Abstract 12208: Microrna-181b Inhibits Thrombin-mediated Activation of Endothelial Cells and Arterial Thrombosis by Targeting Card10 (Best of Basic Science Abstract)

Abstract

Objective: Thrombogenic and inflammatory mediators such as thrombin induce NF-kB-mediated EC activation and dysfunction, which contribute to the pathogenesis of arterial thrombosis. The role of the anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Methods and Results: It has been demonstrated that miR-181b inhibits downstream NF-kB signaling in response to TNF-a. Herein, we reveal that miR-181b can inhibit upstream NF-kB signaling uniquely in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-kB signaling demonstrated by the reduction of phospho-IKK-b, -IkB-a, and p65 nuclear translocation in ECs. MiR-181b also reduced the expression of NF-kB target genes VCAM-1, ICAM-1, E-selectin, PAI-1, and tissue factor. Mechanistically, miR-181b targets Card10, an adaptor protein that participates in the activation of the IKK complex in response to signals transduced from G-protein coupled receptors such as PAR-1. MiR-181b reduced the expression of Card10 mRNA and protein, but not PAR-1. In addition, miR-181b inhibited Card10 3’-UTR luciferase reporter activity, and Argonaute-2 miRNP-IP studies revealed 4-fold enrichment of Card10 mRNA in the RNA-induced silencing complex in the presence of miR-181b, indicating that Card10 is a bona fide direct target. SiRNA-mediated knockdown of Card10 expression phenocopied miR-181b’s effects on NF-kB signaling and targets. Interestingly, knockdown of Card10 does not affect TNF-a-induced activation of NF-kB signaling and target gene expression, suggesting stimulus-specific regulation of NF-kB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries, and prolonged the time to occlusion by 1.6-fold. Conclusions: MiR-181b is an important regulator of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of miRNA-dependent targets in response to ligand-specific signaling in ECs. Delivery of miR-181b or Card10 inhibition may represent a new therapeutic approach to reduce arterial thrombosis by improving vascular EC function.