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Reduction in pre-tetinal neovascularization by ribozymes that cleave the A2b receptor mRNA

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Title:
Reduction in pre-tetinal neovascularization by ribozymes that cleave the A2b receptor mRNA
Creator:
Afzal, Aqeela ( Author, Primary )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
2003
Language:
English

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Subjects / Keywords:
Angiogenesis ( jstor )
Blood vessels ( jstor )
Cells ( jstor )
Endothelial cells ( jstor )
Messenger RNA ( jstor )
Plasmids ( jstor )
Purinergic P1 receptors ( jstor )
Receptors ( jstor )
Retina ( jstor )
RNA ( jstor )

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University of Florida
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University of Florida
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Copyright Afzal, Aqeela. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
9/9/1999

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REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES
THAT CLEAVE THE A2B RECEPTOR mRNA


















By

AQEELA AFZAL


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

UNIVERSITY OF FLORIDA


2003


































To my son, Faris Wasim.















ACKNOWLEDGMENTS

It is a pleasure to thank the many people who have made this dissertation possible. Many people have been a part of my graduate education as friends, teachers and colleagues. My mentor, Maria Grant, has been all of those. It is difficult to overstate my gratitude for her. She has instilled in me the qualities of being a good scientist. Her infectious enthusiasm for clinical research has been a major driving force during my career at the University of Florida. This dissertation is a small tribute to an exceptional woman from a student who is still anxious to learn from her.

My sincerest thanks are also due to Lynn Shaw. He patiently taught me all the techniques I needed to complete my work. He also spent countless hours editing and doing the graphics for this dissertation. His insightful comments were crucial for editing the many drafts into the final dissertation. My thanks are also due to Polyxenie E. Spoerri who taught me all the tissue culture techniques I needed to complete this dissertation. Thanks also to Sergio Caballero, Rehae Miller and past and present members of the Grant lab: Tom Ruzich, Nilanjana Sengupta, Christopher Beadle, Hao Pan

I would like to thank my committee members: Dr. Don. A. Samuelson (Professor of Veterinary Medicine); Dr. Dennis. E. Brooks (Professor of Veterinary Medicine); Dr. John. B. Dame (Professor of Veterinary Medicine); Dr. Donald. A. Armstrong, Dr. Elizabeth C. Uhl (Clinical Assistant Professor of Veterinary Medicine) and Dr. Harm J.









Knot (Assistant Professor of Pharmacology and Therapeutics) for their guidance over the years.

My son, Faris Wasim, has been a great source of inspiration. Being tired of not being able to fulfill his requests when I wanted to and missing him has been the best motivation for completing this dissertation. My husband, Wasim Asghar, has also shared this exciting journey with me. He has provided constant support and encouragement throughout my graduate career.

A very special thanks to the two people to whom I owe everything I am today, my parents, Mohammed Afzal and Mussarat Afzal. Their unwavering faith and confidence in my ability and in me is what has shaped me to be the person I am today. Thank-you for everything. My thanks are also due to my sister, Aneela Afzal, and brother Yaseen Afzal, for their support and countless hours of babysitting. My family opened their hearts to me and my little one and made it possible for me to come to work knowing that he was in good hands.

In addition, I would also like to thank the Department of Pharmacology and

Therapeutics at the College of Medicine at the University of Florida, and the College of Veterinary Medicine at the University of Florida for their financial support during my graduate career.

Thanks also to the men and women who donated their eyes to our research. Their gift has made it possible for us to understand several eye diseases and prevent them in the future.
















TABLE OF CONTENTS


ACKNOW LEDGM ENT S .iii

LIST OF FIGURES . viii

LIST OF ABBREVIATIONS. xii

ABSTRACT . xvii

CHAPTER

I BACKGROUJND ANDJ SIGNIFICANCE .1.

A natom y of the E ye 1.
The Retina. 4 Blood Supply to the Retina. 8 The Blood Retinal Barrier . 9
Retinopathies. 10
Age Related Macular Degeneration . 10
D iabetic R etinopathy 11.
Non-Proliferative Diabetic Retinopathy (NPDR).11.I Proliferative Diabetic Retinopathy (PDR). 15
Retinopathy of Prem aturity . .17
Treatment of Retinopathies . 19
Angiogenesis . 24
Extracellular matrix (ECM) . 26 ECM degradation . 26
Bound Factors. 30
Integrins . 30 EpH receptors . 31 Vascular endothelial (VE) cadherins. 32
Growth Factors. 33
Angiopoeitins. 33 Vascular Endothelial Growth Factor . 35 Fibroblast Growth Factor. 38 Platelet Derived Growth Factor . 39 Transforming Growth Factor-P . 39
Adenosine . 44
Adenosine and the Retina. 47



v









Adenosine Receptors. 49
Pharmacology of the A2B receptors. 59
Distribution of the Adenosine Receptors. 60 Intracellular Pathways Regulated by A2B Receptors. 62
Ribozymes . 64 Self Splicing Introns. 64
Group I Introns . 64 Group 11 Introns . 67 RNase P RNA. 67 Small Self Cleaving Ribozymes. 71 Hepatitis Delta Virus. 71 Hairpin Ribozymes. 71 Hammerhead Ribozymes . 71
Experimental Aim . 77

2 METHODS AND MATERIALS . 85

Defining Location of the Target Sequence . 85 Preparation of the Target Oligo-Nucleotide . 86
Time Course of Cleavage Reactions for Mouse and Human Targets (Hammerhead
Ribozymes) . 86
Multiple Turnover Kinetics. 87 Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector. 88 Sequencing of the Clones. 88 Human Retinal Endothelial Cell (HREC) Tissue Culture .89 LDL Uptake of the HREC . 90 Transfection of HREC using DEAE-Dextran . 91 Transfection Efficiency using DEAE Dextran for HREC's . 92 Cell Migration Assay. 92 Morphology of HEK Cells. 93 Transfection using Lipofectamine on HEK 293 cells . 93 Transfection Efficiency for HEK Cells using Lipofectamine Reagent . 94 cAMP Assay on Transfected HEK 293 Cells . 94 Total Retinal RNA Extraction for PCR. 97 Real Time PCR . 97 Animals . 98 Intraocular Inj ection into the Mouse Model of Oxygen Induced Retinopathy . 98 Statistical Analysis. 99

3 RESULTS. 100

Determining Accessibility of the Target Site. 100 Time Course of Ribozyme cleavage. 103 Multiple Turnover Kinetics . 106 Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector . 109 Sequencing of the Clones .111.II Cell Cultures . 111II









Transfection of HIREC . 114 Transfection Using Lipofectmaine on HEK Cells. 118 CAMP Assay on Transfected ITEK Cells . 121
Real Time PCR. 125
Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model . 125

4 DISCUSSION . 131

Ribozymes As Tools To Study Gene Expression . 132 Delivery Of The Ribozyme In vivo. 135 Promoter Considerations . 139 Future Studies . 142

5 LIST OF REFERENCES . 147

BIOGRAPHICAL SKETCH. 165
















LIST OF FIGURES


Figure 12M4ge

1-1 Cross sectional view of the components of the eye . 2 1-2 The ten layers of the retina . 6 1-3 A fundus shot of ARMD. 12 1-4 Non-proliferative retinopathy. 14 1-5 New blood vessel growth around optic nerve in PDR. 16 1-6 ICROP definition of retinopathy. 18 1-7 The five stages of ROP . 20 1-8 Laser treatment of the eye . 21 1-9 Cartoon showing cryotherapy application to the anterior avascular retina. 23 1-10 The process of angiogenesis . 25 1-11 PAs hydrolyze plasminogen to plasmin. 28 1-12 Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors. 34 1-13 The vascular endothelial cell growth factor (VEGF R2) signaling pathway. 37 1-14 The FGF receptor and signaling pathway. 40 1-15 The PDGF receptor and signaling pathway. 41 1-16 The TGF-3 receptor signaling pathway . 43 1-17 Intracellular and extracellular production of adenosine. 46 1-18 Role of the high and low affinity adenosine receptors. 50 1-19 Homology of the A, receptor for human and mouse . 53 1-20 Homology of the A2A receptor between human and mouse . 54









1-21 Homology of the A2B receptor between the human and the mouse. 55 1-22 The A 2B receptor .5 1-23. The A2A receptor. 58 1-24 The A 2B signaling pathway. 63 1-25 The secondary structure group I introns . 65 1-26 Splicing mechanism of the group I introns. 66 1-27 Secondary structure of Group 11 introns . 68 1-28 The splicing mechanism of the Group 11 introns . 69 1-29 Cleavage of the tRNA 5' leader sequence by Rnase P. 70 1-30 Self-cleaving ribozymes resolve concatemners formed by rolling-circle replication
into individual genomic molecules. 72 1-31 Structure of the hairpin ribozyme. 73 1-32 Structure of the hammerhead ribozyme . 75 1-33 The hammerhead ribozyme cleaves its substrate by a transesterification
reaction . 76 1-34 Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.79 1-35 Target sequences of the human and mouse A2B ribozymes 1 and 2 . 80 1-36 Hammerhead ribozymes for the A 2B RzlI and Rz2 . 81 1-37 The p2lNewhp Vector with the CMV enhancer and beta actin promoter. 82 1-38 Time course for the ROP model . 84 3-1 Theoretical tertiary structures of the active A2B RzlI generated by the infold
program. 101 3-2 Theoretical tertiary structures of the active A2B Rz2 generated by the infold
program. 102 3-3 Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing
products of cleavage of the A2B Rz2 on the mouse target. 104 3-4 Time course analysis data. 105









3-5 Time course of the ribozyme with increasing target concentrations . 107 3-6 Time course cleavage reaction with varying temperatures (37 �C/25�C) and
magnesium concentrations of 20mM/lmM . 108 3-7 K inetic analysis of the ribozym es . 110 3-8 Sequence of the active and inactive versions of the A2B ribozymes at the site of
insertion w ithin the p21N ew H p vector . 112 3-9 Pebble stone m orphology of the HRE C . 113 3-10 . L D L uptake of H R E C . 115 3-11 The GFP plasmid. This plasmid was driven by a CMV enhancer and a chicken
b eta actin p ro m o ter . 1 16 3-12 Transfection efficiency of the H RE C . 117 3-13 T heory of m igration assay . 119 3-14 Migration data for the cells transfected with the active and inactive versions of
the A2B receptor and the vector control. 10% FBS/DMEM is the positive control
and D M EM alone is the negative control . 120

3-15 Transfection Efficiency of HEK cells . 122 3-16 [EK cells transfection efficiency following passage 1 . 123 3-17 cAMP accumulation in HEK cells transfected with the control, active A2B Rz2 and
in a ctiv e A 2B R z 2 . 12 4 3-18 Real time RT-PCR results showing relative levels of the adenosine A2A and A2B
receptor mRNAs isolated from HEK cells transfected with plasmid DNA . 126 3-19 The mice eyes were embedded in paraffin and three hundred serial sections were
d o n e . . 1 2 7

3-20 Injection with the control plasmid prior to exposure to high oxygen shows a high
number of endothelial cell nuclei surrounding blood vessel lumen . 128 3-21 Injection with the active A2B ribozyme prior to high oxygen exposure significantly
reduced the pre-retinal neovascularization . 129 3-22 Injection of the active and inactive versions of the A2B Rz2 and the vector control
in the R O P m ou se m odel . 130 4-1 Entry of the AAV and transferrin into the cell . 137









4-2 Diagram of the expression cassettes fusion protein and alkaline phosphatase
(Alk Phos) . 141

4-3 A2B signaling pathway with theoretical downstream effects, which have yet to be
confirmed . 146















LIST OF ABBREVIATIONS a-LDL Acetylated 1,1'-Dioctdycl-3,3,3',3' tetra methyl indocarbocyanin
perchlorate
ABAM Antibiotic antimycotic mix

A1 Adenosine receptor type 1

A2A Adenosine receptor type 2A

A2B Adenosine receptor type 2B

A2B Rzl Adenosine receptor type 2 ribozyme 1

A2B Rz2 Adenosine receptor type 2 ribozyme 1

A2R Adenosine receptor type 2

A3 Adenosine receptor type 3

ADA Adenosise deaminase

AK Adenosine kinase

AMP Adenosine monophosphate

ANG-1 Angiopoeitin 1

ANG-2 Angiopoeitin 2

ARMD Age Related Macular Degeneration

ARNT Aryl hydrocarbon receptor nuclear translocator

ARVO Association for Research in Vision and Ophthalmology

ATP Adenosine triphosphate

avP3 Alpha v beta 3 integrin









aG35 bFGF BSA

cAMP CAT CGS21680

CHA CHO CMV DMEM DMSO

DNA DPSPX DTT ECM EDTA EGS

Eph receptor FAK FAT

FBS Flt GAGs


Integrin

Basic fibroblast growth factor Bovine serum albumin 3c, 5c-cyclic monophosphate Chloramphenicol acetyltransferase A2A agonist. 2- {4[(2-carboxylethyl)-phenyl]ethylamine}-5'-Nethylcarboxamidoadenosine Cyclohexyladenosine Chinese hamster ovary cells Cytomegalovirus Dubellco's modifeid eagle medium Dimethyl sulfoxide Deoxyribonucleic acid Non-seletive adenosine receptor antagonist. 1,3-dipropyl-8(psulfophenyl)xanthine Dithiothreitol Extracellular matrix Ethylenediamine tetraacetic acid External guide sequence Ephrin receptor Focal adhesion kinase Focal adhesion targeting sequence Fetal bovine serum VEGF fms like tyrosine kinase Glycosaminoglycans









GC GCL GFP GPI HBSS

HDV

HEK 293 HIF HIV HRE HRECs HSPGs IACUC IB-MECA ICROP ILM INL IP3 IPL KDR LAP MMP NAD


Guanosine cytosine content Ganglion cell layer Green fluorescent protein Glycosylphosphatidylinositol Hanks balanced salt solution Hepatitis delta virus Human embryonic kidney cells Hypoxia inducible factor Human immunodeficiency virus Hypoxia response element Human retinal endothelial cells Heparan sulfate proteoglycans Institution Animal Care and Use Committee. Selective A3 adenosine receptor agonist. N6 (3-iodobenzyl)Ado-5'Nmethyl Uronamide International classification of Retinopathy of Prematurity Inner limiting membrane Inner nuclear layer Inositol triphosphate Inner plexiform layer VEGF kinase insert domain Latency associated peptide Metalloproteinases Nicotinamide adenine dinucleotide









NBTI Nitrobenzylthioinosine

NECA N-ethylcarboxyamidoadenosine

NFL Nerve fibre layer

NPDR Non proliferative diabetic retinopathy

5'NT 5' Nucleotodase

OLM Outer limiting membrane

ONL Outer nuclear layer

OPL Outer plexiform layer.

PAs Plasminogen activators

PAl-1 Plasminogen activator inhibitor-i

PAI-2 Plasminogen activator inhibitor-2

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PKC Protein kinase C

PLC Phospholipase C

PDR Proliferative diabetic retinopathy

rAAV Recombinant adeno associated virus

RBCs Red blood cells

ROP Retinopathy of Prematurity

RNA Ribonucleic acid

rRNA Ribosomal RNA

RNasin Ribonuclease inhibitor

RPE Retinal pigment epithelium









R-PIA SAH

TBS TGF Tie 1 and 2 TIMPS TNF-ca tPA tRNA TR uPA VE cadherin VEGF VEGF-R1 VEGF-R2 WBCs XAC XDH XO


Selective Al receptor agonist. R-phenylisopropyl-adenosine S-Adenosylhomocysteine Tris buffered saline Transforming growth factor Angiopoeitin receptors 1 and 2 Tissue inhibitors of matrix metalloproteinases Tumor necrosis factor alpha Tissue type plasminogen activator Transfer RNA Inverted terminal repeats Urokinase type plasminogen inhibitor Vascular endothelial cadherins Vascular endothelial growth factor Vascular endothelial growth factor-receptor 1 Vascular endothelial growth-receptor 2 White blood cells Xanthine amine cogener Xanthine dehydrogenase Xanthine oxidase















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

REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES
THAT CLEAVE THE A2B RECEPTOR MRNA By

Aqeela Afzal

May 2003

Chair: Dr. M.B. Grant
Cochair: Dr. D. Samuelson
Major Department: Veterinary Medical Sciences

Tissue hypoxia and ischemia initiate events that lead to pre-retinal angiogenesis. Adenosine modulates a variety of cellular functions by interacting with specific cell surface G-protein coupled receptors (Ai, A2A, A2B, A3) and is a potential mediator of angiogenesis. The A2B receptor has been implicated in the mediation of angiogenesis. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design and characterize a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Active and inactive ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. HEK 293 cells were transfected with these plasmids, and A2B mRNA levels were determined by quantitative RT-PCR. Human retinal endothelial cells (HREC) were also transfected, and cell migration was examined. The effects of these ribozymes on the levels of pre-retinal neovascularization were determined using a mouse model of oxygen-induced retinopathy. We produced a









ribozyme with a Vmax of 10.8 pmole min1 and a kcat of 36.1 min1. Transfection of HEK 293 cells with the plasmid expressing ribozyme resulted in a reduction of A2B mRNA levels by 45%. Transfection of HREC reduced NECA stimulated migration of the cells by 47%. Intraocular injection of the constructs into the mouse model reduced pre-retinal neovascularization by 54%. Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor, and provide further support for the role of the A2B receptor in retinal angiogenesis.


xviii














CHAPTER 1
BACKGROUND AND SIGNIFICANCE The formation of blood vessels is a fundamental process that can be broken down into two basic pathways. The first is vasculogenesis, which is the formation of new blood vessels such as seen in embryogenesis. The second is angiogenesis, which is the formation of blood vessels from pre-existing blood vessels. Angiogenesis is common in both normal physiological processes (pregnancy, menstruation, wound healing) and disease states (cancer, retinopathies, psoriasis). The focus of this study is the process of angiogenesis in retinopathies, including diabetic retinopathy, the leading cause of blindness in adults, and retinopathy of prematurity (ROP).

Anatomy of the Eye

The two eyes in humans are oriented to facilitate binocular single vision, which results from the forward position of the eyes and the chiasmal crossing from axons of ganglion cells. Axons from the right visual field carry impulses to the left optic tract and vice versa. The eye contains the elements that take in light and converts them to neural signals. For protection, the eye is located within the bone and connective tissue framework of the orbit. The eyelids cover and protect the anterior surface of the eye and contain glands, which produce a lubricating film (tears).1

The globe has three spaces within it: the anterior chamber, posterior chamber and the vitreous chamber. 1 (Figure 1-1)









conjunctiva ciliary body
iris
aqueous
humor pupil I anterior chamber
crystalline
lens
cornea


vitreous humor
Sretina
optic nerve




Smacula

Schoroid


extraocular
muscle


Figure 1-1. Cross sectional view of the components of the eye









The anterior and posterior chambers contain aqueous humour, which is produced by the ciliary body and provides nourishment for the surrounding structures. The vitreous chamber is the largest space in the eye and lies adjacent to the inner retinal layer and contains the gel-like vitreous humor.'

The eye is made up of three layers: an outer fibrous layer, a middle vascular layer and an inner neural layer (retina).' The outer fiber layer is a dense connective tissue that provides protection for structures within, maintains the shape of the eye, and provides resistance to the pressure of the fluids inside the eye. The sclera is the opaque white of the eye, and the cornea is transparent and allows light to enter the eye where the lens refracts it to bring light rays into focus on the retina.'1

The middle layer of the eye is made up of three structures. The iris acts as a

diaphragm to regulate the amount of light entering the pupil. The ciliary body produces components of the aqueous humor and has muscles that control the shape of the lens during accomodation. The choroid is an anastomosing network of blood vessels with a dense capillary network.1

The principle functions of the choroid are to nourish the outer retina and to provide a pathway for the vessels that supply the anterior eye. The choroid is an egress for catabolites from the retina, which diffuse through Bruch' s membrane into the choriocapillaris. The suprachoroidal space provides a pathway for the posterior vessels and nerves that supply the anterior segment. 12The choroid also plays a role in the maintenance of intraocular pressure due to the high blood flow in its vessels. The choroid has the largest sized and the greatest number of vascular channels in the eye, and the amount of blood flowing through these channels at any time has an effect on the









intraocular pressure. The choroid also provides a regular smooth internal surface for the support of the retina. The smoothness of Bruch's membrane is important in maintaining the exact relationship between the retinal pigment epithelium (RPE) and the outer segments of the adjacent photoreceptor rods and cones. 1,3 The Retina

The retina is located between the choroid and the vitreous, and extends from the

circular edge of the optic disc, where the nerve fibers exit the eye, to the ora serrata and is continuous with the epithelial layers of the ciliary body. 1,4 The retina is a thin, delicate and transparent tissue that lines the inner eye. The neural retina is attached loosely to the choroid through the pigment epithelium. Externally, the RPE contacts the collagen and elastic tissue of Bruch's membrane of the choroid. Bruchs membrane is an elastic layer that stabilizes the RPE and the photoreceptors. Internally, the retina lies next to the vitreous. Anteriorly, the RPE gives rise to the ciliary body, and posteriorly, all the retinal layers terminate at the optic disc except the nerve fiber layer. The retina is thickest at the equator and thins at the ora serrata.1

The retina can be divided into the central retina and the peripheral retina. The central retina is thick and includes the macula, fovea and foveola. The macula has a yellow appearance due to xanthophyl (a carotenoid), which is found in the ganglion cells. The peripheral retina includes the remainder of the retina from the macula to the temporal or nasal side. The ora serrata is the extreme periphery of the retina. It is the junction where the retina ends and gives rise to the teeth like processes that form the ciliary body. The peripheral retina ends at the ora serrata and forms the teeth like processes that form the base of the ciliary body.1









Under the light microscope, ten layers of the retina can be differentiated (Figure 12): RPE, rod and cone layer, outer limiting membrane (OLM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fibre layer (NFL) and the inner limiting membrane (ILM). The visual pathway consists of three interconnecting neurons and some receptor cells. The neurons include the bipolar cells, located within the retina; the ganglion cells, located in the inner retina, the axons of which go through the optic nerve to the chiasm and end in the lateral geniculate nucleus; and the third neuron is from the geniculate body to the occipital cortex.1

The rods and cones are the sensory receptors. The outer segments have

photopigments, which are excited by light, resulting in a visual response. The cell bodies of the rods and cones lie in the ONL and the axons synapse with dendrites of bipolar cells in the OPL. The dendrites of the bipolar cells extend to the OPL and synapse with axons of rods and cones; their axons extend to the IPL and synapse with dendrites of the ganglion cells from the NFL. The INL also has horizontal and amacrine cells. The horizontal and amacrine cells in this layer provide horizontal integration.1 The RPE is a single layer of uniform cells. It is located in the outer circumference of the retina and extends from the edge of the optic disc to the ora serrata. The cells of this layer are hexagonal shaped and carry a brown pigment. These cells may be multinucleated, especially in the ora serrata. The RPE provides metabolites to the receptors and removes the outermost ends of external segments of the photoreceptors. If the RPE cells are damaged or diseased, these cells are not replaced; instead, adjacent cells slide laterally to fill the space of the necrotic cells. The RPE cells possess microvilli on










choroid
pigment
epithelium
outer segments

inner segments

outer nuclear
layer (ONL)

outer plexiform N4-" layer (OPL)

inner nuclear
layer (INL)

inner plexiform
. "a er('IPL)
. - layer (CCL)

optic fiber layer
(OFL)


- photoreceptcor


h.1]1 11t. l LIl


Figure 1-2. The ten layers of the retina include: retinal pigment epithelium, rod and cone
layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fibre
layer and the inner limiting membrane









the apical surface that interdigitate with the photoreceptors. The R-PE is critical to vitamin A metabolism and photoreceptor maintenance. 1

The rod and cone layer lies external to the OLM. It has a thick inner segment and thin outer segments joined by a slight constriction. The cell membrane is continuous between the constrictions. The outer segments have parallel processes, which are short in cones and long and thin in rods. 1

The OLM under a light microscope has a thin fenestrated membrane-like

appearance. However, the OLM is not a basement membrane. Electron microscopy revealed it to be a zonula adherens between the photoreceptors and the Mufller cells. The zonula adherens probably serve to keep the highly elongated photoreceptors in place.'

The ONL has the cell bodies of the rods and the cones. The axons of the rods and cones synapse in the OPL with bipolar and horizontal cells.'

The OPL is a reticular structure, which is a transition zone between the receptors (neuroepithelial). The OPL is a layer of synaptic contacts between photoreceptors, bipolar cells and horizontal cells. The axons of the rods end here in spherules (oval shaped) and those of the cones end in pedicles (broad conical swellings). The spherules are invaginated and synapse with bipolar dendrites or horizontal cells, and can make 2-4 contacts. The pedicles, on the other hand, contact many dendrites of horizontal cells and bipolar cells.1

The INL is a band of nuclei belonging to horizontal cells, bipolar cells, and amacrine cells. The Muller cells provide support and nutrition to the retina. They surround capillary walls and extend from the ILM to the extracellular membrane.









The JPL is a junction between the first order neuron (bipolar cells) and the ganglion cell layer. This layer contains the nuclei of displaced ganglion or amacrine cells and processes of the Mifler cells.1

The GCL contains the cell bodies of ganglion cells, which are thin in the nasal area and thicker near the macula. The axons of the ganglion cells run internally and then become parallel to the inner surface of the retina to give rise to the NFL and the optic nerve fibres.1 The NFL has the axons of the ganglion cells and is thickest around the optic nerve.1 Branching processes of the MWller cells and a basal lamina like structure secreted by them forms the JLM. The macula is the center of the retina (area centralis) and is divided into the fovea (cone dominated), parafovea (ganglion cell dominated) and the perifoveal retina (single layer of ganglion cells).1 Blood Supply to the Retina

The retina has the highest rate of metabolism of any tissue in the body and thus has a dual blood supply from the retinal and choroidal capillaries. If either of these sources is interrupted, ischemnia develops and leads to loss of function. The outer retina is supplied by the choriocapillaris and the central retinal artery supplies the remainder. The retinal artery is different from other arteries and does not have an internal elastic lamina but does have a prominently developed muscularis. 1,5

The outer retinal layers receive their nutrition from the choroidal capillary bed; metabolites diffuse through Bruch' s membrane and the R-PE into the neural retina. The central retinal artery provides nutrients to the inner retinal layers. The artery enters the retina through the optic disc, usually slightly nasal of center, and branches into a superior and inferior retinal artery, each of which further divides into nasal and temporal branches, and these vessels continue to bifurcate. The nasal branches run a relatively straight









course toward the ora serrata, but the temporal vessels arch around the macular area en route to the periphery. Two capillary networks exist within the retina. The deepest one lies in the inner nuclear layer near the outer plexiform layer, and the superficial one is in the nerve fiber or ganglion cell layer. The OPL is avascular and thought to receive its nutrients from both retinal and choroidal vessels. 15

Retinal arterial circulation is terminal; therefore there is no direct communication between the retina and other vessel systems. The junctions of endothelial cells in retinal vessels are tight or occluded. Thus, to enter or leave the retina, most substances require active transport across the endothelial cells. The outer retinal layers receive their nutrition from the choroidal capillary bed; the central retinal artery provides nutrients to the inner retinal layers. These vessels are distributed to the four quadrants of the retina.6 The retinal artery and vein to a particular quadrant supply most of the quadrant. If arterial supply to a retinal quadrant is interrupted, infarction of that section occurs. The retinal capillaries supply the inner two thirds of the retina; the choroidal circulation supplies the remaining outer retina via regulated transport across the pigment epithelium. 1,5 The Blood Retinal Barrier

The epithelial portion of the blood-retinal barrier is the retinal pigment epithelium. This barrier separates the choroidal tissue fluid, which is similar to plasma, from the retinal tissue fluid. Tight junctions that exist between the endothelial cells of the retinal vessels and similar tight junctions in the RPE maintain the blood retinal barrier. Thus, the retinal vessels are impermeable to the passage of molecules greater than 20-30 kDa, and small molecules such as glucose and ascorbate are transported by facilitated diffusion through the RPE. 1,5,7









Vascular beds are situated to provide nourishment. To avoid problems with the presence of blood vessels in the outer retina, the outer layers of the retina receive their nourishment from the choriocapillaris.1

Retinopathies

The retina of the eye is uniquely situated to provide optimal vision. Blood supply to the retina is also strategically placed to avoid any hindrance of the visual pathway. Retinopathies (diseases affecting the retina) disrupt this balance and lead to loss of vision. Retinopathies affecting humans include: age related macular degeneration armed) , which primarily affects the aging population; diabetic retinopathy (DR), which primarily affects the working population; and retinopathy of prematurity (ROP) which primarily affects the newborns.

Age Related Macular Degeneration

ARMD is a disease which affects the RPE and leads to blindness in the aged populations.8 There are two forms of ARMD: dry and wet.9-11

Dry ARMD is characterized by the presence of soft drusen and pigmentary

abnormalities. Drusen is an amorphous acellular debris present within the basement membrane of the RPE. It is seen as 'yellow' spots within the macula. Low amounts of drusen are a consequence of age; however, a larger amount present within the retina is indicative of ARMD.10 Drusen leads to mild vision loss and increases the risk of progression of the disease to the wet form of ARMID.8'9

The wet form of ARMD (also known as the exudative or the neovascular phase) is characterized by choroidal neovascularization, RPE detachment and disciform scarring. The wet form of ARMD leads to rapid vision loss. The choroidal neovascularization









(CNV) leads to the formation of immature blood vessels which result in leakage of serum and blood and loss of central vision. 912 (Figure 1-3) Diabetic Retinopathy

Diabetes Mellitus affects millions of people worldwide and is the leading cause of blindness in working age adults. 13,14,15 There are two forms of diabetes mellitus: Type 1, which typically affects juveniles and is known as insulin dependent diabetes mellitus, and type 11, which is the adult onset form of diabetes and is known as non-insulin dependent diabetes mellitus. 15 Diabetes also leads to systemic complications such as kidney failure, hypertension and cardiovascular disease. 1,3DR is the most frequent diabetic complication. Eye problems due to diabetes can be asymptomatic and if left untreated can lead to serious visual loss. The longer a patient has diabetes, the more likely they are to develop diabetic retinopathy. 13161715 Diabetic eye disease can be divided into two phases: background diabetic retinopathy (non-proliferative phase) and proliferative diabetic retinopathy (PDR).

Non-Proliferative Diabetic Retinopathy (NPDR)

In NPDR small retinal blood vessels are damaged. NPDR is the result of two

major processes which affect retinal blood vessels, vessel closure and abnormal vessel permeability. 13 The vessels leak fluid (edema) and later blood (hemorrhage) into the retina. Macular edema is the most common cause of reduced vision in patients with nonproliferative diabetic retinopathy and is seen as milkiness of the retina surrounded with exudates (yellow clumps). 161 These exudates are the result of fat or protein leaking out of the vessels. Water is quickly reabsorbed into the vessels




















































Figure 1-3. A. A fungus shot showing drusen (yellow). B. Wet form of ARMD
showing blood leakage. (National Eye Institute)









or tissue under the retina. However, the fatty material is absorbed very slowly and thus left behind surrounding the leakage site. 16,18 (Figure 1-4)

Vessel closure may be due to blood cell clumping, damaged endothelium, swelling of an abnormally permeable vessel wall or compression of the capillary by surrounding retinal swelling. Diabetic patients have closure/non-perfusion of capillaries, which leads to a decreased oxygen supply. In areas surrounding the area of non-perfusion capillaries dilate to compensate for the decreased oxygen supply. Small focal dilations (microaneurysms) of retinal capillaries also develop due to weakened capillary walls, thus allowing for bulging. 13 When multiple areas of the retina have lost their blood supply, angiogenic factors are released which stimulate proliferation of new blood vessels. These new blood vessels are small and fragile, therefore, cause bleeding and the formation of scar tissue within the retina. Small arterial closures follow capillary closure, and deprive larger regions of the retina of blood supply. This is seen as 'cotton wool spots' on the retina in fluorescein angiography. 16

Blood vessels in the body are usually fenestrated allowing fluid to pass through vessel walls. These openings are small enough to allow water and ions to pass through, while preventing the passage of blood cells and larger proteins. In contrast, retinal blood vessels have tight junctions between the endothelial cells of blood vessel. Therefore, all fluids and molecules exiting the vessels have to pass through the cell. This lack of fenestration helps to keep the retina thin and dehydrated for proper function. These tight junctions form the blood retinal barrier, which partitions the neural retina from the circulation and protects the retina from circulating inflammatory cells. 16 The tight junctions are formed by a number of proteins such as: occludin and claudin. These





















































Figure 1-4. A. Non-proliferative retinopathy. Hemorrhage (arrowhead) short arrow
microaneurysm, larger arrow exudates. B. Macular edema. (National Eye
Institute)









proteins limit the flow of fluid between endothelial cells. Diabetic patients have a lower amount of occludin at the tight junctions in the retinal endothelial cells and can leak fluid. 16

Proliferative Diabetic Retinopathy (PDR).

Proliferative diabetic retinopathy is the stage of diabetes characterized by

angiogenesis on the surface of the retina. 13Patients can have NPDR for years before progressing to PDR. PDR is diagnosed by the presence of proliferating blood vessels within the retina or optic disc. These vessels grow on the retinal surface or into the vitreous cavity and take on a frond-like configuration as they grow. 19 (Figure 1-5) The new blood vessels form due to the closure of retinal capillaries, which leads to ischemnia. As patches of the retina are deprived of oxygen and nutrients, vasoproliferative factors are released which diffuse into the vitreous cavity. These factors stimulate growth of new vessels throughout the retina. 15

The new blood vessels are not located in the same location as the ischemnically

damaged retina and are very fragile and bleed into the vitreous. A small amount of blood may be removed in a few weeks and larger blood hemorrhages may take a few months. If dense blood from multiple recurrent hemorrhages occurs, then vision may not be restored since the residual inflammatory debris and dead cells cannot be removed. Another complication of PDR is traction retinal detachment. New vessels grow and regress and lay down fibrous scar tissue, which contracts and shrinks as it matures. If the neovascularization is on the surface of the retina then contraction of the fibrous scar distorts the retina. However, if the vessels grow into the vitreous and contract, retinal detachment occurs which leads to blindness. 13,15



















































Figure 1-5. New blood vessel growth around optic nerve in PDR (Top). Hemorrhage
from new blood vessel growth (Bottom). (National Eye Institute)









Retinopathy of Prematurity

Retinopathy of prematurity (ROP), also known as retrolental fibroplasia, is a potentially blinding condition affecting the retina of newborns. In the 1950s, it was associated with the use of high oxygen levels in neonatal units.2 Modem neonatal care has curbed the incidence of ROP, but because the survival rate of low-birth-weight infants is increasing, the exposure of surviving babies to high oxygen levels is also increasing and ROP is still a relevant clinical problem.12

ROP causes more blindness among children in the world than all other causes

combined. It begins after removal from high oxygen conditions and may progress rapidly to blindness over a period of weeks.2 Active growth of the fetal eye occurs between the last 12 weeks of full term delivery (28-40 weeks of gestation). At 16 weeks of gestation, blood vessels gradually grow over the surface of the retina. Vessels reach the anterior edge of the retina and stop progressing at about 40 weeks of gestation .201,42

The international classification of ROP (ICROP) defines retinopathy by several

distinct criteria: location, extent, stage, and plus disease .2 Location refers to the location of the damage to the retina relative to the optic nerve. Normally retinal vessels begin growth at the optic nerve and gradually move toward the edge of the retina. Vessels further from the optic nerve are more mature. To standardize the location of ROP, the retina is divided into three zones: Zone I is centered on the optic disc and extends from the optic disc to twice the distance between the disc and macula; Zone 11 is a concentric ring around zone I and extends to the nasal ora serrata (the edge of the retina on the side toward the nose); and zone III is the remaining crescent of retina on the temporal side (side towards the temple) (Figure 1-6). The extent of ROP is described by the clock hours






18 12


Temporal ora serrata






9




Macular
center


Nasal ora serrata Optic nerve


Figure 1-6. ICROP definition of retinopathy. The retina is divided into three zones:
Zone I, Zone II and Zone III.









of the retina involved in the ROP. For example, if the ROP extends from 1:00 to 5:00, the extent of ROP is 4 clock hours .24

ROP is a progressive disease that begins with some mild changes in vessels and may progress on to more severe changes. The five stages of ROP describe the progression of the disease (Figure 1-7). Stage 1 is characterized by a demarcation line between the normal retina (near the optic nerve) and vascularized retina. In stage 2, a ridge of scar tissue rises up from the retina due to growth of abnormal vessels. This ridge forms in place of the demarcation line. In stage 3, the vascular ridge grows due to spread of abnormal vessels and extends into the vitreous. Stages 4 and 5 refer to retinal detachment; stage 4 refers to a partial retinal detachment caused by contraction of the ridge, thus pulling the retina away from the wall of the eye; and stage 5 refers to complete retinal detachment. Plus disease is a very severe form of ROP which is characterized by the abnormal growth of blood vessels near the optic nerve .2 Treatment of Retinopathies.

Spot laser photocoagulation is used for the treatment of ROP .29 This uses an

argon/diode laser to burn spots on the peripheral and middle portions of the retina. When laser light hits blood or pigment, it is absorbed as heat energy and produces a small burn. The laser treatment leads to a decrease in the level of vasoproliferative factors produced by the ischemnic retina. The avascular retina is treated using a small laser spot (Figure 18). The laser spot directly treats the retina and the underlying tissue, thus reducing inflammation and results in less damage to other ocular structures. Destruction of small patches of the ischemnic retina reduces the oxygen demand and decreases the vasoproliferative factor production. Laser treatment also thins the pigmented tissue under


























Stage 1 ROP is characterized by a demarcation Stage 2 ROP; the white line is replaced by a line. The orange vascular retina is on the left ridge of scar tissue R. The arrow shows a tuft and the gray peripheral retina is on the right of new vessels. separtated by the white line.


Stage 3 ROP. The size of the ridge has Plus disease shows dilation and tortuosity of
increased (between arrows) and the growth of blood vessels near the optic nerve. the ridge extends into the vitreous.


Figure 1-7. The five stages of ROP (National Eye Institute)

































Figure 1-8 Laser treatment of the eye. The laser spot directly treats the retina and the
underlying tissue. Laser treatment thins the pigmented tissue under the retina
and allows more oxygen to diffuse in from the vessels under the retina









the retina thus allowing better oxygen diffusion in the retina. Laser treatment increases oxygen supply, lowers the demand for oxygen and lowers the incidents for new vessels growth.30,28 Laser photocoagulation also causes less pain than other therapies. Currently laser treatment is the best option for the treatment of retinopathies. 1431,29,32

Cryotherapy is also one of the treatments available for the treatment of

retinopathies.33'34 This technique involves placing a cold probe on the sclera until an ice ball forms on the retinal surface. Multiple applications are done to cover the entire vascular area (Figure 1-9). This thins the tissue under the retina (by destroying it) and allows easier oxygen diffusion through the retina.31 Due to the pain involved in cryotherapy, anesthesia has to be administered which is a risk factor for premature infants. If no anesthesia is administered, cardiac arrest follows. Another complication is hemorrhage due to excessive bleeding.31'33'35'36

If laser photocoagulation or cryotherapy is unsuccessful, a scleral buckle may be used.37 This involves surgery and is used if there is shallow retinal detachment due to the

contraction of the ridge. A silicone band is tightly placed around the equator of the eye thus producing a slight indentation on the inside of the eye.38 This indentation relieves traction of the vitreous gel and allows the retina to flatten back onto the wall of the eye. The silicone band is then removed a few months later to allow the eye to grow. 14,31,39 If the scleral buckle is not sufficient, vitrectomy may be performed. Small incisions are made into the eye, the vitreous removed and replaced by saline. This technique also has had limited success. Current available therapies for the different types of retinopathies have had limited success. The underlying cause of retinopathies is angiogenesis































Figure 1-9. Cartoon showing cryotherapy application to the anterior avascular retina. A
cold probe is placed on the sclera till an ice ball forms on the retinal surface.
Multiple applications are done to cover the entire vascular area. This
treatment thins the tissue under the retina and allows easier oxygen diffusion
through the retina









(abnormal blood vessel formation). Development of other effective therapies for the diseases requires an understanding of the process of angiogenesis.28,33,40-42 Angiogenesis

Vasulogenesis is the formation of new blood vessels. Precursor cells (angioblasts) differentiate into endothelial cells which later link to form blood vessels. Angiogenesis on the other hand is the sprouting of blood vessels from pre-existing blood vessels.43 The vasculature of the retina undergoes both vasculogenesis and angiogenesis. The superficial retinal vessels, which originate at the optic disc, are formed by the process of vasculogenesis and the process of angiogenesis later forms the capillary beds.

The process of angiogenesis involves endothelial branching, sprouting, migration, proliferation and anastomosing with endothelial cells in existing vessels.43-45 (Figure 110) Vascular endothelial cells form a monolayer throughout the entire vasculature. They are polarized cells with an apical surface and a basal surface, which is surrounded by a

46-49
basal lamina. Mural cells wrap around this structure and are contractile cells, which regulate vessel diameter and consequently blood flow.47'50 On large vessels they are multi layered and referred to as smooth muscle cells. On capillaries mural cells are sparse and usually referred to as pericytes.48'5 The extracellular matrix, bound factors and the soluble growth factors all play an important role in the process of angiogenesis.














Endothelial cells



VEGF Capillary formation
EC proliferation
EC migration
EC protease production


Pericytes-j


+Ang2
+VEGF

Sprouting


+Ang2
VEGF

Regression
;ap~~
e .4 ~


PDGF-B Recruitment of

pericytes


VEGF

TGF- 1 Differentiation

A ngl Stabilization


+Angl
Stabilization





-Angl
Destabilization


Figure 1-10. The process of angiogenesis. The process of angiogenesis involves
endothelial branching, sprouting, migration and proliferation. Vascular
endothelial cells form a monolayer throughout the entire vasculature.
Pericytes wrap around these cells.


I









Extracellular matrix (ECM).

The ECM surrounds and provides mechanical support for blood vessels. The

activated endothelial cells cerate gaps in the basement membrane, which allows them to sprout into the ECM. The ECM is composed of two compartments: the interstitial matrix and the vascular basement membrane.51'52

The interstitial matrix consists of fibrillar collagen and glycoproteins (e.g.

fibronectin, laminin). Fibronectin attaches cells to a variety of ECM components, and laminin anchors cell surfaces to the basal lamina. Collagen provides structural support, is synthesized by fibroblasts and is the most abundant protein comprising the ECM. There are 12 types of collagen and types I, II and III are the most abundant types of collagen in the ECM.51'52 The vascular basement membrane lies between the endothelial cells and pericytes. It is composed of type IV collagen, which forms the basal lamina upon which the endothelium rests, and heparan sulfate proteoglycans.51

Proteoglycans are glycosaminoglycans (GAGs) linked to proteins. Cell surface heparan sulfate proteoglycans (HSPGs) function as endothelial cell receptors that recognize the ECM. They are present in the basement membranes and cell surfaces. These proteoglycans modulate the response of endothelial cells to basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and other heparan binding angiogenic factors by sequestering these molecules in the ECM. Heparatinases trigger the release of these growth factors from the ECM and make them available for angiogenic stimuli .5153

ECM degradation

Endothelial cells degrade the surrounding ECM by the release of plasminogen

activators (PAs) and matrix metalloproteases (MMPs).54 The PAs hydrolyze plasminogen









to plasmin, which is a general protease that can digest most proteins. (Figure 1-11) It also converts latent collagenase into active collagensase which can then degrade collagen type I, II and III. 54 There are two types of PAs: tissue type PA (tPA) and urokinase-type PA (uPA).55 Both PAs utilize the same substrate, plasminogen, and both have two specific inhibitors, plasminogen activator inhibitor-i (PAl-1) and plasminogen activator inhibitor-2 (PAI-2). PAI-1 is produced by endothelial cells to inhibit PA activity to ensure a balanced degradation of the ECM. uPA and PAI-1 are also upregulated by angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)54-56

Matrix metalloproteases (MMPs) are zinc dependent endopeptidases which are secreted as zymogens and proteolytically activated by other MMPs or plasmin.51 MMP expression may also be regulated by growth factors such as VEGF, bFGF and TGF-.5156 MMPs degrade components of the ECM and are subdivided into: collagenases, stromelysins (cleave laminin and fibronectin), matrilysins, gelatinases (cleave collagen type IV), membrane type (MT) MMP and other MMPs. Endothelial cells, smooth muscle cells and fibroblasts produce collagenase 1 (MMP-1), stromelysin (MMP3), gelatinase A (MMP-2), gelatinase B (MMP-9), matrilysin (MMP-7), and MT1-MMP (MMP-14, which has fibrinolytic activity).54'55










tPA
uPA


pro-MMP


(PAI) - -


positive feedback


Plasminogen Plasim
PA


MM]


- TIMPS


pro-Plasminogen MMP




Angiostatin


Fibin Degradation


ECM Degradation


Figure 1-11. PAs hydrolyze plasminogen to plasmin. Plasmin subsequently activates
matrix metalloproteases, which degrade the extra cellular matrix.
PA=plasminogen activator; uPA=urokinase type PA; tPA=tissue type PA;
PAI=plasminogen activator inhibitor; MMP=matrix metalloproteases;
TIMPs=tissue inhibitors of MMPs









Endothelial cells also produce tissue inhibitors of MMPs (TIMPs) that are specific inhibitors of MMPs and modulate the degradation of the ECM. TIMPs are secreted proteins, which inhibit MMPs in a 1:1 stoichiometry. They reversibly interact with the catalytic domain of the MMPs to inhibit their activity. TIMPs differ in their ability to interact with various MMPs. For example, TIMP2 inhibits MT-MMP and TIMP3 inhibits MMP9. TIMPs also bind to the heparan sulfate proteoglycans in the ECM and concentrate them to the specific regions within the tissue. 56

Angiostatin and endostatin are naturally occurring anti-angiogenic molecules,

however, they are also produced by proteolytic cleavage by MMPs from the pro-forms of plasminogen and collagen XVIII, respectively." The production of MMPs, however, is cell and tissue specific. For example, bFGF and VEGF upregulate interstitial collagenase (MMP-1) and also increase the formation of plasmin. Plasmin converts the inactive form of MMP-1 to the active form. Gelatinase A (MMP-2) is upregulated by calcium influx. It is responsible for the angiogenic switch and for the differentiation of the endothelial cells into tubes. MMPs promote capillary tube formation, however, at high concentrations, they have an opposite effect.

ECM degradation produces fragments, which have the opposite effect of the intact molecule. For example, hyaluronan, a GAG found in the ECM, has anti-angiogenic properties. However, when cleaved, it enhances the action of angiogenic factors. Conversely, proteolytic degradation of fibronectin, plasminogen and collagen produces fragments, which have both anti-angiogenic and angiogenic activity. MMP2 undergoes proteolysis to produce PEX, which is the C-terminal non-catalytic domain of MMP2. PEX is anti-angiogenic and inhibits the gelatinolytic activity of MMP2.









Bound Factors.

Activated endothelial cells are anchorage dependent for survival. In addition to degradation of the ECM, the endothelial cells require bound factors to help in migration towards the ischemic stimulus. These bound factors include integrins, Eph receptor Eph/Ephrins complexes, and VE cadherins Integrins.

Integrins provide the scaffolding for the cells to migrate upon and are used by the
58
endothelial cells to recognize the ECM. Integrins play a role in regulating cell growth, differentiation and survival.5963

Integrins are cellular receptors for ECM proteins and are expressed by all adhesive cells.64 Integrins are composed of c and 3 chain heterocomplexes, which are integral membrane glycoproteins. They have long extracellular domains, which are the ligand binding regions. Eighteen different c subunits, and 8 3 subnits have been identified. These subunits can associate in 24 known combinations. A short transmembrane region follows the short intracellular domains of both the c and the 3 subunits and the cytoplasmic tail of the beta subunit links the integrins to cytoskeletal actin of the endothelial cell.62,63,65

Integrins are linked intracellulary to actin filaments by specific actin binding proteins, such as Talin, alpha actinin, vinicluin and paxillin.66 Focal adhesion kinase (FAK) is a protein with tyrosine kinase activity and is composed of a large kinase domain flanked by an amino and carboxyl terminus. A region of the c-terminus, known as focal adhesion targeting sequence (FAT) recruits FAK to paxillin. Integrin mediated cell adhesion occurs when FAK is tyrosine phosphorylated.58









aP3 has a well-characterized role in angiogenesis. It mediates adhesion of cells to vitronection, fibronectin, von Willebrand factor, osteopontin, tenascin and thrombospondin. Although the aP33 integrin is minimally expressed on normal resting blood vessels, it is significantly upregulated in newly formed blood vessels within tumors, in healing wounds and in response to certain growth factors. vP33 expression is

upregulated in endothelial cells exposed to angiogenic factors and those exposed to hypoxia. Integrins also help to target the activity of the MMPs, for example, vP33 interacts with MMP-2 and also regulates signaling via the vascular endothelial growth factor receptor -2 (VEGF-R2). Natural components of the ECM, such as, endostatin, angiostatin, thrombospondin and tumastatin are all anti-angiogenic and exert their effect by binding to the av33 intergrin and disrupting the endothelial cell-ECM interaction. If this integrin is disrupted using an antibody (LM609) or a peptide antagonist (cyclic peptide 203, RGDfv), it results in the disruption of angiogenesis progression. VEGF and bFGF are capable of inducing the expression of cv33 integrin of endothelial cells.67,68 EpH receptors.

To discriminate cell partners from fibroblasts or inflammatory cells, the Eph receptor is utilized. The Eph receptor is the largest receptor of the receptor tyrosine kinase family (RTK) family.69'70 The receptors are divided based on ligand affinity into class A and class B. The extracellular domain of the Eph receptor consists of the ligand binding globular domain, cysteine rich region and 3 fibronectin type II repeats. The cytoplasmic portion of the receptor consists of a juxtamembrane domain, and a carboxyl terminus. The ligands for these receptors are ephrins, which are also divided into subclass A and subclass B. Ephrins subclass A are anchored to the plasma membrane by









glycosylphosphatidylinositol (GPI) anchor and ligands A1-A5 have been identified. Ephrins subclass B have a short transmembrane domain and a short cytoplasmic tail. Only three subclass B ephrins have been identified (B 1-B3). Both the receptor and the ligands are membrane bound and therefore a signal is transduced in the receptor expressing cells and the ligand expressing cells.70,71

Prior to cell-cell contact, the Eph receptor and ehprins ligand are loosely clustered at the cell surface. Following cell-cell contact, the receptor and ligand heterodimerize and tetramerize. These receptors are capable of bi-directional signaling (forward and reverse signaling). Eph A receptor enhances the adhesion of cells and the number of focal adhesion points and is known to be involved in forward signaling. Eph receptor B is phosphorylated in the intracellular domain and is known to be capable of both forward and reverse signaling.72

Vascular endothelial (VE) cadherins

Endothelial cells express at least three cadherins: N-, P and VE cadherin. Ncadherin is diffusely spread across the cell, P cadherin is present in trace amounts and VE cadherin is specifically localized to inter endothelial cell junctions. Beta catenine and plakoglobulin are anchored to the cadherin through actin and a catinine. VE cadherin mediates contact inhibition of endothelial cells by decreasing the amount of proliferation and allows endothelial cell monolayers formation in the vessel wall. VEGF increases endothelial cell permeability by phosphorylation of a tyrosine residue of VE cadherin. This phosphorylation leads to dissociation of the VE cadherin and translocation of the beta catenine/plakoglobulin complex to the nucleus to regulate gene transcritption.73-75









Growth Factors.

The process of angiogenesis requires co-ordination of several growth factors, which play distinct roles in the process, examples include: angiopoeitins, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF) and transforming growth factor (TGF). Angiopoeitins

Angiopoeitins are secreted ligands for the two Tie receptors: Tie 1 and Tie2 (Tek). Both receptors are endothelium specific,73 and have an extracellular domain composed of two immunoglobulin like folds and three fibronectin repeats. The cytoplasmic region has a tyrosine kinase domain interrupted by a short kinase insert.

Angiopoeitin-1 and angipoeitin-2 are ligands for the Tie receptors. Both ligands can bind the receptors, however, only ANG1 can phosphorylate the receptor.76 ANG-2 inhibits Tie2, detaches pericytes and loosens the matrix surrounding the vessel. ANG-1 does not initiate endothelial network organization it stabilizes networks initiated by VEGF by enhancing the interaction between endothelial cells and pericytes Binding of ANG1 to the Tie 2 receptor initiates cell survival through the PI3 kinase, Akt pathway. Akt leads to the upregulation of survivin, which is an apoptosis inhibitor. Phosphorylation of Tie2 leads to the phosphorylation of Dok. Dok then activates the Ras, Nck, and Crk pathway, which are involved in cell migration, proliferation and organization of the cytoskeleton. Molecules interacting with the Tie2 SH2 domains are Grb2, SHP2 which modulate cell growth, differentiation, migration and survival.76 (Figure 1-12)






































I
Organization
of
Cytoskeleton


I
Endothelial Cell Migration


I
Endothelial Cell Survival


Figure 1-12. Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors. Binding of
angiopoeitin 1 to the Tie 2 receptor leads to endothelial cells proliferation,
migration and cell survival. Angiopoeitin 2 inhibits Tie 2. ANGl=angiopoeitin 1; ANG-2=angiopoetin 2.









Vascular Endothelial Growth Factor

VEGF is a heparan binding potent endothelial cell mitogen. It promotes

endothelial cell survival via activation of the phosphatidylinositol 3-kinase (PI3K/Akt) pathway and inhibits apoptosis.77 VEGF undergoes alternative splicing to produce 5 known isoforms: VEGF 121, VEGF 145, VEGF 165, VEGF 189 and VEGF 206.73 The isoforms differ in storage in the ECM and their extracellular pathways.78 VEGF 121 and VEGF 165 are secreted extracellularly, whereas VEGF 189, VEGF 206 and possibly VEGF 165 are either cell or matrix associated due to their affinity for heparan sulfate. VEGF is a mitogen for endothelial cells and each isoform has varying effects during angiogenesis:73 VEGF 189 decreases lumen diameter, 121 and 165 increase lumen diameter and increases vessel length. VEGF 165 binds to the ECM and releases bFGF stored in the ECM, thus, bFGF and VEGF have a synergistic angiogenic effect. 78

There are three tyrosine kinase receptors for VEGF: VEGF R1 (Fltl), VEGF R2 (KDR/Flk-1) and VEGF R3 (Flt3).73 The receptors all have seven immunoglobulin like extracellular domains, a transmembrane domain and an intracellular tyrosine kinase domain, which is interrupted by a kinase insert.78 VEGFR1 and VEGR2 transduce different signals to endothelial cells. VEGFR1 promotes cell migration and VEGFR2 is mitogenic for the endothelial cells and also promotes migration. 73 Hypoxia upregulates VEGFR1 and induces the expression of VEGF by endothelial cells. The increased production of VEGF activates the VEGFR2 receptor phosphorylation and cell proliferation. 78 Ligand binding to VEGFR1 leads to the activation of the small adaptor proteins: Fyn, Yes and GAP. Ligand binding to VEGFR2, however, leads to phosphorylation of the SHP-1 and SHP-2 adaptor proteins and PLC-gamma. PLC gamma hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to form inositol









triphosphate (IP3) and diacylglycerol (DAG). The DAG remains associated with the plasma membrane and activates protein kinase C (PKC). PKC is a soluble cytosolic protein, which is activated by the increase in calcium concentration. Activation of PKC leads to cell proliferation and permeability. VEGFR2 also leads to the activation of the PI3 kinase/Akt pathway, which enhances cell survival. VEGFR2 plays a role in cell migration by recruiting FAK. The MAPK pathway is also activated through Grb2, which is an SH2 adaptor protein. It has two SH3 domains, which bind the guanine nucleotide exchange factor SOS. SOS then leads to the activation of RAS. Activated RAS binds to the N-terminal of RAF which phosphorylates MEK and phosphorylates MAP kinase.78 The activated MAPK pathway then leads to the activation of the intranuclear proteins such as cyclin D which is important in the progression of the cell cycle from the GI phase to the S phase.79 (Figure 1-13) VEGF also increases vascular permeability and allows leakage of plasma proteins, formation of the ECM and upregulates the production of uPA and tPA and PAl-1 by endothelial cells. VEGF production is regulated by local oxygen concentrations. Hypoxia upregulates production of VEGF by binding to the hypoxia inducible factor (HIF).73

During retinal development astrocytes and neuronal precursors migrate away from existing blood pre-existing blood vessels. As the distance between the astrocytes and the pre-existing blood vessel increases, the astrocytes sense a state of hypoxia. Astrocytes are more sensitive to hypoxia than neuronal cells and thus the astrocytes upregulate the production of VEGF, which leads to angiogenesis. This upregulation of VEGF by the astrocytes creates a concentration gradient of VEGF. This stimulates blood









VEGF g
VEGFR


A,-PLCy
DAG SHC



IP3 P13K
Paxillin

feration i
meability AKT
Pathway Cytoskeloton
Rearrangement C e Cell Migration
\ Cell
Survival /


Ras



MAPK
Pathway




Gene Expression Cell Proliferation


I AngiogenesisI


Figure 1-13. The vascular endothelial cell growth factor (VEGF R2) signaling pathway.
VEGFR2 activates several pathways all of which lead to angiogenesis.


PKC







Cell Proli
Vasopern









vessel formation towards the astrocytes, which produce VEGF. In ROP babies are placed in a high oxygen incubator because their lungs are not fully developed. The hyperoxia inhibits the VEGF production by the astrocytes thus causing newly formed blood vessels to regress. Once the babies are taken out of the incubator all the cells of the retina sense hypoxia and upregulate the production of VEGF. This leads to abnormal angiogenesis and unregulated blood vessel growth.78 Fibroblast Growth Factor

Fibroblast growth factor (FGF) is ubiquitously expressed as either basic FGF or acidic FGF. FGF is either in the cytoplasm or bound to the ECM due to its intrinsic
73
affinity for heparan.73 FGF binds to four related receptors, which are expressed on many cells. Ligand binding induces receptor dimerization. Endogenous heparan sulfate in cells is required for the activation of FGF. The receptor for FGF has three immunoglobulin like folds; two intracellular tyrosine kinase domains, a short transmembrane region and a juxtamembrane domain, which is longer than any other receptor.8 The intracellular domain has two phosphorylation sites.81 Ligand binding to the FGF receptor induces tyrosine phosphorylation of an adaptor molecule, FRS2. The phosphorylated FRS2 then allows binding of a small adaptor molecule GRB2. GRB2 is involved in the activation of the GTP binding protein Ras. Since FRS2 does not have an SH2 domain, another adaptor molecule, SHP-2 associates with FRS2 alpha in the active FGF receptor.81 The importance of the association of this molecule with the FRS2 is not well defined. GRB2 exists with SOS, which catalyses the exchange of GDP for GTP on Ras for activation. Therefore, SOS, facilitates the coupling of GRB2 to Ras.81 The activated MAPK then leads to the activation of the intranuclear proteins such as cyclin D which progresses the cell from the GI phase to the S-phase.79 (Figure 1-14) This growth factor induces









processes in endothelial cells and stimulates proliferation and migration of endothelial cells and pericytes, and production of PA by the endothelial cells. bFGF plays a role in

73 '82
blood vessel remodeling by stimulating endothelial cells to form tube like structures.7382 Platelet Derived Growth Factor

Platelet derived growth factor (PDGF) is a mitogen for smooth muscle cells73 and potent chemoattractant factor for smooth muscle cells, monocytes and fibroblasts. PDGF is a dimer consisting of two polypeptide chains: A and B. These chains combine to form

3 PDGF isoforms of PDGF AA or BB or heterodimers of PDGF AB.7383 The PDGF receptor consists of a single transmembrane domain which has intrinsic kinase activity.83 The receptor is also a dimeric mixture of the alpha and beta subunits.73 Ligand binding induces receptor dimerization and transphosphorylates tyrosine residues in the cytoplasmic domain of the receptor.83 Endothelial cells express the beta receptor and are stimulated by PDGF-BB.73,83 PDGF-BB acts through the MAPK/ERK pathway to stimulate c-jun/c-fos related genes in the nucleus to stimulate proliferation.83 PDGF also acts through the PI3kinase pathway to activate PKB, which stimulates cell survival and proliferation. PDGF also plays a role in angiogenic chords formation and stimulates sprout formation. PDGF also mediates proliferation and migration of pericytes along angiogenic sprouts.73 (Figure 1-15)

Transforming Growth Factor-3

Transforming growth factor beta (TGF-3) is produced by almost all cells and thus its activation represents an important control mechanism.84 TGF-3 is hydrolyzed
















FGFR


FGF


III

FRS2c Grb2 Sos Ras/Raf/MEK/MAPK pathway TK 1
Shp2

TK 2 Gene transcription in the nucleus




Figure 1-14. The FGF receptor and signaling pathway. Ligand binding to the FGF
receptor leads to tyrosine phosphorylation of adaptor molecules and activation
of the MAPK pathway. The MAPK pathway leads to endothelial cell
proliferation, and migration.








PDGF-R


PI3-K
/

PKB Survival


Ras-ERK


I Proliferation I


Figure 1-15. The PDGF receptor and signaling pathway. The PDGF receptor acts
through the MAPK pathway to stimulate proliferation and also stimulates
endothelial cell survival through the PI3-kinase/PKB pathway.


0-


- Grb2
-0


Sos


4e









intracellularly by a furin peptidase to produce the carboxyl terminal peptide.73 This peptide associates with the amino terminal to form the latency associated peptide (LAP). The LAP dimerizes to form the mature TGF-P3 which is then secreted in the inactive form.73 Plasmin activates the latent complex. TGF-P3 also produces Pal-I which inhibits plasminogen. Thus, showing that the action of TGF-P3 is self limiting.85

There are three different types of TGF-P3 receptors designated, I, II and III. TGF-P3 binds directly to the TGF-P1II receptor. Binding of the II receptor is followed by the recruitment of the TGF-3 I receptor. Both the receptors then form a stable complex and receptor II then phosphorylates receptor I which induces the signal cascade of the receptor.85 Once the TGF-P3R2 is bound to the TGF-3 1 receptor, the kinase activity of receptor 1 is activated. This leads to the recruitment and the accumulation of the Smad proteins, which are then phosphorylated by the receptor. The name SMAD is derived from the genes encoding them. The genes were first identified in drosophila and C. elegans. The drosophila gene was named MAD (mother against decapentapleigic) and the gene from C. elegans was named SMA (small body size).86'87 (Figure 1-16)

TGF-P3 is a bifunctional regulator. At low levels, TGF-P3 stimulates angiogenesis, and at high levels it inhibits angiogenesis.85 TGF-P3 is found in the ECM, on endothelial cells and on pericytes. It supports the anchorage independent growth of fibroblasts.73 TGF-P3 also controls cell adhesion by regulating the production of ECM and integrins. Endothelial cell migration and formation of tube like structures are regulated by TGF-3. TGF-P3 also upregulates the production of TIMPS, thus has anti-proteolytic activity. TGF-P3 inhibits endothelial cell proliferation73 by blocking the effect of other mitogenic growth factors and enhances pericyte differentiation. It helps to form the vessel


















TGF-j





Type II Type I

va li 1 i


Angiogenic Blood Vessel Growth
A


Smad4


Cell Growth Cell Mobility Angiogenesis


Smad2 Smad4


Figure 1-16. The TGF-3 receptor signaling pathway. Ligand binding to the TGF- 1
receptor II leads to the recruitment of TGF- 0 receptor I. The activated
receptor recruits the Smad proteins and stimulates angiogenesis at low levels
and inhibits angiogenesis at high levels of TGF-3 .


Arrested Growth
A









wall by stimulating the production of the extracellular matrix, strengthens the vessel wall and has matrix modulating effects and also stimulates tube assembly.73'87

Angiogenesis is a complex process, which involves the extracellular matrix, bound factors and soluble factors. Of the soluble factors, VEGF plays an important role in the early phases of angiogenesis. VEGF is an important mediator of compensatory angiogenesis and is a potent mitogen induced by hypoxia and nucleosides such as adenosine. 53,88,89 However, even though the angiogenesis process may solve the nourishment aspect of the outer retinal layers if the choriocapillaris was impaired, it would still cause vision impairment.90,90-94

Tissue hypoxia and ischemia initiate a series of events which lead to the

development of collateral blood vessels, followed by compensatory angiogenesis, which is detrimental and results in aberrant blood vessels that are friable and prone to bleeding.9'95 Mediators of compensatory angiogenesis include VEGF, which is a potent mitogen induced by hypoxia and nucleosides such as adenosine. 95-97 Depending on the character of the ischemic stimulus, adenosine plays two roles: as an intracellular signaling factor which promotes neovascularization following chronic hypoxia or ischemia, and as an endogenous protective factor which is capable of protecting the retina from acute ischemia. Adenosine also upregulates VEGF in retinal endothelial cells. Therefore, adenosine may be a critical signal in the control of gene expression after retinal ischemia.91'98'99

Adenosine

Adenosine is an endogenous nucleoside, which modulates many physiological processes such as cardiac myocyte contractility, modulation of neurosecretion and neurotransmission, cell growth and gene expression, regulation of intestinal tone and









control of vascular tone.00 Adenosine serves as a signal to increase energy supply and demand by affecting cellular metabolic rates and tissue perfusion. Metabolites of adenosine may also have significant physiological and pathological effects. The level of adenosine available for these effects is determined by a number of factors including the rate of production, transport and metabolism.100

Stimuli that mediate the local production of adenosine include hypoxia, ischemia and inflammation. The endothelium is a barrier to adenosine, thus the adenosine formed within the lumen of the blood vessels may be derived from nucleotides released from platelets or endothelial cells. Ischemic parenchymal cells or nucleotides derived from nerves or intestinal mast cells give rise to interstitial adenosine. This adenosine may produce vasodilation via the A2A receptor on vascular smooth muscle cells, which are especially accessible to the interstitial nucleoside. Adenosine may also be derived from adenine nucleotides from many cell types by mechanisms which are not well understood.100'101

Since AMP is derived from the breakdown of ATP, adenosine formation is closely linked to the cellular energy state. Adenosine may be formed intra or extracellularly. (Figure 1-17) The enzyme 5' nucleotidase (5'N) cataylzes the metabolism of ATP to adenosine. S-adenosylhomocysteine hydrolase also catalyses the break down of Sadenosylhomocysteine (SAIl) into adenosine. SAIl contributes significantly to adenosine formation in the heart and ischemic conditions in the brain. Once formed, intracellular adenosine is transported out of the cell to exert effects on specific cell surface receptors. The transport of adenosine is bidirectional.










Intracellular


4E


Extrace


L-Homocystiene SAH Hydroloose


deaminase






Adenosine Ilular deaminase .

Interstitial space


or XO


V1


Figure 1-17. Intracellular and extracellular production of adenosine. SAH hydrolase: Sadenosyl homocysteine hydrolase. 5'NT:5'nucleotidase. XDH:xanthine
dehydrogenase. XO:xanthine oxidase


I',-









Ecto 5'N catalyses the breakdown of 5'AMP to adenosine, thus giving rise to extracellular adenosine. The intracellular and the extracellular adenosine can be differentiated from each other using specific inhibitors of ecto 5'N. 100,101

Adenosine deaminase (ADA) and adenosine kinase (AK) catalyze the breakdown of adenosine in the cytoplasm. Both ADA and AKA are found in the cytoplasm. ADA is a 36 kDa protein which catalyzes the formation of inosine. ADA is heterogeneously distributed in tissues and highest activity is during development. AK is a 38-56 kDa monomeric protein. It is also widely distributed throughout the body. AK catalyses the phosphorylation of adenosine to 5'AMP. If the intracellular adenosine is high, then AK is inhibited.100'101

Five types of adenosine transporters have been classified according to sensitivity to nitrobenzylthioinosone (NBTI), which is an adenosine transport inhibitor. Most of these transporters are sodium dependent and are bidirectional. Following degradation of adenosine, inosine leaves the intracellular environment and forms hypoxanthine. Xanthine dehydrogenase catalyses the oxidation of hypoxanthine to xanthine and subsequently to uric acid. Conversion of xanthine to uric acid also reduces NAD to NADH. Xanthine oxidase genetrates superoxide and hydrogen peroxide, both of which are damaging to cells. Endothelial cells stimulated by ischemia and reperfusion are key sources of xanthine oxidase formation and activity.100 Adenosine and the Retina

Adenosine is heterogeneously distributed throughout the retina of various species, such as rat, guinea pig, monkey, human and mouse.100 Adenosine immunoreactivity is found in the ganglion cell layer, the inner plexiform layer and the inner nuclear layer. 91,102 Under resting conditions, endogenous purines in the retina are in the form of ATP









(70%) and adenosine (2%). During development, the retinal Miller cells provide glycosaminoglycan to the extracellular spaces for angioblasts which provides a scaffold for angioblast migration and organization. In developing and adult mammalian retina Mufller cells express 5' nucleotidase (5'N) ectoenzyme, a glycoprotein. This enzyme catalyzes the hydrolysis of purine nucleotide monophosphates, to the corresponding nucleoside. The 5'NT can metabolize all purine monophosphates, however, the major product is adenosine. Adenosine is an intercellular communication molecule and is a modulator of synaptic transmission in the brain and the retina, and is a local regulator of blood flow in several organs. In the retina, adenosine is released in response to ischemia, thereby modulating the blood flow in the adult and neonates. Adenosine is also chemotactic and a mitogen for endothelial cells, and enhances endothelial cell migration and tube formation. 102 An increase in the 5'NT activity in cerebral ischemia was shown by Braun et al. 103 The pattern of 5'NT changes as the retina develops. In the early stages of development, the greatest activity of 5'NT is found in the inner Mufller cell processes. When the inner retinal vasculature reaches completion (about 22 days of age), the inner retina activity of the enzyme decreases and the activity in the outer retina increases (in both plexiform layers). 102

Lutty et al showed that at days 1-5, an increased adenosine immunorectivity is

found in the inner retina and the edge of the formed vasculature in the neonatal dog. An increase in the adenosine product shifted toward the ora serrata as the vascular development progressed radially. On day 8 the 5'NT is increased in the inner retina, and on day 15 there is an increase in the adenosine immunorecativity in the nerve fibre layer and the inner nuclear layer. When the radial progression of the inner retinal vasculature









is complete on day 22, the 5'NT and adenosine are decreased throughout the nerve fibre layer and increased in the ganglion cell layer, the inner nuclear layer and the photoreceptor inner segments. 102 An increase in adenosine levels at most ages was found to be proportional to an increase in the 5'NT activity. In summary, the 5'NT activity shifts from the nerve fibre layer to the inner plexiform layer during development and the adenosine location is also shifted. Thus the Muller cells provide a glycosaminoglycan rich extracellular milieu for angioblast differentiation and also provide adenosine which is a stimulus for blood vessel formation.100,102 Adenosine Receptors

Adenosine receptors have been implicated in mast cell activation, asthma,

regulation of cell growth, intestinal function, neurosecretion modulation and vasodilation. Adenosine receptors modulate cAMP (adenosine 3c, 5c-cyclic monophosphate) intracellulary. Based on their ability to inhibit or stimulate adenylyl cyclase, the adenosine receptors were initially divided into A1 and A2 subtypes. 100,104,105 The A2 receptor was further divided into 2 subtypes based on the finding of a high affinity A2 receptor in the rat striatum and a low affinity A2R in the brain 106 Both of these receptors activate adenylyl cyclase. The high affinity receptor was designated as A2A and the low affinity receptor was designated A2B.100'107

Adenosine activates four different cell receptors: A1, A2A, A2B and A3. In most cell types, adenosine activates the A1 receptor to lower oxygen demand, and activates the A2 receptors to increase the oxygen supply. Thus the A1 and A2 receptors act to rectify imbalances between oxygen supply and demand.100'10(Figure 1-18)










02 Supply/Demand


Hypoxia


Figure 1-18. Role of the high and low affinity adenosine receptors. The A2 receptors
increase oxygen supply. The A2A receptor leads to vasodilation and the A2B
receptors lead to angiogenesis


Ir4


AaAA ,'map









A1, A2B and A3 adenosine receptors are N-linked glycoproteins, which have sites

for palmitoylation near the carboxyl terminus. Glycosylation has no effect on the affinity of ligands for these receptors, thus these sites may be involved in targeting newly formed receptors to the cell surface. All receptors can be readily deglycosylated upon incubation with glycosidase.101 The molecular pharmacological and physiological relevance of the A1, A2A and A3 receptors is well known. However, the A2B receptor is not as well characterized due to a lack of selective pharmacological probes and because this receptor has a low affinity for adenosine. 100

The A1 receptor was initially cloned from rat, human, bovine and rabbit. The A1

receptor has seven transmembrane domains and is 326 amino acids in length and is about 36-37 kDa. Mutations in the H 274 and H 251 region result in loss of agonist and antagonist binding. Chimeric receptor constructs reveal transmembrane domains 5, 6 and

7 to be important for binding. In the brain, the A1 receptors couple to Gi and G, and inhibit the actions of adenosine.

The A1 receptor decreases membrane potential (by increasing K+ and C1

conductivity), lowers neurotransmitter release (e.g. glutamate and dopamine) and decreases calcium influx by stimulating calcium mobilization via the pertusis toxin sensitive pathway through the activation of PLC beta with G protein 3/y subunit. 101 All of these effects of the A1 receptor lead to a decrease in neuronal excitability and metabolism. Thus, the A1 receptor has a neuroprotective role in ischemic tissue.100

The A2A receptor was initially cloned from canine, rat and human and produced responses which are anti-inflammatory.101 It has seven transmembrane domains consisting of 410-412 amino acids and is about 45 kDa (comparable to A). Mutations in









H 274 and H 251 also lead to loss of agonist and antagonist binding. Adenosine relaxes vascular smooth muscle via the A2 receptor mediated mechanism and thus increases tissue perfusion.

In the retina, the vasodilatory effects of adenosine are mediated by A2 linked to

potassium ATP channels.100,101 Adenosine increases glyconeogenesis via the A2 receptor and thus promotes an increase in the supply and demand ratio for metabolic substrates in the retina. A2A decreases the superoxide release from activated neutrophils and inhibits platelet aggregation. These are all anti-inflammatory actions, the importance of which in retinal response to ischemia has not been established. Ideally, a drug that is an A2A agonist and an A2B antagonist is needed to further understand the two receptors. 100

The A2B receptor was initially cloned from rat hypothalamus109, human

hippocampus110 and mouse mast cells 100 The receptor was found in these tissues by PCR with degenerate DNA oligonucleotides that recognized conserved regions of the G protein coupled receptors. The human, rat and mouse A2B receptors share 86-87% amino acid homology. 109 The human A1 and the human A2A, and A2B receptors share 45% amino acid homology. 100 Closely related species such as rat and mouse share 96% homology. The A1 receptors have 87% amino acid homology in various species (Figure 1-19) 111,112, the A2A receptors have 90% homology (Figurel-20). 113 while the A3 receptors differ significantly between species.111,112 Figure 1-21 shows the homology between the human and the mouse A2B receptor. 100































Figure 1-19. Homology of the A, receptor for human and mouse. The yellow sequences
indicate homology between the human and mouse Al receptor sequences.
The white sequences indicate non-homologous regions and the blue sequences
indicate conserved sequences.





































Figure 1-20. Homology of the A2A receptor between human and mouse. The yellow
sequences indicate homology between the human and mouse A2A receptor sequences. The white sequences indicate non-homologous regions and the
blue sequences indicate conserved sequences.































Figure 1-21. Homology of the A2B receptor between the human and the mouse. The
yellow sequences indicate homology between the human and mouse A2B
receptor sequences. The white sequences indicate non-homologous regions
and the blue sequences indicate conserved sequences.









The membrane structure of the A2B receptors is that of a typical G protein coupled receptor consisting of a 7 transmembrane domains connected via 3 extracellular and 3 intracellular loops. (Figure 1-22)100 110,114

Trans membrane domains have a high degree of amino acid homology in different species. The human, mouse and rat A2B receptors have 2 potential N-glycosylation sites in the second extracellular loop.109 The human N-linked glycosylation sites are Asp 153 and 163 which are in the second extracellular loop. Both of these sites are conserved in all of the A2B sequences of all species that have been cloned. 100,115

The A2A intracellular and the third intracellular loop are involved in coupling A2A receptor to G proteins. 100,111 The third intracellular loop is a 15 peptide portion of the A2A receptor which has 57% amino acid homology with the A2B receptor and also determines the selective coupling with GS.100'116 Both A2A and A2B are coupled to G. The A2A and A1 receptors have 27% amino acid homology and the A1 is not coupled to G. Amino acids in the second intracellular loop may modulate the A2A receptor coupling since lysine and glutamic acid are necessary for efficient A2A adenosine receptor Gs coupling. 100,116 Analogous lysine and glutamic acid residues are also present in the A2B receptor. The major difference between the A2A and the A2B receptor is the long intracellular C-terminal tail of the A2A. (Figure 1-23) This long tail is not involved in G, coupling to the receptor. Removal of the c-terminal tail of the A2A receptor does not inhibit stimulation of adenylyl cyclase when truncated receptor is expressed in CHO cell.100,111,116

Mutational studies of the A2A receptors have shown that the Thr 298 residue of the C-terminal tail of the A2A receptor is located close to the seventh membrane













NHs

-33a %"




~59

3'3

0 d 3 3
0O a
330 33


)-COOH


3333 33~,p Figure 1-22. The A2B receptor is a G protein coupled receptor consisting of a seven
transmembrane domain connected via 3 extracellular and 3 intracellular loops
flanked by an extracellular N- terminal and an intracellular C-terminal.















NH- ~D9O Q a
5 ) 3 as 30
3 3






3, I))


5 30 ~ 13 a 3


33


via 3 13 3l a0nan


t i 33 ann cu47 )-cooH

Figure 1-23. The A2A receptor structure consists of 7 transmembrane domains connected
via 3 extracellular and 3 intracellular loops flanked by an extracellular Nterminal and a long intracellular C-terminal.









span and is essential for the development of rapid agonist mediated desensitization. 100,111,117 The threonine residue is also present in the human A2B (Thr 300), however, its role in receptor desensitization has not been explored. A2B receptors can be coupled to other intracellular signaling pathways in addition to G, and adenylyl cyclase. 100

The A3 receptor was cloned from the human, rat and sheep. It is composed of 320 amino acids and has about 40-50% homology to the A1 and A2 receptors. It has low affinity for alkylxanthine antagonists such as theophylline and caffeine (which is a classic antagonist for A1 and A2). The non specific A3 antagonist IB-MECA inhibits adenyly 1 cylcase and increase PLC, calcium mobilization and decrease TNF-alpha. Higher concentrations of adenosine are required to activate the A3 receptors than are required to activate the A1 or the A2 receptors. 100

Pharmacology of the A2B receptors

Highly selective and potent agonists designed for A1, A2A, and A3 receptors are available and are important tools for the characterization of adenosine receptors. The lack of a potent selective A2B antagonist hampers the characterization of its cellular functions. 95,100 The most potent agonist for A2B is NECA. 100,118-120 At a concentration of 2[LM, NECA produces half the maximal effect (EC50) for stimulation of adenylyl cyclase. 120 NECA is non-selective and thus activates other adenosine receptors with greater affinity. The EC50 for the A1 and A2A receptors is in the low nanomolar range and that of the A3 receptors is in the high nanomolar range. Therefore, the characterization of the A2B receptor depends on the use of compounds, which are potent









selective agonists of other receptor subtypes. Therefore the A2B receptor is usually characterized by exclusion. 100

CGS 21680121 is an A2A selective agonist that can differentiate A2A and A2B

receptors. 100,122-125 The A2A and the A2B receptors are both positively coupled to adenylyl cyclase and are activated by the non selective agonist NECA. CGS 21680, on the other hand, is ineffective on A2B receptors and as potent as NECA when activating A2A receptors.100,120-122,126-128 R-PIA is an A1 selective agonist and the A2B receptor has low affinity for it. 100,119,120

The pharmacological characterization of the adenosine receptors is based on apparent agonist potencies. This is not ideal as it depends on agonist binding to the receptor and multiple processes of signal transduction. Therefore, for receptor subtype identification, selective antagonists are preferable 100,129 Highly selective A2B antagonists are not available. However, it is known that A2B has a low affinity for agonists, but a high affinity for antagonists. Enprofylline (3-n-propylxanthine) is an anti-asthmatic drug, and is the most selective, but not potent, A2B antagonist known. Other potent but nonselective A2B receptor antagonists include 1,3-dipropyl-8 (p-sulfophenyl)xanthine (DPSPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), xanthine amine cogener (XAC) and IPDX 95,119,120

Distribution of the Adenosine Receptors

Initially the A2B receptor mRNA was found in the rat. The highest levels of the receptor was found in the cecum, bowel, bladder, followed by the spinal cord, lung, epidydimus, vas deferens and the pituitary. 100,130 Subsequently more sensitive RT-PCR showed that the A2B receptors were present in all tissues of the rat, with the highest level in the proximal colon and the lowest level in the liver. 131 Primary tissue cultures have









different adenosine receptors present in the cells. This may be because there are different populations of cells and each cell expresses a different type of adenosine receptor. 100,132134 Studies on established cell lines also showed multiple adenosine receptor subtypes on a single target.100,122,124,125 Also, studies on single cells show the presence of one or more adenosine receptor subtype. 135-137 Clonal cell lines also have co-expression of the A2A and the A2B receptors.100,124 However, subsequent studies showed minute amounts of other receptors too! Therefore it is possible that adenosine selective antagonists are needed to better characterize the distribution of these receptors in cells. It is however, unclear why there is simultaneous expression of multiple adenosine receptors in a single cell. Both A1 and A2A receptors have a high affinity for adenosine and need to be blocked before the effects of the A2B receptor can be seen 100,135,136,138 However, this is not always the case and may be a reason for discrepancies published in the literature. Elfman et al showed that glial cells of rat astrocytes have A1 and A2B adenosine receptors which stimulate cAMP. 133,139-141 However, when the cells were stimulated by the non-selective agonist, NECA, cAMP accumulation was seen even though there are A1 receptors present. Therefore it may be that the importance of the A2B receptors is maximal where adenosine receptor levels are high, such as, in tissues with a high metabolic demand or conditions when oxygen is decreased. Both the A1 and the A2 receptors may modulate the response to lower the concentrations of adenosine, lO

The widespread unique localization of adenosine suggests that it is well positioned to serve as mediator of important physiological and pathophysiological processes in the retina. 91,100,142 In the retina, adenosine receptors are localized to the same retinal layers as endogenous adenosine. In the mouse a tritiated A1 agonist, cyclohexyladenosine (CHA)









was used to localize the A1 to the inner retina (over the inner plexiform layer) and the A2 receptor was localized to the RPE (outer retina) and the outer and inner segments of photoreceptors by using tritiated NECA. 100,142 No A3 receptor has been found in the retina. The location of adenosine receptor mRNA transcripts generally correlated with the autoradiographic localization of the Al receptors, but not the A2 receptors. 100 Intracellular Pathways Regulated by A2B Receptors

Adenosine receptors activate a diverse cascade of intracellular signaling. The A1 and A3 receptors inhibit adenylyl cyclase and stimulate PLCP3 by activation of pertussis toxin sensitive G proteins Gi and Go. 143 Adenosine binding to the A2A and the A2B receptors couples them to G, and adenylyl cyclase positively, however, the A2B receptor is also active in other signaling pathways. The A2B receptor coupled to G, can also increase calcium transport into the cells by the cholera toxin sensitive pathway. This pathway is cAMP independent even though it is coupled to G. 144,100 The A2B receptor is also coupled to G q and leads to the activation of two distinct pathways. One of those pathways lead to the activation of the MAPK pathway and the other pathway activates the P13 kinase/PkB pathway. (Figure 1-24)

Angiogenesis is a complex process and is the underlying cause of several

retinopathies. Currently available treatments for retinopathies are painful and have had limited success. Since adenosine exerts its angiogenic effects upstream of VEGF, it is an attractive target for inhibiting the process of angiogenesis. However a lack of selective and potent A2B antagonists requires the use of molecular techniques to target the A2B receptor. One such approach is the use of ribozymes to target receptors at the molecular level.










Muller Cell

5'N




AMP ADO <


Hypoxia


Adenyl A
Cyclase 2A


Figure 1-24. The A2B signaling pathway. The A 2B receptor couples to Gs and Gaq and
leads to an increase in calcium transport and also leads to the activation of the
MAPK pathway









Ribozymes

Ribozymes are catalytic RNA molecules that cleave other RNA molecules. Ribozyme is short for ribonucleotide enzyme, which, catalyze the hydrolysis and phosphopryl exchange at the phosphodiester linkages between RNA bases resulting in cleavage of the substrate. 145,146 146,147

Ribozymes can be classified into 3 main groups based on function and size: self splicing introns, RNase P, small self cleaving ribozymes.

Self Splicing Introns

Group I Introns

Self splicing introns can be divided into 2 classes: Group I intron and Group II

introns, based on the conserved secondary structure and splicing mechanisms (Figure 125). Group I introns are found in a wide number of species, such as, eubacteria, bacteriphages, fungal mitochondria, plant chloroplasts and rRNA of lower eukaryotes.148,149,150 The splicing action consists of two consecutive transphotoesterification reactions. In a transphotoesterification reaction, the number of phosphodiester bonds remain constant, however, the position of the bonds changes. (Figure 1-26)150,151 Only the Tetrahymena large rRNA group I intron has been shown to function without a protein in vivo. All other known group I introns require a single protein co-factor to provide a scaffold that helps position the introns in a catalytic conformation.152


















uAA A A A U
A A
AUAA A-U
P5 UA
A
A-U C-G
AU-GAA UC
U Up
A U
C A
A A
AAC-GAA
U-A 305

P4 u-,
G-c --u
350 6-G CA
A-U .U 440
U V
C-G AA
AG-CA
AU-AA
U-A U-A A-U
P6 u-.A
A-U
U-A
UAC-G
C A
A-U U-A A-U
U-A U-A A-U U-A A-U
AU-A U U-A
A-U
A-U U-A
A-U

AUAU AUAUAA
A U
U A
A A
UA AUAU

400


u A A
c A A
c A G
u A U P1
U A
u U A
u UAU AG-C 224 a 1 U-A
g 1 u-G pl
U a-U
a u-A
acaccagcac.G

C-GAUUAA
A-U


a LlacUUaL a -3
g
u AA709
A U
U U AA
a A A A G 9 A-U A-U u U-A U-A 0 C-G U-A
0 uA A-U P9
a U-A U-A u A-U U-A 9 UA GAG-C +1 u G A
738G U. .
U U
U A U-A
A A i P7
A C C-G
UAUAUA U.A
G-C G
A-U U A
AU-AGAAAG ACUG A
A CCUUUC UGA AG
G A CAAU
AU C AUA
A A
A
A
A


C. . l . ." . ." """ U-A U-A U-A
U-A
U-A G-C
A UAc 268 -P3
UCC U-A
A-U
G-C . A.Y
U-A

U-G U-A
250 C-G 660 A-U
C-G P2 A-U
G-U A-U P8
C-G U-A
UUU A-U
U-A 500
U-A


Figure 1-25. The secondary structure group I introns.











Iguanosine cofactor binds and attacks phosphate at 5'-junction



GI cleavage at 5'-juction is followed by
ligation of guanosine cofactor to 5'-end



G formation of helicies P1 and P10 aligns the
G 3'-end of the intron with the guanosine
binding site


" Nucleophillic attack by hydroxyl group at 3'-end of
I ,,An, , ' uptream exon at eh 3'-phosphate of the intron
liberates the intron and joins exons


Figure 1-26. Splicing mechanism of the group I introns









Group II Introns

Group II introns are self splicing introns found within nuclear pre-mRNA and in the pre-mRNA of organelles from fungi and plants.153 (Figure 1-27) High concentrations of magnesium and potassium ions are essential for their proper folding.153 Group II introns also require a complex of proteins and small nuclear RNAs (SnRNA) for cleavage. These components form the spliceocome. Group II splicing occurs via two consecutive trans photoesterification reactions similar to group I introns. The main difference in the splicing mechanism between the two introns is the nature of the hydroxyl group, which initiates the initial phototransesterification reaction. In group I introns, the reaction is initiated by the 3' hydroxyl group of the exogenous guanosine and in the group II introns, the reaction is initiated by the 2' hydroxyl group of the internal adenosine.154 (Figure 1-28)

RNase P RNA

RNase P is an endoribonuclease which removes the 5' leader sequence from

precursor tRNAs. RNase P has an RNA and a protein unit, both of which are essential to its function. The RNA component is the catalytic component of the complex. The protein subunit enhances the turnover rate of the reaction by acting as a scaffold for the RNA that forces the RNA into a catalytic conformation.155,156 RNase P can recognize and cleave 60 different tRNA substrates. 157 RNase P recognizes the structure of the tRNA and only a minimal tRNA structure is required for the creation of the RNase P cleavage site (Figure 1-29). 158,157




















Domain


Figure 1-27. Secondary structure of Group II introns.
















































Figure 1-28. The splicing mechanism of the Group II introns.











RNase P


00


I


immature tRNA mature tRNA
Figure 1-29. Cleavage of the tRNA 5' leader sequence by Rnase P.









Small Self Cleaving Ribozymes

Small Self cleaving ribozymes are nucleolytic RNA's and are found naturally. They are associated with viruses and satellite RNA and can catalyze RNA cleavage reactions in the absence of protein. 159 There are several types of small ribozymes, the most extensively studied ones include: hepatitis delta virus (HDV), hammerhead and hairpin ribozymes. The hammer head and hairpin ribozymes are derived from tobacco ring spot virus satellite RNA.

Hepatitis Delta Virus

Hepatitis delta virus (HDV) is a short single stranded RNA found in patients infected with human hepatitis B. It has a circular RNA genome, which encodes a ribozyme in both orientations. HDV replicates through a rolling circle mechanism like other self cleaving ribozymes (Figure 1-30), and the ribozyme is required for the cleavage of the HDV genome into discrete units prior to packaging. 160,161 Hairpin Ribozymes

The hairpin ribozyme was originally found in the tobacco ring spot virus satellite RNA. The hairpin ribozyme binds the substrate and forms a structure with 4 helices and

2 loops (Figure 1-3 1). The arms of the hairpin ribozyme hybridize to the substrate molecule to from helix 1 (6 base pair) and helix 4 (4 base pair). Loop A has a BNGUC target sequence required for cleavage, where B is G, C or U, and N is any nucleotide.162 There are no conserved nucleotides in any of the helices. 163,164 Hammerhead Ribozymes

The catalytic domain of the hammerhead ribozyme was discovered by comparing self cleaving RNA sequences of a number of different viroid infectious RNA molecules.









O JO

Minus





Plus O





Figure 1-30. Self-cleaving ribozymes resolve concatemers formed by rolling-circle
replication into individual genomic molecules










1 C 3'-N N N N NA 5'-NNNNNU


U G
A
NN NN
A GA
A


A A
B
A


2
N N-5'
I I
N N A-U-3'
C-G
C-G 3
A-U

GAG-CCA G A


A U
A
A C-GGU
A-U C-G
G-C 4
C-G U-A C-G
G A UA


Figure 1-31. Structure of the hairpin ribozyme. The arrow indicates the site of cleavage.
The hairpin ribozyme binds the substrate and forms a structure with 4 helices
(1-4) and 2 loops (A and B).









Hammerhead ribozymes are small, approximately 34 base RNA molecules and cleave RNA target in trans. The hammerhead ribozymes bind substrate to form a structure, which consists of a stem and three loops and a catalytic core with a conserved nine nucleotide sequence (Figure 1-32). A mutation in any of the conserved nucleotides prevents RNA cleavage. 165

The catalytic core of the hammerhead ribozyme has two functions: it destabilizes the substrate strand by twisting it into a cleavable conformation and binds the metal cofactor needed for catalysis. 166 The hammerhead ribozyme cleaves the substrate by a tranesterification reaction (Figure 1-33). The reaction requires the presence of magnesium and water. The hydrated magnesium ion has two functions, both mediated by water molecules. First, one molecule of water binds to one of the oxygen atoms of the phosphate group, holding it in the proper orientation for the enzymatic mechanism. Secondly, the environment of the active site lowers the pKa of another water molecule so that it can donate a proton to the aqueous environment. In the transition state, five oxygen atoms are arranged in a triangular bipyramid around the phosphorus atom. A bond is formed between the 2' oxygen of cytosine 17 and the phosphorus atom. Simultaneously a bond is broken between the phosphorous atom and the hydroxyl oxygen of the next nucleotide, adenine 1. 1. This leaves the cytosine with a 2'-3' cyclic phosphate group. The 5'nucleotide recovers a proton from the aqueous environment, completing a hydroxyl group. The reaction products diffuse away from the active site leaving the ribozyme free to bind a second substrate molecule and complete another reaction cycle. The hammerhead ribozyme recognizes substrate sequences on either side of a NUX cleavage site, where N is any nucleotide and X is any nucleotide except G.










I


U GCCGA
11


G AA


31 5'
N N
N-N N-N N-N N-N
A-U
x


k (
GUAGU


N NN N NN-3'
N NN N NN-5'


Figure 1-32. Structure of the hammerhead ribozyme. The hammerhead ribozyme binds
substrate to form a structure, which consists of a stem and three ioops and a
catalytic core with a conserved nine nucleotide sequence. Arrow indicates site
of cleavage.


r-I


I










ine 17


IH:OH


H


0
To Adenine 1.1-CH2�


CAosine 17


'90


To Adenine 1. / H-OH


417

H I



H


To Adenine 1.1-CH2


Figure 1-33. The hammerhead ribozyme cleaves its substrate by a transesterification
reaction. A. A molecule of water binds to an oxygen of the phosphate group.
B. Another water molecule donates a proton. A bond is formed between the
2' oxygen of cytosine 17 and the phosphorous atom. C. A bond is broken
between the phosphorous atom and the hydroxyl oxygen of adenine 1.1. D.
Cytosine remains with a 2'3' cyclic phosphate group. The 5' nucleotide
recovers a proton to complete a hydroxyl group. The reaction products then diffuse away from the active site leaving the ribozyme free to bind a second
substrate molecule and complete another reaction.


To Adenine 1









The ribozyme anneals to the substrate mRNA by means of two flanking arms which hybridize to form helices III and 1. Cleavage occurs at the 3' end of the cleavage site. Not all cleavage sites demonstrate the same efficiency. Generally, GUC is the most efficient cleavage site, then CUC, IJUC and AUC. The remainder of the cleavage sites are cleaved at least 10 times less efficiently than the GUC site. The hammerhead and hairpin ribozymes are being examined as gene therapies for a number of different diseases because they are small and can be easily cloned and packaged into many of the existing viral vectors for delivery to target cells. The advantage of the hammerhead ribozyme is that it can recognize a greater number of cleavage sites than HDV or hairpin ribozymes. 167,168

Experimental Aim

Currently, the only available treatment for ROP is laser treatment of the retina,

which has limited success. The aim of this project is to design a hammerhead ribozyme that will specifically target and cleave the A2B receptor mRNA resulting in a reduction in expression of the A2B receptor protein and a reduction of cellular and physiological functions affected by this receptor. We are using a hammerhead ribozyme primarily as a tool to study the pathways that involve A2B. But this ribozyme can also be used as 'proof of concept' for conventional drugs targeting the A2B receptor and, finally there is a possibility that the hammerhead ribozyme itself could be used as a therapeutic agent.

The goal of this project was to examine the effectiveness of ribozymes in the

treatment of ROP. Previously we have shown that proliferative blood vessels have an enhanced expression of the A2B receptor, therefore, there is justification to target this protein in controlling the disease. Ribozymes were designed to specifically cleave the mRNA of the A2B receptor to decrease the expression of the receptor protein. The









underlying hypothesis was that the cleavage of the mRNA of the A2B receptor at the mRNA level would prevent translation of the protein and subsequently progression of angiogenesis in ROP by preventing the growth of abnormal blood vessels. (Figure 1-34) The selected target site was a short region of the mRNA for the A2B receptor. The first step of the project was to design a ribozyme to cleave the sequence of the A2B receptor in the mouse and the human. (Figure 1-35) Two hammerhead ribozymes were developed,

each of which had an inactive version with a single base mutation. (Figure 1-36) The most efficient ribozyme was cloned into an rAAV construct (p21Newhp) for further analysis. (Figure 1-37) The second step of the project was to develop in vitro assays to examine the ability of the ribozyme to cleave the mRNA. These assays were used to determine if the ribozymes would be effective for reducing pre-retinal neovascularization in an oxygen-induced mouse model of retinopathy.

To test the ribozyme in vivo, the A2B Rz2 was intravitreally injected to the mouse model. Several models for oxygen-induced retinopathy have been developed. Dembinska169 and Chowers170 both used a rat model. The rats were placed in alternating hypoxic and hyperoxic environments. The alternating environments lead to severe retinal complications, which were not representative of retinopathy in human babies. Since the timing and duration of the hypoxia was inadequate, it gave inconsistent results. In our study we used a mouse model developed by Louis Smith.171 An advantage of using the mouse retina is that in the newborn mouse the retinal vessel development stage is the same as that of premature human babies. Also, normal retinal vascular development in mice occurs within two weeks of birth, thus illustrating the evolution











A 2 Receptor


+ Ribozyme


Translatio


Figure 1-34. Cleavage of the A2B receptor by a ribozyme prevents translation of the
protein.








Species A2B Rzl Target A2B Rz2 Target
Mouse ACAUGUCUCUUUG CAUUGUCUAUGCC
Human AAGUGUCUCUUUG CAUUGUCUAUGCU

Figure 1-35. Target sequences of the human and mouse A2B ribozymes 1 and 2. Red
indicates a difference in sequence between the human and mouse species.










3'-U-A -5'
G-C U-A A-U
C - G cleavage site

AAA-UC
G UCUUUG-3'
GCGGC I I I I
I AGAAAC -5' GCCG C
U A G U


A2B Ribozyme 1


3'- U - C -5'
G-A
U-U A-U
C - G cleavage site
AA-UC
G UAUGCC-3'
C GGCI I
UU sGeG AUA CGG-5' U GCCGA_ CU U A nU


A2B Ribozyme 2


Figure 1-36. Hammerhead ribozymes for the A2B Rzl and Rz2. The target sequences
are indicated in red. For each ribozyme an inactive version of the ribozyme
was made with a C replaced by the G (arrow).


C
U











CMV enhancer Beta-actin promoter
Exon



Intron
p21NewHp
6568 bp
indl II (192 1) Spel (1931) Hairpin Ribozyme Nsi (2014)
SV40 Poly(A) PYF441 Enhancer
TR HSV-tk

B SV40 Poly(A) neoR


Hidll el Nil
Hammerhead Hairpin
Ribozyme Ribozyme



Hairpin Cleavae
Site
Figure 1-37. A)The p21Newhp Vector with the CMV enhancer and beta actin promoter.
The hammer head ribozyme was cloned between the HindIII and Spel sites.
B) The hammerhead and the hairpin cleavage sites.




Full Text

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REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES THAT CLEAVE THE A2B RECEPTOR mRNA By AQEELA AFZAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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To my son, Faris Wasim.

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iii ACKNOWLEDGMENTS It is a pleasure to thank the many people who have made this dissertation possible. Many people have been a part of my gra duate education as friends, teachers and colleagues. My mentor, Maria Grant, has been al l of those. It is difficult to overstate my gratitude for her. She has in stilled in me the qua lities of being a good scientist. Her infectious enthusiasm for clinical research has been a major driving force during my career at the University of Flor ida. This dissertation is a sm all tribute to an exceptional woman from a student who is sti ll anxious to learn from her. My sincerest thanks are also due to L ynn Shaw. He patiently taught me all the techniques I needed to complete my work. He also spent countless hours editing and doing the graphics for this dissertation. His insightful comments were crucial for editing the many drafts into the final dissertation. My thanks are also due to Polyxenie E. Spoerri who taught me all th e tissue culture tech niques I needed to complete this dissertation. Thanks also to Sergio Ca ballero, Rehae Miller a nd past and present members of the Grant lab: Tom Ruzich, Nilanjana Sengupta, Christopher Beadle, Hao Pan I would like to thank my committee members: Dr. Don. A. Samuelson (Professor of Veterinary Medicine); Dr. Dennis. E. Brooks (Professor of Veterinary Medicine); Dr. John. B. Dame (Professor of Veterinary Medicine); Dr. Donald. A. Armstrong, Dr. Elizabeth C. Uhl (Clinical Assistant Professo r of Veterinary Medicine) and Dr. Harm J.

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iv Knot (Assistant Professor of Pharmacology and Therapeutics) for their guidance over the years. My son, Faris Wasim, has been a great s ource of inspiration. Being tired of not being able to fulfill his requests when I wanted to and missing him has been the best motivation for completing this dissertation. My husband, Wasim Asghar, has also shared this exciting journey with me. He has provided constant supp ort and encouragement throughout my graduate career. A very special thanks to the two people to whom I owe everything I am today, my parents, Mohammed Afzal and Mussarat Afzal Their unwavering faith and confidence in my ability and in me is what has shaped me to be the person I am today. Thank-you for everything. My thanks are also due to my sister, Aneela Afzal, and brother Yaseen Afzal, for their support and c ountless hours of babysitting. My family opened their hearts to me and my little one and ma de it possible for me to come to work knowing that he was in good hands. In addition, I would also like to thank the Department of Pharmacology and Therapeutics at the College of Medicine at th e University of Florida, and the College of Veterinary Medicine at the Un iversity of Florida for their financial support during my graduate career. Thanks also to the men and women who dona ted their eyes to our research. Their gift has made it possible for us to understand se veral eye diseases and prevent them in the future.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF FIGURES.........................................................................................................viii LIST OF ABBREVIATIONS...........................................................................................xii ABSTRACT....................................................................................................................xvi i CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Anatomy of the Eye......................................................................................................1 The Retina.............................................................................................................4 Blood Supply to the Retina....................................................................................8 The Blood Retinal Barrier.....................................................................................9 Retinopathies..............................................................................................................10 Age Related Macular Degeneration....................................................................10 Diabetic Retinopathy..................................................................................................11 Non-Proliferative Diabet ic Retinopathy (NPDR)...............................................11 Proliferative Diabetic Retinopathy (PDR)...........................................................15 Retinopathy of Prematurity.........................................................................................17 Treatment of Retinopathies.................................................................................19 Angiogenesis...............................................................................................................24 Extracellular matrix (ECM).........................................................................26 ECM degradation.........................................................................................26 Bound Factors......................................................................................................30 Integrins........................................................................................................30 EpH receptors...............................................................................................31 Vascular endothelial (VE) cadherins...........................................................32 Growth Factors...........................................................................................................33 Angiopoeitins......................................................................................................33 Vascular Endothelial Growth Factor...................................................................35 Fibroblast Growth Factor....................................................................................38 Platelet Derived Growth Factor...........................................................................39 Transforming Growth Factor...........................................................................39 Adenosine...................................................................................................................44 Adenosine and the Retina....................................................................................47

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vi Adenosine Receptors...........................................................................................49 Pharmacology of the A2B receptors..............................................................59 Distribution of the Adenosine Receptors............................................................60 Intracellular Pathways Regulated by A 2B Receptors .........................................62 Ribozymes..................................................................................................................64 Self Splicing Introns...................................................................................................64 Group I Introns....................................................................................................64 Group II Introns...................................................................................................67 RNase P RNA......................................................................................................67 Small Self Cleaving Ribozymes..........................................................................71 Hepatitis Delta Virus...........................................................................................71 Hairpin Ribozymes..............................................................................................71 Hammerhead Ribozymes.....................................................................................71 Experimental Aim.......................................................................................................77 2 METHODS AND MATERIALS...............................................................................85 Defining Location of the Target Sequence.................................................................85 Preparation of the Target Oligo-Nucleotide...............................................................86 Time Course of Cleavage Reactions fo r Mouse and Human Targets (Hammerhead Ribozymes)............................................................................................................86 Multiple Turnover Kinetics........................................................................................87 Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector.............88 Sequencing of the Clones...........................................................................................88 Human Retinal Endothelial Ce ll (HREC) Tissue Culture..........................................89 LDL Uptake of the HREC..........................................................................................90 Transfection of HREC using DEAE-Dextran.............................................................91 Transfection Efficiency usi ng DEAE Dextran for HRECs.......................................92 Cell Migration Assay..................................................................................................92 Morphology of HEK Cells..........................................................................................93 Transfection using Lipofectamine on HEK 293 cells.................................................93 Transfection Efficiency for HEK Cells using Lipofectamine Reagent......................94 cAMP Assay on Transfected HEK 293 Cells.............................................................94 Total Retinal RNA Extraction for PCR......................................................................97 Real Time PCR...........................................................................................................97 Animals.......................................................................................................................9 8 Intraocular Injection into the Mouse Model of Oxygen Induced Retinopathy...........98 Statistical Analysis......................................................................................................99 3 RESULTS.................................................................................................................100 Determining Accessibility of the Target Site...........................................................100 Time Course of Ribozyme cleavage.........................................................................103 Multiple Turnover Kinetics......................................................................................106 Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector..............109 Sequencing of the Clones.........................................................................................111 Cell Cultures.............................................................................................................111

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vii Transfection of HREC..............................................................................................114 Transfection Using Lipofectmaine on HEK Cells....................................................118 CAMP Assay on Transfected HEK Cells.................................................................121 Real Time PCR..................................................................................................125 Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model...........125 4 DISCUSSION...........................................................................................................131 Ribozymes As Tools To Study Gene Expression.....................................................132 Delivery Of The Ribozyme In vivo..........................................................................135 Promoter Considerations..........................................................................................139 Future Studies...........................................................................................................142 5 LIST OF REFERENCES..........................................................................................147 BIOGRAPHICAL SKETCH...........................................................................................165

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viii LIST OF FIGURES Figure page 1-1 Cross sectional view of the components of the eye...................................................2 1-2 The ten layers of the retina.........................................................................................6 1-3 A fundus shot of ARMD..........................................................................................12 1-4 Non-proliferative retinopathy...................................................................................14 1-5 New blood vessel growth around optic nerve in PDR.............................................16 1-6 ICROP definition of retinopathy..............................................................................18 1-7 The five stages of ROP.............................................................................................20 1-8 Laser treatment of the eye........................................................................................21 1-9 Cartoon showing cryotherapy applicati on to the anterior avascular retina..............23 1-10 The process of angiogenesis.....................................................................................25 1-11 PAs hydrolyze plasminogen to plasmin...................................................................28 1-12 Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors......................................34 1-13 The vascular endothelial cell growth factor (VEGF R2) signaling pathway...........37 1-14 The FGF receptor and signaling pathway................................................................40 1-15 The PDGF receptor and signaling pathway.............................................................41 1-16 The TGFreceptor signaling pathway...................................................................43 1-17 Intracellular and extracellu lar production of adenosine...........................................46 118 Role of the high and low affinity adenosine receptors.............................................50 1-19 Homology of the A1 receptor for human and mouse...............................................53 1-20 Homology of the A2A receptor between human and mouse.....................................54

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ix 1-21 Homology of the A2B receptor between the human and the mouse.........................55 1-22 The A 2B receptor......................................................................................................57 1-23. The A2A receptor......................................................................................................58 1-24 The A 2B signaling pathway.....................................................................................63 1-25 The secondary stru cture group I introns...................................................................65 1-26 Splicing mechanism of the group I introns..............................................................66 1-27 Secondary structure of Group II introns...................................................................68 1-28 The splicing mechanism of the Group II introns......................................................69 1-29 Cleavage of the tRNA 5 leader sequence by Rnase P............................................70 1-30 Self-cleaving ribozymes resolve concatemers formed by rolling-circle replication into individual genomic molecules..........................................................................72 1-31 Structure of the hairpin ribozyme.............................................................................73 1-32 Structure of the hammerhead ribozyme...................................................................75 1-33 The hammerhead ribozyme cleaves it s substrate by a transesterification reaction.....................................................................................................................76 1-34 Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.....79 1-35 Target sequences of the human and mouse A2B ribozymes 1 and 2.........................80 1-36 Hammerhead ribozymes for the A 2B Rz1 and Rz2................................................81 1-37 The p21Newhp Vector with the CMV e nhancer and beta actin promoter...............82 1-38 Time course for the ROP model...............................................................................84 3-1 Theoretical tertiary structures of the active A2B Rz1 generated by the mfold program..................................................................................................................101 3-2 Theoretical tertiary structures of the active A2B Rz2 generated by the mfold program..................................................................................................................102 3-3 Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing products of cleavage of the A2B Rz2 on the mouse target......................................104 3-4 Time course analysis data......................................................................................105

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x 3-5 Time course of the ribozyme with increasing target concentrations......................107 3-6 Time course cleavage reaction with varying temperatures (37 C/25C) and magnesium concentrations of 20mM/1mM...........................................................108 3-7 Kinetic analysis of the ribozymes..........................................................................110 3-8 Sequence of the active and inactive versions of the A2B ribozymes at the site of insertion within the p21NewHp vector..................................................................112 3-9 Pebble stone morphology of the HREC.................................................................113 3-10. LDL uptake of HREC.............................................................................................115 3-11 The GFP plasmid. This plasmid was driven by a CMV enhancer and a chicken beta actin promoter.................................................................................................116 3-12 Transfection efficiency of the HREC.....................................................................117 3-13 Theory of migration assay......................................................................................119 3-14 Migration data for the ce lls transfected with the active and inactive versions of the A2B receptor and the vector control. 10% FBS/DMEM is the positive control and DMEM alone is the negative control...............................................................120 3-15 Transfection Efficiency of HEK cells....................................................................122 3-16 HEK cells transfection efficiency following passage 1.........................................123 3-17 cAMP accumulation in HEK cells tran sfected with the control, active A2B Rz2 and inactive A2B Rz 2....................................................................................................124 3-18 Real time RT-PCR results showing relative levels of the adenosine A2A and A2B receptor mRNAs isolated from HEK cells transfected with plasmid DNA...........126 3-19 The mice eyes were embedded in paraffi n and three hundred serial sections were done........................................................................................................................127 3-20 Injection with the control plasmid pr ior to exposure to high oxygen shows a high number of endothelial cell nuc lei surrounding blood vessel lumen.......................128 3-21 Injection with the active A 2B ribozyme prior to high oxygen exposure significantly reduced the pre-retina l neovascularization.............................................................129 3-22 Injection of the active an d inactive versions of the A2B Rz2 and the vector control in the ROP mouse model........................................................................................130 4-1 Entry of the AAV and transferrin into the cell.......................................................137

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xi 4-2 Diagram of the expression cassettes fusion protein and alkaline phosphatase (Alk Phos)..............................................................................................................141 4-3 A2B signaling pathway with theoretical downs tream effects, which have yet to be confirmed...............................................................................................................146

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xii LIST OF ABBREVIATIONS a-LDL Acetylated 1,1Â’-Dioctdycl3,3,3Â’,3Â’ tetra methyl indocarbocyanin perchlorate ABAM Antibiotic antimycotic mix A1 Adenosine receptor type 1 A2A Adenosine receptor type 2A A2B Adenosine receptor type 2B A2B Rz1 Adenosine receptor type 2 ribozyme 1 A2B Rz2 Adenosine receptor type 2 ribozyme 1 A2R Adenosine receptor type 2 A3 Adenosine receptor type 3 ADA Adenosise deaminase AK Adenosine kinase AMP Adenosine monophosphate ANG-1 Angiopoeitin 1 ANG-2 Angiopoeitin 2 ARMD Age Related Macular Degeneration ARNT Aryl hydrocarbon receptor nuclear translocator ARVO Association for Research in Vision and Ophthalmology ATP Adenosine triphosphate v3 Alpha V beta 3 integrin

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xiii v5 Integrin bFGF Basic fibroblast growth factor BSA Bovine serum albumin cAMP 3c, 5c-cyclic monophosphate CAT Chloramphenicol acetyltransferase CGS21680 A2A agonist. 2-{4[(2-car boxylethyl)-phenyl]ethylamine}-5Â’-Nethylcarboxamidoadenosine CHA Cyclohexyladenosine CHO Chinese hamster ovary cells CMV Cytomegalovirus DMEM DubellcoÂ’s modifeid eagle medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPSPX Non-seletive adenosine r eceptor antagonist. 1,3-dipropyl-8(psulfophenyl)xanthine DTT Dithiothreitol ECM Extracellular matrix EDTA Ethylenediamine tetraacetic acid EGS External guide sequence Eph receptor Ephrin receptor FAK Focal adhesion kinase FAT Focal adhesion targeting sequence FBS Fetal bovine serum Flt VEGF fms like tyrosine kinase GAGs Glycosaminoglycans

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xiv GC Guanosine cytosine content GCL Ganglion cell layer GFP Green fluorescent protein GPI Glycosylphosphatidylinositol HBSS Hanks balanced salt solution HDV Hepatitis delta virus HEK 293 Human embryonic kidney cells HIF Hypoxia inducible factor HIV Human immunodeficiency virus HRE Hypoxia response element HRECs Human retinal endothelial cells HSPGs Heparan sulfate proteoglycans IACUC Institution Animal Care and Use Committee. IB-MECA Selective A3 adenosine recep tor agonist. N6 (3-iodobenzyl)Ado-5Â’Nmethyl Uronamide ICROP International classificatio n of Retinopathy of Prematurity ILM Inner limiting membrane INL Inner nuclear layer IP3 Inositol triphosphate IPL Inner plexiform layer KDR VEGF kinase insert domain LAP Latency associated peptide MMP Metalloproteinases NAD Nicotinamide adenine dinucleotide

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xv NBTI Nitrobenzylthioinosine NECA N-ethylcarboxyamidoadenosine NFL Nerve fibre layer NPDR Non proliferative diabetic retinopathy 5Â’NT 5Â’ Nucleotodase OLM Outer limiting membrane ONL Outer nuclear layer OPL Outer plexiform layer. PAs Plasminogen activators PAI-1 Plasminogen activator inhibitor-1 PAI-2 Plasminogen activator inhibitor-2 PBS Phosphate buffered saline PDGF Platelet derived growth factor PKC Protein kinase C PLC Phospholipase C PDR Proliferative diabetic retinopathy rAAV Recombinant adeno associated virus RBCs Red blood cells ROP Retinopathy of Prematurity RNA Ribonucleic acid rRNA Ribosomal RNA RNasin Ribonuclease inhibitor RPE Retinal pigment epithelium

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xvi R-PIA Selective A1 receptor agonist. R-phenylisopropyl-adenosine SAH S-Adenosylhomocysteine TBS Tris buffered saline TGF Transforming growth factor Tie 1 and 2 Angiopoeitin receptors 1 and 2 TIMPS Tissue inhibitors of matrix metalloproteinases TNFTumor necrosis factor alpha tPA Tissue type plasminogen activator tRNA Transfer RNA TR Inverted terminal repeats uPA Urokinase type plasminogen inhibitor VE cadherin Vascular endothelial cadherins VEGF Vascular endothe lial growth factor VEGF-R1 Vascular endothelial growth factor-receptor 1 VEGF-R2 Vascular endothe lial growth-receptor 2 WBCs White blood cells XAC Xanthine amine cogener XDH Xanthine dehydrogenase XO Xanthine oxidase

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xvii 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 REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES THAT CLEAVE THE A 2B RECEPTOR MRNA By Aqeela Afzal May 2003 Chair: Dr. M.B. Grant Cochair: Dr. D. Samuelson Major Department: Veterinary Medical Sciences Tissue hypoxia and ischemia initiate events that lead to pre-retinal angiogenesis. Adenosine modulates a variety of cellular functions by interacti ng with specific cell surface G-protein coupled receptors (A1, A2A, A2B, A3) and is a potential mediator of angiogenesis. The A2B receptor has been implicated in the mediation of angiogenesis. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design and characterize a hamme rhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its eff ect on retinal angiogenesis. Active and inactive ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. HEK 293 cells were tran sfected with these plasmids, and A2B mRNA levels were determined by quantitative RT-PCR. Human retinal endothelial cells (HREC) were also transfected, and cell migration was examined. The effects of these ribozymes on the levels of pre-retinal neovascularization were determined using a mouse model of oxyge n-induced retinopathy. We produced a

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xviii ribozyme with a Vmax of 10.8 pmole min-1 and a kcat of 36.1 min1. Transfection of HEK 293 cells with the plasmid expressing ribozyme resulted in a reduction of A2B mRNA levels by 45%. Transfection of HREC reduced NECA stimulated migration of the cells by 47%. Intraocular injection of the construc ts into the mouse model reduced pre-retinal neovascularization by 54%. Ou r results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor, and provide further support for the role of the A2B receptor in retinal angiogenesis.

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1 CHAPTER 1 BACKGROUND AND SIGNIFICANCE The formation of blood vessels is a funda mental process that can be broken down into two basic pathways. The first is vasc ulogenesis, which is the formation of new blood vessels such as seen in embryogenesis. The second is angioge nesis, which is the formation of blood vessels from pre-existing blood vessels. Angiogenesis is common in both normal physiological processes (pre gnancy, menstruation, wound healing) and disease states (cancer, retinopath ies, psoriasis). The focus of this study is the process of angiogenesis in retinopathies, including di abetic retinopathy, the leading cause of blindness in adults, and reti nopathy of prematurity (ROP). Anatomy of the Eye The two eyes in humans are oriented to facilitate binocular single vision, which results from the forward position of the eyes and the chiasmal crossing from axons of ganglion cells. Axons from the right visual fi eld carry impulses to th e left optic tract and vice versa. The eye contains the elements that take in light and converts them to neural signals. For protection, the eye is loca ted within the bone and connective tissue framework of the orbit. The ey elids cover and protect the ante rior surface of the eye and contain glands, which produce a lubricating film (tears).1 The globe has three spaces within it: the anterior chamber, posterior chamber and the vitreous chamber. 1 (Figure 1-1)

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2 Figure 1-1. Cross sectional view of the components of the eye

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3 The anterior and posterior chambers contai n aqueous humour, which is produced by the ciliary body and provides nourishment for th e surrounding structures. The vitreous chamber is the largest space in the eye and lies adjacent to the inner retinal layer and contains the gel-like vitreous humor.1 The eye is made up of three layers: an out er fibrous layer, a middle vascular layer and an inner neural layer (retina).1 The outer fiber layer is a dense connective tissue that provides protection for structures within, ma intains the shape of the eye, and provides resistance to the pressure of the fluids inside the eye. Th e sclera is the opaque white of the eye, and the cornea is transparent and a llows light to enter the eye where the lens refracts it to bring light rays into focus on the retina.1 The middle layer of the eye is made up of three structures. The iris acts as a diaphragm to regulate the amount of light en tering the pupil. The ciliary body produces components of the aqueous humor and has musc les that control the shape of the lens during accomodation. The choroid is an an astomosing network of blood vessels with a dense capillary network.1 The principle functions of the choroid are to nourish the outer retina and to provide a pathway for the vessels that supply the anterior eye. The choroid is an egress for catabolites from the retina, which diffuse through BruchÂ’s membrane into the choriocapillaris. The suprachoroidal space provides a pathway for the posterior vessels and nerves that supply the anterior segment.1,2 The choroid also plays a role in the maintenance of intraocular pressure due to the high blood flow in its vessels. The choroid has the largest sized a nd the greatest number of vascul ar channels in the eye, and the amount of blood flowing through these ch annels at any time has an effect on the

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4 intraocular pressure. The choroid also provi des a regular smooth internal surface for the support of the retina. The smoothness of Bruc hÂ’s membrane is important in maintaining the exact relationship between the retinal pigment epithelium (RPE) and the outer segments of the adjacent phot oreceptor rods and cones. 1,3 The Retina The retina is located between the choroi d and the vitreous, and extends from the circular edge of the optic disc, where the nerve fibers exit the eye, to the ora serrata and is continuous with the epithelia l layers of the ciliary body.1,4 The retina is a thin, delicate and transparent tissue that lines the inner eye. The neural retina is at tached loosely to the choroid through the pigment epithelium. Exte rnally, the RPE contacts the collagen and elastic tissue of BruchÂ’s membrane of the chor oid. Bruchs membrane is an elastic layer that stabilizes the RPE and the photoreceptors. Internally, the retina lies next to the vitreous. Anteriorly, the RPE gives rise to the ciliary body, a nd posteriorly, all the retinal layers terminate at the optic disc except the nerve fiber layer. The retina is thickest at the equator and thins at the ora serrata.1 The retina can be divided into the centra l retina and the peripheral retina. The central retina is thick and includes the macu la, fovea and foveola. The macula has a yellow appearance due to xanthophyl (a caroteno id), which is found in the ganglion cells. The peripheral retina includes the remainder of the retina from the macula to the temporal or nasal side. The ora serrata is the extreme periphery of the retina. It is the junction where the retina ends and gives rise to the te eth like processes that form the ciliary body. The peripheral retina ends at the ora serrata and forms the te eth like processes that form the base of the ciliary body.1

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5 Under the light microscope, ten layers of th e retina can be differentiated (Figure 12): RPE, rod and cone layer, outer lim iting membrane (OLM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fi bre layer (NFL) and the inner limiting membrane (ILM). The visual pathway cons ists of three interconnecting neurons and some receptor cells. The neurons include the bi polar cells, located within the retina; the ganglion cells, located in the inner retina, the axons of which go through the optic nerve to the chiasm and end in the lateral genicula te nucleus; and the third neuron is from the geniculate body to th e occipital cortex.1 The rods and cones are the sensory receptors. The outer segments have photopigments, which are excited by light, resul ting in a visual response. The cell bodies of the rods and cones lie in the ONL and the axons synapse with dendr ites of bipolar cells in the OPL. The dendrites of the bipolar cells extend to the OPL and synapse with axons of rods and cones; their axons extend to the IPL and synapse w ith dendrites of the ganglion cells from the NFL. The INL also has horizontal and amacrine cells. The horizontal and amacrine cells in this layer provide horizontal integration.1 The RPE is a single layer of uniform cells. It is located in the outer ci rcumference of the retina and extends from the edge of the optic disc to th e ora serrata. The cells of this layer are hexagonal shaped and carry a brown pigment. These cells may be multinucleated, especially in the ora serrata. The RPE provi des metabolites to the receptors and removes the outermost ends of external segments of the photoreceptors. If the RPE cells are damaged or diseased, these cells are not repl aced; instead, adjacent cell s slide laterally to fill the space of the necrotic cells. The RPE cells possess microvilli on

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6 Figure 1-2. The ten layers of the retina incl ude: retinal pigment epithelium, rod and cone layer, outer limiting membrane, outer nuc lear layer, outer plexiform layer, inner nuclear layer, inne r plexiform layer, gangli on cell layer, nerve fibre layer and the inner limiting membrane

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7 the apical surface that interdigitate with the photoreceptors. The RPE is critical to vitamin A metabolism and photoreceptor maintenance.1 The rod and cone layer lies external to th e OLM. It has a thick inner segment and thin outer segments joined by a slight c onstriction. The cell membrane is continuous between the constrictions. The outer segments have parallel processes, which are short in cones and long and thin in rods. 1 The OLM under a light microscope has a thin fenestrated membrane-like appearance. However, the OLM is not a basement membrane. Electron microscopy revealed it to be a zonula adherens between the photoreceptors and the Mller cells. The zonula adherens probably serve to keep th e highly elongated photoreceptors in place.1 The ONL has the cell bodies of the rods a nd the cones. The axons of the rods and cones synapse in the OPL with bipolar and horizontal cells.1 The OPL is a reticular structure, which is a transition zone be tween the receptors (neuroepithelial). The OPL is a layer of synaptic contacts between photoreceptors, bipolar cells and horizontal cells. The axons of the rods end here in spherules (oval shaped) and those of the cones end in pedicles (broad conica l swellings). The spherules are invaginated and synapse with bipolar dend rites or horizontal cells, and can make 2-4 contacts. The pedicles, on the other hand, c ontact many dendrites of horizontal cells and bipolar cells.1 The INL is a band of nuclei belonging to horizontal cells, bipolar cells, and amacrine cells. The Muller cells provide support and nutrition to the retina. They surround capillary walls and extend from the ILM to the extracellular membrane.1

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8 The IPL is a junction between the first or der neuron (bipolar cells) and the ganglion cell layer. This layer contains the nuclei of displaced ganglion or amacrine cells and processes of the Mller cells.1 The GCL contains the cell bodies of ganglion cells, which are thin in the nasal area and thicker near the macula. The axons of the ganglion cells run internally and then become parallel to the inner surface of the reti na to give rise to the NFL and the optic nerve fibres.1 The NFL has the axons of the ganglion cells and is thickest around the optic nerve.1 Branching processes of the Mller cel ls and a basal lamina like structure secreted by them forms the ILM. The macula is the center of the retina (area centralis) and is divided into the fovea (cone dominate d), parafovea (ganglion cell dominated) and the perifoveal retina (singl e layer of ganglion cells).1 Blood Supply to the Retina The retina has the highest rate of metabol ism of any tissue in the body and thus has a dual blood supply from the retinal and choroidal capillaries. If either of these sources is interrupted, ischemia develops and leads to loss of function. The out er retina is supplied by the choriocapillaris and the cen tral retinal artery supplies the remainder. The retinal artery is different from other arteries and does not have an in ternal elastic lamina but does have a prominently developed muscularis.1,5 The outer retinal layers r eceive their nutrition from th e choroidal capillary bed; metabolites diffuse through BruchÂ’s membrane a nd the RPE into the neural retina. The central retinal artery provides nutrients to the inner retinal layers. The artery enters the retina through the optic disc, us ually slightly nasal of center, and branches into a superior and inferior retinal artery, each of which furt her divides into nasal and temporal branches, and these vessels continue to bifurcate. The nasal branches run a relatively straight

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9 course toward the ora serrata, but the tem poral vessels arch around the macular area en route to the periphery. Two capillary networks exist within the retina. The deepest one lies in the inner nuclear layer ne ar the outer plexiform layer, and the superficial one is in the nerve fiber or ganglion ce ll layer. The OPL is avascu lar and thought to receive its nutrients from both retinal and choroidal vessels.1,5 Retinal arterial circulation is terminal; therefore there is no direct communication between the retina and other ve ssel systems. The junctions of endothelial cells in retinal vessels are tight or occluded. Thus, to enter or leave the retina, most substances require active transport across th e endothelial cells. The outer retina l layers receive their nutrition from the choroidal capillary bed; the central re tinal artery provides nutrients to the inner retinal layers. These vessels are distributed to the four quadrants of the retina.6 The retinal artery and vein to a particular quadrant supply most of the quadrant. If arterial supply to a retinal quadrant is interrupte d, infarction of that section o ccurs. The retinal capillaries supply the inner two thirds of the retina; the choroidal circ ulation supplies the remaining outer retina via regulated transport across the pigment epithelium.1,5 The Blood Retinal Barrier The epithelial portion of the blood-retinal barr ier is the retinal pigment epithelium. This barrier separates the c horoidal tissue fluid, which is similar to plasma, from the retinal tissue fluid. Tight junc tions that exist between the endothelial cells of the retinal vessels and similar tight junctions in the RP E maintain the blood retinal barrier. Thus, the retinal vessels are impermeable to the pa ssage of molecules gr eater than 20-30 kDa, and small molecules such as glucose and asco rbate are transported by facilitated diffusion through the RPE.1,5,7

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10 Vascular beds are situated to provide nourishment. To avoid problems with the presence of blood vessels in the outer retina, the outer layers of th e retina receive their nourishment from the choriocapillaris.1 Retinopathies The retina of the eye is uniquely situat ed to provide optimal vision. Blood supply to the retina is also strategically placed to avoid any hindrance of the visual pathway. Retinopathies (diseases affecting the retina) disrupt this balan ce and lead to loss of vision. Retinopathies affecting humans include: age related macular degeneration (ARMD), which primarily affects the aging population; diabetic retinopathy (DR), which primarily affects the working population; and retinopat hy of prematurity (ROP) which primarily affects the newborns. Age Related Macular Degeneration ARMD is a disease which affects the RPE and leads to blindness in the aged populations.8 There are two forms of ARMD: dry and wet.9-11 Dry ARMD is characterized by the presen ce of soft drusen and pigmentary abnormalities. Drusen is an amorphous acellu lar debris present within the basement membrane of the RPE. It is seen as ‘ye llow’ spots within the macula. Low amounts of drusen are a consequence of age; however, a larger amount present within the retina is indicative of ARMD.10 Drusen leads to mild vision loss and increases the risk of progression of the disease to the wet form of ARMD.8,9 The wet form of ARMD (also known as the exudative or the neovascular phase) is characterized by choroidal neovascularization, RPE detachment and disciform scarring. The wet form of ARMD leads to rapid visi on loss. The choroida l neovascularization

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11 (CNV) leads to the formation of immature bl ood vessels which result in leakage of serum and blood and loss of central vision.9,12 (Figure 1-3) Diabetic Retinopathy Diabetes Mellitus affects millions of peopl e worldwide and is the leading cause of blindness in working age adults.13,14,15 There are two forms of diabetes mellitus: Type I, which typically affects juveniles and is known as insulin dependent diabetes mellitus, and type II, which is the adult onset form of diabetes and is known as non-insulin dependent diabetes mellitus.15 Diabetes also leads to systemic complications such as kidney failure, hypertension and cardiovascular disease.16,13 DR is the most frequent diabetic complication. Eye problems due to diabetes can be asymptomatic and if left untreated can lead to serious visual loss. The longer a patient has diabetes, the more likely they are to develop diabetic retinopathy.13,16,17,15 Diabetic eye disease can be divided into two phases: background diabetic re tinopathy (non-proliferative phase) and proliferative diabetic retinopathy (PDR). Non-Proliferative Diabetic Retinopathy (NPDR) In NPDR small retinal blood vessels are damaged. NPDR is the result of two major processes which affect retinal blood vessels, vessel closure and abnormal vessel permeability.13 The vessels leak fluid (edema) and later blood (hemorrhage) into the retina. Macular edema is the most common cau se of reduced vision in patients with nonproliferative diabetic retinopathy and is seen as milkiness of the retina surrounded with exudates (yellow clumps). 16,15 These exudates are th e result of fat or protein leaking out of the vessels. Water is quickly reabsorbed into the vessels

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12 Figure 1-3. A. A fundus shot showing drusen (yellow). B. Wet form of ARMD showing blood leakage. (National Eye Institute)

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13 or tissue under the retina. However, the fatty material is absorbed very slowly and thus left behind surrounding the leakage site.16,18 (Figure 1-4) Vessel closure may be due to blood cell clumping, damaged endothelium, swelling of an abnormally permeable vessel wall or compression of the capillary by surrounding retinal swelling. Diabetic patients have cl osure/non-perfusion of capillaries, which leads to a decreased oxygen supply. In areas surr ounding the area of non-pe rfusion capillaries dilate to compensate for the decrea sed oxygen supply. Small focal dilations (microaneurysms) of retinal capillaries also develop due to weakened capillary walls, thus allowing for bulging. 13 When multiple areas of the retina have lost their blood supply, angiogenic factors are released wh ich stimulate proliferation of new blood vessels. These new blood vessels are small a nd fragile, therefore, cause bleeding and the formation of scar tissue within the retina. Sm all arterial closures follow capillary closure, and deprive larger regions of the retina of blood supply. This is seen as ‘cotton wool spots’ on the retina in fluorescein angiography.16 Blood vessels in the body ar e usually fenestrated allo wing fluid to pass through vessel walls. These openings are small enough to allow water and ions to pass through, while preventing the passage of blood cells and larger proteins. In contrast, retinal blood vessels have tight junctions be tween the endothelial cells of blood vessel. Therefore, all fluids and molecules exiting the vessels have to pass through the cell. This lack of fenestration helps to keep the retina thin a nd dehydrated for proper function. These tight junctions form the blood retinal barrier, whic h partitions the neur al retina from the circulation and protects the retina from circulating inflammatory cells.16 The tight junctions are formed by a number of protei ns such as: occludin and claudin. These

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14 Figure 1-4. A. Non-proliferative reti nopathy. Hemorrhage (arrowhead) short arrow microaneurysm, larger arrow exudates. B. Macular edema. (National Eye Institute)

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15 proteins limit the flow of fluid between endot helial cells. Diabetic patients have a lower amount of occludin at the tight junctions in the retinal e ndothelial cells and can leak fluid.16 Proliferative Diabetic Retinopathy (PDR). Proliferative diabetic retinopathy is th e stage of diabetes characterized by angiogenesis on the surface of the retina. 13 Patients can have NPDR for years before progressing to PDR. PDR is diagnosed by th e presence of prolif erating blood vessels within the retina or optic disc. These vesse ls grow on the retinal surface or into the vitreous cavity and take on a frond-li ke configuration as they grow.19 (Figure 1-5) The new blood vessels form due to the closure of re tinal capillaries, which leads to ischemia. As patches of the retina are deprived of oxygen and nutrients, vasoproliferative factors are released which diffuse into the vitreous cavity. These factors stimulate growth of new vessels throughout the retina.15 The new blood vessels are not located in the same location as the ischemically damaged retina and are very fragile and bleed into the vitreous. A small amount of blood may be removed in a few weeks and larger blood hemorrhages may take a few months. If dense blood from multiple recurrent hemorrhages occurs then vision may not be restored since the residual inflammatory debris and dead cells cannot be removed. Another complication of PDR is traction retinal det achment. New vessels grow and regress and lay down fibrous scar tissue, which contra cts and shrinks as it matures. If the neovascularization is on the surface of the reti na then contraction of the fibrous scar distorts the retina. However, if the vessels grow into the vitreous and contract, retinal detachment occurs which leads to blindness. 13,15

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16 Figure 1-5. New blood vessel growth around optic nerve in PDR (Top). Hemorrhage from new blood vessel growth (Bot tom). (National Eye Institute)

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17 Retinopathy of Prematurity Retinopathy of prematurity (ROP), also known as retrolental fibroplasia, is a potentially blinding condition affecting the retina of new borns. In the 1950s, it was associated with the use of high oxygen levels in neonatal units.20 Modern neonatal care has curbed the incidence of ROP, but because the survival rate of low-birth-weight infants is increasing, the exposure of surviv ing babies to high oxygen levels is also increasing and ROP is still a relevant clinical problem.21,22 ROP causes more blindness among children in the world than all other causes combined. It begins after removal from high oxygen conditions and may progress rapidly to blindness over a period of weeks.23 Active growth of the fetal eye occurs between the last 12 weeks of full term deliv ery (28-40 weeks of gestation) At 16 weeks of gestation, blood vessels gradually grow over the surface of the retina. Vessels reach the anterior edge of the retina and stop progressi ng at about 40 weeks of gestation.20,21,24,25 The international classification of RO P (ICROP) defines retinopathy by several distinct criteria: location, ex tent, stage, and plus disease.26 Location refers to the location of the damage to the retina relative to the opt ic nerve. Normally retinal vessels begin growth at the optic nerve and gradually move toward the edge of the retina. Vessels further from the optic nerve ar e more mature. To standard ize the location of ROP, the retina is divided into three zones: Zone I is centered on the optic disc and extends from the optic disc to twice the distance between th e disc and macula; Zone II is a concentric ring around zone I and extends to the nasal ora serrata (the edge of the retina on the side toward the nose); and zone III is the remain ing crescent of retina on the temporal side (side towards the temple) (Figure 1-6). The extent of ROP is described by the clock hours

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18 Figure 1-6. ICROP definition of retinopathy. The retina is divided into three zones: Zone I, Zone II and Zone III

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19 of the retina involved in the ROP. For example, if the ROP extends from 1:00 to 5:00, the extent of ROP is 4 clock hours.24,27 ROP is a progressive disease that begins with some mild changes in vessels and may progress on to more severe changes. The five stages of ROP describe the progression of the disease (Figure 1-7). Stag e 1 is characterized by a demarcation line between the normal retina (near the optic nerve) and vascularized retina. In stage 2, a ridge of scar tissue rises up from the retina due to growth of abnormal vessels. This ridge forms in place of the demarcation line. In st age 3, the vascular ridg e grows due to spread of abnormal vessels and extends into the vi treous. Stages 4 and 5 refer to retinal detachment; stage 4 refers to a partial retin al detachment caused by contraction of the ridge, thus pulling the retina away from the wall of the eye; and stage 5 refers to complete retinal detachment. Plus dis ease is a very severe form of ROP which is characterized by the abnormal growth of blood ve ssels near the optic nerve.24,28 Treatment of Retinopathies. Spot laser photocoagulation is used for the treatment of ROP.29 This uses an argon/diode laser to burn spots on the peripheral and middle por tions of the retina. When laser light hits blood or pigment, it is abso rbed as heat energy and produces a small burn. The laser treatment leads to a decrease in th e level of vasoprolifer ative factors produced by the ischemic retina. The avascular retina is treated using a small laser spot (Figure 18). The laser spot directly treats the re tina and the underlying tissue, thus reducing inflammation and results in less damage to othe r ocular structures. Destruction of small patches of the ischemic retina reduc es the oxygen demand and decreases the vasoproliferative factor produc tion. Laser treatment also th ins the pigmented tissue under

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20 Figure 1-7. The five stages of ROP (National Eye Institute)

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21 Figure 1-8 Laser treatment of the eye. The laser spot directly tr eats the retina and the underlying tissue. Laser treatment thin s the pigmented tissue under the retina and allows more oxygen to diffuse in from the vessels under the retina

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22 the retina thus allowi ng better oxygen diffusion in the retin a. Laser treatment increases oxygen supply, lowers the demand for oxygen and lowers the incidents for new vessels growth.30,28 Laser photocoagulation also causes less pain than other therapies. Currently laser treatment is the best option for the treatment of retinopathies.14,31,29,32 Cryotherapy is also one of the treatments available for the treatment of retinopathies.33,34 This technique involves placing a co ld probe on the sclera until an ice ball forms on the retinal surface. Multiple ap plications are done to cover the entire vascular area (Figure 1-9). This thins th e tissue under the retina (by destroying it) and allows easier oxygen diffusion through the retina.31 Due to the pain involved in cryotherapy, anesthesia has to be administer ed which is a risk factor for premature infants. If no anesthesia is administered, ca rdiac arrest follows. Another complication is hemorrhage due to excessive bleeding.31,33,35,36 If laser photocoagulation or cryotherapy is unsuccessful, a scleral buckle may be used.37 This involves surgery and is used if ther e is shallow retinal detachment due to the contraction of the ridge. A silicone band is tightly placed around the equator of the eye thus producing a slight inde ntation on the inside of the eye.38 This indentation relieves traction of the vitreous gel and allows the retina to flatten back onto the wall of the eye. The silicone band is then remove d a few months later to allow the eye to grow.14,31,39 If the scleral buckle is not sufficien t, vitrectomy may be performed. Small incisions are made into the eye, the vitreous removed and replaced by saline. This technique also has had limited success. Current available therapies for the different types of retinopathies have had limited success. The underlying cause of retinopathies is angiogenesis

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23 Figure 1-9. Cartoon showing cr yotherapy application to the anterior avascular retina A cold probe is placed on the sclera till an ice ball forms on the retinal surface. Multiple applications are done to cove r the entire vascular area. This treatment thins the tissue under the re tina and allows easier oxygen diffusion through the retina

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24 (abnormal blood vessel formation). Developmen t of other effective therapies for the diseases requires an understandi ng of the process of angiogenesis.28,33,40-42 Angiogenesis Vasulogenesis is the formation of new bl ood vessels. Precursor cells (angioblasts) differentiate into endothelia l cells which later link to fo rm blood vessels. Angiogenesis on the other hand is the spr outing of blood vessels from pre-existing blood vessels.43 The vasculature of the retina unde rgoes both vasculogenesis and angiogenesis. The superficial retinal vessels, which originate at the optic disc, are formed by the process of vasculogenesis and the process of angiogene sis later forms the capillary beds. The process of angiogenesis involves e ndothelial branching, sprouting, migration, proliferation and anastomosing with e ndothelial cells in existing vessels.43-45 (Figure 110) Vascular endothelial cells form a monolayer throughout the entire vasculature. They are polarized cells with an apical surface and a basal surface, which is surrounded by a basal lamina.46-49 Mural cells wrap around this struct ure and are contra ctile cells, which regulate vessel diameter and consequently blood flow.47,50 On large vessels they are multi layered and referred to as smooth muscle cells. On capillaries mural cells are sparse and usually referred to as pericytes.48,50 The extracellular matrix, bound factors and the soluble growth factors all play an importa nt role in the process of angiogenesis.

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25 Figure 1-10. The process of angiogenesis. The process of angiogenesis involves endothelial branching, sprouting, migrat ion and proliferation. Vascular endothelial cells form a monolayer throughout the entire vasculature. Pericytes wrap around these cells.

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26 Extracellular matrix (ECM). The ECM surrounds and provides mechanic al support for blood vessels. The activated endothelial cells cerate gaps in th e basement membrane, which allows them to sprout into the ECM. The ECM is com posed of two compartments: the interstitial matrix and the vascular basement membrane.51,52 The interstitial matrix consists of fi brillar collagen and glycoproteins (e.g. fibronectin, laminin). Fibr onectin attaches cells to a va riety of ECM components, and laminin anchors cell surfaces to the basal lami na. Collagen provides structural support, is synthesized by fibroblasts and is the most abundant protein comprising the ECM. There are 12 types of collagen and types I, II and I II are the most abundant types of collagen in the ECM.51,52 The vascular basement membrane li es between the endothelial cells and pericytes. It is composed of type IV collagen, which forms the basal lamina upon which the endothelium rests, and heparan sulfate proteoglycans.51 Proteoglycans are glycosaminoglycans ( GAGs) linked to proteins. Cell surface heparan sulfate proteoglycans (HSPGs) func tion as endothelial cell receptors that recognize the ECM. They are present in the basement membranes and cell surfaces. These proteoglycans modulate the response of e ndothelial cells to basic fibroblast growth factor (bFGF), vascular endot helial growth factor (VEGF) and other heparan binding angiogenic factors by sequestering these molecu les in the ECM. Heparatinases trigger the release of these growth factors from the ECM and make them available for angiogenic stimuli.51,53 ECM degradation Endothelial cells degrade the surroundi ng ECM by the release of plasminogen activators (PAs) and matrix metalloproteases (MMPs).54 The PAs hydrolyze plasminogen

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27 to plasmin, which is a general protease that can digest most protei ns. (Figure 1-11) It also converts latent collagenase into active collagensase wh ich can then degrade collagen type I, II and III. 54 There are two types of PAs: tissue type PA (tPA) and urokinase-type PA (uPA).55 Both PAs utilize the same substr ate, plasminogen, and both have two specific inhibitors, plasminogen activator i nhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). PAI-1 is produced by endothelial cells to i nhibit PA activity to ensure a balanced degradation of the ECM. uPA and PAI-1 are also upregulated by angiogenic factors such as basi c fibroblast growth factor (b FGF) and vascular endothelial growth factor (VEGF)54-56 Matrix metalloproteases (MMPs) are zi nc dependent endopeptidases which are secreted as zymogens a nd proteolytically activated by other MMPs or plasmin.51 MMP expression may also be regulated by grow th factors such as VEGF, bFGF and TGF.51,56 MMPs degrade components of the ECM a nd are subdivided into: collagenases, stromelysins (cleave laminin and fibronectin), matrilysins, gelatinases (cleave collagen type IV), membrane type (MT) MMP and ot her MMPs. Endothelial cells, smooth muscle cells and fibroblasts produce collagenase 1 (MMP-1), stromelysin (MMP3), gelatinase A (MMP-2), gelatinase B (MMP-9), matr ilysin (MMP-7), and MT1-MMP (MMP-14, which has fibrinolytic activity).54,55

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28 Figure 1-11. PAs hydrolyze plasminogen to pl asmin. Plasmin subsequently activates matrix metalloproteases, which degrade the extra cellular matrix. PA=plasminogen activator; uPA=urokin ase type PA; tPA=tissue type PA; PAI=plasminogen activator inhibito r; MMP=matrix metalloproteases; TIMPs=tissue inhibitors of MMPs

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29 Endothelial cells also produce tissue inhi bitors of MMPs (TIMPs) that are specific inhibitors of MMPs and modulate the degrad ation of the ECM. TIMPs are secreted proteins, which inhibit MMPs in a 1:1 stoichio metry. They reversibly interact with the catalytic domain of the MMPs to inhibit their activity. TIMPs differ in their ability to interact with various MMPs. For exam ple, TIMP2 inhibits MT-MMP and TIMP3 inhibits MMP9. TIMPs also bind to the he paran sulfate proteogl ycans in the ECM and concentrate them to the specifi c regions within the tissue. 56 Angiostatin and endostatin are naturally occurring anti-angiogenic molecules, however, they are also produced by proteoly tic cleavage by MMPs from the pro-forms of plasminogen and collagen XVIII, respectively.57 The production of MMPs, however, is cell and tissue specific. For example, bFGF and VEGF upregulate in terstitial collagenase (MMP-1) and also increa se the formation of plasmin. Plasmin converts the inactive form of MMP-1 to the active form. Gelatinase A (MMP-2) is upregulated by calcium influx. It is responsible for the angiogenic switch a nd for the differentiation of the endothelial cells into tubes. MMPs promote capill ary tube formation, however, at high concentrations, they have an opposite effect. ECM degradation produces fragments, which have the opposite effect of the intact molecule. For example, hyaluronan, a GAG found in the ECM, has anti-angiogenic properties. However, when cleaved, it enha nces the action of a ngiogenic factors. Conversely, proteolytic degrad ation of fibronectin, plasmi nogen and collagen produces fragments, which have both anti-angiogenic and angioge nic activity. MMP2 undergoes proteolysis to produce PEX, which is the C-terminal non-catalytic domain of MMP2. PEX is anti-angiogenic and inhibits the gelatinolytic activity of MMP2.

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30 Bound Factors. Activated endothelial cells are anchorage dependent for survival. In addition to degradation of the ECM, the endothelial cells require bound fact ors to help in migration towards the ischemic stimulus. These bound factors include integrins, Eph receptor Eph/Ephrins complexes, and VE cadherins Integrins. Integrins provide the scaffolding for the cells to migrate upon and are used by the endothelial cells to recognize the ECM.58 Integrins play a role in regulating cell growth, differentiation and survival.59-63 Integrins are cellular receptors for ECM pr oteins and are expressed by all adhesive cells.64 Integrins are composed of and chain heterocomplexes, which are integral membrane glycoproteins. They have long extracellular domains, which are the ligand binding regions. Eighteen different subunits, and 8 subnits have been identified. These subunits can associate in 24 known co mbinations. A short transmembrane region follows the short intracel lular domains of both the and the subunits and the cytoplasmic tail of the beta subunit links the inte grins to cytoskeletal actin of the endothelial cell.62,63,65 Integrins are linked intracellulary to actin filaments by specific actin binding proteins, such as Talin, alpha actinin, vinicluin and paxillin.66 Focal adhesion kinase (FAK) is a protein with tyrosine kinase activit y and is composed of a large kinase domain flanked by an amino and carboxyl terminus. A region of the c-terminus, known as focal adhesion targeting sequence (FAT) recruits FAK to paxillin. Integrin mediated cell adhesion occurs when FAK is tyrosine phosphorylated.58

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31 v3 has a well-characterized ro le in angiogenesis. It me diates adhesion of cells to vitronection, fibronectin, von Willebra nd factor, osteopontin, tenascin and thrombospondin. Although the v3 integrin is minimally expressed on normal resting blood vessels, it is significantly upregulated in newly formed blood vessels within tumors, in healing wounds and in re sponse to certain growth factors. v3 expression is upregulated in endothelial ce lls exposed to angiogenic factors and those exposed to hypoxia. Integrins also help to target the activity of the MMPs, for example, v3 interacts with MMP-2 and also regulates sign aling via the vascular endothelial growth factor receptor –2 (VEGF-R2). Natural co mponents of the ECM, such as, endostatin, angiostatin, thrombospondin and tumastatin ar e all anti-angiogenic and exert their effect by binding to the v3 intergrin and disrupting the endot helial cell-ECM interaction. If this integrin is disrupted using an anti body (LM609) or a peptide antagonist (cyclic peptide 203, RGDfv), it results in the disrupt ion of angiogenesis pr ogression. VEGF and bFGF are capable of inducing the expression of v3 integrin of endothelial cells.67,68 EpH receptors. To discriminate cell partners from fibr oblasts or inflammatory cells, the Eph receptor is utilized. The Eph receptor is th e largest receptor of the receptor tyrosine kinase family (RTK) family.69,70 The receptors are divided based on ligand affinity into class A and class B. The extracellular domain of the Eph receptor consists of the ligand binding globular domain, cysteine rich region and 3 fibronec tin type II repeats. The cytoplasmic portion of the receptor consists of a juxtamembrane domain, and a carboxyl terminus. The ligands for these receptors are ephrins, which are also divided into subclass A and subclass B. Ephrins subclass A are anchored to the plasma membrane by

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32 glycosylphosphatidylinositol (GPI) anchor a nd ligands A1-A5 have been identified. Ephrins subclass B have a short transmembran e domain and a short cytoplasmic tail. Only three subclass B ephrins have been iden tified (B1-B3). Both the receptor and the ligands are membrane bound and therefore a signal is transduced in the receptor expressing cells and the ligand expressing cells.70,71 Prior to cell-cell contact, the Eph recepto r and ehprins ligand are loosely clustered at the cell surface. Following cell-cell cont act, the receptor and ligand heterodimerize and tetramerize. These receptors are capab le of bi-directional signaling (forward and reverse signaling). Eph A receptor enhances the adhesion of cells and the number of focal adhesion points and is known to be i nvolved in forward signaling. Eph receptor B is phosphorylated in the intracellular domain and is known to be capable of both forward and reverse signaling.72 Vascular endothelial (VE) cadherins Endothelial cells express at least th ree cadherins: N-, P and VE cadherin. Ncadherin is diffusely spread across the cell, P cadherin is present in trace amounts and VE cadherin is specifically localized to inter e ndothelial cell junctions. Beta catenine and plakoglobulin are anchored to the cadherin through actin and catinine. VE cadherin mediates contact inhibition of endothelial cells by decreasi ng the amount of proliferation and allows endothelial cell monolayers formation in the vessel wall. VEGF increases endothelial cell permeability by phosphorylation of a tyrosine residue of VE cadherin. This phosphorylation leads to dissociation of the VE cadherin and translocation of the beta catenine/plakoglobulin complex to the nucleus to regulate gene transcritption.73-75

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33 Growth Factors. The process of angiogenesis requires co-ordin ation of several growth factors, which play distinct roles in the pr ocess, examples include: angi opoeitins, vascular endothelial growth factor (VEGF), fibroblast growth fact or (FGF), platelet derived growth factor (PDGF) and transforming growth factor (TGF). Angiopoeitins Angiopoeitins are secreted ligands for the tw o Tie receptors: Tie 1 and Tie2 (Tek). Both receptors are endothelium specific,73 and have an extracellular domain composed of two immunoglobulin like folds a nd three fibronectin repeats. The cytoplasmic region has a tyrosine kinase domain interr upted by a short kinase insert. Angiopoeitin-1 and angipoeitin-2 are ligands for the Tie receptors. Both ligands can bind the receptors, however, onl y ANG1 can phosphorylate the receptor.76 ANG-2 inhibits Tie2, detaches peri cytes and loosens the matrix surrounding the vessel. ANG-1 does not initiate endothelial network organization it stabi lizes networks initiated by VEGF by enhancing the interaction betw een endothelial cells and pericytes Binding of ANG1 to the Tie 2 receptor in itiates cell survival through th e PI3 kinase, Akt pathway. Akt leads to the upregulation of survivi n, which is an apoptosis inhibitor. Phosphorylation of Tie2 leads to the phosphorylation of Dok. Dok then activates the Ras, Nck, and Crk pathway, which are involved in cell migration, proliferation and organization of the cytoskeleton. Molecules interacting with the Tie2 SH2 domains are Grb2, SHP2 which modulate cell growth, differentiation, migration and survival.76 (Figure 1-12)

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34 Figure 1-12. Angiopoeitins are ligand for th e Tie 1 and Tie 2 receptors. Binding of angiopoeitin 1 to the Tie 2 receptor lead s to endothelial cells proliferation, migration and cell survival. Angi opoeitin 2 inhibits Tie 2. ANG1=angiopoeitin 1; ANG-2=angiopoetin 2.

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35 Vascular Endothelial Growth Factor VEGF is a heparan binding potent endot helial cell mitogen. It promotes endothelial cell survival via activation of the phosphatidylinositol 3-ki nase (PI3K/Akt) pathway and inhibits apoptosis.77 VEGF undergoes alterna tive splicing to produce 5 known isoforms: VEGF 121, VEGF 145, VEGF 165, VEGF 189 and VEGF 206.73 The isoforms differ in storage in the ECM and their extracellular pathways.78 VEGF 121 and VEGF 165 are secreted extracellularly, whereas VEGF 189, VEGF 206 and possibly VEGF 165 are either cell or matrix associated due to their affinity for heparan sulfate. VEGF is a mitogen for endothelial cells and each isoform has varying effects during angiogenesis:73 VEGF 189 decreases lumen diameter, 121 and 165 increase lumen diameter and increases vessel length. VE GF 165 binds to the EC M and releases bFGF stored in the ECM, thus, bFGF and VEGF have a synergistic angiogenic effect. 78 There are three tyrosine kinase receptors for VEGF: VEGF R1 (Flt1), VEGF R2 (KDR/Flk-1) and VEGF R3 (Flt3).73 The receptors all have seven immunoglobulin like extracellular domains, a transmembrane domai n and an intracellular tyrosine kinase domain, which is interrupted by a kinase insert.78 VEGFR1 and VEGR2 transduce different signals to endothelia l cells. VEGFR1 promotes cel l migration and VEGFR2 is mitogenic for the endo thelial cells an d also promotes migration. 73 Hypoxia upregulates VEGFR1 and induces the expr ession of VEGF by endothe lial cells. The increased production of VEGF activates the VE GFR2 receptor phosphorylation and cell proliferation. 78 Ligand binding to VEGFR1 leads to the activation of the small adaptor proteins: Fyn, Yes and GAP. Ligand bi nding to VEGFR2, however, leads to phosphorylation of the SHP-1 and SHP-2 ad aptor proteins and PLC-gamma. PLC gamma hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to form inositol

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36 triphosphate (IP3) and diacylglycerol (DAG) The DAG remains associated with the plasma membrane and activates protein kina se C (PKC). PKC is a soluble cytosolic protein, which is activated by the increase in calcium concentration. Activation of PKC leads to cell proliferation and permeability. VEGFR2 also leads to the activation of the PI3 kinase/Akt pathway, which enhances cell survival. VEGFR2 plays a role in cell migration by recruiting FAK. The MAPK path way is also activated through Grb2, which is an SH2 adaptor protein. It has two SH 3 domains, which bind the guanine nucleotide exchange factor SOS. SOS then leads to th e activation of RAS. Activated RAS binds to the N-terminal of RAF which phosphorylat es MEK and phosphoryl ates MAP kinase.78 The activated MAPK pathway then leads to th e activation of the intranuclear proteins such as cyclin D which is important in the progression of the cell cycle from the G1 phase to the S phase.79 (Figure 1-13) VEGF also incr eases vascular permeability and allows leakage of plasma proteins, forma tion of the ECM and upregulates the production of uPA and tPA and PAI-1 by endothelial cells VEGF production is regulated by local oxygen concentrations. Hypoxia upregulates production of VEGF by binding to the hypoxia inducible factor (HIF).73 During retinal development astrocytes and neuronal precursors migrate away from existing blood pre-existing blood vessels. As the distance between the astrocytes and the pre-existing blood vessel increase s, the astrocytes sense a st ate of hypoxia. Astrocytes are more sensitive to hypoxia than neuronal cells and thus the astrocytes upregulate the production of VEGF, which lead s to angiogenesis. This upregulation of VEGF by the astrocytes creates a concentration gradie nt of VEGF. This stimulates blood

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37 Figure 1-13. The vascular endothelial cell growth factor (VEGF R2) signaling pathway. VEGFR2 activates several pathways a ll of which lead to angiogenesis.

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38 vessel formation towards the astrocytes, wh ich produce VEGF. In ROP babies are placed in a high oxygen incubator because th eir lungs are not fully developed. The hyperoxia inhibits the VEGF production by th e astrocytes thus causing newly formed blood vessels to regress. Once the babies are ta ken out of the incubato r all the cells of the retina sense hypoxia and upregul ate the production of VEGF. This leads to abnormal angiogenesis and unregulat ed blood vessel growth.78 Fibroblast Growth Factor Fibroblast growth factor (FGF ) is ubiquitously expresse d as either basic FGF or acidic FGF. FGF is either in the cytopl asm or bound to the ECM due to its intrinsic affinity for heparan.73 FGF binds to four related receptors, which are expressed on many cells. Ligand binding induces receptor dimeri zation. Endogenous hepara n sulfate in cells is required for the activation of FGF. The receptor for FGF has three immunoglobulin like folds; two intracellular tyrosine kinase domains, a short transmembrane region and a juxtamembrane domain, which is lo nger than any other receptor.80 The intracellular domain has two phosphorylation sites.81 Ligand binding to the FGF receptor induces tyrosine phosphorylation of an adaptor mol ecule, FRS2. The phosphorylated FRS2 then allows binding of a small adaptor molecule GR B2. GRB2 is involved in the activation of the GTP binding protein Ras. Since FRS2 doe s not have an SH2 domain, another adaptor molecule, SHP-2 associates with FR S2 alpha in the active FGF receptor.81 The importance of the association of this molecule with the FRS2 is not well defined. GRB2 exists with SOS, which catalyses the exch ange of GDP for GTP on Ras for activation. Therefore, SOS, facilitates the coupling of GRB2 to Ras.81 The activated MAPK then leads to the activation of the intranuclear pr oteins such as cyclin D which progresses the cell from the G1 phase to the S-phase.79 (Figure 1-14) This growth factor induces

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39 processes in endothelial cells and stimulates proliferation and migration of endothelial cells and pericytes, and production of PA by th e endothelial cells. bFGF plays a role in blood vessel remodeling by stimulating endothelia l cells to form tube like structures.73,82 Platelet Derived Growth Factor Platelet derived growth factor (PDGF) is a mitogen for smooth muscle cells73 and potent chemoattractant factor for smooth muscle cells, monocytes and fibroblasts. PDGF is a dimer consisting of two pol ypeptide chains: A and B. These chains combine to form 3 PDGF isoforms of PDGF AA or BB or heterodimers of PDGF AB.73,83 The PDGF receptor consists of a single transmembran e domain which has intrinsic kinase activity.83 The receptor is also a dimeric mixture of the alpha and beta subunits.73 Ligand binding induces receptor dimerization and trans phosphorylates tyrosine residues in the cytoplasmic domain of the receptor.83 Endothelial cells express the beta receptor and are stimulated by PDGF-BB.73,83 PDGF-BB acts through the MAPK/ERK pathway to stimulate c-jun/c-fos related genes in the nucleus to stimulate proliferation.83 PDGF also acts through the PI3kinase path way to activate PKB, which s timulates cell survival and proliferation. PDGF also plays a role in angiogenic chords formation and stimulates sprout formation. PDGF also mediates pr oliferation and migration of pericytes along angiogenic sprouts.73 (Figure 1-15) Transforming Growth FactorTransforming growth factor beta (TGF) is produced by almost all cells and thus its activation represents an important control mechanism.84 TGFis hydrolyzed

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40 Figure 1-14. The FGF receptor and signa ling pathway. Ligand binding to the FGF receptor leads to tyrosine phosphorylation of adaptor molecules and activation of the MAPK pathway. The MAPK pathway leads to endothelial cell proliferation, and migration.

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41 Figure 1-15. The PDGF receptor and signa ling pathway. The PDGF receptor acts through the MAPK pathway to stimulate proliferation and also stimulates endothelial cell survival thr ough the PI3-kinase/PKB pathway.

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42 intracellularly by a furin peptidase to produce the carboxyl terminal peptide.73 This peptide associates with the amino terminal to form the latency associated peptide (LAP). The LAP dimerizes to form the mature TGFwhich is then secreted in the inactive form.73 Plasmin activates the latent complex. TGFalso produces PaI-1 which inhibits plasminogen. Thus, showing that the action of TGFis self limiting.85 There are three different types of TGFreceptors designated, I, II and III. TGFbinds directly to the TGFII receptor. Binding of the II receptor is followed by the recruitment of the TGFI receptor. Both the receptors then form a stable complex and receptor II then phosphorylates receptor I which induces the signal cascade of the receptor.85 Once the TGFR2 is bound to the TGF1 receptor, the kinase activity of receptor 1 is activated. This leads to the recruitment and the accu mulation of the Smad proteins, which are then phosphorylated by th e receptor. The name SMAD is derived from the genes encoding them. The genes we re first identified in drosophila and C. elegans. The drosophila gene was named MAD (mother against decapentapleigic) and the gene from C. elegans was named SMA (small body size).86,87 (Figure 1-16) TGFis a bifunctional regulat or. At low levels, TGFstimulates angiogenesis, and at high levels it inhibits angiogenesis.85 TGFis found in the ECM, on endothelial cells and on pericytes. It supports the anchorage independe nt growth of fibroblasts.73 TGFalso controls cell adhesi on by regulating the production of ECM and integrins. Endothelial cell migration and formation of tube like structures are regulated by TGF. TGFalso upregulates the pr oduction of TIMPS, thus has anti-proteolytic activity. 73 TGFinhibits endothelial cell proliferation73 by blocking the effect of other mitogenic growth factors and enhances pericyte differentiation. It helps to form the vessel

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43 Figure 1-16. The TGFreceptor signaling pathway. Ligand binding to the TGFreceptor II leads to th e recruitment of TGFreceptor I. The activated receptor recruits the Smad proteins and stimulates angiogenesis at low levels and inhibits angiogenesis at high levels of TGF.

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44 wall by stimulating the production of the extrace llular matrix, strengthens the vessel wall and has matrix modulating effects and also stimulates tube assembly.73,87 Angiogenesis is a complex process, whic h involves the extra cellular matrix, bound factors and soluble factors. Of the soluble f actors, VEGF plays an important role in the early phases of angiogenesis. VEGF is an important mediator of compensatory angiogenesis and is a potent mitogen i nduced by hypoxia and nucleosides such as adenosine. 53,88,89 However, even though the angiogenesis process may solve the nourishment aspect of the outer retinal layers if the choriocapillaris was impaired, it would still cause vision impairment.90,90-94 Tissue hypoxia and ischemia initiate a se ries of events which lead to the development of collateral blood vessels, fo llowed by compensatory angiogenesis, which is detrimental and results in aberrant bl ood vessels that are friable and prone to bleeding.92,95 Mediators of compensatory angiogene sis include VEGF, which is a potent mitogen induced by hypoxia and nucleosides such as adenosine.95-97 Depending on the character of the ischemic stimulus, adenosin e plays two roles: as an intracellular signaling factor which promotes neovasc ularization following chronic hypoxia or ischemia, and as an endogenous protective fact or which is capable of protecting the retina from acute ischemia. Adenosine also upreg ulates VEGF in retinal endothelial cells. Therefore, adenosine may be a critical signal in the control of gene expression after retinal ischemia.91,98,99 Adenosine Adenosine is an endogenous nucleoside, which modulates many physiological processes such as cardiac myocyte contra ctility, modulation of neurosecretion and neurotransmission, cell growth and gene expression, regulatio n of intestinal tone and

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45 control of vascular tone.100 Adenosine serves as a signal to increase energy supply and demand by affecting cellular metabolic rate s and tissue perfusi on. Metabolites of adenosine may also have significant physiologica l and pathological effects. The level of adenosine available for these effects is dete rmined by a number of factors including the rate of production, transport and metabolism.100 Stimuli that mediate the local production of adenosine includ e hypoxia, ischemia and inflammation. The endothelium is a barrie r to adenosine, thus the adenosine formed within the lumen of the blood vessels may be derived from nucleotides released from platelets or endothelial cells. Ischemic pare nchymal cells or nucleotides derived from nerves or intestinal mast cells give rise to interstitial ad enosine. This adenosine may produce vasodilation via the A2A receptor on vascular smoot h muscle cells, which are especially accessible to the interstitial nucle oside. Adenosine may also be derived from adenine nucleotides from many cell t ypes by mechanisms which are not well understood.100,101 Since AMP is derived from the breakdown of ATP, adenosine formation is closely linked to the cellular energy state. Adenos ine may be formed intra or extracellularly. (Figure 1-17) The enzyme 5Â’ nucleotidase (5Â’N) cataylzes the metabolism of ATP to adenosine. S-adenosylhomocysteine hydrol ase also catalyses the break down of Sadenosylhomocysteine (SAH) into adenosin e. SAH contributes significantly to adenosine formation in the heart and ischemic conditions in the brain. Once formed, intracellular adenosine is transported out of the cell to exert e ffects on specific cell surface receptors. The transport of adenosine is bidirectional.

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46 Figure 1-17. Intracellular a nd extracellular production of adenosine. SAH hydrolase: Sadenosyl homocysteine hydrolase. 5Â’NT:5Â’nucleotidase. XDH:xanthine dehydrogenase. XO:xanthine oxidase

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47 Ecto 5Â’N catalyses the breakdown of 5Â’AM P to adenosine, thus giving rise to extracellular adenosine. The intracellula r and the extracellula r adenosine can be differentiated from each other using specific inhibitors of ecto 5Â’N.100,101 Adenosine deaminase (ADA) and adenosin e kinase (AK) catalyze the breakdown of adenosine in the cytoplasm. Both ADA a nd AKA are found in the cytoplasm. ADA is a 36 kDa protein which catalyzes the forma tion of inosine. ADA is heterogeneously distributed in tissues and hi ghest activity is during deve lopment. AK is a 38-56 kDa monomeric protein. It is al so widely distributed thr oughout the body. AK catalyses the phosphorylation of adenosine to 5Â’AMP. If the intracellular ade nosine is high, then AK is inhibited.100,101 Five types of adenosine transporters have been classified according to sensitivity to nitrobenzylthioinosone (NBTI), which is an aden osine transport inhibitor. Most of these transporters are sodium dependent and are bidirectional. Following degradation of adenosine, inosine leaves the intracellular environment and forms hypoxanthine. Xanthine dehydrogenase catalyses the oxi dation of hypoxanthine to xanthine and subsequently to uric acid. Conversion of xa nthine to uric acid also reduces NAD to NADH. Xanthine oxidase genetrates supe roxide and hydrogen peroxide, both of which are damaging to cells. Endothelial cells stim ulated by ischemia and reperfusion are key sources of xanthine oxidase formation and activity.100 Adenosine and the Retina Adenosine is heterogeneously distributed th roughout the retina of various species, such as rat, guinea pig, monkey, human and mouse.100 Adenosine immunoreactivity is found in the ganglion cell layer, the inner pl exiform layer and the i nner nuclear layer. 91,102 Under resting conditions, e ndogenous purines in the retin a are in the form of ATP

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48 (70%) and adenosine (2%). During devel opment, the retinal Mller cells provide glycosaminoglycan to the extracellular spaces for angioblasts which provides a scaffold for angioblast migration and organization. In developing and adult mammalian retina Mller cells express 5Â’ nucleotidase (5Â’N) ectoenzyme, a glycoprotein. This enzyme catalyzes the hydrolysis of purine nucle otide monophosphates, to the corresponding nucleoside. The 5Â’NT can metabolize a ll purine monophosphates, however, the major product is adenosine. Adenosine is an in tercellular communication molecule and is a modulator of synaptic transmission in the brai n and the retina, and is a local regulator of blood flow in several organs. In the retina, ad enosine is released in response to ischemia, thereby modulating the blood flow in the a dult and neonates. Adenosine is also chemotactic and a mitogen for endothelial ce lls, and enhances endothelial cell migration and tube formation.102 An increase in the 5Â’NT activit y in cerebral ischemia was shown by Braun et al.103 The pattern of 5Â’NT changes as the retina develops. In the early stages of development, the greatest act ivity of 5Â’NT is found in the inner Mller cell processes. When the inner retinal vasculature reaches completion (about 22 days of age), the inner retina activity of the enzyme decreases and th e activity in the outer retina increases (in both plexiform layers).102 Lutty et al showed that at days 1-5, an increased adenosine immunorectivity is found in the inner retina and the edge of the formed vasculature in the neonatal dog. An increase in the adenosine product shifted toward the ora serrat a as the vascular development progressed radially. On day 8 the 5Â’NT is increased in the inner retina, and on day 15 there is an increase in the adenos ine immunorecativity in the nerve fibre layer and the inner nuclear layer. When the radial progression of the inne r retinal vasculature

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49 is complete on day 22, the 5Â’NT and adenos ine are decreased throughout the nerve fibre layer and increased in the ganglion cell layer, the i nner nuclear layer and the photoreceptor inner segments.102 An increase in adenosine le vels at most ages was found to be proportional to an incr ease in the 5Â’NT activity. In summary, the 5Â’NT activity shifts from the nerve fibre layer to the i nner plexiform layer during development and the adenosine location is also shifted. Thus the Mller cells provide a glycosaminoglycan rich extracellular milieu for angioblast differentiation and also provide adenosine which is a stimulus for blood vessel formation.100,102 Adenosine Receptors Adenosine receptors have been impli cated in mast cell activation, asthma, regulation of cell growth, inte stinal function, neurosecretion modulation and vasodilation. Adenosine receptors modulate cAMP (adenosine 3c, 5c-cyclic monophosphate) intracellulary. Based on their ability to i nhibit or stimulate adenylyl cyclase, the adenosine receptors were initially divided into A1 and A2 subtypes. 100,104,105 The A2 receptor was further divided into 2 subtypes based on the finding of a high affinity A2 receptor in the rat striatum and a low affinity A2R in the brain 106 Both of these receptors activate adenylyl cyclase. The high affinity receptor was designated as A2A and the low affinity receptor was designated A2B.100,107 Adenosine activates four di fferent cell receptors: A1, A2A, A2B and A3. In most cell types, adenosine activates the A1 receptor to lower oxygen demand, and activates the A2 receptors to increase the oxygen supply. Thus the A1 and A2 receptors act to rectify imbalances between oxygen supply and demand.100,108(Figure 1-18)

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50 Figure 1-18. Role of the high and low affinity adenosine receptors. The A2 receptors increase oxygen supply. The A2A receptor leads to vasodilation and the A2B receptors lead to angiogenesis

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51 A1, A2B and A3 adenosine receptors are N-linked glycoproteins, which have sites for palmitoylation near the carboxyl terminus. Glycosylation has no effect on the affinity of ligands for these receptors, thus these si tes may be involved in targeting newly formed receptors to the cell surface. All receptors can be readily deglycosylated upon incubation with glycosidase.101 The molecular pharmacological a nd physiological relevance of the A1, A2A and A3 receptors is well known. However, the A2B receptor is not as well characterized due to a lack of selective pha rmacological probes and because this receptor has a low affinity for adenosine.100 The A1 receptor was initially cloned from rat, human, bovine and rabbit. The A1 receptor has seven transmembrane domains and is 326 amino acids in length and is about 36-37 kDa. Mutations in the H 274 and H 251 region result in loss of agonist and antagonist binding. Chimeric receptor construc ts reveal transmembrane domains 5, 6 and 7 to be important for bind ing. In the brain, the A1 receptors couple to Gi and Gs and inhibit the actions of adenosine. The A1 receptor decreases membrane potential (by increasing K+ and Clconductivity), lowers neurotransmitter release (e.g. glutamate and dopamine) and decreases calcium influx by stimulating calci um mobilization via the pertusis toxin sensitive pathway through the activat ion of PLC beta with G protein / subunit.101 All of these effects of the A1 receptor lead to a decrease in neuronal excitability and metabolism. Thus, the A1 receptor has a neuroprotective role in ischemic tissue.100 The A2A receptor was initially cloned from canine, rat and human and produced responses which are anti-inflammatory.101 It has seven transmembrane domains consisting of 410-412 amino acids and is about 45 kDa (comparable to A1). Mutations in

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52 H 274 and H 251 also lead to loss of agonist and antagonist binding. Adenosine relaxes vascular smooth muscle via the A2 receptor mediated mechanism and thus increases tissue perfusion. In the retina, the vasodilatory eff ects of adenosine are mediated by A2 linked to potassium ATP channels.100,101 Adenosine increases glyconeogenesis via the A2 receptor and thus promotes an increase in the supply and demand ratio for metabolic substrates in the retina. A2A decreases the superoxide release from activated neutrophils and inhibits platelet aggregation. These ar e all anti-inflammatory actions, the importance of which in retinal response to ischemia has not been established. Ideally, a drug that is an A2A agonist and an A2B antagonist is needed to fu rther understand the two receptors.100 The A2B receptor was initially cloned from rat hypothalamus109, human hippocampus110 and mouse mast cells 100 The receptor was found in these tissues by PCR with degenerate DNA oligonucleotides that recognized conserved regions of the G protein coupled receptors. The human, rat and mouse A2B receptors share 86-87% amino acid homology.109 The human A1 and the human A2A, and A2B receptors share 45% amino acid homology.100 Closely related species such as rat and mouse share 96% homology. The A1 receptors have 87% amino acid hom ology in various species (Figure 1-19) 111,112, the A2A receptors have 90% homology (Figure1-20). 113 while the A3 receptors differ significantly between species.111,112 Figure 1-21 shows the homology between the human and the mouse A2B receptor.100

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53 1 50 A1 human (1) VPAMPPSISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCF A1 mouse (1) ---MPPYISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCF 51 100 A1 human (51) IVSLAVADVAVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILA A1 mouse (48) IVSLAVADVAVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILA 101 150 A1 human (101) LLAIAVDRYLRVKIPLRYKMVVTPRRAAVAIAGCWILSFVVGLTPMFGWN A1 mouse (98) LLAIAVDRYLRVKIPLRYKTVVTQRRAAVAIAGCWILSLVVGLTPMFGWN 151 200 A1 human (151) NLSAVERAWAANGSMGEPVIKCEFEKVISMEYMVYFNFFVWVLPPLLLMV A1 mouse (148) NLSEVEQAWIANGSVGEPVIKCEFEKVISMEYMVYFNFFVWVLPPLLLMV 201 250 A1 human (201) LIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALILFLFALSW A1 mouse (198) LIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALILFLFALSW 251 300 A1 human (251) LPLHILNCITLFCPSCHKPSILTYIAIFLTHGNSAMNPIVYAFRIQKFRV A1 mouse (248) LPLHILNCITLFCPTCQKPSILIYIAIFLTHGNSAMNPIVYAFRIHKFRV 301 329 A1 human (301) TFLKIWNDHFRCQPAPPIDEDLPEERPDD A1 mouse (298) TFLKIWNDHFRCQPKPPIEEDIPEEKADD Figure 1-19. Homology of the A1 receptor for human and mous e. The yellow sequences indicate homology between the human and mouse A1 receptor sequences. The white sequences indicate non-homol ogous regions and the blue sequences indicate conserved sequences.

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54 1 50 A2A human (1) --MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSL A2A mouse (1) VASPAMGSSVYIMVELAIAVLAILGNVLVCWAVWINSNLQNVTNFFVVSL 51 100 A2A human (49) AAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAI A2A mouse (51) AAADIAVGVLAIPFAITISTGFCAACHGCLFFACFVLVLTQSSIFSLLAI 101 150 A2A human (99) AIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQ A2A mouse (101) AIDRYIAIRIPLRYNGLVTGMRAKGIIAICWVLSFAIGLTPMLGWNNCSQ 151 200 A2A human (149) PKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYL A2A mouse (151) KDEN--STKTCGEGRVTCLFEDVVPMNYMVYYNFFAFVLLPLLLMLAIYL 201 250 A2A human (199) RIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLP A2A mouse (199) RIFLAARRQLKQMESQPLPGERTRSTLQKEVHAAKSLAIIVGLFALCWLP 251 300 A2A human (249) LHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQT A2A mouse (249) LHIINCFTFFCSTCQHAPPWLMYLAIILSHSNSVVNPFIYAYRIREFRQT 301 350 A2A human (299) FRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWAN A2A mouse (299) FRKIIRTHVLRRQEPFRAGGSSAWALAAHSTEGEQVSLRLNGHPLGVWAN 351 400 A2A human (349) GSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPP A2A mouse (349) GSAPHSGRRPNGYTLGPGGGGSTQGSPG-----DVELLTQEHQEGQ-EHP 401 423 A2A human (399) GLDDPLAQDGAGVS--------A2A mouse (393) GLGDHLAQGRVGTASWSSEFAPS Figure 1-20. Homology of the A2A receptor between human and mouse. The yellow sequences indicate homology betw een the human and mouse A2A receptor sequences. The white sequences in dicate non-homologous regions and the blue sequences indicate conserved sequences.

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55 1 50 A2B human (1) MLLETQDALYVALELVIAALSVAGNVLVCAAVGTANTLQTPTNYFLVSLA A2B mouse (1) MQLETQDALYVALELVIAALAVAGNVLVCAAVGASSALQTPTNYFLVSLA 51 100 A2B human (51) AADVAVGLFAIPFAITISLGFCTDFYGCLFLACFVLVLTQSSIFSLLAVA A2B mouse (51) TADVAVGLFAIPFAITISLGFCTDFHGCLFLACFVLVLTQSSIFSLLAVA 101 150 A2B human (101) VDRYLAICVPLRYKSLVTGTRARGVIAVLWVLAFGIGLTPFLGWNSKDSA A2B mouse (101) VDRYLAIRVPLRYKGLVTGTRARGIIAVLWVLAFGIGLTPFLGWNSKDSA 151 200 A2B human (151) TNNCTEPWDGTTNESCCLVKCLFENVVPMSYMVYFNFFGCVLPPLLIMLV A2B mouse (151) TSNCTELGDGIANKSCCPLTCLFENVVPMSYMVYFNFFGCVLPPLLIMLV 201 250 A2B human (201) IYIKIFLVACRQLQRTELMDHSRTTLQREIHAAKSLAMIVGIFALCWLPV A2B mouse (201) IYIKIFMVACKQLQSMELMDHSRTTLQREIHAAKSLAMIVGIFALCWLPV 251 300 A2B human (251) HAVNCVTLFQPAQGKNKPKWAMNMAILLSHANSVVNPIVYAYRNRDFRYT A2B mouse (251) HAINCITLFHPALAKDKPKWVMNVAILLSHANSVVNPIVYAYRNRDFRYS 301 333 A2B human (301) FHKIISRYLLCQADVKSGNGQAGVQPALGVGLA2B mouse (301) FHKIISRYVLCQAETKGGSGQAGAQSTLSLGLFigure 1-21. Homology of the A2B receptor between the human and the mouse. The yellow sequences indicate homology between the human and mouse A2B receptor sequences. The white sequences indicate non-homologous regions and the blue sequences i ndicate conserved sequences.

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56 The membrane structure of the A2B receptors is that of a typical G protein coupled receptor consisting of a 7 transmembrane dom ains connected via 3 extracellular and 3 intracellular loops. (Figure 1-22)100,110,114 Trans membrane domains have a high de gree of amino acid homology in different species. The human, mouse and rat A2B receptors have 2 potential N-glycosylation sites in the second extracellular loop.109 The human N-linked glycosylation sites are Asp 153 and 163 which are in the second extracellular loo p. Both of these sites are conserved in all of the A2B sequences of all specie s that have been cloned.100,115 The A2A intracellular and the third intracellu lar loop are involved in coupling A2A receptor to G proteins. 100,111 The third intracellular loop is a 15 peptide portion of the A2A receptor which has 57% amino acid homology with the A2B receptor and also determines the selective coupling with GS.100,116 Both A2A and A2B are coupled to Gs. The A2A and A1 receptors have 27% amino acid homology and the A1 is not coupled to Gs. Amino acids in the second intr acellular loop ma y modulate the A2A receptor coupling since lysine and glutamic acid are necessary for efficient A2A adenosine receptor Gs coupling.100,116 Analogous lysine and glutamic acid residues are also present in the A2B receptor. The major difference between the A2A and the A2B receptor is the long intracellular C-terminal tail of the A2A. (Figure 1-23) This long tail is not involved in Gs coupling to the receptor. Removal of the c-terminal tail of the A2A receptor does not inhibit stimulation of adenylyl cyclase wh en truncated receptor is expressed in CHO cell.100,111,116 Mutational studies of the A2A receptors have shown that the Thr 298 residue of the C-terminal tail of the A2A receptor is located close to the seventh membrane

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57 Figure 1-22. The A 2B receptor is a G protein coupled receptor consisting of a seven transmembrane domain connected via 3 ex tracellular and 3 intracellular loops flanked by an extracellular Nterminal and an intracellular C-terminal.

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58 Figure 1-23. The A2A receptor structure consists of 7 transmembrane domains connected via 3 extracellular and 3 intracellula r loops flanked by an extracellular Nterminal and a long intr acellular C-terminal.

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59 span and is essential for the deve lopment of rapid agonist mediated desensitization.100,111,117 The threonine residue is also present in the human A2B (Thr 300), however, its role in receptor desensitization has not been explored. A2B receptors can be coupled to other intracellular signaling pathways in addition to Gs and adenylyl cyclase.100 The A3 receptor was cloned from the human, rat and sheep. It is composed of 320 amino acids and has about 40-50% homology to the A1 and A2 receptors. It has low affinity for alkylxanthine antagonists such as theophylline and caffeine (which is a classic antagonist for A1 and A2). The non specific A3 antagonist IB-MECA inhibits adenyly l cylcase and increase PLC, calcium mobilization and decrease TNF-alpha. Higher concentrations of adenosine are required to activate the A3 receptors than are required to activate the A1 or the A2 receptors.100 Pharmacology of the A2B receptors Highly selective and potent agonists designed for A1, A2A, and A3 receptors are available and are important tools for the char acterization of adenosine receptors. The lack of a potent selective A2B antagonist hampers the characterization of its cellular functions. 95,100 The most potent agonist for A2B is NECA.100,118-120 At a concentration of 2 M, NECA produces half the maximal effect (EC50) for stimulation of adenylyl cyclase.120 NECA is non-selective and thus ac tivates other adenosine receptors with greater affinity. The EC50 for the A1 and A2A receptors is in the low nanomolar range and that of the A3 receptors is in the high nanom olar range. Therefore, the characterization of the A2B receptor depends on the use of compounds, which are potent

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60 selective agonists of other receptor subtypes. Therefore the A2B receptor is usually characterized by exclusion.100 CGS 21680121 is an A2A selective agonist that can differentiate A2A and A2B receptors.100,122-125 The A2A and the A2B receptors are both positively coupled to adenylyl cyclase and are activated by the non selec tive agonist NECA. CGS 21680, on the other hand, is ineffective on A2B receptors and as potent as NECA when activating A2A receptors.100,120-122,126-128 R-PIA is an A1 selective agonist and the A2B receptor has low affinity for it.100,119,120 The pharmacological characte rization of the adenosine receptors is based on apparent agonist potencies. Th is is not ideal as it depends on agonist binding to the receptor and multiple processes of signal transduction. Therefore, for receptor subtype identification, selective an tagonists are preferable 100,129 Highly selective A2B antagonists are not available. However, it is known that A2B has a low affinity for agonists, but a high affinity for antagonists. Enprofylline (3 -n-propylxanthine) is an anti-asthmatic drug, and is the most selective, but not potent, A2B antagonist known. Other potent but nonselective A2B receptor antagonists include 1,3-dipropyl-8 (p-sulfophenyl)xanthine (DPSPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), xanthine amine cogener (XAC) and IPDX 95,119,120 Distribution of the Adenosine Receptors Initially the A2B receptor mRNA was found in the rat. The highest levels of the receptor was found in the cecum, bowel, bladder, followed by the spinal cord, lung, epidydimus, vas deferens and the pituitary.100,130 Subsequently more sensitive RT-PCR showed that the A2B receptors were present in all tissues of the rat, with the highest level in the proximal colon and the lowest level in the liver.131 Primary tissue cultures have

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61 different adenosine receptors present in the cell s. This may be because there are different populations of cells and each cell expresses a different type of adenosine receptor.100,132134 Studies on established cell lines also show ed multiple adenosine receptor subtypes on a single target.100,122,124,125 Also, studies on single cells s how the presence of one or more adenosine receptor subtype.135-137 Clonal cell lines also ha ve co-expression of the A2A and the A2B receptors.100,124 However, subsequent studies showed minute amounts of other receptors too! Therefore it is possible that adenosine selective antagonists are needed to better characterize the distribution of these rece ptors in cells. It is however, unclear why there is simultaneous expression of multiple adenosine receptors in a single cell. Both A1 and A2A receptors have a high affini ty for adenosine and need to be blocked before the effects of the A2B receptor can be seen 100,135,136,138 However, this is not always the case and may be a reason for discrepancies published in the literature. El fman et al showed that glial cells of ra t astrocytes have A1 and A2B adenosine receptors which stimulate cAMP.133,139-141 However, when the cells were st imulated by the non-selective agonist, NECA, cAMP accumulation was se en even though there are A1 receptors present. Therefore it may be that the importance of the A2B receptors is maximal where adenosine receptor levels are high, such as, in tissues with a high metabolic demand or conditions when oxygen is decreased. Both the A1 and the A2 receptors may modulate the response to lower the concentrations of adenosine.100 The widespread unique localization of ade nosine suggests that it is well positioned to serve as mediator of important physiologi cal and pathophysiologi cal processes in the retina. 91,100,142 In the retina, adenosine receptors are lo calized to the same retinal layers as endogenous adenosine. In the mouse a tritiated A1 agonist, cyclohexyladenosine (CHA)

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62 was used to localize the A1 to the inner retina (over the inner plexiform layer) and the A2 receptor was localized to the RPE (outer re tina) and the outer and inner segments of photoreceptors by using tritiated NECA.100,142 No A3 receptor has been found in the retina. The location of adenosine receptor mRNA transcripts generally correlated with the autoradiographic localization of the A1 receptors, but not the A2 receptors.100 Intracellular Pathways Regulated by A 2B Receptors Adenosine receptors activate a diverse cascade of intracellular signaling. The A1 and A3 receptors inhibit adenylyl cyclase and stimulate PLC by activation of pertussis toxin sensitive G proteins Gi and Go.143 Adenosine binding to the A2A and the A2B receptors couples them to Gs and adenylyl cyclase pos itively, however, the A2B receptor is also active in other signaling pathways. The A2B receptor coupled to Gs can also increase calcium transport into the cells by the cholera toxin sensitive pathway. This pathway is cAMP independent even though it is coupled to Gs. 144,100 The A2B receptor is also coupled to G q and leads to the activation of two distinct pathways. One of those pathways lead to the activat ion of the MAPK pathway and the other pathway activates the PI3 kinase/PkB pathway. (Figure 1-24) Angiogenesis is a complex process and is the underlying cause of several retinopathies. Currently avai lable treatments for retinopathie s are painful and have had limited success. Since adenosine exerts its a ngiogenic effects upstream of VEGF, it is an attractive target for inhibiting the process of angiogenesis. However a lack of selective and potent A2B antagonists requires the use of mo lecular techniques to target the A2B receptor. One such approach is the use of ri bozymes to target receptors at the molecular level.

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63 Figure 1-24. The A 2B signaling pathway. The A2B receptor couples to Gs and G q and leads to an increase in calcium transport and also leads to the activation of the MAPK pathway

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64 Ribozymes Ribozymes are catalytic RNA molecules that cleave other RNA molecules. Ribozyme is short for ribonucleotide en zyme, which, catalyze the hydrolysis and phosphopryl exchange at the phosphodiester li nkages between RNA bases resulting in cleavage of the substrate. 145,146 146,147 Ribozymes can be classified into 3 main groups based on function and size: self splicing introns, RNase P, small self cleaving ribozymes. Self Splicing Introns Group I Introns Self splicing introns can be divided into 2 classes: Group I intron and Group II introns, based on the conserved secondary st ructure and splicing mechanisms (Figure 125). Group I introns are found in a wide number of species, such as, eubacteria, bacteriphages, fungal mitochondria, pl ant chloroplasts and rRNA of lower eukaryotes.148,149,150 The splicing action c onsists of two consecutive transphotoesterification reactions. In a tr ansphotoesterification reaction, the number of phosphodiester bonds remain constant, however the position of the bonds changes. (Figure 1-26)150,151 Only the Tetrahymena large rRNA group I intron has been shown to function without a protein in vivo. All ot her known group I introns require a single protein co-factor to provide a scaffold th at helps position the introns in a catalytic conformation.152

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65 Figure 1-25. The secondar y structure group I introns.

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66 Figure 1-26. Splicing mechanism of the group I introns

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67 Group II Introns Group II introns are self sp licing introns found within nuclear pre-mRNA and in the pre-mRNA of organelles from fungi and plants.153 (Figure 1-27) High concentrations of magnesium and potassium ions are essential for their proper folding.153 Group II introns also require a complex of protei ns and small nuclear RNAs (SnRNA) for cleavage. These components form the sp liceocome. Group II spli cing occurs via two consecutive trans photoesterifi cation reactions similar to group I introns. The main difference in the splicing mechanism between the two introns is the nature of the hydroxyl group, which initiates the initial phot otransesterification reaction. In group I introns, the reaction is ini tiated by the 3Â’ hydroxyl group of the exogenous guanosine and in the group II introns, the reac tion is initiated by the 2Â’ hydr oxyl group of the internal adenosine.154 (Figure 1-28) RNase P RNA RNase P is an endoribonuclease which removes the 5Â’ leader sequence from precursor tRNAs. RNase P has an RNA and a pr otein unit, both of which are essential to its function. The RNA component is the ca talytic component of the complex. The protein subunit enhances the turnover rate of the reaction by acting as a scaffold for the RNA that forces the RNA into a catalytic conformation.155,156 RNase P can recognize and cleave 60 different tRNA substrates.157 RNase P recognizes the structure of the tRNA and only a minimal tRNA structure is required fo r the creation of the RNase P cleavage site ( Figure 1-29 ). 158,157

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68 Figure 1-27. Secondary st ructure of Group II introns.

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69 Figure 1-28. The splicing mech anism of the Group II introns.

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70 Figure 1-29. Cleavage of the tRNA 5Â’ leader sequence by Rnase P.

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71 Small Self Cleaving Ribozymes Small Self cleaving ribozymes are nucle olytic RNAÂ’s and are found naturally. They are associated with viruses and sa tellite RNA and can catalyze RNA cleavage reactions in the absence of protein. 159 There are several types of small ribozymes, the most extensively studied ones include: he patitis delta virus (HDV), hammerhead and hairpin ribozymes. The hammer head and ha irpin ribozymes are derived from tobacco ring spot virus satellite RNA. Hepatitis Delta Virus Hepatitis delta virus (HDV) is a short single stranded RNA found in patients infected with human hepatitis B. It ha s a circular RNA genome, which encodes a ribozyme in both orientations. HDV replicat es through a rolling circle mechanism like other self cleaving ribozymes (Figure 1-30), and the ribozyme is required for the cleavage of the HDV genome into discrete units prior to packaging. 160,161 Hairpin Ribozymes The hairpin ribozyme was originally found in the tobacco ring spot virus satellite RNA. The hairpin ribozyme binds the substr ate and forms a structure with 4 helices and 2 loops (Figure 1-31). The arms of the hairpin ribozyme hybridi ze to the substrate molecule to from helix 1 (6 base pair) and helix 4 (4 base pair). Loop A has a BNGUC target sequence required for cleavage, where B is G, C or U, and N is any nucleotide.162 There are no conserved nucleoti des in any of the helices. 163,164 Hammerhead Ribozymes The catalytic domain of the hammerhead ribozyme was discovered by comparing self cleaving RNA sequences of a number of diff erent viroid infectious RNA molecules.

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72 Figure 1-30. Self-cleaving ribozymes resolve concatemers formed by rolling-circle replication into individual genomic molecules

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73 Figure 1-31. Structure of the ha irpin ribozyme. The arrow indi cates the site of cleavage. The hairpin ribozyme binds the substrate and forms a structure with 4 helices (1-4) and 2 loops (A and B).

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74 Hammerhead ribozymes are small, appr oximately 34 base RNA molecules and cleave RNA target in trans. The hammerh ead ribozymes bind substrate to form a structure, which consists of a stem and thr ee loops and a catalytic core with a conserved nine nucleotide sequence (Figure 1-32). A mu tation in any of the conserved nucleotides prevents RNA cleavage.165 The catalytic core of the hammerhead ri bozyme has two functions: it destabilizes the substrate strand by twisting it into a cleavable conformation and binds the metal cofactor needed for catalysis.166 The hammerhead ribozyme cleaves the substrate by a tranesterification reaction (Figure 1-33). The reacti on requires the presence of magnesium and water. The hydrated magnesi um ion has two functions, both mediated by water molecules. First, one molecule of water binds to one of the oxygen atoms of the phosphate group, holding it in the proper orie ntation for the enzymatic mechanism. Secondly, the environment of the active site lo wers the pKa of another water molecule so that it can donate a proton to the aqueous envi ronment. In the transition state, five oxygen atoms are arranged in a triangular bipyramid around the phosphorus atom. A bond is formed between the 2Â’ oxygen of cytosine 17 and the phosphorus atom. Simultaneously a bond is broken between th e phosphorous atom and the hydroxyl oxygen of the next nucleotide, adenine 1.1. This leaves the cytosine with a 2Â’-3Â’ cyclic phosphate group. The 5Â’nucleotide recovers a proton from the aqueous environment, completing a hydroxyl group. The reaction products diffuse away from the active site leaving the ribozyme free to bind a second substrate molecule and complete another reaction cycle The hammerhead ribozyme recognizes substrate sequences on either side of a NUX cleavage site, where N is any nucle otide and X is any nuc leotide except G.

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75 Figure 1-32. Structure of the hammerhead ribozyme. The hammerhead ribozyme binds substrate to form a structure, which c onsists of a stem and three loops and a catalytic core with a conserved nine nuc leotide sequence. Arrow indicates site of cleavage.

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76 A B C D Figure 1-33. The hammerhead ribozyme cleav es its substrate by a transesterification reaction. A A molecule of water binds to an oxygen of the phosphate group. B. Another water molecule donates a proton. A bond is formed between the 2Â’ oxygen of cytosine 17 and the phosphorous atom. C. A bond is broken between the phosphorous atom and th e hydroxyl oxygen of adenine 1.1. D Cytosine remains with a 2Â’3Â’ cyc lic phosphate group. The 5Â’ nucleotide recovers a proton to complete a hydr oxyl group. The reaction products then diffuse away from the active site leav ing the ribozyme fr ee to bind a second substrate molecule and complete another reaction.

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77 The ribozyme anneals to the substrate mR NA by means of two flanking arms which hybridize to form helices III and I. Cleavage oc curs at the 3’ end of the cleavage site. Not all cleavage sites demonstrate the same efficiency. Generally, GUC is the most efficient cleavage site, then CUC, UUC and AUC. The remainder of the cleavage sites are cleaved at least 10 times less efficientl y than the GUC site. The hammerhead and hairpin ribozymes are being examined as gene therapies for a number of different diseases because they are small and can be easily cloned and packaged into many of the existing viral vectors for delivery to target cells. The advantage of the hammerhead ribozyme is that it can recognize a greater num ber of cleavage sites than HDV or hairpin ribozymes.167,168 Experimental Aim Currently, the only available treatment for ROP is laser treatment of the retina, which has limited success. The aim of this pr oject is to design a hammerhead ribozyme that will specifically target and cleave the A2B receptor mRNA resulting in a reduction in expression of the A2B receptor protein and a reduc tion of cellular and physiological functions affected by this receptor. We are using a hammerhead ribozyme primarily as a tool to study the path ways that involve A2B. But this ribozyme can also be used as ‘proof of concept’ for conventio nal drugs targeting the A2B receptor and, finally there is a possibility that the hammerhead ribozyme itself could be used as a therapeutic agent. The goal of this project was to examine the effectiveness of ribozymes in the treatment of ROP. Previously we have s hown that proliferative blood vessels have an enhanced expression of the A2B receptor, therefore, there is justification to target this protein in controlling the disease. Ribozymes were designed to specifically cleave the mRNA of the A2B receptor to decrease the expression of the receptor protein. The

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78 underlying hypothesis was that the cleavage of the mRNA of the A2B receptor at the mRNA level would prevent translation of th e protein and subseque ntly progression of angiogenesis in ROP by preventing the growth of abnormal blood vessels. (Figure 1-34) The selected target site was a s hort region of the mRNA for the A2B receptor. The first step of the project was to design a ri bozyme to cleave the sequence of the A2B receptor in the mouse and the human. (Figure 1-35) Two hammerhead ribozymes were developed, each of which had an inactive version with a single base mutation. (Figure 1-36) The most efficient ribozyme was cloned into an rAAV construct (p21Newhp) for further analysis. (Figure 1-37) The s econd step of the project was to develop in vitro assays to examine the ability of the ribozyme to cleav e the mRNA. These assays were used to determine if the ribozymes would be effectiv e for reducing pre-retinal neovascularization in an oxygen-induced mouse model of retinopathy. To test the ribozyme in vivo, the A2B Rz2 was intravitreally injected to the mouse model. Several models for oxygen-indu ced retinopathy have been developed. Dembinska169 and Chowers170 both used a rat model. The rats were placed in alternating hypoxic and hyperoxic environments. The alterna ting environments lead to severe retinal complications, which were not representative of retinopathy in huma n babies. Since the timing and duration of the hypoxia was inadequate, it gave inconsistent results. In our study we used a mouse model developed by Louis Smith.171 An advantage of using the mouse retina is that in the newborn mouse the retinal vesse l development stage is the same as that of premature human babies. Also, normal retinal vascular development in mice occurs within two weeks of bi rth, thus illustra ting the evolution

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79 Figure 1-34. Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.

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80 Figure 1-35. Target sequences of the human and mouse A2B ribozymes 1 and 2. Red indicates a difference in sequence be tween the human and mouse species.

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81 Figure 1-36. Hammerhead ribozymes for the A 2B Rz1 and Rz2. The target sequences are indicated in red. For each ribozym e an inactive version of the ribozyme was made with a C replaced by the G (arrow).

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82 Figure 1-37. A)The p21Newhp Vector with th e CMV enhancer and beta actin promoter. The hammer head ribozyme was cloned between the HindIII and SpeI sites. B) The hammerhead and the hairpin cleavage sites.

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83 of the vascular bed. Mouse retinal vessels develop from spindle-cell precursors, which are found in the superficial layer of the retina and the deeper retinal vessels develop later. The same developmental pattern is observed in the human retina. Thus the similarities between the mouse retinal vasculature and th at of the human premature babies makes it amenable for use in a model of ROP to better understand the human form of the disease. In the Smith model, seven day old mouse pups are placed in a 75% oxygen chamber for 5 days. Upon return to normal air, these mice developed retinal neovascularization. Five days following retu rn to normoxia (day 17), the animals are sacrificed and the eyes rem oved (Figure 1-38). Smith described two methods to quantify the neovascularization in the mice retina while minimizing the error and making the model applicable to retinopathy.171 First, Smith recommended fluorescein labeled dextran perfusion of the animals into the left ventri cle followed by flat mounting of the retinas. The perfusion technique delin eated the blood vessels, however, it was not permanent as the fluorescein is sensitive to light. Thus, perfusion was recommended for a quick survey of the blood vessels present in the retina following expos ure to hyperoxia. A second method that Smith recommended was counting the vascular nuclei in paraffin cross sections of the retina. Serial cross sections were stained with hematoxylin and eosin and the blood vessels were quantified by counting th e number of vascular cell nuclei on the vitreal side of the inner limiting membrane This method is more time consuming, however, it was reported by Smith to be mo re sensitive, reliable and reproducible.171 This model has also been used by our la b for other studies pertaining to retinal development. For example, Mino et al used this model to examine the effects of various adenosine antagonists on neovascularization. 95

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84 Figure 1-38. Time course for the ROP model. Seven day old pups are placed in a 75% oxygen chamber for 5 days. On day 12, the mice are returned to normal air and their eyes enucleated on day 17.

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85 CHAPTER 2 METHODS AND MATERIALS Defining Location of the Target Sequence It is important to define the region of target mRNA where the ribozyme will bind and cleave. For hammer h ead ribozymes the highest kcat (enzymatic rate of catalysis) has been observed for a GUC ribonucleotide sequence. Thus, it is important to locate all the GUC triplets within the target mRNA. To decide where in the mRNA to target the ribozyme, Genbank was used to obtain sequences for the human A2B receptor and for the mouse A2B receptor. The results were transferred into Vector NTI and all of the GUC sequences identified. All the sites containing the GUC triplet were considered to be potential targ et regions, however, additional criteria were used to narrow thes e potential sites. Target regions with six nucleotides on either side of the arms consis ting of a 50% GC content were selected since an ideal length for the target region is betw een 6-7 nucleotides. The presence of a U/A at the 3Â’ cleavage site also enhances the kcat ten fold. Therefore, a pplication of these criteria reduced the potential number of target sequences to those containing GUCU/GUCA regions. Selected target regions were examined using the RNA folding algorithum designed by Micheal Zucker.172 The folding program was used to fold 00-200 nucleotides on either side of the GUC target to determine whether the ribozyme binding location was acceptable. A blast search was also conducted to en sure that the ribozyme did not cleave another known mRNA sequence within th e known mouse or human sequences.

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86 Preparation of the Target Oligo-Nucleotide. The target oligonucleotide to be used in the cleavage reactions was radioactively labeled at the 5Â’end using T4 polynucleot ide kinase (New England Biochmeicals; Beverly, MA). The reactions were set up as follows: 2 l of the RNA oligo (10pmol/ l, 20pmol total) was added to a mixture contai ning 1ul of 10X polynucleotide kinase buffer (Promega, Madison, WI), 1ul RNASin (Pro mega, Madison, WI), 1ul 0.1M DTT (Sigma, St. Louis, MO), 3 l water, 1 l (gamma 32P) dATP (10uci) (ICN Santa Clara CA) and 1 l of polynucleotide kinase (5 units) (Sig ma, St. Louis, MO). The reaction was incubated at 37 C for 30 minutes. 90 l of TE (Fisher, Swanee, GA) was added to the reaction prior to extraction of the unincorporated nucleotides. A spin column (1ml syringe) was prepared with sterile glass wool and loaded with sephadex (Sigma, St. Louis, MO) saturated in water. The column was centrifuged at 1000 RPM for 5 minutes to remove any excess water and to further pack the sephadex. The 32P labeled target (100 l) was loaded on to the column. The column was sealed with parafilm and centrifuged again at 1000 RP M for 5 minutes. The labeled elute was collected in a 1.5ml Eppendorf tube (Fishe r, Swanee, GA) and was stored at -20 C. Time Course of Cleavage Reactions fo r Mouse and Human Targets (Hammerhead Ribozymes) To evaluate the time course of cleavage of the ribozyme for the target, a cleavage reaction was set up as follows: 13 l of 400mM Tris-HCL (Fisher, Swanee, GA), pH 7.47.5 was added to 1ul ribozyme (2pmol) and 88ul of water. The mixture was incubated at 65 C for 2 minutes and then left at room temperature for 10 minutes. 13 l of a 1:10 ratio of RNASEin:0.1M DTT was added to the reaction mixture along with 13 l of 200mM Magnesium chloride (20mM final) (S igma, St. Louis, MO). The reaction was

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87 then incubated at 37 C for 10 minutes. 1 l of the 32P labeled target (0.2 pmol) and 1 l of the cold target (20pmol total) were prem ixed and added to the reaction mixture at 37C. Time points were take n at 0, 1, 2, 3, 4, 5, 10, 15, and 30 minutes and at 1, 2, and 3 hours and overnight. For each time point, 10 l of the reaction mixture was removed from 37 C and added to a tube containing 10ul of formamide dye mix (90% formamide (Sigma, St. Louis, MO), 50mM ehtylenediam ine tetra acetic acid (EDTA) pH 8 (Fisher, Swanee, GA), 0.05% bromophenol blue (Sigma, St. Louis, MO), and 0.05% xylene cyanol (Sigma, St. Louis, MO). The sample s were initially placed on ice and then heat denatured at 90 C for 3 minutes. The denatured sa mples were cooled on ice before loading 6 l onto a 10% PAGE-8M urea gel to sepa rate the products. Bromophenol blue was run about 2/3 down the gel. The gels were analyzed on a molecular dynamics phosphoimager. Multiple Turnover Kinetics Multiple turnover kinetics were perfor med on the ribozymes. Reactions were performed in a final volume of 20 l. Ribozyme (0.3 picomol, 15nM final) in 40mM Tris –HCL (pH 7.5) was incubated at 65 C for 2 minutes and then incubated at 25 C for 10 minutes. DTT (20mM final) and magnesium chloride (20mM final) and 4 units of RNasin were added. The reac tions were incubated at 37 C for 10 minutes, and cleavage was initiated by the addition of increasing con centrations of the targ et oligonucleotide (0300 picomol; 0-1500 nM final). The reactions were incubated at 37 C for a fixed interval determined in the time course analysis of cleavage. A vari ation on this protocol was incubation at 25 C in 1mM MgCl2. Reactions were terminated by the addition of 20 l of formamide stop buffer and held on ice. The samples were then heat denatured at 95

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88 C for 2 minutes, placed on ice and th e reaction products separated on 10% polyacrylamide-8M urea gels. The gels were analyzed on a molecular dynamics phosphoimager. Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector Two complimentary DNA oligonucleotides (Invitrogen, Carlsbad, CA) were annealed in order to produce a double stranded DNA fragment coding for each hammerhead ribozyme. All DNA oligonucleotid es were synthesized with 5Â’phosphate groups. The DNA oligonucleotides were designed to generate a cut Hi ndIII site at the 5Â’ end and a cut SpeI site at the 3Â’ end afte r annealing. The DNA oligonucleotides were incubated at 65 C for 2 minutes and annealed by slow cooling to room temperature for 30 minutes. The resulting double stranded DNA fragment was ligated into the HindIII and SpeI sites of the rAAV vector pTRUF-21 (UF vector Core, http://www.gtc.ufl.edu/gtc-home.htm). A self cleaving hairpin ribozyme has been cloned downstream of the inserted hammerhead ribozy mes into the SpeI and NsiI sites. This vector has the cytomegalovirus (CMV) beta -actin chimeric enhancer-promoter and results in the hairpin ribozyme cleaving ei ght bases downstream of the 3Â’end of the hammerhead ribozymes. The ligated plasmids were transformed into SURE electroporation competent cells (Stratagene, La Jolla, CA) in order to maintain the integrity of the inverted terminal repeat s. The ribozyme clones were verified by sequencing. Sequencing of the Clones Prior to sequencing the hammerhead inserts, the integrity of the inverted terminal repeats (TRÂ’s) was verified by digestion w ith the restriction en donuclease SmaI. This

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89 digest also served to determine the appr oximate concentration of the plasmid. The hammerhead inserts were verified by sequencing using the Ladderman Dideoxy sequencing kit (TaKaRa Shuzo Co, Japan) ac cording to the manu facturers protocol. Human Retinal Endothelial Cell (HREC) Tissue Culture HREC were isolated from human donor eyes within 24 hours of death. The eyes were placed on a sterile ga uze pad (Johnson and Johnson Me dical Supplies, Arlington, Texas) in a laminar flow hood and washed with 5ml betadine (Courtesy, Shands Hospital, Gainesville, Fl). Sterile s calpels (No. 1, Feather Industr ies limited, Japan) and tweezers were used to dissect the eyes and remove th e neural retina from th e posterior portion of both eyes. The RPE layer was not included in the harvested retina. The retinae were placed on a 53 micron mesh nylon membrane (T etko Inc, Lab Pack, Kansas City, MO) and the remainder of the ocular components discarded. The retinas were washed with phosphate buffered saline (PBS) containing 2% antibiotic/antimycotic mix (ABAM) (Sigma, St. Louis, MO). A sterile wooden sp atula was used to grind the retina over the nylon membrane while washing. The remaining re tinae were aspirated into a sterile 10ml pipette and added to a 20ml Erlenmeyer flask containing 10ml of PBS with antibiotics. Approximately 1mg of collagenase (342 u/m g, Worthington Biomedical Corporation, Lakewood, NJ) was added to the flask and placed in a 37 C water bath for 15 minutes. The flask contents were mixed every 5 minut es to keep the collagenase dissolved. Following the 15 minute incubation in the 37 C water bath, 20ml of complete endothelial cell media was a dded to the flask. (250ml DulbelcoÂ’s Modified Eagle Medium (DMEM) low glucose, 250 ml HAMÂ’s F12, 10% fetal bovine serum, 15% endothelial cell growth supplement, 15% insu lin/transferring/selen ium, 2% L-glutamic

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90 acid, 2% antibiotic/antimycotic mix). The cells were washed twice with media and plated onto a T25 flask (Fisher, Springfiel d, NJ) coated with 1% gelatin (Sigma, St. Louis, MO). The cells were allowed to gr ow and attach for 48-72 hours before changing the media and adding fresh antibiotics. Once th e T25 flask was confluent, the cells were passed and split into T75 flasks using 5ml of trypsin EDTA solution for endothelial cells (Sigma, St. Louis, MO). For passing the cells the cells were washed twice with PBS and then 5ml of trypsin added. Th e flask was placed into the CO2 incubator for 45 seconds. The trypsin was neutralized usi ng 2x volume of the complete endothelial cell media. The cells were centrifuged at 1000 RPM in an Eppendorf CT 5810R. The pellet was resuspended in 6ml of complete endothelial cell media and plated in a T75 flask (Fisher, Swanee, GA) containing 15ml of complete endothelial ce ll growth media and fresh antibiotics. LDL Uptake of the HREC HREC were seeded in a 6 well tissue cultu re plate (Corning Incorporated, Corning, NY) and allowed to attach and grow to a bout 80-85% confluency. Morphology of the HRECs was observed and recorded using a Ca rl Zeiss Microscope (Zeiss, Goettingen, Germany). The HREC were washed twice with Hanks Balanced salt solution (HBSS) (Sigma, St. Louis, MO). 50 g/ml of Dil labeled (1,1Â’-di octdycl-3,3,3Â’,3Â’-tetramethylindocarbocyanin perchlorate) acetylated LDL (Molecular probes, Eugene, OR) was added to the cells overnight in serum free me dium. The cells were then washed with HBSS and examined by fluorescence microscopy for the uptake of the label. The cells were quantified as a percentage of labeled cells/total cells.

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91 Transfection of HREC using DEAE-Dextran HREC were transfected at 70% confluency on an 150 mm tissue culture dish (BioRad, Hercules, CA). The cells were washed twice with PBS (Biowhittaker, Walkersville, Maryland) and 10.5ml of 10% Nuserum (B io-Rad, Bedford, MA) was added to the culture dish. (10% Nuserum has all the ingr edients of endothelial cell complete media except for the fetal bovine serum). 10 g of DNA in Tris buffered saline (TBS) (BioRad, Hercules, CA) (total volume of 108 l) was prepared in an eppendorf tube. 216 ul of DEAE dextran (diethylami noethyl-dextran) (Sigma, St. Louis, MO) (10mg/ml) were added dropwise to the tube while co nstantly mixing on a vortex mixer. 324 l of this mixture was added to the 150mm tissue cultur e dish containing 10.5ml of 10% Nuserum. The mixture was added dropwise across the pl ate and the plate swirled to distribute completely. Immediately, 8.1 l of chloroquine (100 mM) (Sigma, St. Louis, MO) was added to the plate and swirled. Chloroquine prevents the lysosm es from releasing DNAses that would destroy the DNA. The plates were incubated at 37 C for 4 hours in a CO2 incubator and the plates were mixed ever y 15 minutes to ensure even distribution of the plasmid. After incubation, the cells were treated with 15ml of 10% dimethly sulfoxide (DMSO) (Sigma, St. Louis, MO) in PBS for 1 minute. The cells were then washed twice with PBS. 20ml of complete e ndothelial cell media was added to the plates and the plates held in a CO2 incubator at 37 C. Media with fresh antibiotics was replaced after 24 hours. The cells were then allowed to grow for about 48 hours following replacement of media before harv esting the cells for further analysis.

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92 Transfection Efficiency using DEAE Dextran for HRECÂ’s To determine the efficiency of HREC transfection, HREC we re transfected as described above, using a plasmid expressing GFP (PTRUF11) obtained from the Vector Core at the University of Florida. The level of GFP expression was observed using a Carl Zeiss fluorescent microscope (Zeiss, Goettingen, Germany). Cell Migration Assay HREC were grown in 150mm tissue culture dishes and transfected using the DEAE dextran method. The HREC were washed twice with PBS. 7ml of trypsin was added to the culture dish and incubated at 37 C in a CO2 incubator for 45 seconds. The cells were observed during this period to ensure maximal detachment of the cells. The trypsin was neutralized using 14ml of complete endothelial cell media containing fetal bovine serum. The cells were then centrifuged at 1000 RPM for 5 minute. The supernatant was discarded and the cells washed three times with basal media. The number of cells was determined with a hemacytometer (Hausser Scientific Horsham, PA). The cells were suspended in 50 l of PBS to a final concentration of 10,000 cell/ l. 30ul of the resuspended cells were loaded into the wells of a Chemotaxis Chamber (Neuro Probe Inc, Gaithersburg, MD ). The wells were covered with a 12uM porous polycarbonate membrane (Neuro Probe Inc, Gaithersburg, MD) pre-coated with 10% bovine collagen. The chemotaxis chamber was then sealed and placed upside down in a 37 C CO2 incubator. This allowed the cells to attach to the lower side of the 12 M porous membrane. After 4 hours, the chamber was removed, and the cells stimulated with 1, 10 or 100 M concentrations of NECA (dissolv ed in endothelial cell basal media (serum free). 50ul of the positive control (c omplete endothelial cell media), negative

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93 control (endothelial cell basa l media), or NECA concentratio ns were added to the top of the chamber. The chamber was then incubated at 37 C in a CO2 incubator for 12 hours, to allow the attached cells to migrate thr ough the porous membrane. The chamber was disassembled and the porous membrane removed from the chamber. The dull side of the membrane (c ontaining all the attached cells) was scraped using a cell scraper. The membrane was then stained using a diff Quik R stain set (Dade Behring Inc, Newark, DE) and mounted onto a gl ass slide. The migrated cell nuclei per well were counted per high power field. Morphology of HEK Cells HEK 293 cells were obtained from Amer ican Type Culture Collection (ATCC) (Manassas, VA) and plated onto a 150 mm tissu e culture dish. The cells were allowed to attach and grow to about 80% confluency. To ensure a homogenous population of HEK cells, the morphology of the HEK cells was r ecorded using a Zeiss microscope (Zeiss, Goettingen, Germany). Transfection using Lipofectamine on HEK 293 cells HEK 293 cells (ATCC, Manassas, VA) were seeded onto a 150mm tissue culture dish and allowed to attach and grow to 85-90% confluency. The HEK cells were fed with 1X high glucose DubellcoÂ’s modified Eagle medium (Gibco, Carlsbad, CA) containing 2% fetal bovine serum and 2% ABAM Prior to transfection, the media was removed and 20.1 ml of high glucose DMEM w ith 2% FBS was added to the cultured cells. The antibiotics were not added fo r the duration of th e transfection assay. 33.5g of DNA was dissolved in 2,010 l of optimem I reduced serum media (Gibco, Carlsbad, CA) (final volume). 134ul of lipofectamine 2000 reagent (Invitrogen,

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94 Carlsbad, CA) was also diluted in 2,010 l of optimem (final volume) and incubated at room temperature for 5 minutes. The d iluted DNA was then added to the diluted lipofectamine reagent and incubated at room temperature for a further 20 minutes. The entire mixture was then added to the cultu red HEK 293 cells and gently swirled. The cells were incubated at 37 C in a CO2 incubator. After 24 hours, the media was changed and the antibiotics replaced. Transfection Efficiency for HEK Cells using Lipofectamine Reagent The HEK cells were transfected as ab ove with the plasmid pTRUF-11 which expresses GFP. GFP expression was determin ed using a fluorescence microscope at: 6hr, 24 hr, 48hr, 72 hr, 96hr, P1 and P2. cAMP Assay on Transfected HEK 293 Cells HEK cells transfected with the plasmids expressing either the active or inactive ribozymes, or the control plasmid were ha rvested for the cAMP assay. A Bradford protein assay (Bio-Rad) was used to determin e the amount of protei n in the transfected cells. A stock solution of 1mg/ml of BSA (S igma, St. Louis, MO) was used to prepare the standard curve. 100ul final volum e of protein standards containing 50 g, 100 g, 200 g and 300 g of BSA were prepared in water. The unknown samples were diluted 1:10 with water to a final volume of 100 l. 2ml of protein assay dye reagent (Bio-Rad, Hercules, CA) diluted 1:5 with water wa s added to all the standards and unknown samples. The absorbance of standards and samples at 595nm was determined using a Beckman spectrophotometer. The concentration of protei n in the unknown samples was determined by comparison to the linear regressi on plot of the standard curve. Based on the protein concentration in the unknown samp les, the unknowns were diluted to a final

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95 concentration of 1.5mg/ml with Hanks balanc ed salt solution (HBSS) (Gibco, Carlsbad, CA.). Unknown samples were diluted immediat ely prior to the addi tion of the adenosine agonist. For each transfected dish, a total of 16 Eppendorf tubes were set up and the cells stimulated with NECA. Basal levels were determined in duplicate in the absence of NECA. Seven concentrations of NECA were prepared from a 1x10-2 stock solution (1x10-4, 5x10-5, 1x10-5, 5x10-6, 1x10-6, 1x10-7, 1x10-8 (final concentrations in HBSS). Each concentration of NECA was done in duplicate. Tubes cont ained 150ul of HBSS and 50ul of cells (1.5mg/ml). Cells were pre-warmed in a 37 C water bath for 5 minutes. Following the incubation, 50ul of a phosphodiestera se inhibitor (10 M roliprom (Sigma, RBI, St. Louis, MO) (100 mM final concentration ) was added to all the tubes including the basal tubes. 5ul of the NECA concentra tions were added in duplicate to all 16 tubes. No NECA was added to the basal tubes. The tubes were incubated for a further 10 minutes at 36 C water bath. To end the reacti on, all tubes were placed in a boiling water bath for minutes. The tubes were cooled to room temperature and centrifuged at 2000 rpm for 2 minutes. The s upernatants were then assayed for cAMP content. A cAMP standard curve ranging from 1 nm ol to 25 pmol was prepared using a stock solution of 1x10-3 M cAMP (Sigma, St Louis, MO). Each unknown was assayed in duplicate. Additional tubes used to dete rmine total binding and non-specific binding were also included. The resultant cAMP sta ndard curve ranged from the non-specific to the total binding and was a sigmoidal curve. The samples were expected to fall within the linear range of the sigmoidal curve.

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96 For samples, 50 l of the supernatant was added to a glass test tube. To the nonspecific tube, 50 l of the highest cAMP concentration was added. 50 l of (125I) ScAMP (adenosine 3Â’5Â’ cyclic phos phoric acid 2Â’) -succinyl (125I) iodotyrosine methyl ester, courtesy Dr. John Shyrock, Dept of Pharm acology and Therapeutics, University of Florida, Gainesville) was added to all the tubes including the cAMP standard curve. 50 l of the cAMP antibody (Accurate Chemical Company, Westburg, NY) was also added to all tubes including the standard curve. The tubes were then covered with parafilm and incubated at 4 C overnight for at least 12 hours. Next, 75 l of a hydroxyapatite solution (Biome dical Research and Development Laboratories), diluted 1:3 with water, was added to the tubes. The cAMP and the antibody bound to the hydroxyapatitie. The t ubes were then washed with cold 10mM Tris-HCl pH 7.0 using a cell ha rvester (Brandell, Gathersburg, Maryland). A glass fiber filter, grade No.32 (pore size 2.3 um) (Schleicher and Schuell, Keena, NH) was used to trap the complexes bound to the hydroxyapatite. The individual filters were then placed in a Beckman G5500 counter which measured the 125I emissions in a one minute window. The results were analyzed with the statisti cal analysis and Graphics software, Prism (Graphpad software, San Diego, CA) was used. The unknown sample concentrations were determined from the standard curve as pmole per sample of cAMP accumulated. The cAMP per mg of protein was determined. Basal values were subtracted from the NECA stimulated tubes to obt ain a net response of NECA. Based on these values, a dose response graph was plotted. Maximum binding was determined by the (125I) ScAMP bound in the absence of additional non-radi oactive cAMP. Non-specific binding was

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97 determined by the addition of 500 pmoles cA MP and was subtracted from all the other readings before plotting the standard curve. By subtracting non specific binding from the total binding, specific binding for the assay wa s determined. The percent inhibition of (125 I) ScAMP binding to the antibody by additi onal cAMP (1nmol to 25 pmoles) was graphed. As the cAMP c oncentraion increased, less 125 I was detected. An EC 50 for the effect of NECA was also calcu lated. EC 50 is the effective concentration that displays a 50% response in cAMP production. Total Retinal RNA Extraction for PCR Total retinal RNA was isolated from HEK 293 cells using TRizol LS reagent (Invitrogen, Carlsbad, CA) followi ng the manufacturers protocol. Real Time PCR The cDNA was synthesized using either 2 or 4 g of total RNA and TaqMan R reverse transcription reagents (PE App lied Biosystems, Foster City, CA) in 100 l RT reactions. TaqMan R real time PCR analysis was app lied using 1ul cDNA per reaction and SYBR R Green PCR core reagents on AB I prism sequence detection system 5700 (PE Applied Biosystem, Foster City, CA). In each experiment, a standard curve for each primer pair was obtained using a serial dilution of total RNA samples prepared from cells that over expressed A2B adenosine receptor. At the end of the PCR cycle, a dissociation curve was generated to ensure the amplific ation of a single product and the threshold cycle time (ct values) for each gene was determined. Relative mRNA levels were calculated based on the ct values and normalized to a house-keeping gene: cyclophilin (100%).

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98 Animals All animals were treated in accordance w ith the IACUC of the University of Florida and the ARVO statement for the use of animals in ophthalmic and vision research. All animal protocols were approve d by the IACUC at the University of Florida prior to experimentation. C5BL6/J pregna nt mice on gestation day 14 were obtained from Jackson Laboratory (Bar harbor, ME). The mice were housed in the University of Florida Health Science Animal Resources facili ties. A Total of 24 animals were used (8 animals per plasmid) Animals were sacrificed by injecting a lethal dose of ketamine followed by spinal dislocation. Intraocular Injection into the Mouse Model of Oxygen Induced Retinopathy One day following birth, the mouse pups r eceived a 0.5ul intrav itreal injection of plasmid (2mg/ml) OD (right eye). In th e neonatal mouse model of oxygen induced retinopathy, 7 day old mice were placed with their nursing dams in a 75% oxygen atmosphere for 5 days. The oxygen chamber was monitored with an oxygen sensor within the closed chamber. The chamber oxyge n level was maintained for 5 days at 75%. After the fifth day, the oxygen chamber was sl owly returned to 21% oxygen over a period of one hour. The animals were removed from the oxygen chamber and placed in clean bedding. Upon return to normal air, these mice developed retinal neovascularization, with peak development occurring 5 days after their return to normoxia. After the fifth day following return to normoxia (day 17), the animals were sacrificed and the eyes removed and fixed in 4% paraformaldehyde and embedded in paraffin. Three hundred serial sections (6 m) were cut sagitally through the co rnea parallel to the optic disc. Every thirtieth section was placed on slid es and stained with hematoxylin and eosin (H&E). This resulted in te n sections from each eye being scored in a masked fashion

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99 using light microscopy by c ounting endothelial cell nucl ei extending beyond the inner limiting membrane into the vitreous. The e fficacy of treatment with each plasmid was then calculated as the percent average nuclei pe r section in the injected eye versus the uninjected control. Statistical Analysis. Student T-test was used to evaluate the da ta generated for all the experiments. A p value of less than 0.05 was considered to be significant.

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100 CHAPTER 3 RESULTS The expression of the A2B receptor upregulates VEGF expression which in turn leads to angiogenesis. The hypothesis underl ying this research project was that a decrease in the A2B receptor expression may prevent or decrease the severity of the angiogenesis. In this study, two differen t ribozymes were desi gned to cleave the A2B receptor mRNA. Two corresponding inactive versions of the ribozyme were also made. Determining Accessibility of the Target Site Success of ribozyme therapy depends on the id entification of an RNA target site. The m-fold program analysis was used to determine if the chos en target would be accessible to the ribozyme for binding. Figure 31 shows the theoretical tertiary structure of the active A2B Rz1 and Figure 3-2 shows the active A2B Rz2 mRNA under cellular conditions. Ribozymes generally fold into one of four secondary structure types. Type A, B, C and D. Type A structures have the highest activity based on the delta G values. The targeting arms in the type A structure do not interact to form a secondary structure and thus are readily available for target bindi ng. Types B and C compete with the type A structure and reduce the activity of the ri bozyme. However, these structures are catalytically active in vitro and should thus be effective in vivo. Ribozymes that form

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101 Figure 3-1. Theoretical tertiary structures of the active A2B Rz1 generated by the mfold program.

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102 Figure 3-2. Theoretical tertiary structures of the active A2B Rz2 generated by the mfold program.

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103 structures which are significantly more stable than the type A, such as type D, can completely inhibit the ribozymes catalytic ac tivity and should not be considered for in vitro and in vivo studies. The m-fold analysis showed that the A2B Rz1 could fold into two structures, types A and C, with dGÂ’s of -7.6 and 7.0 kcal/moles respectively. The A2B Rz2 analysis by mfold produced three structures types A, B and C with dGÂ’s of -7.6, -7.0 and -6.9 kcal/mol respectively. Based on this analysis both of the A2B ribozymes should be effective in vitro, and, therefore we deci ded to test both ribozymes. Time Course of Ribozyme cleavage Time course of cleavage analysis was done for the A2B Rz1 and the A2B Rz 2 as described in the Methods section. Figur e 3-3 is an autoradiograph from a 10% polyacrylamide-8M urea gel used to sepa rate the products of cleavage of the A2B Rz2 on the mouse target for reactions performed at 37 C and at 20 mM MgCl2. The autoradiograph shows an increase in the 5Â’cleaved product over tim e and a corresponding decrease in the target. Significant produc t accumulation was found at 1 minute after the addition of the target to the ribozyme. Fi gure 3-4 shows the graphical representation of the data in Figure 3-3 in addition to the data for the A2B Rz1 on the mouse target. The A2B Rz2 appears to have the hi gher rate of cleavage. Kinetic analysis on the hammerhead riboz ymes is performed at the time point where no more than 15% of the target has been cleaved. We select this point because the ribozyme cleavage rate is at a maximum and is linear at this point and the ribozyme is saturated by the target. Figur e 42 shows that this time point is less than one minute for the A2B Rz2. Since we do our kinetic analysis manually, as opposed to using a flow

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104 Figure 3-3. Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing products of cleavage of the A2B Rz2 on the mouse target. The autoradiogarph shows an increase in the 5Â’ cleava ge product over time and a corresponding decrease in target.

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105 0.0 0.2 0.4 0.6 0.8 1.0 020406080100120 Time (minutes)Fraction of Target Cleaved A2B Rz1 A2B Rz2 Figure 3-4 Time course analys is data. Graphical representation of the cleavage for the A2B Rz1 and A2B Rz2 appears to have a higher rate of cleavag e with 15% of the target being cleaved in less than one minute.

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106 cytometer, we decided to reduce the rate of cleavage of both ribozymes in order to find a time point where manual kinetic analysis could be easily completed. Time course reactions were performed with increasing concentrations of the target (1:10, 1:40, 1:66, 1:100; Rz:Tar). (Figure 3-5) Increasing the target concentrations also did not slow the ribozyme down enough to asse ss the time point at which 15% of the cleavage occurs. Next the temperature and Mg Cl2 concentration were varied for the time course of cleavage reactions. Figure 3-6 shows the results of this analysis for the A2B Rz2 which yielded a time point of about 1 minute where the amount of target was reduced by 15%. Based on this type of analys is we did kinetic analysis on the A2B Rz1 at 37 C at 20mM MgCl2 at a time point of 6 minutes, and on the A2B Rz2 at 25 C at 1 mM MgCl2 at a time point of 1 minute. Multiple Turnover Kinetics To ensure cleavage of the RNA substr ate in vivo, it is important to design ribozymes with the highest possible catalytic activity and therefore this turnover number (kcat) and the Michaelis constant (Km) of the ribozymes were determined. This analysis relies on several assumptions: first, measuremen t of the initial rate of the reaction ensures that changes in the formation of the product and depletion of substr ate do not affect the rate of reaction. Conventionally, kinetic m easurements are made when no more than 15% of the substrate is converted to the products. A second assumption is that the concentration of the ribozyme is lower than the Km. A low ratio of the ribozyme to target ensures that the

PAGE 125

107 0 0.2 0.4 0.6 0.8 1 0102030405060 Time (minutes)Fraction of Target Cleaved 1:10 (Rz:Tar) 1:40 (Rz:Tar) 1:66(Rz:Tar) 1:100 (Rz:Tar) Figure 3-5. Time course of the ribozyme with increasing target concentrations.

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108 Figure 3-6. Time course cleavage reaction wi th varying temperatur es (37 C/25C) and magnesium concentrations of 20mM/1mM. 0.00 0.20 0.40 0.60 0.80 1.00 0246810 Time (minutes)Fraction of Target Cleaved 37/20 25/1

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109 velocity of the reaction is pr oportional to the concentration of the ribozyme thus allowing the turnover rate of the reaction to be determine d. The initial rate (v0) is determined by dividing the concentration of the reaction product over time. A liner regression curve is then plotted of 1/v vs 1/s and the kinetic para meters (Figure 3-7) can thus be determined from the equation for the line generated from the data (1/VMAX=abs/y at X=0, 1Km= abs/X at y=0). Each analysis was performe d a minimum of 3 times. The reactions were done at 37C and terminated at 6 minutes for the A2B Rz1 and at 1 minute for the A2B Rz2. At 37 C and 20 mM MgCl2, the A2B Rz1 has a Vmax of 27.3 nM/min, km of 8.3 M and a kcat of 1.8/min. Under the same conditions, the A2B Rz2 had a Vmax of 515 nM/min, Km of 4.3 M and a kcat of 36.1/min. At 25 C and 1mM MgCl2, the A2B Rz2 had a Vmax of 16.9 M/min, a km of 14.4 M, and a kcat of 1.1/min. At 37 C and 20 mM MgCl2, the A2B Rz2 had the highest catalytic activity of the two ribozymes and based on these results we dropped the A2B Rz1 and only cloned the A2B Rz2 for further testing. Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector Ribozymes injected directly into an in vivo model are suscepti ble to endonuclease attack. Modification of the ribozyme by a dding phosphorothioate groups would protect the ribozyme from the endonucleases. Howe ver, modifications such as these are expensive and potentially toxic. Therefore, the A2B Rz2 was cloned into an rAAV vector, p21Newhp. These vectors are suitable for injectio n of the plasmid directly into an in vivo model and for the HREC transfection. This ve ctor allows us the option to package into AAV for subsequent experiment s in vitro and in vivo.

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110 Figure 3-7. Kinetic anal ysis of the ribozymes.

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111 Sequencing of the Clones Once the A2B Rz2 was cloned into the rAAV vect or, it was sequenced to confirm the sequences of the active A2B Rz2 ribozyme, inactive Rz2 and the p21Newhp vector alone. Figure 3-8 shows the sequencing resu lts. The C/G base difference between the active and the inactive ribozyme are indicated. Cell Cultures Human retinal endothelial cells (HREC) were grown and assesed for purity of culture. A special mesh pore nylon membrane was used to isolate these cells. This membrane retains the retinal endothelial cells but allows passage of the retinal components. Morphologically, the HREC usually have a ‘pebble stone’ morphology (Figure 3-9). The pebble stone morphology is not always easily distinguishable. If the culture flask is coated with 1% gelatin, it help s the cells to adhere to the culture flask and display their morphological charac teristics. They have a rounded nucleus which fills up most of the cell and a scanty cytoplasm. It is not always possibl e to have a 100% pure culture of the retinal endothel ial cells, and the presence of some pericytes is not unusual. To confirm the purity of the HREC the abil ity of the endothelial cells to take up acetylated LDL was assesed. The human LDL complex delivers cholesterol to the cells by receptor mediated endocytosis. The complex consists of a core of ester and triglycerides surrounded by a thic k shell of phospholip ids, unesterified cholesterol and an apoprotein B unit. Once internalized, LDL disso ciates from its receptors and appears in lysosomes. The lysine residues of LDL’ s apoprotein B can be acetylated to form acetylated LDL. Once acetylated, the LDL comp lex no longer binds to the LDL receptor

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112 Figure 3-8. Sequence of the active and inactive versions of the A2B ribozymes at the site of insertion within the p 21NewHp vector. The G to C change in the catalytic core at position 15 in the upper panel is also noted on the 6% polyacrylamide8M urea gel in the lower panel. The yellow sequences indicate the HindIII and SpeI sites. The blue sequences indicate the target. 1 56 10 20 30 40 (1) A A G C T T G G C A T A C T G A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G Tp21A2Bactive(1) A A G C T T G G C A T A C T C A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G Tp21A2Binactive(1) A A G C T T G C A T G C C T G C A G A C T A G Tp21NewHp(1) A A G C T T G G C A T A C T A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G TConsensus(1) p21A2Bactive p21A2Binactivep21NewHp

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113 Figure 3-9. Pebble stone morphology of the HREC

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114 and is taken up by scavenger re ceptors specific for modified LDL. Endothelial cells and macrophages have these scavenger recep tors. The acetylated LDL complexes accumulate within the cells diffusely in the cytoplasm (Figure 3-10). To asses the purity of the culture, the number of cells that take up the LDL are expressed as a percentage of the total cells. Transfection of HREC The HREC were transfected with the ribozyme constructs to asses the ribozymes ability to cleave the target mRNA. HREC we re initially transfected with a rAAV vector PTRUF11. This plasmid is similar to th e p21NewHp, and, it expresses GFP downstream of a beta-actin CMV promoter (Figure 3-11). HR EC were transfected with this plasmid to determine the transfection efficiency using th e DEAE dextran protocol. The cells were transfected and observed under a fluorescent microscope for expression of GFP in the HREC over time. The number of cells expressi ng GFP increased with time as expected. However, the maximal expression of GFP wa s not seen until 3 w eeks post transfection. (Figure 3-12). Once the efficiency of the protocol was established, HREC were transfected with the active and inactive versions of the A2B Rz2, and the vector c ontrol (p21Newhp) using the DEAE dextran protocol. Following transf ection, the cells were allowed to grow for 72 hours. After 72 hours, the cells were not able to sustain normal morphology or characteristics of HRECs. Thus, the cells were harvested at 72 hours post transfection for further analysis.

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115 Figure 3-10. LDL uptake of HREC.

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116 Figure 3-11. The GFP plasmid. This plas mid was driven by a CMV enhancer and a chicken beta actin promoter pTR-UF117200 bp ApR ColE1 ori f1(+) origin TR TR GFPh neoR PYF441 enhancer CMV ie enhancer Intron SV40 poly(A) bGH poly(A) HSV-tk Chiken b-actin promot e Exon1 Chicken -actin p romote r

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117 Figure 3-12. Transfection efficiency of the HREC. A)24hrs. B)48hrs. C)72hrs. D)96hrs. E)3 weeks and F)Passage 1 post transfection.

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118 A migration assay was used to assess the levels of the A2B receptor in the transfected HRECs. For a migration assay, ce lls are placed in a chemotaxis chamber and covered with a porous membrane and allowed to attach to the lower side of this membrane. A stimulus is provided to the ce lls. The cells respond to the stimulus and migrate through the pores of the membrane. For this experiment, we placed the transfected cells into the wells of the chemot axis chamber and stimulated the cells with increasing concentrations of NECA. NE CA is an adenosine analogue and cells transfected with the active A2B Rz2 were not expected to re spond to NECA as readily as the inactive transfected, or the vector transf ected cells. The cells transfected with the active A2B Rz2 are expected to have the A2B receptor mRNA cleaved and thus have a decreased dose-dependent response to NECA th an the other cells. (Figure 3-13) Cells transfected with the pl asmid coding for active A2B Rz 2 reduced migration of cells by an average of 39% when compared to the p21NewHp control at increasi ng concentrations of NECA (10 and 100ng/ml), and cells transfected with plasmid coding for the inactive A2B Rz2 reduced migration of cells by an average of 15%. (Figure 3-14) Transfection Using Lipofectmaine on HEK Cells Human embryonic kidney cells (HEK) 293 cells were also transfected with the same plasmids for further in vitro analysis. Since these cells were transfected using a different protocol than the HREC the transf ection efficiency had to be determined for these cells also. To assess the transfection efficiency, the HEK cells were transfected with the GFP plasmid and the cells observed for expr ession of GFP under the fluorescence

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119 Figure 3-13. Theory of migration assay. Cells are plated into a well and covered with a porous membrane. Cells migrate through the membrane in response to a stimulus (eg. NECA). When A2B Rz2 transfected cells ar e placed in the well, fewer cells respond to the same stimulus.

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120 0 20 40 60 80 100 120 140 160 020406080100 NECA ( M)Average number of cells Active Inactive Control 10% FBS/DMEM DMEM Figure 3-14. Migration data fo r the cells transfected with th e active and inactive versions of the A2B receptor and the vector control. 10% FBS/DMEM is the positive control and DMEM alone is the negative control

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121 microscope. In this case, expression of th e GFP plasmid was observed within 24 hours of transfection. The number of ce lls expressing the GFP increased with time. (Figure 3-15) After 96 hours post-transfection, the cells were passaged to assess the level of expression. The HEK cells continued to e xpress GFP following passage P 1. (Figure 3-16) The level of expression of GFP also continued to increas e with time. Ninety six hours after the first passage, the cells were passaged again to determine if the GFP expression was maintained with repeated passaging. The HEK cells showed a considerably diffe rent pattern of expression of the same GFP plasmid used for the tran sfection of the HRECs. Th ere are a number of possible explanations for this observation. First and foremost, the HEK cells were obtained from an established cell line and the purity of th eir culture guaranteed. The HEK cells are much smaller than the HRECs and the transfection protocol of the HEK cells was completely different from that of the HRECs. CAMP Assay on Transfected HEK Cells Once the transfection efficiency of the HEK cells was established, the A2B RZ2 plasmids were used to transfect the cells. Seventy two hours post-transfection, the cells were harvested for a cAMP assay. The transf ected cells were stimulated with NECA, the adenosine analogue. Adenosine binding to receptors leads to pr oduction of cAMP. Figure 3-17 shows the results of the cAMP assay. There is a significant reduction of cAMP production in the HEK cells transfected with the active A2B Rz2. However, this effect was not reproducible.

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122 Figure 3-15. Transfection Efficiency of HE K cells. A)6hrs, B)24hr s, C)48hrs, D)72hrs and E)96hrs post transfection.

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123 Figure 3-16. HEK cells transfection effici ency following passage 1. A)24hrs, B)48hrs, C)72hrs, and D)96hrs following passage 1.

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124 0 100 200 300 400 500 600 700 ControlActiveInactivecAMP accumulation (pmole/mg) ** Figure 3-17. cAMP accumulation in HEK cells transfected with the control, active A2B Rz2 and inactive A2B Rz 2.

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125 Real Time PCR Figure 3-18 shows the results of TaqMan real time RT-PCR on mRNA isolated from HEK cells transfected with the active and inactive versions of the A2B Rz2, and with the control plasmid. The active A2B Rz2 reduces mRNA levels by 43%. The inactive A2B ribozyme shows no significant reduction in mRNA levels as expected for the catalytically inert ribozyme. The leve l of mRNA detected were normalized to cyclophilin. Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model Active and inactive A2B ribozymes and the cloning vect or p21NewHp were injected intra-ocularly on post natal day one in th e right eye of mouse pups. There was no injection in the left eyes (which served as controls), and the pups and their dams were taken through the oxygen-induced model of retinopathy. On day 17 the mice were euthanized and their eyes enucleated and fi xed in 4% paraformaldehyde. The eyes were embedded in paraffin and three hundred serial, 6uM sections were done Every thirtieth section was placed on a slide and stained with hematoxylin and eosin. (Figure 3-19). The extent of angiogenesis was determined by counting the pre-retinal endothelial cell nuclei surrounding a blood vesse l lumen. The cloning vector p21NewHp showed marked angiogenesis. (Figure 3-20) The active A2B Rz2 reduced the average number of nuclei per section on average by 55% (Figure 3-21) The inactive A2B Rz2 reduced the average number of nuclei on aver age by 5%. (Figure 3-22)

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126 0 0.2 0.4 0.6 0.8 1 1.2 p21 NewHp (control) p21A2BRz2 (active) p21A2BRz2i (inactive)Normalized RNA Level (Cyclophilin is 100%) A2A A2B Figure 3-18. Real time RT-PCR results showi ng relative levels of the adenosine A2A and A2B receptor mRNAs isolated from HEK ce lls transfected with plasmid DNA.

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127 Figure 3-19. The mice eyes were embedded in paraffin and three hundr ed serial sections were done. Every thirtieth section was placed on a slide and stained with hematoxylin and eosin. This figure s hows a representative mouse eye that was stained. Arrows indicate the cornea, sclera and the lens of the mouse eye.

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128 Figure 3-20. Injection with the control plas mid prior to exposure to high oxygen shows a high number of endothelial cell nuclei surrounding blood vessel lumen. This indicates that the control plasmid di d not reduce the amount of pre-retinal angiogenic vessels.

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129 Figure 3-21. Injection w ith the active A 2B ribozyme prior to high oxygen exposure significantly reduced the preretinal neovascularization.

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130 Figure 3-22. Injection of the activ e and inactive versions of the A2B Rz2 and the vector control in the ROP mouse model

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131 CHAPTER 4 DISCUSSION The overall goal of this project was to develop a hammerhead ribozyme targeted against the adenosine A2B receptor that would cleave the A2B mRNA and reduce the expression of this receptor in cell cult ure and in a mouse model of oxygen-induced retinopathy. The value of this ribozyme is that it allows us to study the involvement of the A2B receptor in the complex physiological path way of angiogenesis. Secondary to this is the potential of using this ribozyme as a therapy in the treatment of pathologies that produce abnormal neovascularization such as retinopathies and tumorogenesis. Our results demonstrate that we have developed a hammerhead ribozyme that specifically cleaves the mouse and human adenosine A2B receptor mRNA. We have demonstrated that this ribozyme reduces the expression and function of the A2B receptor in HREC and HEK cells and we have shown that this ribozyme reduces abnormal retinal neovascularization in the mouse model of oxygen-induced retinopathy. The ability of the active ribozyme to inhibit the expression of the A2B mRNA in HEK cells was clearly shown. Reduction in A2B mRNA signal in cells transfected with the active ribozyme was 43% compared to cell s transfected with the control plasmid. The chemotactic migration of HRECs acro ss a porous membrane toward solutions containing increasing concentrations of NE CA is dependent on the presence of A2B receptors on the cell surface. The reduction of this migration in HREC transfected with a plasmid expressing the active A2B ribozyme suggests that cell surface levels of the A2B receptor have been reduced due to inhibition of expression of the A2B mRNA by the

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132 ribozyme. A reduction of 39% in the numbe r of migrating cells was found in cells expressing the active ribozyme. Our result s show that approximately 60% of this reduction in expression is due to cleavage of the message while the remaining inhibition results from an antisense effect of ribozyme binding to the target. These results also show that ribozyme cleavage of the A2B mRNA reduces expression of the protein in cultured cells to a level that significantly inhibits the cellular function of the A2B receptor. Therefore, inhibition of the A2B receptor as it affects othe r components of this pathway can be quantitatively examined. In addition this ribozyme inhibits pre-re tinal neovascularization in vivo in a mouse model of oxygen-induced retinopathy, where a re duction in pre-retina l neovascularization of 55% in eyes injected with the plasmi d expressing the active ribozyme was achieved and only a small portion of this reduction, ap proximately 5%, is due to an anti-sense effect. These results suggest that the reduc tion in neovascularizati on is the result of ribozyme inhibition of the expression of the A2B receptor and demonstrate that this ribozyme will also be useful for in vivo studies of A2B receptor function including studies on retinopathies. Ribozymes As Tools To Study Gene Expression There are a number of reports on the ab ility of the hammerhead ribozymes to control expression of specifi c genes in cell culture. For example, a hammerhead ribozyme designed to cleave mRNA encoding CHa Ras mutation inhibited formation of foci of transformed cells by 50%.173 Hammerhead ribozymes have also been used to target anticancer therapies. For example, hammerhead ribozymes were used to target mRNA of a BCR/ABL fusion protein, which is responsible for chronic myelogenous leukemia. The hammerhead ribozyme elim inated expression of the protein in K562

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133 cells.174 Hammerhead ribozymes targeted to surviv in, which is expressed in carcinoma cells were also able to re duce survivin mRNA by 74%.175 In another study, a hammerhead ribozyme was used to target bc l-2 mRNA in tumors, which overexpress the protein. The ribozyme was tested in a lympho ma cell line and showed a decrease in the bcl-2 mRNA and protein following transfection with the ribozyme.176 In yet another study, ribozymes were designed as HIV therap ies by a number of groups. A hammerhead ribozyme expressed in Hum7 cells decreas ed the hepatitis B viral production by 83%.177 Naturally occurring RNAs have distin ct advantages over DNA. RNA has a 2Â’OH group associated with the sugar moiety of RNA which participates dir ectly in a chemical reaction which enhances the reactivity of the adjacent 3Â’OH group. The 2Â’OH group is also a hydrogen donor and acceptor, thus re ndering RNA more versatile then DNA in tertiary structure formation.178 DNA is predominantly a base paired duplex of complementary strands and RNA is folded from a single strand. RNA, can thus form extensive secondary structures due to pairing of imperfect complementary sequences in the RNA strand. Single stranded regions punc tuate through this s econdary structure leading to a variety of RNA conformations. RNA also has a uridine base instead of a thymidine allowing it to form unique secondary st ructures. For example, a uridine turn in tRNA and hammerhead ribozymes causes an abrupt turn in direction of the polynucleotide backbone and allows formation of complex tertiary interactions. The uridine turn is stabilized by hydrogen bonding and Van Der Waals interactions between uridine and the surr ounding nucleotides. Antisense therapy has been used by a number of investig ators to inhibit angiogenesis. For example, Robinson et al used an antisense oligonucelotide against the

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134 mouse VEGF to inhibit re tinal neovascularization.179 The oligos were injected into the mouse eye intravitreally and then exposed to hypoxia. Treatment with the anti-sense oligoÂ’s resulted in a 25% decr ease in angiogenic vessel growt h. This study was able to confirm the importance of VEGF in the angi ogenic pathway, however, the antisense. olinucleotides were not succe ssful at potently inhibiting th e growth of abnormal vessels. 179 Another study used an o ligonucleotide targetting the human and rat VEGF forms and were only able to lower the incidence of choroidal neovascularization by 30%. Antisense therapy is effective when the targeted mRNA is not abundant. Antisense nucleotides are complementary DNA or RNA sequences whic h hybridize to specific mRNA. These nucleotides are specific to the target, easy to design and synthesize. However, antisense nucleotides used for therapy of disease have a poor uptake, are unstable. The nucleotides are also sensitive to degradation by exogenous and endogenous nucleases. Antisense nucleotides are toxic to cells because a higher concentration of nucleotides is required to significantly affect the expression of a gene. The stability of anti sense oligonucleotides is enhanced by phosphorothioate bonds, wh ich are toxic to mammalian cells. 180,181 An advantage of using ribozymes instead of antisense oligonucleotides is their small size making them inexpensive and easy to produce. Their small size allows them to be inserted into gene encoding ribozymes into viral vectors used for their delivery to cells in vivo. Trans acting ribozymes inactivate or m odify multiple mRNA molecules, which is a distinct advantage over anti-sense o ligonucleotides, which act stoichiometrically. Ribozymes also do not require host cellular machinery to degrade the substrate. Therefore trans-acting ribozymes inactivate targ et RNA more efficiently than anti-sense oligonucleotides. In comparison to the conve ntional antisense RNAs, ribozymes provide

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135 the potential of turnover, with a single molecu le being able to inactivate multiple target RNAs. 181 Ribozymes and protein enzymes share simila rities in their mode of action. Both, ribozymes and protein enzymes require a comp lex secondary and tertiary structure for catalysis.166,182,183 They also share similar mechanisms of acid base catalysis to carry out a reaction184 and stabilize a transitio n state between substrate and product formation. Catalysis is driven by intrinsic binding energy resulting from interactions between enzyme and its substrate at sites different from the catalytic core.185 Ribozymes can recognize RNA substrates via base pairing, thus their specificity is easy to manipulate. Ribozymes also do not evoke an immune re sponse to the same degree as (foreign) proteins evoke. RNA is a natural component of the cell and has a low half-life, thus low toxicity. Proteins, on the ot her hand require the host ce ll machinery to degrade the substrate. Delivery Of The Ribozyme In vivo The plasmid used for the expression of the A2B Rz2 has inverted terminal repeats (ITRs) at either ends of the ribozyme. ITRs contain the sequences, which are required for replication, packaging and inte gration of the plasmid into an rAAV vector. AAV is a single stranded, encapsulated DNA virus. AAV contains ITRs, which flank the AAV genome. This region of the genome is replaced with the gene of inte rest to generate the rAAV. 186 rAAV has been successfully used to deliv er transgenes into the eyes. While the A2B ribozyme was cloned into this vector for potential packaging into AAV, we have demonstrated a significant response of the Active A2B Rz2 naked plasmid and thus did not proceed to packing the ribozyme into an rAAV virus to enhance the effect.

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136 There are several advantages to using plasmid DNA versus rAAV. The plasmid DNA is easier to propagate and is usually of higher quality. 187 Plasmids can also carry larger DNA sequences as compared to limited 5kb capacity of rAAVs.188 Plasmid DNA can also be repeatedly administered du e to its low immunogenicity and toxicity. 189,190 Another method for the delivery of the ribozyme involves packaging the plasmid into a liposomal mixture that would bind to cell surface receptors and enhance uptake of the plasmid. 191,192 Liposomes are microscopic vesse ls composed of an aqueous compartment surrounded by a lipid layer. The transferrin receptor can be used as a vehicle to carry liposome/DNA complex into the cell. Transferrin binds to iron extracellulary and subsequently binds to the transferrin receptor on the cell surface. The receptor and transferrin are taken up by endocytos is into the cell. The iron is released and the transferrin and the receptor are recycled out of the cell. The transferrin helps the uptake of plasmid DNA by the cell. (Figure 41) Transferrin complexes can also be used to deliver cancer chemotherapeutic drugs to the tumor directly. For example, doxorubin, a cytotoxic drug was complexed with transferrin in liposomes and delivered to the site of tumor development. In another study, li posomal mediated delivery of the alpha interferon to murine bladder tumor cell line MBT2 was shown to increase the uptake of alpha interferon and enhance proliferative activ ity. Liposomal mediat ed delivery is safe, non-immunogenic, can be re-administered wi thout harm and an unlimited size of DNA can be delivered. However, a major drawb ack using the liposome /transferrin route of delivery is the poor transfection e fficiency of liposomal vectors. 193

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137 Figure 4-1. Entry of the AAV into the cell involves binding of the virus particle to heparan receptors on the cell surface follo wed by uptake of the virus into the cell and eventual release of viral DNA. In the transferrin cycle, transferrin after binding to the cell, it binds to th e transferrin receptor on the cell surface and is taken into the cell where the ir on is released and transferrin and its receptor are recycled out of the cell.

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138 Our lab has also tested the delivery of liposomes versus the naked DNA in an ROP model. A GFP plasmid was packed into a liposome with transferrin and suspended in HEPES buffer. Naked DNA resuspended in HEPES buffer was also used. Both formulations were tested in an ROP model of retinopathy in the mice. The mice were sacrificed and their eyes flat mounted to observe the expressi on of GFP. Results showed that the naked plasmid DNA injection had higher levels of GFP expression, thus supporting the results that naked DNA plasmid injection is sufficient. Use of the ribozyme in tissue cultures has shown a decrease in the expression of the A2B receptor in HREC. However, the efficiency of the transfection might be improved with increasing the amounts of the ribozyme. The transfection efficiency was determined by the transfection efficiency of a similar pl asmid containing a GFP expression site. GFP is a unique fluorophore, which forms intracellulary. It does not require additional cofactors and the emitted fluorescent intensity is proportional to the GFP expression levels within the cells. Thus GFP intensity can be measured at a single cell level. The GFP plasmid is also driven by a CMV promoter and thus would be taken up by any type of cell present in the culture. Using this GFP plasmid it was shown that the cells expressed GFP for up to three passages in the HEK ce lls and up to two passages in the HREC. However, this plasmid was not exactly the same as the one used to carry the ribozyme. To further enhance the chance of transfecting more cells, it would be better to place GFP upstream of the ribozyme and th en transfect the cells. The tissue culture can then be monitored for cells that expre ss the GFP. Any cells expres sing the GFP can be analyzed by flow cytometry which, can simultaneously measure and analyze multiple physical characteristics of cells as they flow in a fluid stream thr ough a beam of light. Properties

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139 it can measure include cell size, granularity a nd relative fluorescence. The cells can also be separated by a fluorescence activated cell so rter (FACS) and subsequently grown as pure culture. The pure culture would allow be tter characterization of the effects of the ribozyme. Sorenson et al 194 transfected H9 cells with a GFP marker located upstream of a retroviral vector. They were able to sort cells into two distinct populations by measuring the expression and the intensity of GFP expression. The sorted cells were viable for culture following sorting and the presence of GFP did not affect the cells. Promoter Considerations A CMV promoter was used to drive the expression of the plasmid, which carried the ribozyme. The CMV promoter is used for mammalian promoter strength to enhance the level of transient transgene expression in a majority of mammalian cells. The promoter is ubiquitous and affects most cells types. To better understand the pathophysiology of retinal angiogenesis, the CMV/beta actin promoter needs to be replaced with a proliferating endothelial cell specific promoter which is under the control of cell cycle gene switch. Such a promoter would only be expre ssed in proliferating endothelial cells in animals undergoing angi ogenesis. A promoter has been designed with a cdc6 cell cycle promoter and endot helin elements. Cdc6 is expressed in proliferating cells195,196 and the expression of endotheli n on mainly endothelial cells makes it an attractive targ et for endothelial cells.197,198,199 The cdc6 promoter was inserted into a plasmid and a mouse multimerized endothelin enhancer was inserted upstream of the cdc6 promoter. A five-fold increase in the expression of activity for the endothelin enhancer/cdc6 promoter was obser ved in diving cells ve rsus the non-diving endothelial cells. However, th is promoter was not found to be sufficient for adequately expressing ribozymes. Therefore a GAL4 DN A binding protein was used. The trans-

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140 activating protein was fused to the NF-kB p65 transactivation domain. Expression of the fusion protein was driven by a CMV enhancer promoter (Figure 4-2). To test the specificity of the endothelin/cdc6 promoter fo r endothelial proliferating cells, mice were implanted with SCCVII tumor cells. The tumo r was allowed to develop for a few days following and IV injection w ith liposome/plasmid complexes. The tumor and lung tissues were harvested at 18 hours and 4 days post injection and assayed for CAT expression. At 18hours post injection, the tumo r did not have any Cat activity present, and the lung tissue had detectable levels of CAT. However, at 4 days post injection, the tumor had ten times the activity of CAT and the lung tissue had less amounts. These results indicated that the CAT expressi ng promoter was repressed in non-dividing endothelial cells of the lung but was very high in the dividing cells of the tumor. It was however, interesting to note that there was no expression of CAT following 18hours of injection into the tumor. It is possible that endothelial cells need time to pass through at least one round of cell divisi on for the promoter to become activated. Experiments are currently underway to clone the A2B Rz2 downstream of this promoter. Thus far the distribution of the A2B receptors in different tissues has been based on the characterization of the receptors based on agonist binding. Antagonists would have been more preferable to determine the loca lization of these receptors, however, none are available. The A2B receptor has also been shown to be present in fibroblasts, which are present at sites of angiogenesis. Th erefore, the widespread pattern of A2B receptor distribution, based on agonist bind ing studies, may be misleading.200 Cloning of the A2B

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141 Figure 4-2. Diagram of the expression casse ttes fusion protein and alkaline phosphatase (Alk Phos). UT12 refers to the consen sus untranslated region of the message. IVS8 is a consensus splice site for mRNA processing. HGHpA is the human growth hormone poly A sequence. +1 is the transcriptional start site.

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142 Rz2 into the cdc6/endothelin promoter woul d allow better localizat ion and distribution patterns of the A2B receptor. The purity of the HRECs was important for the success of this project and for determining the presence of contaminating cell s such as pericytes. The purity of the culture was assessed either based on the morphology of the cells or the uptake of acetylated LDL by scavenger receptors. Both of these techniques are reliable for assessing the purity of the cultu res. In addition, a functional assay, Matrigel, can also be performed. This assay involves plating the ce lls onto a commercially available Matrigel. Matrigel is a basement membrane composed of collagens, laminin and proteoglycans. It also contains matrix degrading enzymes, TI MPs and growth factors. Endothelial cells plated onto a Matrigel helps them to form tube like structures. The Matrigel assay is time consuming and would not have provided any additional information to help in determining the purity of the HRECs culture. Future Studies Transfection with the active A2B ribozyme showed a 43% decrease in the levels of the A2B receptor by real time PCR. VEGF is a potent contributor to the process of angiogenesis and it has alrea dy been shown that the A2B receptor works upstream of VEGF. Hypoxia has been shown to upregulat e the expression of the VEGF receptor and the production of VEGF. Therefore, cells tr ansfected with the act ive form of the A2B ribozyme should also show a decrease in the levels of VEGF receptor and VEGF production. Therefore, it woul d be important to assess how down regulation of the A2B receptor affects the subsequent growth factors, which also play an important role in angiogenesis.

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143 It also remains to be ascertained whether down-regulation of the A2B receptor affects the other adenosine receptors. It is possible that down-regulation of one of the receptors causes the other receptors to enha nce their effects to compensate for the deficiency. The data from the real-tim e PCR shows that the levels of the A2A receptor mRNA remain unaffected following transfection with the A2B Rz2. Even though the mRNA for the A2B receptor has been shown to be decreased, it is possible that the amount of protein being produced is unaffected. Therefore, it would be useful to measure the A2B receptor protein by wester n blotting to confirm that a reduction in the amount of mRNA corresponds to a reduction in the am ount of protein. Currently, however, a suitable antibody for western blotting purposes is unavailable for the A2B receptor. Since a suitable antibody is available for the A2A receptor, it would be interesting to see if transfection of HRECs with the A2B Rz2 affects the level of protein production of the A2A receptor. Our lab has also made ribozymes to othe r components of the angiogenesis pathway, for example, VEGF and the integrins. Thes e ribozymes are made similar to the methods described for the A2B ribozymes. Sufficient in vivo and in vitro testing of the ribozymes has also been carried out. It would be interes ting to see the effect of a combination of the ribozymes might have on the angiogenesis pathway. Theoreticall y, a combination of ribozymes targeting various aspects of the a ngiogenic pathway would have a more potent effect. However, it is al so possible that down regulati on of too many of the growth factors may also hinder the deve lopment of normal blood vessels too. Downstream signaling of the A2B receptors is not well established. For example, the A2B receptors in xenopus have been shown to stimulate PLC, which in turn can

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144 activate calcium dependent chloride conductance. 201 This effect has not been shown in the retinal endothelial cells. The A2B receptor also leads to th e activation of the MAPK pathway, which may stabilize HIF-1 which, in turn may lead to the mitogenic effects of VEGF.108 The MAPK pathway may also stab ilize hypoxia inducible factor (HIF-1 ) by preventing its proteasomal degradation. HI F-1 is a heterodimeric protein, which is activated by hypoxia and regulates the transcription of many genes. It consists of constitutive HIF-1 and the rate limiting HIF-1 .202-204 Under normoxia, HIF-1 is regulated by the removal of the HIF-1 subunit by ubiquination and proteasomal degradation. HIF-1 is the aryl hydrocarbon receptor nuclear translocator (ARNT) which heterodimerizes with the aryl hydrocarbon receptor. 204 Hypoxia inhibits the removal of the HIF-1 subunit by the proteasome thus preventing HIF-1 destruction. The prevention of proteasomal degradation of HIF-1 is poorly understood. However, it is thought that the hydroxylation of two proline residues in the HIF-1 subunit prevent its degradation by the proteasome.203 The stabilized HIF-1 translocates to the nucleus and dimerizes with the HIF-1 (ARNT). The stabilized HIF-1 / subunit subsequently binds to the hypoxia response element (HRE). The HRE is a gene promoter which upregulates the expression of VEGF.205 Hypoxia also leads to the stab ilization of the short lived VEGF mRNA directly, thereby augm enting the effect of HIF-1 alpha.206 Glycogen synthase kinase-3 (GSK-3) is an enzyme, which catalyzes the breakdown of glycogen synthase. It is a pro-apoptotic enzyme and is constitutively active in cells. Phosphorylation of GSK-3 inhibits its activity. 207 The PI3 kinase pathway has been shown to phosphorylate GSK-3 and hen ce inhibit its activity. The A2B receptor couples to G q and activates the PI-3 kinase pathwa y. However, it has not been shown yet

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145 whether the PI-3 kinase pathway leads to the phosphorylation of GSK-3 by the A2B receptor. 207 Coupling of the A2B receptor to G q also leads to the act ivation of the MAPK pathway. It is possible that th e MAPK pathway stabilizes HIF-1 by preventing its proteasomal degradation. 207 Therefore it may also be hypothesized that phosphorylation of GSK-3 and subsequent inhibi tion of its activity also stabilizes the HIF-1 subunit. This pathway would contri bute to the upregulation of VEGF and angiogenic blood vessel growth (Figure 4-3) It is important to be able to dissect the signaling pathway of the receptor to better understand the angiogenic pathway. The active A2B ribozyme in combination with inhibito rs of some of the common kinases may be an important tool in studying this pathway.

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146 Figure 4-3. A2B signaling pathway with theoretical downstream effects, which have yet to be confirmed

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147 LIST OF REFERENCES 1. Hogan M. Histology of theHuman Eye Philadelphia: WB Saunders Co; 1976. 2. Snell R, Lemp, MA. Clinical Anatomy of the Eye Chicago, IL: Blackwell Scientific Publications.; 1989. 3. Gelatt K. Veterinary Ophthalmology NY: Lippincott and Williams; 1996. 4. Forrester J. The eye. Basic Sciences inPpractice Philadelphia, PA: WB Saunders Co Ltd; 1996. 5. Wise G, Dollery CT, Henkind P. The Retinal Circulation. NY: Harper & Row Publishers; 1971. 6. Hart W. Adler's Physiology of the Eye. 9th. ed. NY: Blackwell Scientific publications.; 1992. 7. Rubin LL, Staddon JM. The cell bi ology of the blood-brain barrier. Annu Rev Neurosci 1999;22:11-28. 8. Hyman L, Neborsky R. Risk factors fo r age-related macular degeneration: an update. Curr Opin Ophthalmol 2002;13:171-5. 9. Stone EM, Sheffield VC, Hageman GS. Molecular genetics of age-related macular degeneration. Hum Mol Genet 2001;10:2285-92. 10. Gottlieb JL. Age-related macular degeneration. Jama 2002;288:2233-6. 11. Harvey PT. Common eye diseases of el derly people: identifying and treating causes of vision loss. Gerontology 2003;49:1-11. 12. Campochiaro PA. Retinal and choroidal neovascularization. J Cell Physiol 2000;184:301-10. 13. Cai J, Boulton M. The pathogenesis of di abetic retinopathy: ol d concepts and new questions. Eye 2002;16:242-60.

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148 14. Aiello LP. The potential role of PKC beta in diabetic retinopathy and macular edema. Surv Ophthalmol 2002;47 Suppl 2:S263-9. 15. Sulochana K, Ramakrishnan, S., Rajesh M., Coral, K., and Badrinath, SS. Diabetic retinoapthy:moelcular mechanism, present regime of treatment and future perspectives. Current Science 2001;80:133-142. 16. Gardner TW, Antonetti DA, Barber AJ LaNoue KF, Levison SW. Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 2002;47 Suppl 2:S25362. 17. Rosenbaum JT. Sugar creates a sticky business: round up the usual suspects. Am J Pathol 2002;160:1547-50. 18. Gardner TW, Antonetti DA, Barber AJ Lieth E, Tarbell JA. The molecular structure and function of the inner bl ood-retinal barrier. Penn State Retina Research Group. Doc Ophthalmol 1999;97:229-37. 19. Miller JW, Adamis AP, Aiello LP. Vascul ar endothelial growth factor in ocular neovascularization and prolif erative diabetic retinopathy. Diabetes Metab Rev 1997;13:37-50. 20. Weinberger B, Laskin DL, Heck DE, La skin JD. Oxygen toxicity in premature infants. Toxicol Appl Pharmacol 2002;181:60-7. 21. McColm JR, Fleck BW. Retinopa thy of prematurity: causation. Semin Neonatol 2001;6:453-60. 22. Wesolowski E, Smith LE. Effect of light on oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994;35:112-9. 23. Hack M, Flannery DJ, Schluchter M, Cart ar L, Borawski E, Klein N. Outcomes in young adulthood for very-low-birth-weight infants. N Engl J Med 2002;346:14957. 24. Wheatley CM, Dickinson JL, Mackey DA, Craig JE, Sale MM. Retinopathy of prematurity: recent advances in our understanding. Br J Ophthalmol 2002;86:696-700. 25. Kotecha S. Oxygen therapy for infants with chronic lung disease. Arch Dis Child Fetal Neonatal Ed. 2002;87:F11-4.

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165 BIOGRAPHICAL SKETCH Aqeela Afzal was born in Lahore, Pakistan in 1972. She was raised in England (UK), California (USA), and Kuwait. She received her Bachel ors degree in 1995 in Medical Technology from Kuwait University. Sh e then went to the State University of New York at Buffalo and received a Masters in Clinical Laboratory Sciences in 1997. In 1999, she came to Gainesville, Florida and joined the Ph.D. program in Veterinary Medical Sciences. List of Publications. Dandona, P., Mohanty, P., Ghanim, H., A ljada, A., Browne, R., Hammouda, W., Prabhala, A, Afzal, A, Garg, R. The Suppr essive effect of di etary restriction and weight loss in the obese on the generati on of reactive oxygen species by leucocytes, lipid peroxidation and protein carbonylation. J. Clin. Metab 2001. Jan 86(1):35562. Lewis, P., Afzal, A. Human Ocular Histology. Advance 2001; 53-56. Afzal, M., Afzal, A., Jones, A., Armstrong, D. A rapid method for the quantification of GSH and GSSG in biological samples. Methods. Mol. Biol. 2002; 186:117-22 Afzal, A., Afzal, M., Jones, A., Armstrong, D. Rapid determination of glutamate using HPLC technology. Methods. Mol. Biol 2002; 186:111-5. Shaw, LC., Afzal, A., Lewin, AS., Timmers, A ., Spoerri, PE., Grant, MB. Reduction of the expression of the IGF-1 receptor by ri bozyme cleavage results in reduction of pre-retinal angiogenesis. Invest. Ophthal. Vis. Res Accepted. Afzal, A., Shaw, LC., Caballer o, S., Spoerri, P., Lewin, AS., Zeng, D., Bellardinelli, L., Grant, MB. Reduction in pre-retinal neova scularization by ribozymes that cleave the A2B adenosine receptor mRNA. Circ Res Submitted.

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166 Abstracts Afzal, A., Iwabuchi, S., Ellis, EA., Tama i, K., Samuelson, D., Armstrong, D. Localization of lipid peroxides at sites of oxidative stress in ocular tissues by the tetramethylbenzidine reaction. Invest. Ophthalmol. Vis. Sci. 41:S904, 2000 Armstrong, D., Aljada, A., Higa, H., Ghanim H., Afzal, A., Iwai, S., Browne, R., Dandona, P. Activation of signal transducti on in the retina by lip id hydroperoxide. Invest. Ophthalmol. Vis. Sci 42:S243, 2001. Iwai, S., Cahallers, S., Higa, A., Afzal, A., Ue da, T., Fukuda, S., Iwabuchi, S., Grant, M., Armstrong, D. Increased MMP activity in rabbit virtreous following exposure to lipid hydroperoxide (LHP). Invest. Ophthalmol. Vis. Sci 42:S573, 2001. Harding, R.J., Kallberg, M., Lewis, PA., Ellis, EA., Afzal, A., Samuelson, D. Immunocytochemistry of endothelin-1 recepto rs A and B in iridocorneal angles of the dog and monkey. Invest. Ophthalmol. Vis. Sci 42:S328, 2001. Afzal, A., Shaw, LC., Caballero, S., Ellis EA., Grant, MB. The development of hammerhead ribozymes that specifically cleave the A2B receptor mRNA. Invest. Ophthal. Vis. Sci 2002. 43:E-Abstract 3711 Samuleson, D., Kallberg, M., Lewis, P., Ellis, A., Afzal, A. Locali zation of endothelin-a receptor in iridocorneal angl es of glaucomatous dogs. Invest. Ophthal. Vis.Res. 2002. 43. 43 E-abstract 1053. Afzal, A., Shaw, LC., Caballero, S., Ellis, A., Zeng, D., Bellard inelli, L., Grant, MB. The development of hammerhead ribozym es that specifically cleave the A2B receptor mRNA. American Diabetes Association, San Francisco, CA, Jun 2002. Spoerri, PE., Shaw, LC., Bead le, C., Afzal, A., Pan, H., Grant, MB. IGF-1R and VEGFR-1 hammerhead ribozymes affect gl ucose induced tight junction protein modifications in cultured huma n retinal endothelial cells. Invest. Ophthal. Vis. S ci. 2003. 44; E-Abstract 3911.