Retinal Edema & Mode of action of anti-VEGF therapies

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

Pathogenesis of DME 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 Role of genetic factors?Sustained hyperglycaemia Macular edema  AGE  ET  VEGF Hypoxia  IL-6 Destabilization of vitreous Abnormalities in collagen cross- linking MMP activity  PPVP  DAG  PKC Vasoconstriction  Histamine ET-receptors on pericytes Oxidative damage  LPO, NO, NADH/NAD + Antioxidant enzymes RAS activation Vitreomacular traction Accumulation of cytokeratin and glial fibrillary acidic protein Phosphorylation of tight junction proteins Disorganization of BRB  AII

RVO Pathology All types of RVO are multifactorial in origin and their pathology includes one or more of the following 1 –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 present 2 1 Hayreh. Indian J Ophthalmol 1994; 42: Klein et al. Trans Am Ophthalmol Soc 2000; 98:

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:

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: Hayreh. Indian J Ophthalmol 1994; 42:

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 Dvorak 1 and purified / cloned in 1989 by N Ferrara 2 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 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, Photo used courtesy of the AREDS Research Group. Fundus photo of normal retina

Pathologic VEGF-A secreted by RPE Hypoxia Accumulation of lipid metabolic byproducts 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) Initiating stimuli for VEGF release Witmer et al, Prog Retin Eye Res, 2003; Ferrara et al, Nat Med,

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

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

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

The Angiogenic Cascade (cont) Proliferation Migration Proteolysis VEGFFGF Other Angiogenic Growth Factors Vascular Endothelial Cell Enzymes permeabilize the basement membrane Hypoxia Basement Membrane

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

The angiogenic cascade in AMD

Characteristics of new vessels

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 VEGF 165 is VEGF 164 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.

Ferrara et al, Nat Med. 2003; 9: Most abundant isoform expressed in humans & largest contributor to angiogenesis - Sequestered in the extracellular matrix Highly diffusible and bioactive isoform VEGF-A Highest molecular weight isoform bound to extracellular matrix VEGFR Binding DomainHeparin Binding Domain VEGF-A VEGF-A VEGF-A VEGF-A isoforms

VEGF-A 110 Soluble & bioactive plasmin cleavage product Plasmin 1651 VEGF-A 165 VEGF-A VEGF Receptor Binding Domain Keyt et al, J Biol Chem. 1996; 271: 7788 VEGF Receptor Binding Domain Heparin Binding Domain Targeted binding site

Rationale for anti-VEGF therapy

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

Ranibizumab inhibits biologically active plasmin cleavage product of VEGF-A isoforms Keyt et al, J Biol Chem 1996; 271: 7788 Plasmin 1651 VEGF-A 165 VEGF-A – –89 VEGF Receptor Binding Domain VEGF Receptor Binding Domain Heparin Binding Domain Ranibizumab binding site Pegaptanib binding site

Mechanisms of anti-VEGF therapy Blood Vessel VEGF Receptor VEGF Signal Signaling Pathways New Vessel Formation Anti-VEGF 2,3 Pegaptanib Ranibizumab Bevacizumab Vascular Endothelial Cell Proliferation Migration Proliferation Migration Signaling Pathways

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

AMD Therapies: Mechanisms of action Blood Vessel VEGF Receptor VEGF Signal Signaling Pathways New Vessel Formation Vascular Endothelial Cell Proliferation Migration Proliferation Migration Signaling Pathways Steroids 2,3 Anecortave Triamcinolone Photodynamic Therapy 4,5 Visudyne Causes thrombosis and vessel closure. Leads to increased VEGF Anti-VEGF Pegaptanib Ranibizumab