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.

Figure 1.

miR-181b reduces thrombin-induced expression of adhesion molecules in ECs. A) HUVECs were transfected with 10 nM miRNA negative control (NS-m), or miR-181b mimics (181b-m); 50 nM miRNA inhibitor negative control (NS-i), or miR-181b inhibitor (181b-i) for 24 h. Cells were treated with thrombin (5 U/ml) or PBS for 4 h and harvested for quantitative PCR analysis; n = 3 per group. B) HUVECs were transfected with NS-m (10 nM), 181b-m (10 nM), NS-i (50 nM), or 181b-i (50 nM) for 24 h, treated with thrombin (5 U/ml) or PBS for 8 h, and harvested for Western blot analysis. C) Quantification of results from panel B; n = 3 independent experiments. Data are given as means ± sem. *P < 0.05.

Figure 2.

miR-181b inhibits thrombin-induced upstream NF-κB signaling in ECs. HUVECs were transfected with 10 nM NS-m or 181b-m. A) Cells were treated with thrombin (5 U/ml) for 1.5 h, and p65 expression was examined in the nuclear fraction by Western blot analysis; n = 3 independent experiments. B) Cells were treated with thrombin (5 U/ml) for 0, 30, 60, and 120 min and harvested for Western blot analysis of phospho-IKK-β, -IKK-γ, and -IκB-α expression. Ratios of phosphorylated protein to total protein were quantified; n = 3 independent experiments. Values are given as means ± sem. *P < 0.05.

Figure 3.

miR-181b directly targets Card10 and reduces its expression. A) Quantitative PCR analysis of Card10 expression in NS-m and 181b-m transfected HUVECs; n = 3 independent experiments. B) Western blot analysis of Card10 expression in NS-m and 181b-m transfected HUVECs and quantification of Card10 expression normalized by β-actin; n = 3 independent experiments. C) Luciferase reporter assays of Card10 3′-UTR in HUVECs; n = 3 independent experiments. D) Microribonucleoprotein immunoprecipitation analysis showed enrichment of Card10 mRNA in HUVECs transfected with NS-m or 181b-m; n = 3 per group. E, F) Quantitative PCR (E) and Western blot (F) analysis of NF-κB target genes in HUVECs transfected with pCMV-Card10 or control vector. Card10 overexpresssion blocked reduction of NF-κB target genes by miR-181b; n = 3 independent experiments. Ctl, control; N.S., not significant. Data are given as means ± sd. *P < 0.05.

Figure 4.

Card10 knockdown reduces thrombin-induced gene expression and NF-κB signaling in ECs. HUVECs were transfected with 30 nM control siRNA or Card10 siRNA. A) Cells were treated with thrombin (5 U/ml) for 8 h and harvested for Western blot analysis of VCAM-1 and ICAM-1. B) Expression of p65 and p50 in the nuclear fraction of HUVECs treated with thrombin (5 U/ml, 1.5 h) was examined by Western blot analysis; quantifications were normalized by USF-2; n = 3 independent experiments. C) Cells were treated with thrombin (5 U/ml) for 0, 30, 60, and 120 min and harvested for Western blot analysis of phospho-IKK-β, -IKK-γ, and -IκB-α expression. Ratios of phosphorylated protein to total protein were calculated. Ctl, control; USF-2, upstream transcription factor 2. Values are given as means ± sem; n = 3 independent experiments. *P < 0.05.

Figure 5.

Card10 knockdown has no effect on TNF-α–induced gene expression and NF-κB signaling in ECs. HUVECs were transfected with 30 nM control siRNA or Card10 siRNA. A) Cells were treated with TNF-α (10 ng/ml) for 8 h and harvested for Western blot analysis. Expression of VCAM-1 and ICAM-1 was normalized with β-actin. B) Expression of p65 and p50 in the nuclear fraction of HUVECs treated with TNF-α (10 ng/ml) for 4 h was examined by Western blot analysis. C, D) HUVECs were treated with TNF-α (10 ng/ml) for 0, 30, 60, and 120 min followed by Western blot analysis of phospho-IKK-β and -IκB-α expression. Ratios of phosphorylated protein to total protein were calculated. Ctl, control; N.S., not significant; USF-2, upstream transcription factor 2. Values are given as means ± sem; n = 3 independent experiments.

Figure 6.

Systemic delivery of miR-181b or Card10 siRNA delays development of thrombosis in vivo. C57BL/6 mice were administered with NS-m, 181b-m, control siRNA, or Card10 siRNA by tail-vein injection on 3 consecutive days. Photochemical injury was performed to induce development of thrombosis in carotid arteries. A) Occlusion times of the carotid arteries in NS-m– or miR-181b–treated mice (left). Two representative curves show blood flow of carotid arteries after injury in NS-m– and miR-181b–treated mice, respectively (right). Values are given as means ± sem; n = 8 per group. B) Thrombus formation revealed by hematoxylin and eosin staining of cross-sections of injured carotid arteries. Quantifications show ratios of thrombus to lumen areas. Data represent means ± sem; n = 4–5 per group. C) Frozen sections of carotid arteries were stained for anti-Card10 (red) and CD31 (green). Arrows indicate differential Card10 expression in ECs. Card10 expression was quantified in vascular ECs reflecting NS-m (n = 28 ECs) and 181b-m (n = 52 ECs), respectively. D) Occlusion times of carotid arteries in control siRNA– or Card10 siRNA–treated mice (left). Two representative curves show blood flow of carotid arteries after injury in control siRNA– and Card10 siRNA–treated mice, respectively (right). Data are given as means ± sem; n = 9–10 for each group. E) Schema of ligand-specific regulation of NF-κB signaling by miR-181b. In response to TNF-α, miR-181b inhibits importin-α3, an effect that inhibits downstream cytoplasmic-to-nuclear NF-κB accumulation in vascular endothelium during sepsis and atherosclerosis. In contrast, in response to thrombin, miR-181b inhibits Card10, an effect that inhibits upstream NF-κB signaling, activation of the IKK complex, and arterial thrombosis. Ctl, control. *P < 0.05.