Abstract
Abstract: :
Purpose: Vascular endothelial growth factor (VEGF)–mediated retinal angiogenesis includes both VEGF induction and transduction signaling cascades. To date, much more effort has been spent on understanding and intervening in VEGF downstream signaling than on the upstream induction pathway. In order to study the mechanism of retinal VEGF induction by ischemia–induced hypoxia, we have established an in vitro model of VEGF induction in Müller cell cultures. Methods: Primary and transformed, immortalized Müller cells were exposed to a range of hypoxic conditions. VEGF production in culture media was quantified with colorimetric ELISA assay. Immunocytochemical staining was used to confirm cell identity and glial fibrillary acidic protein (GFAP) expression. Results: Exposure to an oxygen concentration of ≤2% for 24 hours consistently and significantly induced VEGF protein production by two–fold in primary Müller cells. This VEGF induction was further increased by longer exposure periods, and it was inhibited by a Src–selective inhibitor, PP2. Only mild impact on cell growth or death was observed. The hypoxia treatment also induced a nearly six–fold induction of VEGF production in rMC–1 cultures, however, there was resistance to PP2 inhibition. Cell growth retardation and death were also substantial in rMC–1. Both primary Müller cells and rMC–1 showed no increase of GFAP expression after 24 hours of hypoxia treatment. Conclusions: Primary rat Müller cells can be used as an in vitro model to study the VEGF induction pathway in ischemia–induced retinal angiogenesis. rMC–1 is a potential candidate as well, particularly due to its substantial response of VEGF induction to hypoxia exposure, although the fragility of the cells must be accommodated. This model will facilitate elucidation of the VEGF induction pathway and development of future therapeutic strategies aimed at limiting VEGF levels in hypoxic tissues.
Keywords: Muller cells • hypoxia • retinal neovascularization