Yong-Yuan Guan

Deficiency in ClC-3 Chloride Channels Prevents Rat Aortic Smooth Muscle Cell Proliferation

Abstract— Recent growing evidence suggests that chloride (Cl−) channels are critical to the cell cycle. In cultured rat aortic vascular smooth muscle cells (VSMCs), we have previously found that Cl− channel blockers inhibit endothelin-1 (ET-1)–induced cell proliferation. The present study was designed to further identify the specific Cl− channels responsible for VSMC proliferation. Due to the lack of a specific blocker or opener of any known Cl− channels, we used the antisense strategy to investigate the potential role of ClC-3, a member of the voltage-gated Cl− channel gene family, in cell proliferation of cultured rat aortic VSMCs. With [3H]-thymidine incorporation and immunoblots, we found that ET-1–induced cell proliferation was parallel to a significant increase in the endogenous expression of ClC-3 protein. Transient transfection of rat aortic VSMCs with antisense oligonucleotide specific to ClC-3 caused an inhibition in ET-1–induced expression of ClC-3 protein and cell proliferation of VSMCs in the same concentration- and time-dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 protein expression and cell proliferation. These results strongly suggest that ClC-3 may be the Cl− channel involved in VSMC proliferation and thus provide compelling molecular evidence linking a specific Cl− channel to cell proliferation. The full text of this article is available at http://www.circresaha.org.

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.

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.

A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Pretreatment with xyloketal B (0.63-40 microM) significantly improved oxLDL (150 microg/ml)-induced injury in human umbilical vein endothelial cells (HUVECs) without either toxic or proliferative effects. Xyloketal B concentration-dependently attenuated oxLDL-induced ROS generation, peroxynitrite formation and decrease of Bcl-2 expression. In addition, xyloketal B significantly inhibited NADPH oxidase activity, as well as mRNA expression of gp91phox and p47phox. Furthermore, xyloketal B alone augmented the production of nitric oxide (NO). Collectively, these data indicate that xyloketal B protects against oxLDL-induced endothelial oxidative injury probably through inhibiting NADPH oxidase-derived ROS generation, promoting NO production and restoring Bcl-2 expression, making it a promising compound for further evaluation in the treatment of atherosclerosis.

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.

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl− channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl− channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl− channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively.

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.