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.

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.

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.