Ftsz Inhibitor”]

On day cells were washed and received medium, free of genistein containing buffer only.Duplicate wells were counted at the indicated times.In lowdensity cultures of proliferating endothe lial cells, genistein induced marked morphological changes: at concentrations up to jimolL, genistein induced a highly spread morphology compat ible with growth arrest; cell densities always ex ceeded those determined at seeding, indicating that cell death was not involved.When exposed to genistein concentrations above mol L, the cells acquired an elongated morphology, and eventually died. Reversibility experiments, in which lowden sity cultures of endothelial cells were exposed to bFGF, increasing concentrations of genistein, and medium without genistein, confirmed this assumption. The result was the same on uncoated and gelatin coated substrata.In con trast, confluent, quiescent endothelial cells did not exhibit toxicity signs even at genistein concentra tions up to tmolL. These data clearly suggest that genistein targets only proliferating cells leaving quiescent, nondividing cells unaffected.This property is important with respect to possible use of the compound in therapeutic applications, as fewer side effects are likely to be expected.Angiogenesis is a complex process requiring the coordinated, sequential involvent of a number of cellular events other than prolif eration.Indeed, formation of new capillaries begins with a localized breakdown of the basement membrane of the parent vessel, through the finely tuned elabo ration of Bacitracin proteolytic enzymes and their inhibitors, followed by migration of endothelial cells and invasion of the surrounding matrix.The initial sprout further elongates as a result of continued migration and endothelial cell replica tion.Once a lumen has been formed, the capillary fuses with the tip of another maturing sprout forming a functional capillary loop. Inhibition of pro duction of proteolytic enzymes and migration of en dothelial cells by genistein, therefore, represents a more complex interference of the compound with im portant early events of angiogenesis other than en dothelial cell proliferation.The combined effects of genistein on proliferation, proteolytic enzyme production and migration on en dothelial cells prompted us to investigate the effects of the compound on an experimental in vitro system that mimics angiogenesis in vivo.As previously shown invade the gels when exposed to bFGF and form capillary like tubes beneath the gel surface. Genistein alone at umol L concentrations had no effect on confluent BME cul tures. However, when added together with bFGF, it inhibited their ability to invade the gels and generate capillary like structures. BME cells seeded at X IO per mL received basic fibroblast growth factor concentrations of genistein. When seeded at a density of X mL, BME cells were first allowed to grow to confluence and then received either. Though we have not addressed the mechanism of action of genistein, there are several known properties of genistein pointing to ward the possible molecular events affected by it.Indeed, genistein has been shown to be a competitive inhibitor of ATP binding to the catalytic domain of tyrosine Atazanavir sulfate kinases receptor tyrosine kinase activities both in intact cells and in vitro.This appears as an attractive hypothesis, because highaf finity FGF receptors are tyrosine kinases and because vanadate, an inhibitor of phosphotyrosine phosphatases induces angiogenesis in the collagen assay.

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