Ming-Ming Ma

Blood pressure (BP) is positively correlated with the activity of volume-regulated chloride channels (VRCCs) in vascular smooth muscle cells (VSMCs) during hypertension, and LRRC8A has recently been identified as the key molecular component of VRCCs. However, the role of LRRC8A in BP regulation remains unclear. Here, we generated SMC-specific LRRC8A knockout (CKO) mice and performed bulk RNA-sequencing, ChIP-sequencing, and coimmunoprecipitation coupled with mass spectrometry to elucidate the underlying mechanisms. We found that systolic BP was largely reduced in CKO mice under basal conditions as well as in those with angiotensin II- or DOCA-salt-induced hypertension. Aortas from CKO mice exhibited decreased vasoconstriction, whereas vasodilation and cardiac function remained unaffected. Mechanistically, LRRC8A deletion reduced STAT1 degradation and enhanced Calcrl transcription, thereby strengthening the inhibitory effect of Calcrl on myosin light chain phosphorylation and identifying LRRC8A deficiency as a negative regulator of VSMC contractility. Moreover, LRRC8A deficiency promoted the interaction between CSRP2 and PIAS1 and subsequently suppressed STAT1 degradation. Pharmacological inhibition of STAT1 with fludarabine abolished the protective effects of LRRC8A deficiency against hypertension and vasoconstriction. In summary, our findings reveal an essential role for LRRC8A in BP regulation and highlight a potential therapeutic strategy for hypertension.

Vascular smooth muscle cells (VSMCs) are essential for maintaining arterial wall integrity, with their associated apoptosis critically involved in cardiovascular diseases. Intracellular chloride anion (Cl ⁻ ) dysregulation is closely linked to apoptosis. However, the underlying mechanism via chloride‐sensitive kinases remains unclear. Here, we show that hypotonic stimulation activates volume‐regulated chloride channels (VRCCs) and induces the phosphorylation of the chloride‐sensitive kinase WNK1. Activated WNK1 protects VSMCs against H ₂ O ₂ ‐induced apoptosis by maintaining the Bcl‐2/Bax balance, preserving mitochondrial membrane potential and inhibiting caspase‐9/3 activation. These findings uncover a novel protective role of WNK1 in VRCC‐mediated Cl ⁻ signalling and provide new perspectives for targeting chloride‐sensitive kinases in the treatment of hypertension and related cardiovascular diseases.

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.

Background and Purpose: Atherosclerosis induced by cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) pathway, is a major concern after organ transplantation. However, the atherosclerotic mechanisms of CsA remain obscure. We previously demonstrated that calcineurin/NFAT signalling inhibition contributes to atherogenesis via suppressing microRNA‐204 (miR‐204) transcription. We therefore hypothesised that miR‐204 is involved in the development of CsA‐induced atherosclerosis.Experimental Approach: ApoE^−/− mice with macrophage‐miR‐204 overexpression were generated to determine the effects of miR‐204 on CsA‐induced atherosclerosis. Luciferase reporter assays and chromatin immunoprecipitation sequencing were performed to explore the targets mediating miR‐204 effects.Key Results: CsA alone did not significantly affect atherosclerotic lesions or serum lipid levels. However, it exacerbated high‐fat diet‐induced atherosclerosis and hyperlipidemia in C57BL/6J and ApoE^−/− mice, respectively. miR‐204 levels decreased in circulating monocytes and plaque lesions during CsA‐induced atherosclerosis. The upregulation of miR‐204 in macrophages inhibited CsA‐induced atherosclerotic plaque formation but did not affect serum lipid levels. miR‐204 limited the CsA‐induced foam cell formation by reducing the expression of the scavenger receptors SR‐BII and CD36. SR‐BII was post‐transcriptionally regulated by mature miR‐204‐5p via 3′‐UTR targeting. Additionally, nuclear‐localised miR‐204‐3p prevented the CsA‐induced binding of Ago2 to the CD36 promoter, suppressing CD36 transcription. SR‐BII or CD36 expression restoration dampened the beneficial effects of miR‐204 on CsA‐induced atherosclerosis.Conclusion and Implications: Macrophage miR‐204 ameliorates CsA‐induced atherosclerosis, suggesting that miR‐204 may be a potential target for the prevention and treatment of CsA‐related atherosclerotic side effects.

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.

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 (H₂O₂) 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 H₂O₂ 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 H₂O₂‐induced apoptosis via modulation of mitochondrial membrane permeability in VSMCs. This study establishes TMEM16A as a target for therapy of several remodelling‐related diseases.

Ca ²⁺ -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 Ca ²⁺ -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.