426-P: ROCK2 Regulates Monocytic Migration and Cell-to-Cell Adhesion in Vascular Endothelial Cells

Abstract

A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the RhoA/Rho-kinase signaling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). LPA levels are elevated in the circulation of patients with acute coronary syndrome and have also been detected inside human atherosclerotic specimens. However, the involvement of Rho-kinase isoforms (i.e., ROCK1, ROCK2) in LPA-mediated expression of chemokines and CAMs in endothelial cells is not fully understood. In this study, we investigated isoform-specific roles of Rho-kinase in LPA-mediated MCP-1 and E-selectin expression using human aortic endothelial cells (HAEC). Exposure of HAEC to LPA resulted in an increase of MCP-1 and E-selectin expression at both mRNA and protein levels. Rho-kinase inhibitor abolished MCP-1 and E-selectin protein expression, indicating that LPA induces MCP-1 and E-selectin expression via Rho-kinase activation. From a mechanistic standpoint, phosphorylation of IκBα, a well-characterized initial step in NF-κB activation, was decreased by Rho-kinase inhibition in the LPA-stimulated cells. Furthermore, using isoform specific siRNAs, we found that Rho-kinase isoform ROCK2 is the key mediator of LPA-induced MCP-1 and E-selectin expression. Consistently, ROCK2 but not ROCK1 controlled LPA-induced monocytic migration and adhesion in functional assays using HAECs and human monocytic cells (THP-1). In the present study, we demonstrate that endothelial ROCK2 mediates monocyte migration and cell adhesion induced by lipid mediator. In order to further explore endothelial-specific role of ROCK2 in vivo, we generated endothelial ROCK2 knockout mouse (EC-ROCK2KO) by mating of ROCK2 flox mice with VE-cadherin-cre mice. Further investigation using HFD (high-fat diet) mouse model is ongoing. ROCK2 could be an important therapeutic target against atherosclerosis. Disclosure Y. Takeda: None. K. Matoba: Research Support; Self; Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Sanofi, Shionogi & Co., Ltd., Takeda Pharmaceutical Company Limited. D. Kawanami: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Nagai: None. K. Utsunomiya: None. Funding Japan Society for the Promotion of Science; Uehara Memorial Foundation; MSD Life Science Foundation