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.

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.

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.

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.

Endophilin A2 Influences Volume-Regulated Chloride Current by Mediating ClC-3 Trafficking in Vascular Smooth Muscle Cells.

BACKGROUND: Previous research has demonstrated that ClC-3 is responsible for volume-regulated Cl-current (ICl.vol) in vascular smooth muscle cells (VSMCs). However, it is still not clear whether and how ClC-3 is transported to cell membranes, resulting in alteration ofICl.vol.Methods and Results:Volume-regulated chloride current (ICl.vol) was recorded by whole-cell patch clamp recording, and Western blotting and co-immunoprecipitation were performed to examine protein expression and protein-protein interaction. Live cell imaging was used to observe ClC-3 transporting. The results showed that an overexpression of endophilin A2 could increaseICl.vol, while endophilin A2 knockdown decreasedICl.vol. In addition, the SH3 domain of endophilin A2 mediated its interaction with ClC-3 and promotes ClC-3 transportation from the cytoplasm to cell membranes. The regulation of ClC-3 channel activity was also verified in basilar arterial smooth muscle cells (BASMCs) isolated from endophilin A2 transgenic mice. Moreover, endophilin A2 increase VSMCs proliferation induced by endothelin-1 or hypo-osmolarity. CONCLUSIONS: The present study identified endophilin A2 as a ClC-3 channel partner, which serves as a new ClC-3 trafficking insight in regulatingICl.volin VSMCs. This study provides a new mechanism by which endophilin A2 regulates ClC-3 channel activity, and sheds light on how ClC-3 is transported to cell membranes to play its critical role as a chloride channel in VSMCs function, which may be involved in cardiovascular diseases. (Circ J 2016; 80: 2397-2406).

Endophilin-A2-mediated increase in scavenger receptor expression contributes to macrophage-derived foam cell formation.

BACKGROUND AND AIMS: Macrophage-derived foam cell formation (MFCF) is a crucial step in the pathogenesis of atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors is indispensable for MFCF. Endophilin-A2 has been reported to regulate clathrin-mediated endocytosis (CME). In this study, we tested the hypothesis that endophilin-A2 regulates oxLDL uptake and MFCF by mediating CME of oxLDL-scavenger receptor complexes. METHODS: In vitro MFCF was induced by oxLDL treatment. Involvement of endophilin-A2 in oxLDL cytomembrane binding, cellular uptake, and MFCF was evaluated by manipulation of endophilin-A2. RESULTS: Endophilin-A2 was involved in MFCF via scavenger receptor CD36 and scavenger receptor-A (SR-A)-mediated positive feedback pathways. We observed that oxLDL triggered interaction of endophilin-A2 with CD36 or SR-A, and induced an endophilin-A2-dependent activation of the apoptosis signal-regulating kinase-1 (ASK1)/Jun N-terminal kinase (JNK)/p38 signaling pathway. The activation of ASK1-JNK/p38 signal increased expression of both CD36 and SR-A, which promoted oxLDL cytomembrane binding, cellular uptake, and MFCF. In the absence of oxLDL, endophilin-A2 up-regulated the expression of receptors and Dil-oxLDL binding and uptake, but not the intracellular accumulation of lipids. In the presence of oxLDL, the CME inhibitors pitstop2 and ikarugamycin mimicked the inhibiting effect of endophilin-A2 knockdown and eliminated the elevating effect of endophilin-A2 overexpression on oxLDL uptake and MFCF. CONCLUSIONS: Endophilin-A2 was identified as a novel molecule regulating MFCF by mechanisms attributable to CME and beyond CME.

Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl(-) current and migration in cultured vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl(-) current, but the underlying mechanism is not clear. EXPERIMENTAL APPROACH: The A10 cell line and primary cultures of VSMC from control, ClC-3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole-cell patch clamp, co-immunoprecipitation, site-specific mutagenesis and Western blotting, KEY RESULTS: In VSMC, AngII induced Cl(-) currents was carried by the chloride ion channel ClC-3. This current was absent in VSMC from ClC-3 channel null mice. The AngII-induced Cl(-) current involved interactions between ClC-3 channels and Rho-kinase 2 (ROCK2), shown by N- or C-terminal truncation of ClC-3 protein, ROCK2 siRNA and co-immunoprecipitation assays. Phosphorylation of ClC-3 channels at Thr(532) by ROCK2 was critical for AngII-induced Cl(-) current and VSMC migration. The ClC-3 T532D mutant (mutation of Thr(532) to aspartate), mimicking phosphorylated ClC-3 protein, significantly potentiated AngII-induced Cl(-) current and VSMC migration, while ClC-3 T532A (mutation of Thr(532) to alanine) had the opposite effects. AngII-induced cell migration was markedly decreased in VSMC from ClC-3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII-induced cerebrovascular remodelling was decreased in ClC-3 null mice, possibly by the ROCK2 pathway. CONCLUSIONS AND IMPLICATIONS: ClC-3 protein phosphorylation at Thr(532) by ROCK2 is required for AngII-induced Cl(-) current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.

ClC-3 deficiency prevents atherosclerotic lesion development in ApoE-/- mice.

BACKGROUND: Recent evidence suggested that ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, plays a critical role in regulation of a variety of physiological functions. However, remarkably little is known about whether ClC-3 is involved in atherosclerosis. This study aims to establish the involvement and direct role of ClC-3 in atherogenesis and underlying mechanisms by using ClC-3 and ApoE double null mice. METHODS AND RESULTS: After a 16-week western-type high-fat diet, the ClC-3(+/+)ApoE(-/-) mice developed widespread atherosclerotic lesions in aorta. However, the lesion size was significantly reduced in aorta of ClC-3(-/-)ApoE(-/-) mice. Compared with the ClC-3(+/+) controls, there was significantly decreased ox-LDL binding and uptake in isolated peritoneal macrophages from ClC-3(-/-) mice. Moreover, the expression of scavenger receptor SR-A, but not CD36, was significantly decreased in both ClC-3(-/-) peritoneal macrophages and aortic lesions from ClC-3(-/-)ApoE(-/-) mice. These findings were further confirmed in ox-LDL-treated RAW264.7 macrophages, which showed that silence of ClC-3 inhibited SR-A expression, ox-LDL accumulation and foam cell formation, whereas overexpression of ClC-3 produced the opposite effects. In addition, ClC-3 siRNA significantly inhibited, whereas ClC-3 overexpression increased, the phosphorylation of JNK/p38 MAPK in ox-LDL-treated RAW264.7 foam cells. Pretreatment with JNK or p38 inhibitor abolished ClC-3-induced increase in SR-A expression and ox-LDL uptake. Finally, the increased JNK/p38 phosphorylation and SR-A expression induced by ClC-3 could be mimicked by reduction of [Cl(-)]i by low Cl(-) solution. CONCLUSIONS: Our findings demonstrated that ClC-3 deficiency inhibits atherosclerotic lesion development, possibly via suppression of JNK/p38 MAPK dependent SR-A expression and foam cell formation.