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L. Leconte, E. Lejmi, I. Bouras, W. Raoul, G. Tobelem, F. Sennlaub, J. Plouet; Netrin-4 Inhibits Angiogenesis via Neogenin and Unc5b Receptors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3299.
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© ARVO (1962-2015); The Authors (2016-present)
Originally identified as axonal guidance molecules, netrins have been recently shown as angiogenesis modulators. We investigated the role of Netrin-4 (N4) in endothelial cells (EC) and identified the receptors involved in N4 anti-angiogenic activity.
A subtractive hybridization strategy identified N4 as a gene up-regulated in EC upon Vascular Endothelial Growth Factor (VEGF) exposure. Migration was studied by the wound healing assay and tubulogenesis by counting the branching of Human Umbilical Arterial Endothelial Cells (HUAEC) seeded in Matrigel. N4 was injected intravitreally at days 7 and 10 after laser injury. The extent of choroidal neovascularization was assessed by systemic dextran injection and count of CD31+ EC. N4 receptor studies were performed using qPCR, siRNA and blocking antibodies in HUAEC.
N4 is faintly detected in adult choroidal and retinal EC but is up-regulated in choroidal neovessels after laser injury. N4 inhibited VEGF-mediated HUAEC migration. Knock-down of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. EC express 3 of the 6 Netrin-1 cognate receptors: Neogenin, Unc5B and Unc5C. In contrast to Netrin-1, N4 bound only to neogenin but not to Unc5B or C. Immunoneutralization of Neogenin abolished the chemotactic effect of N4. Furthermore, the silencing of either Neogenin or Unc5B abolished N4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Co-immunoprecipitation experiments demonstrated that N4 increased the association between Unc5B and Neogenin on VEGF or FGF-2 stimulated EC. Finally, we showed that Netrin-4 significantly inhibited angiogenesis in the model of laser-induced choroïdal neovascularization.
We demonstrate that N4 acts as an anti-angiogenic factor through binding to Neogenin and recruitment of Unc5B. Moreover, N4 is a potent inhibitor of choroidal neovascularization and may be applied in the treatment of vaso-proliferative retinal diseases such as diabetic retinopathy or ARMD.
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