Angiogenic Inhibitor Plasminogen Kringle 5 Down-Regulates the Expression of Vascular Endothelial Growth Factor in Müller Cells

Abstract

Abstract: : Purpose: Vascular endothelial growth factor (VEGF) plays an important role in retinal neovascularization. Müller cells are a major source of VEGF in the retina. Plasminogen kringle 5 (K5) is known to inhibit retinal neovascularization. This study was designed to determine if K5 regulates VEGF in Müller cells. Methods: A rat Müller cell line (rMC-1) was treated with cobalt chloride (CoCl₂ 200 µM) to induce VEGF expression and K5 was administered at different concentrations (50, 100, 200, 400 nM) in a serum-free medium. Western blot analysis was used to measure intracellular VEGF levels, and ELISA was used to measure secreted VEGF in the medium. The intracellular VEGF level was normalized by ß-actin and the secreted VEGF level by the total protein concentration. To determine the in vivo effect, rats with oxygen-induced retinopathy (OIR) received intravitreal injections of K5 with different doses (0.3, 1, 3, 10 µg/eye). Immunohistochemistry was performed to determine the expression of VEGF in the retinal Müller cells. Results: In the Müller cell line, CoCl₂ significantly increased VEGF levels both in the cells and in the medium. Western blot analysis and ELISA both showed that the expression of VEGF induced by CoCl₂ was inhibited by K5 in a concentration-dependent manner. In OIR rats, immunohistochemistry showed that K5 decreased the expression of VEGF in Müller cells. This in vivo effect also appeared to be K5 dose-dependent. Conclusions: These results indicate that K5 decreases the VEGF expression induced by CoCl₂ in a Müller cell line and in retinal Müller cells of OIR rats. The K5-induced down-regulation of VEGF in Müller cells may be responsible for its anti-angiogenic activity.

Keywords: hypoxia • Muller cells • growth factors/growth factor receptors