The role of calcium in the regulation of ion channels, downstream signaling, and gene expression in endothelial cells has been under investigation. CAI, an inhibitor of nonvoltage-gated calcium influx, has been used as a tool to dissect calcium-mediated signaling. We have focused on the role of calcium on the regulation of endothelial cell function and have demonstrated that CAI inhibits endothelial cell proliferation, adhesion, motility, and collagenase expression. Intracellular free cytosolic calcium increases after attachment to type IV collagen, during the spreading process. This is regulated by changes in intracellular calcium due to calcium influx and we have shown that this calcium influx is necessary at the level of RhoA activation for formation of actin stress fibers. We have linked our calcium-regulation of focal contact formation with the stress fiber observations by demonstration of a calcium influx sensitive partnership between FAK and p190RhoGAP. A different approach was taken to identify other genes involved in angiogenesis in vitro through the study investigated gene expression in HUVEC cells undergoing tubular reorganization on Matrigel. cDNA microarray analysis identified several differentially expressed genes, as well expression of expected angiogenesis-associated genes. One gene, caldesmon, interacts with the RhoA pathway and may be a downstream target of calcium influx regulation. Studies are planned to investigate this link. Further studies are ongoing to expand our dissection of the signaling events occurring during endothelial cell spreading on basement membrane collagen. We have further found that endothelial cells respond to calcium as a ligand with generation of IP3 and mobilization of intracellular calcium. Cotreatment with VEGF caused a synergistic signaling effect. However, the proliferative signal of calcium or VEGF as independent ligands was signficantly abrogated by co-exposure to VEGF and calcium. Studies are ongoing to dissect the pathways and functions of this calcium activity.
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