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.

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.

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.

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization.

PURPOSE: Adeno-associated viral (AAV) vectors have been used to express several different proteins in the eye. The purpose of this study was to determine whether AAV-mediated intraocular gene transfer of pigment epithelium-derived factor (PEDF) inhibits the development of choroidal neovascularization (CNV) in a murine model. METHODS: C57BL/6 mice were given intravitreous or subretinal injections of a PEDF expression construct packaged in an AAV vector (AAV-chicken beta-actin promoter-exon 1-intron 1[CBA]-PEDF) or control vector (AAV-CBA-green fluorescent protein[GFP]). After 4 or 6 weeks, the Bruch's membrane was ruptured by laser photocoagulation at three sites in each eye. After 14 days, the area of CNV at each rupture site was measured by image analysis. Intraocular levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS: Four to six weeks after intraocular injection of AAV-CBA-PEDF, levels of PEDF in whole-eye homogenates were 6 to 70 ng. The average area of CNV at sites of the Bruch's membrane rupture showed no significant difference in eyes injected with AAV-CBA-PEDF compared with uninjected eyes. In contrast, 4 to 6 weeks after intraocular injection of 1.5 x 10(9) or 2.0 x 10(10) particles of AAV-CBA-PEDF, the area of CNV at the Bruch's membrane rupture sites had significantly decreased compared with CNV area at rupture sites in eyes injected with AAV-CBA-GFP. CONCLUSIONS: These data suggest that intraocular expression of PEDF or other antiangiogenic proteins with AAV vectors may provide a new treatment approach for ocular neovascularization.

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.

Adeno-associated virus type-2 expression of pigmented epithelium-derived factor or Kringles 1-3 of angiostatin reduce retinal neovascularization.

Neovascular diseases of the retina include age-related macular degeneration and diabetic retinopathy, and together they comprise the leading causes of adult-onset blindness in developed countries. Current surgical, pharmaceutical, and laser therapies for age-related macular degeneration (AMD) rarely result in improved vision, do not significantly prevent neovascularization (NV), and often result in at least some vision loss. To address this therapeutic gap, we determined the efficacy of recombinant adeno-associated viral (rAAV) serotype-2-mediated expression of pigment epithelium-derived factor (PEDF) or Kringle domains 1-3 of angiostatin (K1K3) in reducing aberrant vessel formation in a mouse model of ischemia-induced retinal NV. Both PEDF and K1K3 are potent inhibitors of NV when injected directly, hence expression of these therapeutic factors from rAAV may provide long-term protection from neovascular eye disease. rAAV vectors expressing the therapeutic gene were injected into one eye of postnatal day 0 (P0) newborn mouse pups. Retinal NV was induced in P7 mice by exposure to elevated oxygen for 5 days followed by room air for another five days. Retinal NV was quantified by the number of vascular-endothelial-cell nuclei above the inner-limiting membrane in P17 eyes. The number of such vascular endothelial cell nuclei in eyes treated with rAAV-PEDF or rAAV-K1K3 was significantly reduced (both P < 0.0000002) compared with control eyes. Ocular protein levels detected by ELISA correlate well with the reduction in NV and confirm that expression of antineovascular agents from rAAV vectors may be a therapeutically useful treatment of retinal or choroidal neovascular disease.