In adult life, takes place generally from preexisting vessels, in direct response to tissue demands, by true Alvocidib Flavopiridol sprouting or by splitting angiogenesis. It is now known that endothelial progenitor cells mobilize from the bone marrow in response to a variety of signalling molecules and can target sites of angiogenesis in ischaemic peripheral vasculature, myocardium or induced ocular injury. In healthy adults, angiogenesis does not normally occur, except during the female ovarian cycle. However, neo angiogenesis may occur in several pathophysiological conditions, including wound healing, chronic inflammatory diseases and solid tumours. Endothelial cell proliferation, adhesion and migration are early essential events for mediating angiogenesis.
It is well documented Danusertib that the induction of endothelial cell proliferation, migration and adhesion in response to numerous intracellular or extracellular stimuli is a tightly regulated process requiring the coordination of a complex set of inward and outward signals involving the ECM, the integrins and the actin cytoskeleton associated molecules. We have previously reported that baicalein exhibited a strong antiproliferative effect in rat heart endothelial cells. In this study, we investigated the effect of baicalein on endothelial cell migration and adhesion. Since cell migration and adhesion are associated with regulation of actin dynamics and cell surface integrins, the intracellular cytoskeletal architecture and surface receptor integrins were also investigated.
We found that baicalein upregulated the expression of the integrins, a5b1 and avb3, and of vinculin, which mediated intracellular signalling through interaction with fibronectin and vitronectin, and promoted the reorganization of actin fibres and increased focal contact formation and adhesion, as well as reducing migration of endothelial cells. Methods Cell culture Endothelial cells were isolated from rat heart as described previously. Cells were maintained in minimal essential medium with 10% foetal bovine serum, antibiotics and 50 mgml 1 endothelial cell growth supplements. Endothelial cells were identified by their typical cobblestone appearance and CD31 immunostaining. Cell migration assay Migration assays were performed following two standard protocols using a wound repair assay and Transwell chemotaxis chambers.
The mechanical injury of confluent endothelial cells and lesion repair assay were performed as described elsewhere. Briefly, confluent endothelial cells were wounded by scraping with a pipette tip, denuding a strip of the monolayer. Cultures were washed twice with phosphate buffered saline and incubated with serumcontaining medium supplemented with the baicalein or vehicle, as indicated. The rate of wound closure was measured and photographed over 48 h. The progression of cell migration was assessed with a calibrated ocular grid. The modified Boyden,s chamber assay was performed by using cell culture inserts composed of a porous 8 mm membrane. Briefly, baicaleinpretreated or untreated rat heart endothelial cells were washed and trypsinized to induce cell detachment. Cells were then suspended in 50 ml of serum free medium, with or without baicalein, and seeded in the upper compartment. Vascular endothelial growth fac