Abstract-Nitric oxide generated by endothelial nitric oxide synthase (eNOS) plays an important role in maintaining cardiovascular homeostasis. Under various pathological conditions, abnormal expression of eNOS contributes to endothelial dysfunction and the development of cardiovascular diseases. A variety of pathological stimuli has been reported to decrease eNOS expression mainly through decreasing eNOS mRNA stability by regulating the binding of several cytosolic proteins to the cis-acting sequences within eNOS mRNA 3′ untranslated regions. However, the detailed mechanisms remain elusive. Because microRNAs inhibit gene expression through binding to the 3′ untranslated regions of their target mRNAs, microRNAs may be the important posttranscriptional modulators of eNOS expression. Here, we provided evidence that eNOS is a direct target of miR-155. Overexpression of miR-155 decreased, whereas inhibition of miR-155 increased, eNOS expression and NO production in human umbilical vein endothelial cells and acetylcholineinduced endothelium-dependent vasorelaxation in human internal mammary arteries. Inflammatory cytokines including tumor necrosis factor-α increased miR-155 expression. Inhibition of miR-155 reversed tumor necrosis factor-α-induced downregulation of eNOS expression and impairment of endothelium-dependent vasorelaxation. Moreover, we observed that simvastatin attenuated tumor necrosis factor-α-induced upregulation of miR-155 and ameliorated the effects of tumor necrosis factor-α on eNOS expression and endothelium-dependent vasodilation. Simvastatin decreased miR-155 expression through interfering mevalonate-geranylgeranyl-pyrophosphate-RhoA signaling pathway. These findings indicated that miR-155 is an essential regulator of eNOS expression and endothelium-dependent vasorelaxation.Inhibition of miR-155 may be a new therapeutic approach to improve endothelial dysfunction during the development of cardiovascular diseases.

Background-The Ca 2ϩ -activated chloride channel (CaCC) plays an important role in a variety of physiological functions. In vascular smooth muscle cells, CaCC is involved in the regulation of agonist-stimulated contraction and myogenic tone. The physiological functions of CaCC in blood vessels are not fully revealed because of the lack of specific channel blockers and the uncertainty concerning its molecular identity. Methods and Results-Whole-cell patch-clamp studies showed that knockdown of TMEM16A but not bestrophin-3 attenuated CaCC currents in rat basilar smooth muscle cells. The activity of CaCC in basilar smooth muscle cells isolated from 2-kidney, 2-clip renohypertensive rats was decreased, and CaCC activity was negatively correlated with blood pressure (nϭ25; PϽ0.0001) and medial cross-sectional area (nϭ24; PϽ0

Recent evidence suggested that ClC-3 channel/antiporter is involved in regulation of nuclear factor (NF)-κB activation. However, the mechanism explaining how ClC-3 modulates NF-κB signaling is not well understood. We hypothesized that ClC-3-dependent alteration of intracellular chloride concentration ([Cl(-)](i)) underlies the effect of ClC-3 on NF-κB activity in endothelial cells. Here, we found that reduction of [Cl(-)](i) increased tumor necrosis factor-α (TNFα)-induced expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and adhesion of monocytes to endothelial cells (P<0.05; n=6). In Cl(-) reduced solutions, TNFα-evoked IκB kinase complex β and inhibitors of κBα phosphorylation, inhibitors of κBα degradation, and NF-κB nuclear translocation were enhanced. In addition, TNFα and interleukin 1β could activate an outward rectifying Cl(-) current in human umbilical vein endothelial cells and mouse aortic endothelial cells. Knockdown or genetic deletion of ClC-3 inhibited or abolished this Cl(-) conductance. Moreover, Cl(-) channel blockers, ClC-3 knockdown or knockout remarkably reduced TNFα-induced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocytes to endothelial cell adhesion, and NF-κB activation (P<0.01; n=6). Furthermore, TNFα-induced vascular inflammation and neutrophil infiltration into the lung and liver were obviously attenuated in ClC-3 knockout mice (P<0.01; n=7). Our results demonstrated that decrease of [Cl(-)](i) induced by ClC-3-dependent Cl(-) efflux promotes NF-κB activation and thus potentiates TNFα-induced vascular inflammation, suggesting that inhibition of ClC-3-dependent Cl(-) current or modification of intracellular Cl(-) content may be a novel therapeutic approach for inflammatory diseases.

MicroRNAs have emerged as important post-transcriptional regulators of gene expression and are involved in diverse diseases and cellular process. Decreased expression of miR-181a has been observed in the patients with coronary artery disease, but its function and mechanism in atherogenesis is not clear. This study was designed to determine the roles of miR-181a-5p, as well as its passenger strand, miR-181a-3p, in vascular inflammation and atherogenesis. We found that the levels of both miR-181a-5p and miR-181a-3p are decreased in the aorta plaque and plasma of apoE −/− mice in response to hyperlipidemia and in the plasma of patients with coronary artery disease. Rescue of miR-181a-5p and miR-181a-3p significantly retards atherosclerotic plaque formation in apoE −/− mice. MiR-181a-5p and miR-181a-3p have no effect on lipid metabolism but decrease proinflammatory gene expression and the infiltration of macrophage, leukocyte and T cell into the lesions. In addition, gain-of-function and loss-of-function experiments show that miR-181a-5p and miR-181a-3p inhibit adhesion molecule expression in HUVECs and monocytes-endothelial cell interaction. MiR-181a-5p and miR-181a-3p cooperatively receded endothelium inflammation compared with single miRNA strand. Mechanistically, miR-181a-5p and miR-181a-3p prevent endothelial cell activation through blockade of NF-κB signaling pathway by targeting TAB2 and NEMO, respectively. In conclusion, these findings suggest that miR-181a-5p and miR-181a-3p are both antiatherogenic miRNAs. MiR-181a-5p and miR-181a-3p mimetics retard atherosclerosis progression through blocking NF-κB activation and vascular inflammation by targeting TAB2 and NEMO, respectively. Therefore, restoration of miR-181a-5p and miR-181a-3p may represent a novel therapeutic approach to manage atherosclerosis.