Angiopoietin–1 promotes pericyte survival and activation

Abstract

Abstract: : Purpose:To determine if the angiopoietin–1/Tie–2 system can play a role in the survival and activation of pericytes in the vasculature of the diabetic retina at varying stages of retinopathy. Methods:Expression of angiopoietin–1 (Ang–1) and Tie–2 in confluent cultures of bovine retinal pericytes was determined by immunoprecipitation and Western blotting. Annexin V–FITC flow cytometry was performed to determine the effect of Ang–1 on TNFα–induced apoptosis. Pericyte activation plays a critical role in the maturation of new vessels. The ability of Ang–1 to induce pericyte activation was determined by monitoring expression of Aminopeptidase N (a marker for activated pericytes) by western blotting. Tie–2 antisense treatment was used to verify that Ang–1 was acting through the Tie–2 receptor. Cell proliferation in response to Ang–1 was determined using the crystal violet assay. Observations were correlated with the immunolocalisation of Ang–1 and Tie–2 in human retinas with varying stages of retinopathy and post scatter photocoagulation surgery. Results:Tie–2 was expressed in both the lysates and supernatant from normal cultured pericytes. By contrast Ang–1 was not detected in either the supernatant or cell lysates. Ang–1 was capable of a dose–dependent inhibition of TNFα–induced apoptosis in retinal pericytes which could be blocked by knocking out the Tie–2 receptor. Ang–1 had no effect on retinal proliferation but did significantly, albeit weakly, promote pericyte migration. Aminopeptidase N expression in pericytes was increased by addition of angiopoietin–1 in a dose–dependent manner. Moreover, Tie–2–knockout treatment prevented Ang–1 activation of retinal pericytes. Immunostaining for Ang–1 was apparent in retinas from both non–diabetic and diabetic donors with staining intensity being highest in intraretinal vessels of retinas with proliferative diabetic retinopathy. After apparently successful laser treatment angiopoietin–1 staining was reduced. Tie–2 staining intensity appeared similar between non–diabetic and diabetic retinas irrespective of the stage of retinopathy. Conclusions:Ang–1 production by endothelial cells can promote pericyte survival in quiescent vessels. In addition, the Ang–1/Tie–2 system may play an important role in the activation and recruitment of pericytes towards newly–formed microvessels during the maturation of new vessels. Furthermore, the secretion of soluble Tie–2 may act to buffer the angiopoietin system.

Keywords: growth factors/growth factor receptors • vascular cells • neovascularization