Retinal Edema & Mode of action of anti-VEGF therapies

1. Retinal Edema &Mode of action of anti-VEGF therapies

2. Pathogenesis of neovascular AMD The ageing eye UV light exposure Thickening Bruch’s membrane Thinning choriocapillaris Oxidative stress and related tissue damage RPE dysfunction Drusen formation Complement activation IL-1, IL-6, IL-8, MCP-1 Stimulation of C5a receptor Inflammatory mediators (C3a and C5a) Associated with genetic polymorphism in CFH VEGF Macrophages Disruption of Bruch’s membrane Neovascularizationand invasion of subretinal space Advanced AMD and vision loss Augustin AJ, Kirchhoff J. Expert Opin Ther Targets 2009;13:641–651 Kijlstra A et al. In Uveitis and immunological disorders. 2009. p73–85 CFH, complement factor H; IL, interleukin; MCP, monocyte chemoattractant protein; RPE, retinal pigment epithelium

3. Pathogenesis of DME Sustained hyperglycaemia Role of genetic factors? LPO, NO, NADH/NAD+ Antioxidant enzymes DAG Histamine AGE RAS activation ET PKC ET-receptors on pericytes Vasoconstriction Oxidative damage Destabilization of vitreous Abnormalities in collagen cross-linking MMP activity  PPVP Hypoxia IL-6 VEGF AII Accumulation of cytokeratin and glial fibrillary acidic protein Phosphorylation of tight junction proteins Disorganization of BRB Macular edema Vitreomacular traction Bhagat N et al. Surv Ophthalmol 2009;54:1–32 AII, angiotensin II; AGE, advanced glycation end; BRB, blood–retinal barrier; DAG, diacylglycerol; ET, endothelin; LPO, lypoxygenase; MMP, matrix metallo-proteinases; NO, nitric oxide; PKC, protein kinase C; PPVP, posterior precortical vitreous pocket; RAS, renin-angiotensin system

4. RVO Pathology • All types of RVO are multifactorial in origin and their pathology includes one or more of the following1 • narrowing of the retinal vein due to external pressures • sclerotic adjacent structures • secondary endothelial proliferation • primary venous wall disease • hemodynamic disturbances • In both CRVO and BRVO, the development of new vessels and macular edema result in variable loss of vision • In one study, nearly 10% of eyes with BVRO had new vessels present and another 10% had macular edema present2 1Hayreh. Indian J Ophthalmol 1994; 42: 109-132 2Klein et al. Trans Am Ophthalmol Soc 2000; 98: 133-141

5. CRVO • Non-ischemic CRVO • site of occlusion is distal to the lamina cribrosa or the adjacent retrolaminar region • sluggish retinal circulation due to fall in perfusion pressure resulting from a rise in proximal venous pressure • Ischemic CVRO • site of occlusion is in the region of the lamina cribrosa (or immediately posterior) • marked rise in venous pressure • retinal hemorrhage due to rupture of ischemic capillaries Hayreh. Indian J Ophthalmol 1994; 42: 109-132

6. BRVO • Defined by the site of occlusion • major BVRO (occlusion within one of the major branch retinal veins) • macular BVRO (occlusion within one of the macular venules) • Pathogenesis of BRVO may be due to a combination of three primary mechanisms • compression of the vein at the A/V crossing • degenerative changes of the vessel wall • abnormal hematologic factors Rehak & Rehak. Curr Eye Res 2008; 33: 111-131 Hayreh. Indian J Ophthalmol 1994; 42: 109-132

7. Angiogenesis • Angiogenesis • Growth of blood vessels

8. Angiogenesis – A Natural Process • Physiological angiogenesis • Embryonic development • Wound healing • Endometrium, ovary

9. Angiogenesis – A Pathologic Problem • Pathological angiogenesis • Cancer • Eye disease ie. ARMD

10. What is VEGF-A? • First described as vascular permeability factor by Dvorak1 and purified / cloned in 1989 by N Ferrara2 • Homo-dimeric glycoprotein • A member of a family of angiogenic and lymphangiogenic growth factors: • VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor • VEGF-A is mainly responsible for angiogenesis

11. VEGF-A binds to dimeric VEGF receptors (VEGFR1 & VEGFR2) VEGFR bindingsite VEGFRbindingsite

12. Role of VEGF-A in angiogenesis • Stimulates angiogenesis • Increase permeability • Chemotactic factor for inflammatory cells – Promotes inflammation

13. VEGF-A is present in the healthy eye • VEGF and its receptors naturally expressed in healthy eye • High concentrations of VEGFin RPE • Receptors primarily located on vascular endothelial cells • In healthy eye, VEGF may play a protective role in maintaining adequate blood flow (choroidal) to RPE and photoreceptors Fundus photo of normal retina Witmer et al, Prog Retin Eye Res, 2003; Adamis and Shima, In press; Kim et al, Invest Ophthalmol Vis Sci, 1999; Ambati et al, Surv Ophthalmol, 2003;Zarbin, Arch Ophthalmol, 2004. Photo used courtesy of the AREDS Research Group.

14. Initiating stimuli for VEGF release • Hypoxia • Accumulation of lipid metabolicbyproducts • Oxidative stress to retina & RPE • Alterations in Bruch’s membrane • Drusen (Reduction in the choriocapillaries blood flow and block diffusion of oxygen and nutrients to RPE and photoreceptors) Pathologic VEGF-A secreted by RPE Witmer et al, Prog Retin Eye Res, 2003; Ferrara et al, Nat Med, 2003. 14

15. The Angiogenic Cascade Hypoxia • Hypoxia stimulates production of VEGF and other angiogenic growth factors in the subretinal space

16. The Angiogenic Cascade (cont) Other Angiogenic Growth Factors Hypoxia VEGF FGF • VEGF and other angiogenic factors bind to endothelial cells of nearby capillaries and activate them Vascular Endothelial Cell

17. The Angiogenic Cascade (cont) Proliferation Proteolysis Migration Other Angiogenic Growth Factors Hypoxia VEGF FGF • Activated endothelial cells proliferate, migrate, and release proteases Vascular Endothelial Cell

18. The Angiogenic Cascade (cont) Basement Membrane Proliferation Proteolysis Migration Other Angiogenic Growth Factors Hypoxia VEGF FGF • Enzymes permeabilize the basement membrane Vascular Endothelial Cell

19. The Angiogenic Cascade (cont) Basement Membrane Proliferation Proteolysis Migration Other Angiogenic Growth Factors Hypoxia VEGF FGF • Migrating endothelial cells form new blood vessels in formerly avascular space Vascular Endothelial Cell

20. The angiogenic cascade in AMD

21. Characteristics of new vessels

22. VEGF-A isoforms

23. VEGF-A isoforms • VEGF-A is a single gene that codes for distinct protein isoforms • Human VEGF-A isoforms include: 121, 165, 189 and 206 • Isoform number refers to number of amino acids contained in the mature, secreted proteins • Murine (rodent) isoforms contain 1 less amino acid than human isoforms • Thus, murine equivalent of VEGF165 is VEGF164 Neufeld et al, FASEB J, 1999; Robinson and Stringer, J Cell Sci, 2001; Ferrara et al, Endocr Rev, 1992; Adamis and Shima, In press, 2004; Shima et al, J Biol Chem, 1996.

24. VEGF-A isoforms 86-89 VEGF-A206 VEGF-A189 86-89 VEGF-A165 86-89 86-89 VEGF-A121 0 VEGFR Binding Domain Heparin Binding Domain 1 206 - Highest molecular weight isoform bound to extracellular matrix 1 189 - Sequestered in the extracellular matrix 1 165 - Most abundant isoform expressed in humans & largest contributor to angiogenesis 1 121 - Highly diffusible and bioactive isoform Ferrara et al, Nat Med. 2003; 9: 669

25. VEGF-A110 Soluble & bioactive plasmin cleavage product 0 Plasmin HeparinBindingDomain VEGF Receptor Binding Domain 1 110 121 165 VEGF-A165 86-89 Targetedbinding site 110 1 VEGF-A110 86-89 VEGF Receptor Binding Domain Keyt et al, J Biol Chem. 1996; 271: 7788

27. Rationale for anti-VEGF therapy

29. Ranibizumab inhibits all biologically active isoforms of VEGF-A 86–89 VEGF-A206 VEGF-A189 86–89 VEGF-A165 86–89 86–89 VEGF-A121 VEGFR Binding Domain Heparin Binding Domain 1 206 - Highest molecular weight isoform bound to extracellular matrix 1 189 - Sequestered in the extracellular matrix 1 165 - Most abundant isoform expressed in humans & largest contributor to angiogenesis 1 121 - Highly diffusible and bioactive isoform Ranibizumab binding site Ferrara et al, Nat Med 2003; 9: 669 Ferrara et al, Nat Med. 2003; 9: 669

30. Plasmin Ranibizumab inhibits biologically active plasmincleavage product of VEGF-A isoforms HeparinBindingDomain VEGF Receptor Binding Domain 1 110 121 165 VEGF-A165 86–89 Pegaptanib binding site Ranibizumabbinding site 110 1 VEGF-A110 86–89 VEGF Receptor Binding Domain Keyt et al, J Biol Chem 1996; 271: 7788

31. Mechanisms of anti-VEGF therapy Signal Signaling Pathways New Vessel Formation Blood Vessel VascularEndothelial Cell VEGFReceptor VEGF SignalingPathways Proliferation Migration • Anti-VEGF2,3 • Pegaptanib • Ranibizumab • Bevacizumab

32. AMD Therapies: Mechanisms of action Block VEGF: Macugen, Lucentis Inhibit VEGF production: siRNA Block Integrins Prevent Extracellular Matrix Dissolution: Steroids Thrombose vessels: Visudyne Burn vessels: Thermal Laser Steroids stop vessel leakage