| The endothelium is a multifunctional disseminated tissue, which lines the entire vascular system. Its structural and functional integrity is fundamental to vessel wall homeostasis. Endothelial cell (EC) dysfunction, including apoptosis and activation, is critical in the pathogenesis of diverse vascular diseases. My research has focused on the pathways regulating EC survival and activation in order to understand the mechanisms by which ECs are activated or undergo apoptosis. Endothelial cells are anchorage-dependent cells and their interaction with extracellular matrix (ECM) is fundamental for their survival. This adhesion-dependent survival is mediated by integrin receptors, which interact with both ECM proteins and cytoskeletal proteins. The small G-protein RhoA and its effector RhoA-kinase are key regulators of integrin-mediated stress fiber formation and focal adhesion. Protein prenylation is necessary for RhoA function. Therefore, we hypothesized that the RhoA/RhoA-kinase pathway promotes EC survival and that inhibition of RhoA signaling would induce EC apoptosis. We showed that inhibition of protein prenylation by the HMG-CoA reductase inhibitor, lovastatin, or the geranylgeranyltransferase inhibitor, GGTI-298, induced EC death via caspase-3-dependent apoptosis. Specific inhibition of RhoA pathway by C3 exoenzyme or RhoA-kinase inhibitors also caused apoptotic cell death. Inhibition of RhoA pathway did not inhibit endogenous survival pathways, such as NF-κB, ERK, or PI3-kinase/Akt. However, the proapoptotic protein, p53, was induced by blockade of RhoA signaling, suggesting that death was mediated by a p53-dependent apoptotic signaling. PI3-kinase and nuclear factor κB (NF-κB) are two important intracellular signaling pathways that mediate diverse cellular functions, including activation and survival. The signaling pathways by which PI3-kinase is activated have not been fully elucidated, and the role of PI3-kinase in NF-κB activation by Toll-like receptors (TLRs) has not been determined. We showed that inhibition of PI3-kinase by dominant-negative p85 subunit inhibited NF-κB luciferase activity, IκBα degradation and IL-6 production induced by TLR agonists, lipopolysaccharide (LPS) and interleukin-1 (IL-1). Activation of PI3-kinase and NF-κB by LPS and IL-1 was mediated by TLR adapter proteins MyD88 and IRAK-1. These findings provide new information on the regulation of EC survival and activation by inflammatory stimuli. |
