Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

Increased intracellular Cl- concentration mediates neutrophil extracellular traps formation in atherosclerotic cardiovascular diseases.

Neutrophil extracellular traps (NETs) play crucial roles in atherosclerotic cardiovascular diseases such as acute coronary syndrome (ACS). Our preliminary study shows that oxidized low-density lipoprotein (oxLDL)-induced NET formation is accompanied by an elevated intracellular Cl- concentration ([Cl-]i) and reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in freshly isolated human blood neutrophils. Herein we investigated whether and how [Cl-]i regulated NET formation in vitro and in vivo. We showed that neutrophil [Cl-]i and NET levels were increased in global CFTR null (Cftr-/-) mice in the resting state, which was mimicked by intravenous injection of the CFTR inhibitor, CFTRinh-172, in wild-type mice. OxLDL-induced NET formation was aggravated by defective CFTR function. Clamping [Cl-]i at high levels directly triggered NET formation. Furthermore, we demonstrated that increased [Cl-]i by CFTRinh-172 or CFTR knockout increased the phosphorylation of serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and generation of intracellular reactive oxygen species in neutrophils, and promoted oxLDL-induced NET formation and pro-inflammatory cytokine production. Consistently, peripheral blood samples obtained from atherosclerotic ApoE-/- mice or stable angina (SA) and ST-elevation ACS (STE-ACS) patients exhibited increased neutrophil [Cl-]i and SGK1 activity, decreased CFTR expression, and elevated NET levels. VX-661, a CFTR corrector, reduced the NET formation in the peripheral blood sample obtained from oxLDL-injected mice, ApoE-/- atherosclerotic mice or patients with STE-ACS by lowering neutrophil [Cl-]i. These results demonstrate that elevated neutrophil [Cl-]i during the development of atherosclerosis and ACS contributes to increased NET formation via Cl--sensitive SGK1 signaling, suggesting that defective CFTR function might be a novel therapeutic target for atherosclerotic cardiovascular diseases.

Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl- concentration and activation of SGK1 signaling pathway.

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells.

ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) plays important roles in arterial functions and the fate of cells. To further understand its function in vascular remodeling, we examined whether CFTR directly regulates platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) proliferation and migration, as well as the balloon injury-induced neointimal formation. The CFTR adenoviral gene delivery was used to evaluate the effects of CFTR on neointimal formation in a rat model of carotid artery balloon injury. The roles of CFTR in PDGF-BB-stimulated VSMC proliferation and migration were detected by mitochondrial tetrazolium assay, wound healing assay, transwell chamber method, western blot, and qPCR. We found that CFTR expression was declined in injured rat carotid arteries, while adenoviral overexpression of CFTR in vivo attenuated neointimal formation in carotid arteries. CFTR overexpression inhibited PDGF-BB-induced VSMC proliferation and migration, whereas CFTR silencing caused the opposite results. Mechanistically, CFTR suppressed the phosphorylation of PDGF receptor ß, serum and glucocorticoid-inducible kinase 1, JNK, p38 and ERK induced by PDGF-BB, and the increased mRNA expression of matrix metalloproteinase-9 and MMP2 induced by PDGF-BB. In conclusion, our results indicated that CFTR may attenuate neointimal formation by suppressing PDGF-BB-induced activation of serum and glucocorticoid-inducible kinase 1 and the JNK/p38/ERK signaling pathway.

Low Chloride-Regulated ClC-5 Contributes to Arterial Smooth Muscle Cell Proliferation and Cerebrovascular Remodeling.

BACKGROUND: Low serum chloride (Cl-) level is considered an independent predictor of cardiovascular mortality associated with chronic hypertension. However, the underlying mechanisms are unknown. ClC-5, a member of the Cl- channel family, is sensitive to changes in intracellular and extracellular Cl- concentration and conducts outwardly rectifying Cl- currents. The aims of this study were to determine if ClC-5 is regulated by low extracellular Cl-, clarify its putative roles in hypertension-induced cerebrovascular remodeling, and elucidate the associated underlying mechanisms. METHODS: Whole-cell patch technique, intracellular Cl- concentration measurements, flow cytometry, Western blot, Clcn5 knockdown (Clcn5-/y), and adenovirus-mediated ClC-5 overexpression mice, 2-kidney, 2-clip, and angiotensin II infusion-induced hypertensive models were used. RESULTS: We found that low extracellular Cl- evoked a ClC-5-dependent Cl- current that was abolished by ClC-5 depletion in basilar artery smooth muscle cells (BASMCs). ClC-5 was upregulated in the arterial tissues of rats and patients with hypertension. Low Cl--induced current and ClC-5 protein expression positively correlated with basilar artery remodeling during hypertension. ClC-5 knockdown ameliorated hypertension-induced cerebrovascular remodeling and smooth muscle cell proliferation, whereas ClC-5 overexpression mice exhibited the opposite phenotype. ClC-5-dependent Cl- efflux induced by low extracellular Cl- activated WNK1 (lysine-deficient protein kinase 1) which, in turn, activated AKT (protein kinase B), and culminated in BASMC proliferation and vascular remodeling. CONCLUSIONS: ClC-5 mediates low extracellular Cl-induced Cl- currents in BASMCs and regulates hypertension-induced cerebrovascular remodeling by promoting BASMC proliferation via the WNK1/AKT signaling pathway.

Blockade of TMEM16A protects against renal fibrosis by reducing intracellular Cl- concentration.

BACKGROUND AND PURPOSE: Renal fibrosis is the final common outcome in most forms of chronic kidney disease (CKD). However, the underlying causal mechanisms remain obscure. The present study examined whether transmembrane member 16A (TMEM16A), a Ca2+ -activated chloride channel, contributes to the progression of renal fibrosis. EXPERIMENTAL APPROACH: Masson staining, western blot and immunohistochemistry were used to measure renal fibrosis and related proteins expression. MQAE was used to evaluate the intracellular Cl- concentration. KEY RESULTS: TMEM16A expression was significantly up-regulated in fibrotic kidneys of unilateral ureteral obstruction (UUO) and high-fat diet murine models and in renal samples of IgA nephropathy patients. In vivo knockdown of TMEM16A with adenovirus harbouring TMEM16A-shRNA or inhibition of TMEM16A channel activity with inhibitors CaCCinh-A01 or T16Ainh-A01 effectively prevented UUO-induced renal fibrosis and decreased protein expression of fibronectin, α-SMA and collagen in the obstructed kidneys. In cultured HK2 cells, knockdown or inhibition of TMEM16A suppressed TGF-ß1-induced epithelial-mesenchymal transition, reduced snail1 expression and phosphorylation of Smad2/3 and ERK1/2, whereas overexpression of TMEM16A showed the opposite effects. TGF-ß1 increased [Cl- ]i in HK2 cells, which was inhibited by knockdown or inhibition of TMEM16A. Reducing [Cl- ]i significantly blunted TGF-ß1-induced Smad2/3 phosphorylation and profibrotic factors expression. The profibrotic effects of TGF-ß1 were also reduced by inhibition of serum- and glucocorticoid-inducible protein kinase 1 (SGK1). SGK1 was also suppressed by reducing [Cl- ]i. CONCLUSION AND IMPLICATIONS: Blockade of TMEM16A prevented the progression of kidney fibrosis, likely by suppressing [Cl- ]i/SGK1/TGF-ß1 signalling pathway. TMEM16A may be a potential new therapeutic target against renal fibrosis.

LRRC8A is essential for volume-regulated anion channel in smooth muscle cells contributing to cerebrovascular remodeling during hypertension.

OBJECTIVES: Recent studies revealed LRRC8A to be an essential component of volume-regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A-dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension. MATERIALS AND METHODS: siRNA-mediated knockdown and adenovirus-mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle-specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling. RESULTS: LRRC8A is essential for volume-regulated chloride current (ICl, Vol ) in BASMCs. Overexpression of LRRC8A induced a voltage-dependent Cl- current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K-p85/AKT axis. Smooth muscle-specific upregulation of LRRC8A aggravated Angiotensin II-induced cerebrovascular remodeling in mice. CONCLUSIONS: LRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension.

SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway.

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

TMEM16A ameliorates vascular remodeling by suppressing autophagy via inhibiting Bcl-2-p62 complex formation.

Rationale: Transmembrane member 16A (TMEM16A) is a component of calcium-activated chloride channels that regulate vascular smooth muscle cell (SMC) proliferation and remodeling. Autophagy, a highly conserved cellular catabolic process in eukaryotes, exerts important physiological functions in vascular SMCs. In the current study, we investigated the relationship between TMEM16A and autophagy during vascular remodeling. Methods: We generated a transgenic mouse that overexpresses TMEM16A specifically in vascular SMCs to verify the role of TMEM16A in vascular remodeling. Techniques employed included immunofluorescence, electron microscopy, co-immunoprecipitation, and Western blotting. Results: Autophagy was activated in aortas from angiotensin II (AngII)-induced hypertensive mice with decreased TMEM16A expression. The numbers of light chain 3B (LC3B)-positive puncta in aortas correlated with the medial cross-sectional aorta areas and TMEM16A expression during hypertension. SMC-specific TMEM16A overexpression markedly inhibited AngII-induced autophagy in mouse aortas. Moreover, in mouse aortic SMCs (MASMCs), AngII-induced autophagosome formation and autophagic flux were blocked by TMEM16A upregulation and were promoted by TMEM16A knockdown. The effect of TMEM16A on autophagy was independent of the mTOR pathway, but was associated with reduced kinase activity of the vacuolar protein sorting 34 (VPS34) enzyme. Overexpression of VPS34 attenuated the effect of TMEM16A overexpression on MASMC proliferation, while the effect of TMEM16A downregulation was abrogated by a VPS34 inhibitor. Further, co-immunoprecipitation assays revealed that TMEM16A interacts with p62. TMEM16A overexpression inhibited AngII-induced p62-Bcl-2 binding and enhanced Bcl-2-Beclin-1 interactions, leading to suppression of Beclin-1/VPS34 complex formation. However, TMEM16A downregulation showed the opposite effects. Conclusion: TMEM16A regulates the four-way interaction between p62, Bcl-2, Beclin-1, and VPS34, and coordinately prevents vascular autophagy and remodeling.

SGK1 mediates the hypotonic protective effect against H2O2-induced apoptosis of rat basilar artery smooth muscle cells by inhibiting the FOXO3a/Bim signaling pathway.

Serum- and glucocorticoid-inducible kinease-1 (SGK1) is a serine/threonine kinase regulated by hypotonic stimuli, which is involved in regulation of cell cycle and apoptosis. Our previous study shows that activation of volume-regulated Cl- channels (VRCCs) protects rat basilar artery smooth muscle cells (BASMCs) against hydrogen peroxide (H2O2)-induced apoptosis. In the present study, we investigated whether SGK1 was involved in the protective effect of VRCCs in BASMCs. We showed that hypotonic challenge significantly reduced H2O2-induced apoptosis, and increased SGK1 phosphorylation, but did not affect SGK1 protein expression. The protective effect of hypotonic challenge against H2O2-induced apoptosis was mediated through inhibiting mitochondria-dependent apoptotic pathway, evidenced by increased Bcl-2/Bax ratio, stabilizing mitochondrial membrane potential (MMP), decreased cytochrome c release from the mitochondria to the cytoplasm, and inhibition of the activation of caspase-9 and caspase-3. These protective effects of hypotonic challenge against H2O2-induced apoptosis was diminished and enhanced, respectively, by SGK1 knockdown and overexpression. We further revealed that SGK1 activation significantly increased forkhead box O3a (FOXO3a) phosphorylation, and then inhibited the translocation of FOXO3a into nucleus and the subsequent expression of Bcl-2 interacting mediator of cell death (Bim). In conclusion, SGK1 mediates the protective effect of VRCCs against H2O2-induced apoptosis in BASMCs via inhibiting FOXO3a/Bim signaling pathway. Our results provide compelling evidences that SGK1 is a critical link between VRCCs and apoptosis, and shed a new light on the treatment of vascular apoptosis-associated diseases, such as vascular remodeling, angiogenesis, and atherosclerosis.

Endophilin A2 regulates calcium-activated chloride channel activity via selective autophagy-mediated TMEM16A degradation.

TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 µM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.

Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition.

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca2+-activated Cl- channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-ß1/Smad3, and non-canonical TGF-ß1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl--sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-ß1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Clcn3 deficiency ameliorates high-fat diet-induced obesity and adipose tissue macrophage inflammation in mice.

Obesity induces accumulation of adipose tissue macrophages (ATMs) and ATM-driven inflammatory responses that promote the development of glucose and lipid metabolism disorders. ClC-3 chloride channel/antiporter, encoded by the Clcn3, is critical for some basic cellular functions. Our previous work has shown significant alleviation of type 2 diabetes in Clcn3 knockout (Clcn3-/-) mice. In the present study we investigated the role of Clcn3 in high-fat diet (HFD)-induced obesity and ATM inflammation. To establish the mouse obesity model, both Clcn3-/- mice and wild-type mice were fed a HFD for 4 or 16 weeks. The metabolic parameters were assessed and the abdominal total adipose tissue was scanned using computed tomography. Their epididymal fat pad tissue and adipose tissue stromal vascular fraction (SVF) cells were isolated for analyses. We found that the HFD-fed Clcn3-/- mice displayed a significant decrease in obesity-induced body weight gain and abdominal visceral fat accumulation as well as an improvement of glucose and lipid metabolism as compared with HFD-fed wild-type mice. Furthermore, the Clcn3 deficiency significantly attenuated HFD-induced ATM accumulation, HFD-increased F4/80+ CD11c+ CD206- SVF cells as well as HFD-activated TLR-4/NF-κB signaling in epididymal fat tissue. In cultured human THP-1 macrophages, adenovirus-mediated transfer of Clcn3 specific shRNA inhibited, whereas adenovirus-mediated cDNA overexpression of Clcn3 enhanced lipopolysaccharide-induced activation of NF-κB and TLR-4. These results demonstrate a novel role for Clcn3 in HFD-induced obesity and ATM inflammation.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 µmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-ß-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

Transmembrane member 16A participates in hydrogen peroxide-induced apoptosis by facilitating mitochondria-dependent pathway in vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Transmembrane member 16A (TMEM16A), an intrinsic constituent of the Ca2+ -activated Cl- channel, is involved in vascular smooth muscle cell (VSMC) proliferation and hypertension-induced cerebrovascular remodelling. However, the functional significance of TMEM16A for apoptosis in basilar artery smooth muscle cells (BASMCs) remains elusive. Here, we investigated whether and how TMEM16A contributes to apoptosis in BASMCs. EXPERIMENTAL APPROACH: Cell viability assay, flow cytometry, Western blot, mitochondrial membrane potential assay, immunogold labelling and co-immunoprecipitation (co-IP) were performed. KEY RESULTS: Hydrogen peroxide (H2 O2 ) induced BASMC apoptosis through a mitochondria-dependent pathway, including by increasing the apoptosis rate, down-regulating the ratio of Bcl-2/Bax and potentiating the loss of the mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytoplasm. These effects were all reversed by the silencing of TMEM16A and were further potentiated by the overexpression of TMEM16A. Endogenous TMEM16A was detected in the mitochondrial fraction. Co-IP revealed an interaction between TMEM16A and cyclophilin D, a component of the mitochondrial permeability transition pore (mPTP). This interaction was up-regulated by H2 O2 but restricted by cyclosporin A, an inhibitor of cyclophilin D. TMEM16A increased mPTP opening, resulting in the activation of caspase-9 and caspase-3. The results obtained with cultured BASMCs from TMEM16A smooth muscle-specific knock-in mice were consistent with those from rat BASMCs. CONCLUSIONS AND IMPLICATIONS: These results suggest that TMEM16A participates in H2 O2 -induced apoptosis via modulation of mitochondrial membrane permeability in VSMCs. This study establishes TMEM16A as a target for therapy of several remodelling-related diseases.

WNK1 is required for proliferation induced by hypotonic challenge in rat vascular smooth muscle cells.

Hypotonic challenge evoked vascular cell proliferation through activation of volume-regulated Cl- channel (VRCC), leading to a decrease in the intracellular Cl- concentration ([Cl-]i). We hypothesize that the decrease in [Cl-]i may activate one or several Cl--sensitive kinases, resulting in a subsequent signaling cascade. In this study we demonstrated that WNK1, a Cl--sensitive kinase, was involved in VRCC-induced proliferative signaling pathway in A10 vascular smooth muscle cells in vitro. A10 cells were exposed to a hypotonic challenge (225 mosmol·kg-1·H20), which caused significantly increase in WNK1 phosphorylation without altering WNK1 protein expression. WNK1 overexpression significantly increased hypotonic-induced A10 cell proliferation, whereas silencing of WNK1 caused an opposite action. WNK1 mutation did not affect hypotonic-induced WNK1 phosphorylation and cell proliferation. Silencing of WNK1 caused cell cycle arrest at G0/G1 phase and prevented transition from G1 to S phase, whereas the WNK1 overexpression accelerated cell cycle transition from G1 to S phase. Silencing of WNK1 significantly inhibited cyclin D1/cyclin E1 expression and increased p27kip/p21cip expression. WNK1 overexpression significantly increased cyclin D1/cyclin E1 expression and reduced p27KIP/p21CIP expression. In addition, WNK1 knockdown or overexpression significantly attenuated or increased the hypotonic-induced phosphorylation of Akt and PI3K respectively.In conclusion, the reduction in [Cl-]i caused by hypotonic challenge-induced VRCC opening evokes WNK1 phosphorylation in A10 VSMCs, which mediates cell cycle transition from G0/G1 to S phase and proliferation through the PI3K-Akt signaling pathway.

Ca2+/Calmodulin-Dependent Protein Kinase II γ-Dependent Serine727 Phosphorylation Is Required for TMEM16A Ca2+-Activated Cl- Channel Regulation in Cerebrovascular Cells.

BACKGROUND: TMEM16A is a critical component of Ca2+-activated chloride channels (CaCCs) and mediates basilar arterial smooth muscle cell (BASMC) proliferation in hypertensive cerebrovascular remodeling. CaMKII is a negative regulator of CaCC, and four CaMKII isoforms (α, ß, γ and δ) are expressed in vasculature; however, it is unknown which and how CaMKII isoforms affect TMEM16A-associated CaCC and BASMC proliferation.Methods and Results:Patch clamp and small interfering RNA (siRNA) knockdown of different CaMKII isoforms revealed that only CaMKIIγ inhibited native Ca2+-activated chloride currents (ICl.Ca) in BASMCs. The TMEM16A overexpression evoked TMEM16A Cl-current and inhibited angiotensin II (Ang II)-induced proliferation in BASMCs. The co-immunoprecipitation and pull-down assay indicated an interaction between CaMKIIγ and TMEM16A protein. TMEM16A Cl-current was modulated by CaMKIIγ phosphorylation at serine residues in TMEM16A. Serine525 and Serine727 in TMEM16A were mutated to alanine, and only mutation at Ser727 (S727A) reversed the CaMKIIγ inhibition of the TMEM16A Cl-current. Phosphomimetic mutation S727D markedly decreased TMEM16A Cl-current and reversed TMEM16A-mediated suppression of BASMC proliferation, mimicking the inhibitory effects of CaMKIIγ on TMEM16A. A significant increase in CaMKIIγ isoform content was observed in parallel to the decrease of TMEM16A and ICl.Cain basilar artery proliferative remodeling in Ang II-infused mice. CONCLUSIONS: Serine 727 phosphorylation in TMEM16A by CaMKIIγ provides a new mechanism for regulating TMEM16A CaCC activity and Ang II-induced BASMC proliferation.

Xyloketal B alleviates cerebral infarction and neurologic deficits in a mouse stroke model by suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. We previously demonstrated that pretreatment with Xyl-B exerted neuroprotective effects and attenuated hypoxic-ischemic brain injury in neonatal mice. In the present study we investigated the neuroprotective effects of pre- and post-treatment with Xyl-B in adult mice using a transient middle cerebral artery occlusion (tMCAO) model, and explored the underlying mechanisms. Adult male C57 mice were subjected to tMCAO surgery. For the pre-treatment, Xyl-B was given via multiple injections (12.5, 25, and 50 mg·kg-1·d-1, ip) 48 h, 24 h and 30 min before ischemia. For the post-treatment, a single dose of Xyl-B (50 mg/kg, ip) was injected at 0, 1 or 2 h after the onset of ischemia. The regional cerebral perfusion was monitored using a laser-Doppler flowmeter. TTC staining was performed to determine the brain infarction volume. We found that both pre-treatment with Xyl-B (50 mg/kg) and post-treatment with Xyl-B (50 mg/kg) significantly reduced the infarct volume, but had no significant hemodynamic effects. Treatment with Xyl-B also significantly alleviated the neurological deficits in tMCAO mice. Furthermore, treatment with Xyl-B significantly attenuated ROS overproduction in brain tissues; increased the MnSOD protein levels, suppressed TLR4, NF-κB and iNOS protein levels; and downregulated the mRNA levels of proinflammatory cytokines, including IL-1ß, TNF-α, IL-6 and IFN-γ. Moreover, Xyl-B also protected blood-brain barrier integrity in tMCAO mice. In conclusion, Xyl-B administered within 2 h after the onset of stroke effectively protects against focal cerebral ischemia; the underlying mechanism may be related to suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.

Ca2+-activated Cl- channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca2+-activated Cl- channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases.

Suppression of Kv1.5 protects against endothelial apoptosis induced by palmitate and in type 2 diabetes mice.

AIMS: Palmitate, a common saturated free fatty acid, induces endothelial apoptosis in vitro in culture endothelial cells and in vivo in type 2 diabetes mellitus (T2DM) patients. The present study aimed to investigate whether Kv1.5 regulates palmitate-induced endothelial apoptosis and endothelial dysfunction in T2DM. MAIN METHODS: In vitro experiments were carried out in primary human HUVECs. Apoptosis was analyzed by flow cytometry. Cell viability was determined by Cell Counting Assay Kit-8. The siRNA transfection was employed to knockdown Kv1.5 protein expression. Intracellular and mitochondrial ROS, and mitochondrial membrane potential were detected using fluorescent probes. Male C57BL/6 mice fed with high-sucrose/fat diet were injected with streptozotocin (35mg/kg body weight) to establish T2DM animal model. KEY FINDINGS: We found that palmitate-induced endothelial apoptosis was parallel to a significant increase in endogenous Kv1.5 protein expression in endothelial cells. Silencing of Kv1.5 with siRNA reduced palmitate-induced endothelial apoptosis, intracellular ROS generation, mitochondrial ROS generation and membrane potential (Δψm) alteration and cleaved caspase-3 protein expression; while increased cell viability and ratio of Bcl-2/Bax. Furthermore, we observed that Kv1.5 protein expression increased in endothelial cells of thoracic aorta of T2DM mice. Silencing of Kv1.5 significantly improved the endothelium-dependent vasodilation in thoracic aortic rings of T2DM mice. SIGNIFICANCE: These results demonstrate that suppression of Kv1.5 protects endothelial cells against palmitate-induced apoptosis via inhibiting mitochondria-mediated excessive ROS generation and apoptotic signaling pathway, suggesting that Kv1.5 may serve as a therapeutic target of treatment for endothelial dysfunction induced by palmitate and lipid metabolism in T2DM patients.