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

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


Proliferation

Migration

Proteolysis



Cell

• Activated endothelial cells proliferate, migrate, and release proteases

Hypoxia


Proliferation

Migration

Proteolysis



Cell

• Enzymes permeabilize the basement membrane

Hypoxia

BasementMembrane


Proliferation

Migration

Proteolysis



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


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



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

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

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