Retinal Edema &Mode of action of anti-VEGF
therapies
Pathogenesis of neovascular AMD
Augustin AJ, Kirchhoff J. Expert Opin Ther Targets 2009;13:641–651Kijlstra 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
Thinning choriocapillarisUV light exposure
Thickening Bruch’s membrane
Advanced AMD and vision loss
The ageing eye
Oxidative stress and related tissue damage
RPE dysfunction Drusen formationComplement 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
VEGFHypoxia IL-6 Destabilization of vitreousAbnormalities in collagen cross-linking MMP activity PPVP
DAG
PKC
Vasoconstriction
Histamine
ET-receptors on pericytes Oxidative damage
LPO, NO, NADH/NAD+
Antioxidant enzymesRAS 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 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: 109-1322Klein 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: 111-131Hayreh. 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)
VEGFRbinding
siteVEGFRbinding
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 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
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.
Fundus photo of normal retina
PathologicVEGF-A secreted by RPE
• 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)
Initiating stimuli for VEGF release
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)
VEGF FGFOther AngiogenicGrowth 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
VEGF FGFOther AngiogenicGrowth Factors
Vascular Endothelial
Cell
• Activated endothelial cells proliferate, migrate, and release proteases
Hypoxia
The Angiogenic Cascade (cont)
Proliferation
Migration
Proteolysis
VEGF FGFOther AngiogenicGrowth Factors
Vascular Endothelial
Cell
• Enzymes permeabilize the basement membrane
Hypoxia
BasementMembrane
The Angiogenic Cascade (cont)
Proliferation
Migration
Proteolysis
VEGF FGFOther AngiogenicGrowth Factors
Vascular Endothelial
Cell
• Migrating endothelial cells form new blood vessels in formerly avascular space
Hypoxia
BasementMembrane
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.
Ferrara et al, Nat Med. 2003; 9: 669
1651
- Most abundant isoform expressed in humans & largest contributor to angiogenesis
- Sequestered in the extracellular matrix
1 189
- Highly diffusible and bioactive isoform
VEGF-A121 86-89
1 121
1 206
- Highest molecular weight isoform bound to extracellular matrix
VEGFR Binding Domain Heparin Binding Domain
VEGF-A206 86-89
VEGF-A189 86-89
VEGF-A165 86-89
VEGF-A isoforms
VEGF-A110 Soluble & bioactive plasmin cleavage product
Plasmin
1651 VEGF-A165
VEGF-A110
86-89 121110
1 11086-89
VEGF ReceptorBinding Domain
Keyt et al, J Biol Chem. 1996; 271: 7788
VEGF ReceptorBinding Domain
HeparinBindingDomain
Targetedbinding site
Rationale for anti-VEGF therapy
Ranibizumab inhibits all biologically active isoforms of VEGF-A
Ferrara et al, Nat Med 2003; 9: 669
1651
- Most abundant isoform expressed in humans & largest contributor to angiogenesis
- Sequestered in the extracellular matrix
1 189
- Highly diffusible and bioactive isoform
VEGF-A121 86–89
1 121
1 206
- Highest molecular weight isoform bound to extracellular matrix
VEGFR Binding Domain Heparin Binding Domain
VEGF-A206 86–89
VEGF-A189 86–89
VEGF-A165 86–89
Ranibizumab binding siteFerrara et al, Nat Med. 2003; 9: 669
Ranibizumab inhibits biologically active plasmincleavage product of VEGF-A isoforms
Keyt et al, J Biol Chem 1996; 271: 7788
1651 VEGF-A165
VEGF-A110
86–89 121110
1 11086–89
VEGF ReceptorBinding Domain
VEGF ReceptorBinding Domain
HeparinBindingDomain
Ranibizumabbinding site
Pegaptanibbinding site
Mechanisms of anti-VEGF therapy
Blood Vessel
VEGFReceptorVEGF
Signal Signaling Pathways New Vessel Formation
Anti-VEGF2,3
• Pegaptanib• Ranibizumab• Bevacizumab
VascularEndothelial Cell
VascularEndothelial Cell
Proliferation
Migration
Proliferation
Migration
SignalingPathwaysSignalingPathways
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