Role of Ca^<2+> signaling and reactive oxygen species in endothelial dysfunction induced by lysophosphatidylcholine

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Lysophosphatidylcholine (LPC) is an atherogenic compound of oxidized low density lipoprotein which evokes endothelial dysfunction. In this research project, we investigated the roles of Ca^<2+> and reactive oxygen species (ROS) in signal transduction induced by LPC in endothelial cells (Ecs).LPC is known to increase intracellular Ca^<2+> concentration ([Ca^<2+>]_i) Ecs. [Ca^<2+>]_i was determined in cultured human aortic Ecs by fura-2 assay and membrane current was measured by whole-cell patch-clamp. HMG-CoA reductase inhibitors (statins) markedly decreased the [Ca^<2+>]_i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho-kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly activates RhoA. Statins prevented the GTP/GDP excha … More nge of RhoA and its membrane translocation from the cytoplasm caused by LPC and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca^<2+> mobilization. LPC also induced a nonselective cation current (NSC) after a lag. Statins prolonged the lag and decreased the current amplitude and GGPP abolished the inhibitory effect of statins on the current. In summary of this part, LPC induced Ca^<2+> mobilization and membrane current via a Rho activation-dependent PLC pathway in Ecs and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca^<2+> movement (Yokoyama K, Ishibashi T et al. Circulation. 2002 ; 105 : 962-967). In addition, the effect of LPC on membrane current and its signaling were examined in cardiomyocytes (Li L et al. Mol Pharmacol. 2002 : 62 ; 602-607).We also investigated the association of ROS with Ca^<2+> influx in LPC stimulation. The [Ca^<2+>]_i increase caused by LPC was suppressed by radical scavengers such as superoxide dismutase (SOD) and catalase. In fact, we detected the generation of hydrogen peroxide (H_2O_2) 60 seconds after LPC stimulation. When external Ca^<2+> was present, the suppressive effect of catalase was greater than that of SOD. Catalase, but not SOD, suppressed the amplitude of NSC induced by LPC. H_2O_2 indeed induced NSC in Ecs. We show that in LPC stimulation H_2O_2 contributes to NSC in Ecs, suggesting that H_2O_2 generated by LPC may play a role in Ca^<2+> signaling mediated via NSC.Furthermore, we studied the role of RhoA activation in the gene expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in human macrophages (Atherosclerosis. 2002 ; 163 : 39-47, BBA. 2002 ; 1590 : 123-130). Less