MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10

Abstract

Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB 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 time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA-dependent targets in response to ligand-specific signaling in ECs.-Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Keywords: Card10; NF-κB; endothelial cells.