Netrin 4 Induces Apoptosis Of Retinal Endothelial Cells And Inhibits Choroidal Neovascularization

Abstract

Purpose: : It is well known that angiogenic endothelial cells (EC) have an increased sensititivity to apoptosis mediated by growth factors withdrawal. We searched to identify genes up–regulated by VEGF in retinal EC (BREC)and assayed their bioactivity in vitro and in vivo.

Methods: : BREC were cultured in the presence or absence of VEGF for at least 3 weeks and a subtractive hybridisation strategy was used to identify genes up–regulated in angiogenic BREC. To identifie those of the VEGF–target genes which are specifically expressed in BREC, their expression was assayed in RPE cells exposed or not to VEGF. The candidate proteins were tested in vitro on BRECs. Furthermore, one candidate (Netrin 4) was tested on LASER induced subretinal neovascularization in the rat.

Results: : We identified 5 genes up–regulated in BREC but not in RPE stimulated by VEGF. 4 genes exert an anti–angiogenic activity and we choosed to focus on netrin–4. Recombinant Netrin 4 inhibited the mitogenic and haptotatic effects of VEGF on BREC. Although Netrin 4 induced a modest effect on EC apoptosis, it totally reversed the anti–apoptotic effect of VEGF or FGF2. These effects were inhibited by neutralizing antibodies directed against neogenin, a known netrin receptor. Subretinal injection of netrin 4 inhibited significantly (50%) the laser–induced choroidal neovascularization in a rat model.

Conclusions: : These results demonstrate that VEGF induces the endothelial expression of anti–angiogenic genes such as netrin 4. Moreover, Netrin 4 is an encouraging new avenue in the control of ocular neovascularization.

Keywords: growth factors/growth factor receptors • choroid: neovascularization • cell-cell communication