Jia-Guo Zhou

Essential Role of MicroRNA-155 in Regulating Endothelium-Dependent Vasorelaxation by Targeting Endothelial Nitric Oxide Synthase

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 acetylcholine-induced endothelium-dependent vasorelaxation in human internal mammary arteries. Inflammatory cytokines including tumor necrosis factor-&agr; increased miR-155 expression. Inhibition of miR-155 reversed tumor necrosis factor-&agr;–induced downregulation of eNOS expression and impairment of endothelium-dependent vasorelaxation. Moreover, we observed that simvastatin attenuated tumor necrosis factor-&agr;–induced upregulation of miR-155 and ameliorated the effects of tumor necrosis factor-&agr; 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.

Downregulation of TMEM16A Calcium-Activated Chloride Channel Contributes to Cerebrovascular Remodeling During Hypertension by Promoting Basilar Smooth Muscle Cell Proliferation

Background— The Ca2+-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 (n=25; P<0.0001) and medial cross-sectional area (n=24; P<0.0001) in basilar artery during hypertension. Both upregulation of CaMKII activity and downregulation of TMEM16A expression contributed to the reduction of CaCC in the hypertensive basilar artery. Western blot results demonstrated that angiotensin II repressed TMEM16A expression in basilar smooth muscle cells (n=6; P<0.01). Knockdown of TMEM16A facilitated and overexpression of TMEM16A inhibited angiotensin II–induced cell cycle transition and cell proliferation determined by flow cytometry and BrdU incorporation (n=6 in each group; P<0.05). TMEM16A affected cell cycle progression mainly through regulating the expression of cyclin D1 and cyclin E. Conclusions— TMEM16A CaCC is a negative regulator of cell proliferation. Downregulation of CaCC may play an important role in hypertension-induced cerebrovascular remodeling, suggesting that modification of the activity of CaCC may be a novel therapeutic strategy for hypertension-associated cardiovascular diseases such as stroke.

Regulation of Intracellular Cl- Concentration through Volume-regulated ClC-3 Chloride Channels in A10 Vascular Smooth Muscle Cells

We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented rat aortic smooth muscle cell proliferation, which was related to cell volume regulation. In the present study, we further characterized the regulation of intracellular Cl- concentrations ([Cl-]i) via volume-regulated ClC-3 Cl- channels in an embryo rat aortic vascular smooth muscle cell line (A10 cell) and ClC-3 cDNA-transfected A10 cells (ClC-3-A10) using multiple approaches including [Cl-]i measurement, whole cell patch clamp, and application of ClC-3 antisense and intracellular dialysis of an anti-ClC-3 antibody. We found that hypotonic solution decreased [Cl-]i and evoked a native ICl.vol in A10 cells. The responses of [Cl-]i and ICl.vol to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (ICl.vol) and in ClC-3-A10 cells (ICl.ClC-3) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl-]i and activation of ICl.vol and ICl.ClC-3 in A10 and ClC-3-A10 cells, respectively, were significantly inhibited by activation of protein kinase C (PKC) by phorbol-12,13-dibutyrate (PDBu) and inhibition of tyrosine protein kinase by genistein. Sodium orthovanadate (vanadate), a protein-tyrosine phosphatase inhibitor, however, enhanced the cell swelling-induced reduction in [Cl-]i, accompanied by the activation of ICl.vol and ICl.ClC-3 in a voltage-independent manner. Our results suggest that the volume-regulated ClC-3 Cl- channels play important role in the regulation of [Cl-]i and cell proliferation of vascular smooth muscle cells.

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G1/S phase arrest in rat basilar arterial smooth muscle cells

Abstract.  Objectives: Previously, we have found that the ClC‐3 chloride channel is involved in endothelin‐1 (ET‐1)‐induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC‐3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC‐3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET‐1‐induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC‐3 protein. Silence of ClC‐3 by siRNA inhibited expression of ClC‐3 protein, prevented an increase in BrdU incorporation and cell number induced by ET‐1. Silence of ClC‐3 also caused cell cycle arrest in G0/G1 phase and prevented the cells’ progression from G1 to S phase. Knockdown of ClC‐3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin‐dependent kinase inhibitors (CDKIs) p27KIP and p21CIP expression. Furthermore, ClC‐3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase‐3β (GSK‐3β) induced by ET‐1. Conclusion: Silence of ClC‐3 protein effectively suppressed phosphorylation of the Akt/GSK‐3β signal pathway, resulting in down‐regulation of cyclin D1 and cyclin E, and up‐regulation of p27KIP and p21CIP. In these BASMCs, integrated effects lead to cell cycle G1/S arrest and inhibition of cell proliferation.

Alteration of Volume-Regulated Chloride Movement in Rat Cerebrovascular Smooth Muscle Cells During Hypertension

The cerebrovascular remodeling is a prominent feature of hypertension and considered a major risk factor for stroke. Cerebrovascular smooth muscle cells meet volume challenge during this pathophysiological process. Our previous studies suggest that volume regulated chloride channels may be critical to the cell cycle of vascular smooth muscle cells. However, it is unknown whether the volume-regulated chloride movement is altered in hypertension. Therefore, we directly measured the concentration of intracellular chloride ([Cl−]i) in rat basilar arterial smooth muscle cells isolated from control rats and rats that were made hypertensive for 1 to 12 weeks after partial renal artery constriction (2-kidney, 2-clip method) using a 6-methoxy-N-ethylquinolinium iodide fluorescence probe. The [Cl−]i in isotonic solution showed no difference in all of the groups. After hypotonic perfusion, the reduction in [Cl−]i was more prominent in hypertensive cerebrovascular smooth muscle cells than in sham control cells. Genistein, a protein tyrosine kinase inhibitor, inhibited hypotonic-induced reduction in [Cl−]i, whereas sodium orthovanadate, a protein–tyrosine phosphatase inhibitor, enhanced hypotonic-induced reduction in [Cl−]i in both groups. The percentage inhibition of reduction in [Cl−]i by genistein on volume-regulated chloride movement has a positive correlation with blood pressure levels in the 2-kidney, 2-clip hypertensive group, as is the case for the percentage increase of reduction in [Cl−]i by sodium orthovanadate. Antihypertensive therapy with the angiotensin-converting enzyme inhibitor captopril completely reversed abnormal volume-regulated chloride movement in hypertensive rats. We conclude that volume-regulated chloride movement is augmented in rat cerebrovascular smooth muscle cells in proportion to the severity of hypertension.

Involvement of Chk1-Cdc25A-cyclin A/CDK2 pathway in simvastatin induced S-phase cell cycle arrest and apoptosis in multiple myeloma cells.

Statins have been demonstrated to effectively inhibit proliferation and induce apoptosis in cancer cells by inhibition of geranylgeranylation, however its novel molecular mechanism remains to be determined. Recently simvastatin has been found to result in the synergistic induction of apoptosis with 7-hydroxystaurosporine (UCN-01) (a Chk1 inhibitor) in myeloma cells. Therefore we hypothesized that Chk1 plays a role in the anti-myeloma effect of simvastatin. Interestingly, we found that simvastatin caused a dose-dependent increase in S phase cell cycle and induced significant apoptosis. The results of western blot showed that simvastatin-induced S-phase cell cycle arrest was associated with activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression. Additionally, simvastatin-induced apoptosis was accompanied by diminished Bcl-2 protein expression, increased cytosolic cytochrome c level, and activation of caspase 9 and caspase 3. Further investigation revealed that silence of Chk1 expression by Chk1 specific siRNA inhibited simvastatin-induced activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression, and diminished S phase cell cycle arrest. Additionally, inhibition of Chk1 expression enhanced simvastatin-induced downregulation of Bcl-2, caspase 9 cleavage and subsequent apoptosis. These results suggested that the Chk1-Cdc25A-cyclin A/CDk2 pathway was involved in simvastatin-induced S-phase cell cycle arrest and apoptosis in multiple myeloma cell lines.

Ginsenoside-Rd, a new voltage-independent Ca2+ entry blocker, reverses basilar hypertrophic remodeling in stroke-prone renovascular hypertensive rats

The total saponins of Panax notoginseng have been clinically used for the treatment of cardiovascular diseases and stroke in China. Our recent study has identified ginsenoside-Rd, a purified component of total saponins of P. notoginseng, as an inhibitor to remarkably inhibit voltage-independent Ca(2+) entry. We deduced a hypothesis that the inhibition of voltage-independent Ca(2+) entry might contribute to its cerebrovascular benefits. Ginsenoside-Rd was administered to two-kidney, two-clip (2k2c) stroke-prone hypertensive rats to examine its effects on blood pressure, cerebrovascular remodeling and Ca(2+) entry in freshly isolated basilar arterial vascular smooth muscle cells (BAVSMCs). Its effects on endothelin-1 induced Ca(2+) entry and cellular proliferation were assessed in cultured BAVSMCs. The results showed that, in vivo, ginsenoside-Rd treatment attenuated basilar hypertrophic inward remodeling in 2k2c hypertensive rats without affecting systemic blood pressure.During the development of hypertension, there were time-dependent increases in receptor-operated Ca(2+) channel (ROCC)-, store-operated Ca(2+) channel (SOCC)- and voltage dependent Ca(2+) channel (VDCC)-mediated Ca(2+) entries in freshly isolated BAVSMCs. Ginsenoside-Rd reversed the increase in SOCC- or ROCC- but not VDCC-mediated Ca(2+) entry. In vitro, ginsenoside-Rd concentration-dependently inhibited endothelin-1 induced BAVSMC proliferation and Mn(2+) quenching rate within the same concentration range as required for inhibition of increased SOCC- or ROCC-mediated Ca(2+) entries during hypertension. These results provide in vivo evidence showing attenuation of hypertensive cerebrovascular remodeling after ginsenoside-Rd treatment. The underlying mechanism might be associated with inhibitory effects of ginsenoside-Rd on voltage-independent Ca(2+) entry and BAVSMC proliferation, but not with VDCC-mediated Ca(2+) entry.

Decrease of Intracellular Chloride Concentration Promotes Endothelial Cell Inflammation by Activating Nuclear Factor-κB Pathway

Recent evidence suggested that ClC-3 channel/antiporter is involved in regulation of nuclear factor (NF)-&kgr;B activation. However, the mechanism explaining how ClC-3 modulates NF-&kgr;B 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-&kgr;B activity in endothelial cells. Here, we found that reduction of [Cl−]i increased tumor necrosis factor-&agr; (TNF&agr;)-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, TNF&agr;-evoked I&kgr;B kinase complex &bgr; and inhibitors of &kgr;B&agr; phosphorylation, inhibitors of &kgr;B&agr; degradation, and NF-&kgr;B nuclear translocation were enhanced. In addition, TNF&agr; and interleukin 1&bgr; 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 TNF&agr;-induced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocytes to endothelial cell adhesion, and NF-&kgr;B activation (P<0.01; n=6). Furthermore, TNF&agr;-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-&kgr;B activation and thus potentiates TNF&agr;-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.

Ginsenoside-Rd, a purified component from panax notoginseng saponins, prevents atherosclerosis in apoE knockout mice

Recently, it was revealed that the dysfunction of transmembrane Ca(2+) transport, results in an increase in intracellular Ca(2+)[Ca(2+)](i), which is involved in the process of atherosclerosis. We previously demonstrated that ginsenoside-Rd, a purified component from panax notoginseng, is a voltage-independent Ca(2+) channels blocker. In this study, we investigated the effects of ginsenoside-Rd on atherosclerosis and the underlying mechanisms in apolipoprotein E deficient (apoE(-/-)) mice and RAW264.7 cells. Atherosclerotic plaques were stained by Red oil O staining. Ca(2+) influx was measured by Fura-2 dyed Mn(2+) quenching. Intracellular cholesterol and uptake of lipid was assayed by enzymatic, fluorometric method and DiI-labeled Ox-LDL. Western blot was used to determine protein expression. We found that Ginsenoside-Rd (20mg/kg/day. i.p.) significantly reduced the atherosclerotic plaque areas, oxidized low-density lipoprotein (ox-LDL) uptake and thapsigargin and l-oleoyl-2-acetyl-glycerol (OAG, membrane-permeable diacylglycerol analog)-induced Ca(2+) influx in macrophages from high-fat diet apoE(-/-) mice. In vitro, 20μM ginsenoside-Rd significantly inhibited ox-LDL-induced foam cell formation and the increase of thapsigargin- and OAG-induced Ca(2+) influx. Ox-LDL induced an increase in scavenger receptor A (SR-A) expression, and ginsenoside-Rd inhibited this effect of ox-LDL significantly. The results suggest that ginsenoside-Rd prevents the development of atherosclerosis. The underlying mechanism may be related to the inhibition of Ca(2+) influx through voltage-independent Ca(2+) channels, resulting in the inhibition of SR-A activity and expression, followed by reductions of ox-LDL uptake and cholesterol accumulation in macrophages.

Simvastatin Ameliorates Rat Cerebrovascular Remodeling During Hypertension via Inhibition of Volume-Regulated Chloride Channel

Statins have pleiotropic actions against the development of vascular remodeling and the incidence of ischemic stroke. Although previous studies have suggested that posttranslational modification of several proteins, such as Rho by mevalonate-derived isoprene groups, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, underlie the pleiotropic effects of statins, the detailed mechanisms remain elusive. Recent growing evidence demonstrated that ClC-3 volume-regulated chloride channel plays an important role in cell proliferation, and the activity of this channel is increased in basilar smooth muscle cells from a hypertensive rat. We hypothesized that inhibition of volume-regulated chloride channel may contribute to the beneficial effects of statins on cerebrovascular remodeling during hypertension. Our study here demonstrated that simvastatin ameliorated hypertension-caused cerebrovascular remodeling. In rat basilar smooth muscle cells, simvastatin inhibited cell proliferation and activation of volume-regulated chloride channel, and these effects of simvastatin were abolished by pretreatment with mevalonate or geranylgeranyl pyrophosphate. In addition, Rho A inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 both reduced cell proliferation and activation of volume-regulated chloride channel. Moreover, ClC-3 overexpression decreased the suppressive effect of simvastatin on cell proliferation and increased estimated IC50 of simvastatin on endothelin 1- and hypo-osmolarity-induced cell proliferation from 3.40±0.08 and 3.50±0.10 &mgr;mol/L to 5.30±0.70 and 5.60±0.70 &mgr;mol/L, respectively (P<0.01; n=6). Furthermore, the expression of ClC-3 was increased in basilar artery during hypertension, and simvastatin normalized the upregulation of ClC-3. Our data suggested that simvastatin ameliorates cerebrovascular remodeling in the hypertensive rat through inhibition of vascular smooth muscle cell proliferation by suppression of volume-regulated chloride channel.