Rho/Rho-Kinase Pathway Contributes to C-Reactive Protein–Induced Plasminogen Activator Inhibitor-1 Expression in Endothelial Cells

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Abstract
Objective—Rho/Rho-kinase pathway plays pivotal roles in cardiovascular diseases including arteriosclerosis and hypertension. Recently it has become evident that C-reactive protein (CRP), a powerful marker for cardiovascular events, has direct proatherothrombotic effects on vascular cells. However, its molecular mechanism has not been fully investigated. We examined the involvement of Rho/Rho-kinase signaling in CRP-induced plasminogen activator inhibitor-1 (PAI-1) expression in bovine aortic endothelial cells (BAECs).Methods and Results—PAI-1 expression was determined by Western blotting. RhoA activation was determined by an affinity pull-down assay using Rho-binding fragment of rhotekin. NF-κB activity was determined using the luciferase reporter gene. Incubation of BAECs with human recombinant CRP (≥25 μg/mL) induced a significant increase in PAI-1 expression. Stimulation of BAECs with CRP significantly increased RhoA activation. Pretreatment with TAT-C3 (a membrane-permeable RhoA inhibitor) and Y-27632 (Rho-kinase inhibitor) significantly inhibited CRP-induced PAI-1 expression. NF-κB activity was markedly enhanced by CRP and pretreatment with Y-27632 inhibited its activation. Parthenolide, SN50, and BAY 11-7082 (NF-κB inhibitors) significantly blocked CRP-mediated PAI-1 expression.Conclusions—These data suggested that CRP activates Rho/Rho-kinase signaling, which in turn activates NF-κB activity, resulting in PAI-1 expression in BAEC. These observations provide evidence for the possible involvement of Rho/Rho-kinase signaling in CRP-induced atherothrombogenesis.