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Retinal Edema & Mode of action of anti-VEGF therapies Pathogenesis of neovascular AMD The ageing eye UV light exposure Thickening Bruch’s membrane RPE dysfunction IL-1, IL-6, IL-8, MCP-1 VEGF Macrophages Neovascularization and invasion of subretinal space CFH, complement factor H; IL, interleukin; MCP, monocyte chemoattractant protein; RPE, retinal pigment epithelium Thinning choriocapillaris Drusen formation Oxidative stress and related tissue damage Complement activation Stimulation of C5a receptor Disruption of Bruch’s membrane Inflammatory mediators (C3a and C5a) Associated with genetic polymorphism in CFH 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 Pathogenesis of DME Sustained hyperglycaemia DAG Histamine RAS activation LPO, NO, NADH/NAD+ Antioxidant enzymes ET PKC ET-receptors on pericytes Vasoconstriction Hypoxia AGE Role of genetic factors? IL-6 Oxidative damage VEGF AII Accumulation of cytokeratin and glial fibrillary acidic protein Phosphorylation of tight junction proteins Disorganization of BRB AII, angiotensin II; AGE, advanced glycation end; BRB, blood–retinal barrier; DAG, diacylglycerol; ET, endothelin; LPO, lypoxygenase; MMP, matrix metalloproteinases; NO, nitric oxide; PKC, protein kinase C; PPVP, posterior precortical vitreous pocket; RAS, renin-angiotensin system Macular edema Destabilization of vitreous Abnormalities in collagen crosslinking MMP activity PPVP Vitreomacular traction Bhagat N et al. Surv Ophthalmol 2009;54:1–32 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. 2Klein Indian J Ophthalmol 1994; 42: 109-132 et al. Trans Am Ophthalmol Soc 2000; 98: 133-141 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 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 Angiogenesis •Angiogenesis – Growth of blood vessels Angiogenesis – A Natural Process Physiological angiogenesis – Embryonic development – Wound healing – Endometrium, ovary Angiogenesis – A Pathologic Problem Pathological angiogenesis – Cancer – Eye disease ie. ARMD 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 VEGF-A binds to dimeric VEGF receptors (VEGFR1 & VEGFR2) VEGFR binding site VEGFR binding site Role of VEGF-A in angiogenesis • Stimulates angiogenesis • Increase permeability • Chemotactic factor for inflammatory cells – Promotes inflammation VEGF-A is present in the healthy eye • VEGF and its receptors naturally expressed in healthy eye – High concentrations of VEGF in RPE – Receptors primarily located on vascular endothelial cells Fundus photo of normal retina • In healthy eye, VEGF may play a protective role in maintaining adequate blood flow (choroidal) to RPE and photoreceptors 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. Initiating stimuli for VEGF release • Hypoxia • Accumulation of lipid metabolic byproducts • Oxidative stress to retina & RPE • Alterations in Bruch’s membrane • Drusen (Reduction in the Pathologic VEGF-A secreted by RPE choriocapillaries blood flow and block diffusion of oxygen and nutrients to RPE and photoreceptors) Witmer et al, Prog Retin Eye Res, 2003; Ferrara et al, Nat Med, 2003. 14 The Angiogenic Cascade Hypoxia • Hypoxia stimulates production of VEGF and other angiogenic growth factors in the subretinal space The Angiogenic Cascade (cont) Hypoxia VEGF FGF Other Angiogenic Growth Factors • VEGF and other angiogenic factors bind to endothelial cells of nearby capillaries and activate them Vascular Endothelial Cell The Angiogenic Cascade (cont) Hypoxia • Activated endothelial cells proliferate, migrate, and release proteases VEGF FGF Proliferation Other Angiogenic Growth Factors Proteolysis Migration Vascular Endothelial Cell The Angiogenic Cascade (cont) Hypoxia VEGF • Enzymes permeabilize the basement membrane Proliferation FGF Other Angiogenic Growth Factors Proteolysis Migration Vascular Endothelial Cell Basement Membrane The Angiogenic Cascade (cont) Hypoxia • Migrating endothelial cells form new blood vessels in formerly avascular space VEGF FGF Proliferation Other Angiogenic Growth Factors Proteolysis Migration Vascular Endothelial Cell Basement Membrane The angiogenic cascade in AMD Characteristics of new vessels VEGF-A isoforms 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. VEGF-A isoforms VEGFR Binding Domain Heparin Binding Domain 1 206 VEGF-A206 86-89 - Highest molecular weight isoform bound to extracellular matrix 1 189 VEGF-A189 86-89 - Sequestered in the extracellular matrix 1 165 VEGF-A165 86-89 - Most abundant isoform expressed in humans & largest contributor to angiogenesis 1 121 VEGF-A121 86-89 - Highly diffusible and bioactive isoform Ferrara et al, Nat Med. 2003; 9: 669 VEGF-A110 Soluble & bioactive plasmin cleavage product Heparin Binding Domain VEGF Receptor Binding Domain 1 VEGF-A165 86-89 110 121 Plasmin Targeted binding site 1 VEGF-A110 86-89 VEGF Receptor Binding Domain Keyt et al, J Biol Chem. 1996; 271: 7788 165 110 Rationale for anti-VEGF therapy Ranibizumab inhibits all biologically active isoforms of VEGF-A VEGFR Binding Domain Heparin Binding Domain 1 206 VEGF-A206 86–89 - Highest molecular weight isoform bound to extracellular matrix 1 189 VEGF-A189 86–89 - Sequestered in the extracellular matrix 1 165 VEGF-A165 86–89 - Most abundant isoform expressed in humans & largest contributor to angiogenesis 1 121 VEGF-A121 86–89 - Highly diffusible and bioactive isoform Ferrara et al, Nat Med. 2003; 9: 669 Ranibizumab binding site Ferrara et al, Nat Med 2003; 9: 669 Ranibizumab inhibits biologically active plasmin cleavage product of VEGF-A isoforms Heparin Binding Domain VEGF Receptor Binding Domain 1 VEGF-A165 86–89 110 121 Pegaptanib binding site Ranibizumab binding site 1 VEGF-A110 86–89 165 110 VEGF Receptor Binding Domain Keyt et al, J Biol Chem 1996; 271: 7788 Mechanisms of anti-VEGF therapy Signal Signaling Pathways New Vessel Formation Blood Vessel VEGF VEGF Receptor Vascular Endothelial Cell Signaling Pathways Anti-VEGF2,3 • Pegaptanib • Ranibizumab • Bevacizumab Proliferation Migration 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