100 cells for each treatment were imaged at random and analysed by image analysis software. Results showed that the number of focal adhesion contacts per cell markedly increased in baicalein treated cells compared to untreated PD-183805 CI-1033 cells. This result is consistent with the increased cellular adhesion described above. It is well documented that integrins are present in focal contacts in cells. Next, we carried out double labelled immunofluorescent experiments to examine the distribution of vinculin compared to integrin avb3, avb5 and integrina5b1. In untreated endothelial cells, integrin av and a5 staining was generally restricted to distinct spots. When endothelial cells were exposed to baicalein, integrin staining was detected throughout the cell.
However, in both untreated and baicalein treated endothelial cells, av and a5 appeared to colocalize with vinculin. The expressed levels of vinculin, av and a5 present in focal adhesions in endothelial cells exposed to baicalein appeared to increase. These data indicate that the initial baicalein induced formation of focal adhesion might be due SGX-523 to the increase in the cellular levels of integrins and vinculin expression. Effects of LOX products on baicalein mediated endothelial proliferation, adhesion and migration It has been reported that baicalein is a potent inhibitor of 5, 12, and 15 lipooxygenase. To address whether the LOX metabolic pathway was involved in these baicalein mediated endothelial responses, cells were exposed to baicalein in the absence or presence of exogenously added LOX metabolites, 5 HETE, 12 HETE, and 15 HETE.
Modulation of endothelial cell proliferation, adhesion and migration by these HETEs was then assessed. Over a range of concentrations the HETEs exhibited concentration related effects on the baicalein mediated biological responses. We chose to use 100 nM as the test concentration for our further studies. At this concentration, treatment with each HETE alone significantly increased endothelial cell proliferation, adhesion and migration. However, exogenous 5 HETE, 12 HETE, and 15 HETE could partially reverse the inhibitory effects of baicalein on endothelial proliferation and migration, and slightly suppress baicalein induced endothelial adhesion. Discussion Baicalein is a relatively selective 12 LOX inhibitor that also possesses many LOX unrelated effects such as blocking calcium mobilization and acts as an antioxidant.
Our previous study demonstrated that baicalein markedly inhibited proliferation of rat heart endothelial cells. In the present study, we show that pretreatment of rat heart endothelial cells with baicalein for 48 h significantly enhanced cell adhesion while suppressing cell migration. Short term baicalein treatment of rat endothelial cells, however, had no effect on migration and adhesion, suggesting that longer exposure to baicalein is essential for its stimulation of adhesion and its antimigratory actions. Our observations also indicate that baicalein exhibits slight effects on quiescent endothelial cells, but has significant effect on endothelial cells activated by serum or VEGF. A previous study demonstrated that phenylephrine induced cell proliferation and migration was inhibited by baicalein in vascular smooth muscle cells. This report suggests that