Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5−/−) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5−/− mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5−/− and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5−/− retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5−/− retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5−/− retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/ Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. IntroductionPhysiologic angiogenesis is tightly regulated. However, in proliferative retinopathy and cancer, there is excessive and disorganized growth of pathologic blood vessels. We hypothesized that there are endogenous angiostatic regulators that restrain pathologic vascular growth triggered by massive inflammatory and growth factor angiogenic stimuli. In the present study, we examined suppressor of cytokine signaling-3 (SOCS3) as such an endogenous angiostatic regulator.SOCS proteins are known negative feedback regulators of inflammation and growth factor signaling. 1,2 SOCS3 is transiently induced by inflammatory mediators such as lipopolysaccharide, 3 IL-6, 3 and TNF␣. 4 SOCS3 inhibits cytoplasmic effectors such as the JAK/STAT kinases and deactivates tyrosine kinase receptor signaling, including the IGF-1 receptor. 1,5 It also regulates endothelial cell (EC) apoptosis. 4 However, the role of SOCS3 in regulating angiogenesis in vivo is unknown. If SOCS3 were angiostatic, as has been hypothesized, then its suppression would increase neovascularization in pathologic conditions (see Figure 1A).Systemic deletion of Socs3 is embryonically lethal. 6,7 Therefore, in the present study, we generated Tie2-specific conditional Socs3 knock-out mice (Tie2-Socs3 ko ) 8 to compare developmental and pathologic angiogenic responses with littermate controls (Socs3 flox/flox ). In oxygen-induced retinopathy (OIR), 9-12 which mimics proliferative retinopathy, and in 2 tumor models, vascular deletion of Socs3 increased pathologic angiogenesis. Lack of SOCS3-mediated suppression of STAT3 and mTOR activation promoted EC proliferation and vascular sprouting. SOCS3 is therefore a newly identified endogenous negative regulator of angiogenesis acting on both inflammation and growth factormediated vessel formation specifically in pathologic contexts. Methods Animal modelsExperiments adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals 13 and were approved by the Boston Children's Hospital Animal Care and Use Committee. Tie2-Cre-expressing C57Bl/6 mice were crossed with Socs3 flox/flox mice (Dr Yoshimura, Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan 14 ) to generate Tie2-Socs3 ko and littermate control mice (Socs3 flox/flox ). In OIR, mice were exposed to 75% oxygen from P7-12. Retinas were dissected at the disease peak (postnatal day 17 [P17]) and neovascularization analyzed using SWIFT_NV. 15 Mice Ͻ 5 g (P17) ...

Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity.