. To date, this is the first in depth examination into IGFBP-3s effects on retinal vascular permeability. IGFBP-3 Increases NO and Activates SR-B 1 to Mediate its Protective Effects NO is an essential signaling molecule that promotes revascularization and vascular remodeling. Endogenous NO generation by BMDCs is critical for their migration, which in turn is required for their reparative function. IGFB P-3 increased eNOS phosphorylation, leading to increased NO generation and subsequent VASP redistribution promoting cell migration. Previous work has shown IGFBP-3 promotes ce ll migration in EPCs. In this study IGFBP-3 treatment led to the redistri bution of VASP to focal adhesi ons and pseudopodia. The rapid increase in VASP (within 15 minutes) suggests that VASP redistribution occu rred, rather than a change in VASP protein expression. Moreover, IGFBP-3 was shown to mediat e IGFBP-3 induced NO generation and this occurred independent of HDL. Activation of eNOS by phosphorylation at Serine 1177 by IGFBP-3 is dependent on activation of SR-B 1 and the downstream signaling pathway involving PI3K and Akt activity. NO released by IGFBP-3 in circulating EPCs and resident endothelial cells may modulate the function of BMDCs in an autocrine, as well as paracrine manner, thus contributing to va scular repair. In agreement with Granata et al. in HUVECs, the studies conducted in CD34+ cells support the notion that IGFBP-3 mediated NO ge neration is also depe ndent on activation of Sphk1, because NO generation is blocked by Sphk inhibition. S1P-mediated NO generation occurs by activation of endothelial differentiati on genes, also known as S1P receptors. S1P, 106

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much like IGFBP-3, increases NO generation, thus promoting migration of cells. Moreover, S1P, like IGFBP-3 has direct vascular protective effects. In the bl ood, S1P is associated with lipoproteins including low-dens ity lipoprotein, very high density lipoprotein, and HDL, with the majority of S1P being bound to HDL. The studies performed support that in EPCs, both IGFBP3 and HDL activate Sphk1, because inhibiting S phk1 with N,N-dimethylsphingosine resulted in a loss of NO generation in response to either agents. Although there was no observed increase in S1P in vivo in response to IGFBP-3, measurements were performed at 3 weeks following retinal injury. It is plausible any acute rise in S1P would have returned to baseline levels by this point. Despite the inability to detect S1P changes in the retina, changes in sphingolipids still persisted at 3 weeks. Most notably, the ceramide/sphingomyelin lipid ratio was completely normalized by IGFBP-3 to that of control levels. This is in contrast to the laser-trea ted eyes, which had a higher ceramide/sphingomyelin ratio, consistent with an infl ammatory, pro-apoptotic state. This suggests IGFBP-3 mediates vascular protection through i nhibition of inflammation, allo wing IGFBP-3 to promote the recruitment of reparative BMDC population, EP Cs, rather than a deleterious inflammatory population. Basically, the data collected supports the hypothesis that IGFBP-3 mediates functional revascularization in the retinal by promoting the homing of beneficial EPCs, while reducing the number of detrimental inflammatory cells. Once EPCs are routed to areas of damage IGFBP-3 enhances their incorporation and differentiation into endothelial cells, which facilitates vessel formation and stabilization. The studies conducted suggest that these bene ficial effects may be mediated by increased NO generation, which occurs via IGFBP-3 binding of SR-B1 and subsequent activation of the PI3K-Akt path way. Furthermore, the studies support a second 107

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mechanism by which IGFBP-3 stimulated NO gene ration by activation of Sphk-1 and generation of S1P. IGFBP-3 can also influence vascul ar permeability via sphingomyelinase, which is downstream of S1P. IGFBP-3 effects on vasc ular permeability can augment its vascular stabilization and reparative cap acity. Hence, IGFBP-3 can modul ate interactions between the scavenger receptor system and the S1P recepto r system, which may serve to regulate both physiological and pathological angiogenesis, as well as vascular remodeling. Figure 4-1 summarizes the results showing the mechanism of action by which IGFBP-3 acts to increase NO generation in both EPCs and endothelial cells via SR-B1 and S1P to elicit its cytoprotective and reparative effects in injured m ouse retinal vasculature. IGFBP-3: New Perspectives and Remaining Questions Although this study has identified a new recepto r system that mediates the effects of IGFBP-3 with respect to NO generation, IGFB P-3 has been shown to bind several other receptors including transforming growth factorreceptor, transferrin receptor, and low-density lipoprotein receptor-related protein (LRP). Als o, IGFBP-3 has numerous binding partners such as plasminogen, plasmin, fibrin, humanin, and even caveolin.164 Therefore, the diverse physiological effects of IGFBP-3 are likely mediat ed by multiple receptor systems. It is likely IGFBP-3 will be implicated in activation of other receptors due to its multi-faceted roles in being proand anti angiogenic as well as proand an ti-apoptotic. S1PRs have already been linked to IGFBP-3s pro-angiogenic effects and additional new findings will likely surface in the future between S1P receptors and IGFBP-3. The role of IGFBP-3 and S1PRs in vascular permeability and their role in IGFBP-3 me diated nitric oxide generati on especially require greater investigation. Through the use of IGFBP-3, S1PR and eNOS knockout mice, these studies can be completed. 108

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IGFBP-3 as a Therapeutic in the Future IGFBP-3 can still interact with a wide range of binding partners and activate multiple receptors, which can mediate its IGF independent e ffects. IGFBP-3 will continue to stimulate the interest of researchers. The studies described su pport IGFBP-3 as a potential therapeutic to treat ischemic ocular disorders, but IGFBP-3 is like ly to be suitable for modulation of various vascular and endothelial dysfunctions. Furtherm ore, IGFBP-3s effects on EPCs will be of further interest. The EPC biology field is beginning to undergo tremendous growth and exploration with regard to understanding the mechanisms by which EPCs home, engraft, and participate in repair. There are a plethora of systemic and local fact ors that influence EPC behavior. IGFBP-3 is just one of the many factors, which have a role in recruiting EPCs. It is possible other IGFBPs may also play a role in EPC recruitment and mobilization. A recent study by Bartling et al. suggests IGFBP-2 and IGFBP-4 can enhance the migration of human CD34-/CD133+ hematopoietic stem and progenitor cells.228 They evaluated at IGFBP-2 and IGFBP-4 in lung epithelial cancer cells and showed CD34-/CD133+ cells can be mediated in part by IGFindependent action of IGFBP-2 and -4. There is still much to uncover regarding IGFBPs and their effects on EPCs. Delivery, therapeutic dosing, and as well as how to best treat EPCs with IGFBPs are major clinical questions researchers must tackle before IGFBPs can be utilized in the clinical setting. Currently, there are ongoing clinical trials w ith IGFBP-3 administered to premature babies to alleviate the effects of retinopathy of prematurity.64 However, many unanswered questions remain such as the delivery method for IGFBP-3. In the case of the ey e, local delivery may be appropriate, but for other vascular complications systemic delivery may be optimal. Also, whether a patients EPCs should be pre-treated with IGFBP-3 in a way to prime them to increa se their therapeutic reparative capacity is another ke y question that merits study or whether treating the injured tissue 109

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with IGFBP-3 is the most effective approach. Basically, IGFBP-3 has th e potential to be an effective vascular therapeutic. Cell based therapies involving IG FBP-3 are likely in the future, particularly for patients who suffer from ischem ic vascular complications, such as retinopa thies. Conclusions IGFBP-3 is clearly more than just a binding pr otein. It has significan t vascular protective effects and has a profound impact on EPC migrati on and homing. Cell migration is vital for stem cell based therapies to be effective. Thus, IG FBP-3 has many positive attributes and qualities, which make it an ideal therapeutic for EPC mediat ed repair. IGFBP-3 will continue to captivate the interest of researchers. IGFBP-3 is still en igmatic in many ways and there is still much to learn about IGFBP-3. The re sults discussed here, as well as previous reports, provide a platform upon which to build greater knowledge about IGFBP-3s effects on EPC homing and recruitment. Overall, IGFBP-3 is a complex factor, but one with great promise and hope for stem cell based therapies for ischemic vascular diseases. 110

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111 Figure 4-1. Mechanisms of action of IGFBP-3. IGFBP-3 can activate th e SR-B1 or S1PR to increase NO production and release via the PI3K/Akt cell survival pathway. IGFBP-3 induces VASP re-distribution, which promot es cell migration. IGFBP-3 increases NO production in both EPCs and endothelial cells, which allows these ce lls to migrate to areas of needed repair or vascular instability. Likewise, NO production and release by these cells can also attract other cells to promote vascular remodeling in areas of vascular damage. IGFBP-3 can influence vascular permeability leading to vessel stabilization and increased blood vessel in tegrity. IGFBP-3 has vascular protective and reparative effects.

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BIOGRAPHICAL SKETCH Jennifer Kielczewski was born and raised in Maryland. She attended Hood College in Frederick, MD where she obtained her B.A. in biology in 2001 with Honors. She then attended Johns Hopkins University in Baltimore, MD where she was awarded her masters degree in biotechnology in 2005. She enrolled at the University of Florid a in August 2006 to pursue her doctorate degree in biomedical sciences in the College of Medicine. She was awarded her doctorate degree in May 2010 from the University of Florida in Medical SciencesPhysiology and Pharmacology. 130