Abstract
BackgroundMishandling of intracellular chloride (Cl−) concentration ([Cl−]i) in cerebrovascular smooth muscle cells is implicated in several pathological processes, including hyperplasia and remodeling. We investigated the effects of ClC-2-mediated Cl− efflux on the proliferation of human brain vascular smooth muscle cells (HBVSMCs) induced by angiotensin II (AngII).MethodsCell proliferation and motility were determined using the CCK-8, bromodeoxyuridine staining, wound healing and invasion assays. ClC-2, PCNA, Ki67, survivin and cyclin D1 expression, and β-catenin and GSK-3β phosphorylation were examined using western blotting. Histological analyses were performed using hematoxylin and eosin staining and α-SMA staining.ResultsOur results showed that AngII-induced HBVSMC proliferation was accompanied by a decrease in [Cl−]i and an increase in ClC-2 expression. Inhibition of ClC-2 by siRNA prevented AngII from inducing the efflux of Cl−. AngII-induced HBVSMC proliferation, migration and invasion were significantly attenuated by ClC-2 downregulation. The inhibitory effects of ClC-2 knockout on HBVSMC proliferation and motility were associated with inactivation of the Wnt/β-catenin signaling pathway, as evidenced by inhibition of β-catenin phosphorylation and nuclear translocation, and decrease of GSK-3β phosphorylation and survivin and cyclin D1 expression. Recombinant Wnt3a treatment markedly reversed the effect of ClC-2 knockdown on HBVSMC viability. An in vivo study revealed that knockdown of ClC-2 with shRNA adenovirus ameliorated basilar artery remodeling by inhibiting Wnt/β-catenin signaling in AngII-treated mice.ConclusionThis study demonstrates that blocking ClC-2-mediated Cl− efflux inhibits AngII-induced cerebrovascular smooth muscle cell proliferation and migration by inhibiting the Wnt/β-catenin pathway. Our data indicate that downregulation of ClC-2 may be a viable strategy in the prevention of hyperplasia and remodeling of cerebrovascular smooth muscle cells.
Highlights
Mishandling of intracellular chloride (Cl−) concentration ([Cl−]i) in cerebrovascular smooth muscle cells is implicated in several pathological processes, including hyperplasia and remodeling
Cl− channel (ClC)-2 knockdown reversed Angiotensin II (AngII)-induced decrease in [Cl−]i levels To explore the relationship between cerebrovascular proliferation and [Cl−]i, human brain vascular smooth muscle cells (HBVSMCs) were treated with various concentrations of AngII and the cell viability and [Cl−]i were measured
The results showed that AngII markedly increased ClC-2 expression, suggesting that ClC-2 may be involved in AngII-mediated changes in [Cl−]i (Fig. 1d and e)
Summary
Mishandling of intracellular chloride (Cl−) concentration ([Cl−]i) in cerebrovascular smooth muscle cells is implicated in several pathological processes, including hyperplasia and remodeling. We investigated the effects of ClC-2-mediated Cl− efflux on the proliferation of human brain vascular smooth muscle cells (HBVSMCs) induced by angiotensin II (AngII). Cerebrovascular smooth muscle cell hyperplasia and direct migration from the media into the intima in the basilar artery play an important role in the pathogenesis of vascular remodeling, hypertension and strokes [1]. Angiotensin II (AngII) plays a role in the cellular growth and survival of vascular smooth muscle cells (VSMCs) [3, 4]. Several studies have demonstrated that dysregulated intracellular Cl− concentration ([Cl−]i) is implicated in endothelial cell apoptosis, foam cell formation, vascular inflammation, hypertension and atherosclerosis [7,8,9,10,11]. A recent study reported that reduction in serum chloride is an independent prognostic factor for acute heart failure [12]
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