DOWN–REGULATION OF PLASMINOGEN KRINGLE 5 RECEPTOR IN MÜLLER CELLS UNDER HYPOXIA AND IN THE DIABETIC RETINA

Abstract

Abstract: : Purpose:Plasminogen kringle 5 (K5) down–regulates an endogenous angiogenic stimulator, vascular endothelial growth factor (VEGF) and up–regulates an angiogenic inhibitor, pigment epithelium–derived factor (PEDF) in endothelial cells and retina. The purpose of this study is to determine whether the K5 effect is mediated by the K5 receptor (voltage–dependent anion channel 1, VDAC1) and to measure the expression of the K5 receptor in hypoxia and diabetes. Methods: K5 binding and competition assays were performed by incubating rat Müller cells with ¹²⁵I–labeled and un–labeled K5. To induce hypoxia, Müller cells were treated with cobalt chloride (CoCl₂ 200 mM) or cultured in a chamber with 5% oxygen. The expressions of the K5 receptor in Müller cells under hypoxia and in the retina from rats with oxygen–induced retinopathy (OIR) and streptozotocin (STZ)–induced diabetes were analyzed by quantitative real–time reverse transcription PCR. Results:The binding assay showed that K5 bound to Müller cells in a dose–dependent manner and the binding is saturable at 100 nM of ¹²⁵I–K5. ¹²⁵I–K5 binding to Müller cells was inhibited by increasing un–labeled K5 concentration, but not by angiostatin (kringle1–4). The mRNA levels of the K5 receptor in normal rat retina and untreated Müller cells were higher than those of GAPDH. The 10–hour treatment with CoCl₂ and low concentration oxygen significantly reduced the expression of the K5 receptor in Müller cells (to approximately 20% of normal control levels). The K5 receptor mRNA levels in the retina were decreased by 50% in rats with OIR and in those with STZ–diabetes, compared to age–matched normal controls. Conclusion: Müller cells contain high levels of the K5 receptor on the membrane. The K5 receptor expression is down–regulated by hypoxia, which may contribute to retinal neovascularization in diabetes.

Keywords: receptors • retinal neovascularization • Muller cells