OBJECTIVE: To test the hypothesis that Hcy impairs angiogenic outgrowth through an iNOS-dependent mechanism. METHODS: Adult C57Bl/6 mouse choroid explants were used in angiogenic outgrowth assays. Mouse microvascular endothelial cells were studied in culture during scrape-induced migration and dispersed cell locomotion experiments. Activity of iNOS was manipulated with pharmacology (1400 W), siRNA, and by use of choroid explants from iNOS knockout mice (iNOS(-/-)). RESULTS: Hcy (20 μM) significantly decreased the area of endothelial outgrowth without altering the number of cells in the choroid explant angiogenic assay, resulting in more densely packed outgrowth. Hcy prevented the outward orientation of actin filaments and decreased the number of actin projections along the leading edge of outgrowth. Hcy also slowed outgrowth from the edge of a scraped endothelial monolayer and in cultures of dispersed cells, Hcy impaired cell locomotion without affecting proliferation. Inhibition of iNOS activity rescued the effect of Hcy on area of explant outgrowth, cell density, number of projections, cell locomotion, and rate of outgrowth following scraping. CONCLUSIONS: Hcy impairs microvascular endothelial outgrowth, but not proliferation, by disrupting cell locomotion through an iNOS-dependent mechanism.
OBJECTIVE: To test the hypothesis that Hcy impairs angiogenic outgrowth through an iNOS-dependent mechanism. METHODS: Adult C57Bl/6 mouse choroid explants were used in angiogenic outgrowth assays. Mouse microvascular endothelial cells were studied in culture during scrape-induced migration and dispersed cell locomotion experiments. Activity of iNOS was manipulated with pharmacology (1400 W), siRNA, and by use of choroid explants from iNOS knockout mice (iNOS(-/-)). RESULTS:Hcy (20 μM) significantly decreased the area of endothelial outgrowth without altering the number of cells in the choroid explant angiogenic assay, resulting in more densely packed outgrowth. Hcy prevented the outward orientation of actin filaments and decreased the number of actin projections along the leading edge of outgrowth. Hcy also slowed outgrowth from the edge of a scraped endothelial monolayer and in cultures of dispersed cells, Hcy impaired cell locomotion without affecting proliferation. Inhibition of iNOS activity rescued the effect of Hcy on area of explant outgrowth, cell density, number of projections, cell locomotion, and rate of outgrowth following scraping. CONCLUSIONS:Hcy impairs microvascular endothelial outgrowth, but not proliferation, by disrupting cell locomotion through an iNOS-dependent mechanism.
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