Paul McGuire

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Purpose: Hepatocyte growth factor (HGF), acting through its receptor, c-met, is a cytokine whose role in promoting cell migration has been extensively documented. HGF and c-met have been implicated in the progression of angiogenesis in many cancer models and in the mouse model of retinal neovascularization. However, HGF mediation of protease expression and of an endothelial cell migratory phenotype in the retina has not been explored. We determined if HGF and/or hypoxia effects cell migration and protease levels in retinal microvascular endothelial cells and in a mouse model of retinal neovascularization. Methods: Bovine retinal microvascular endothelial cells (BRMVEC) were grown to near confluence and then stimulated with HGF (10ng/ml and 30 ng/ml) under normoxic and/or hypoxic conditions. At varying time points, conditioned media and cell extracts were collected and analyzed by zymography or by reverse-transcription PCR. Stable hypoxic conditions were achieved by adding 100\uf06dM CoCl2 to the cell media. Migration assays were performed by growing BRMVECs to confluence with or without HGF and wounding the monolayer. After 24 hours, the amount of cellular migration into the wound was analyzed. Retinal neovascularization was induced in newborn mice by exposure to 75% oxygen followed by room air. Eyes were collected at day 17 following removal from high oxygen, fixed in 10% formalin and embedded in paraffin. Sections were cut, and immunohistochemistry was performed to localize retinal c-met expression. Results: After 24 hours of HGF stimulation there is a hypoxia-dependent, and dose-dependent increase in the 54 kDa form of urokinase in endothelial cells. This expression pattern correlated with the PCR analysis of uPA and uPAR mRNA. There is also a hypoxia-dependent and dose dependent increase in the active form of MMP 2. The wound assay demonstrated that HGF significantly increased BRMVEC cell migration compared to control samples. Immunohistochemical staining showed localized c-Met expression in the retina, which was increased in experimental animals compared to control. Conclusion: HGF, acting through its receptor c-met, may play an important role in the initial stages of retinal angiogenesis by stimulating a migratory phenotype and endothelial cell protease production.'

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