Abstract 5574: Cooperation of MT1-MMP and LOX-1 for RhoA- and Rac1-dependent Signaling Pathways in Oxidized LDL-mediated Endothelial Dysfunction and Atherosclerosis
Background: Membrane type 1-matrix metalloproteinase (MT1-MMP) functions not only as a proteolytic enzyme but also as a signaling molecule. Lectin-like oxidized-LDL receptor-1 (LOX-1) is a major receptor for oxidized LDL (ox-LDL). RhoA and Rac1 play crucial roles in endothelial dysfunction including eNOS downregulation and NADPH oxidase-dependent reactive oxygen species (ROS) generation. Hypothesis: MT1-MMP modifies ox-LDL-triggered RhoA- and Rac1-dependent endothelial dysfunction and atherosclerosis.
Methods: GTP-loading of RhoA and Rac1 were assessed by pull-down assays. The activity of MT1-MMP was evaluated by fluorescent assay. A neutral antibody to LOX-1 (TS92) was used for inhibition of LOX-1, while MT1-MMP was silenced by siRNA. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence. Fluorescent immunohistochemistry and immunoprecipitation were performed using the cultured endothelial cells (ECs) and the aortae of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits.
Results: Ox-LDL activated RhoA and Rac1 within 5 and 15 minutes, as well as the MT1-MMP activity of membrane fractions after 15 minutes in ECs, which was inhibited by TS92. Ox-LDL-induced activation of RhoA and the downstream events including endothelial cell invasion and eNOS downregulation were blocked by silencing of MT1-MMP. Knockdown of MT1-MMP also markedly attenuated ox-LDL-triggered Rac1 activation, NADPH oxidase activity and ROS generation. Interestingly, LOX-1 and MT1-MMP were partly co-localized in ECs by fluorescent immunohistochemistry, and immunoprecipitation demonstrated that MT1-MMP formed a complex with LOX-1. Furthermore, immunohistochemistry revealed that the two molecules were co-localized in ECs and in the aortic atherosclerotic lesions of WHHLMI rabbits. In addition, immunoprecipitation demonstrated that MT1-MMP and LOX-1 formed a complex in cultured ECs and in the atherosclerotic aortae of WHHLMI rabbits.
Conclusions: We show new insights into modulation of endothelial dysfunction and atherosclerosis by an MT1-MMP-LOX-1 axis, suggesting that MT1-MMP is a cell surface modifier of ox-LDL-triggered RhoA and Rac1-activated signaling pathways through LOX-1.