Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease.

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a panphosphatidyl inositol 3-kinase -PI3K -inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad. . These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo. Neurotrophins (NTs) were initially considered for their prosurvival and regenerative actions on the nervous system. These beneficial actions of NTs are mediated by their tropomyosin-related kinase receptors (trks), which are tyrosin kinases.1,2 Recent studies have unraveled the potential of nerve growth factor (NGF) and other NTs for vascular biology.3-7 Our group discovered that NGF protects endothelial cells and skeletal myocytes from apoptosis and promotes neovascularization in murine ischemic limb muscles and diabetic skin wounds. 3,5,6 We demonstrated the involvement of protein kinase B (Akt) in NGF-induced angiogenesis. 3Although others already tracked the presence of NGF in the heart and isolated cardiomyocytes, [8][9][10][11] the hypothesis that NGF controls cardiomyocyte survival was, to the best of our knowledge, never investigated.In noncardiovascular cells, NGF, via Akt, promotes the nuclear exclusion of Foxo-3a and Foxo-1, two members of the Forkhead box, class O (Foxo) subfamily of Forkhead transcription factors, which stimulate cell death.12 Nuclear localization of Foxo activates several Foxo-responsive genes, including the proapoptotic TNFa, Fas ligand, and Bim. [13][14][15][16] Akt-dependent Foxo phosphorylation inactivates the Foxo nuclear translocation signal and promotes the interaction of Foxo with 14-3-3 proteins, thus k...

Rationale The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown. Objective We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation. Methods and Results We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a−/− mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a−/− mice. Conclusions Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.