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Norbert Kociok, Sabrina Klein, Yong Liang, Sergio Crespo-Garcia, Nadine Reichart, Sergej Skosyrski, Christina Nuernberg, Olaf Strauss, Manuel Koch, Antonia M Joussen; The role of netrin-4 in pathologic neovascularization in the eye . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3397.
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© ARVO (1962-2015); The Authors (2016-present)
Netrins function as guidance signals in a broad range of pathologies including cancer and artheriosclerosis. To answer the question whether netrin-4 acts either pro- or anti-angiogenic under pathological conditions, angiogenesis in the retina was assessed in netrin-4 knock-out mice (Ntn4-/-) with oxygen-induced retinopathy (OIR) resembling hypoxia-mediated neovascularization and laser-induced choroidal neovascularisation (CNV), resembling inflammatory mediated angiogenesis.
Wild-type and Ntn4-/- mice were exposed to hyperoxia from P7 to P12 (75% O2) and examined at P17, 21, and 28. Control groups were kept at room air. An argon laser was used to set four to five distinct 50μm wide spots (120mW, 100ms, 532nm) around the optic disc. Retinal flatmounts from P6 to P21 were used to visualize the developing vessels by staining with Alexa-488-labeled isolectin IB4. Immunohistochemical stainings from retina flatmounts and paraffin retina sections were analyzed by image-processing and subsequent statistical evaluation. mRNA expression by quantitative PCR and protein expression by western blotting were assessed at the respective time points. Retinal function was monitored by means of electroretinography (ERG).
At P6 Netrin-4 localization started at the basal membrane of the large retinal blood vessels and was limited to mature vessels during vessel devoloment. Netrin-4 but not netrin-1 mRNA expression decreased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retinal vessel morphology were found in Ntn-4-/- mice. In OIR Ntn-4-/- mice at first displayed larger avascular areas which recovered faster to revascularization. Correspondingly, in Ntn-4-/- mice VEGF expression peaked an earlier time-point than in wild-type mice. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn4-/- mice. Expression of putative netrin-4 receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Unc5H2 was up-regulated after oxygen treatment. Pericytes and Laminin γ3 were coexpressed in pathological vessels. In response to laser-induced damage Nnt-4-/- mice did not react to their controls.
Our results indicate the role for netrin-4 is an anti-angiogenic factor in O2-dependent vascular homeostasis but not in inflammatory or physiologic retinal angiogenesis.
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