Brian J. Raisler

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.

Toward a Higher Fidelity Model of AMD

Age-related macular degeneration (AMD) is the leading cause of acquired blindness in the elderly population of industrialized countries (Klein et al., 1997; Vingerling et al., 1995; Leibowitz et al., 1980). Characterized clinically in two stages; ‘dry’ AMD consists of deposition of drusen under the RPE, particularly in the macular region, while ‘wet/exudative’ AMD consists of formation of new, invasive blood vessels originating from the choriodal vascular bed. Dry AMD can lead to vision loss due to geographic atrophy and photoreceptor loss, but most patients with drusen do not suffer loss of vision. Exudative AMD is the less common form, but is by far the greater cause of vision loss. Retina scarring and macular edema due to poorly formed and leaky choriodal vessels contribute to visual loss in advanced AMD.

Adeno‐associated virus mediated gene therapy for vascular retinopathies

Publisher Summary The use of gene based therapy for the treatment of ocular neovascular disease offers advantages over conventional methods. By using a viral vector with a selective promoter to express the antiangiogenic protein or factor locally, expression can be limited to a specific cell type or subset of cell types within the retina. This reduces the safety concerns relative to systemic administration of antiangiogenic agents. Delivery of the vector to discreet compartments within the eye by sub-retinal or intravitreous injection may allow additional control of expression to only those local vessels that are affected. Choice of the appropriate viral vector for delivery of the therapeutic gene allows modulation of the duration of expression. Adeno-associated viral vectors appear to provide extended, perhaps even life-long, expression of therapeutic proteins within the eye. Several alternative viralvectored approaches have been reported. An adenoassociated viral vector (AAV) encoding the soluble vascular endothelial growth factor receptor 1, sFlt-1, shows promise for long-term inhibition of two types of ocular neovascularization. Another gene-based approach to treating neovascular disease involves the virally mediated intraocular expression of antiangiogenic agents. The chapter demonstrates that recombinant AAV vectors incorporating a CBA promoter are capable of producing sustained therapeutic levels of pigment epithelium derived factor (PEDF) and Kringle domains 1 through 3 of angiostatin (K1K3) in the mouse eye.

Gene Therapy for Neovascular Retinopathies

Normal control of retinal angiogenesis, the formation of new blood vessels in either the retinal or choroidal beds from preexisting vasculature, is essential for vision. Conversely, pathological neovascularization (NV) of retinal and choroidal vessels is a key process leading to vision loss in several prevalent ocular diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and age-related macular degeneration (AMD). PDR and AMD are the leading causes of blindness in developed countries, and ROP is the leading cause of infant blindness. Proper regulation of retinal vascularization is thought to depend on an equilibrium between ocular vascular growth factors, primarily vascular endothelial growth factor (VEGF) (1), and natural inhibitors of angiogenesis, primarily pigment epithelium-derived factor (PEDF) (2). When this balance becomes disturbed—as may happen, for example, during and after the hyperoxic treatment of premature infants—pathological angiogenesis often occurs that ultimately leads to vision loss.

Drusen complement components C3a and C5a promote choroidal neovascularization

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.