Transcript Ferrara et al, Nat Med 2003 - Kashyap Memorial Eye Hospital

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