ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

Abstract

Background: 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).

Methods: Cell 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.

Results: Our 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.

Conclusion: This 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.

Keywords: Angiotensin II; Cerebrovascular smooth muscle cells; Chloride; ClC-2; Proliferation; Wnt/β-catenin signaling.

Fig. 1

ClC-2 knockdown inhibited the AngII-induced efflux of Cl⁻ in HBVSMCs. a HBVSMCs were treated with angiotensin II (AngII) at different concentrations (10^− 9, 10^− 8 10^− 7 and 10^− 6 M) for 48 h. Cell viability was determined using the CCK-8 assay. b Intracellular Cl⁻ concentration [Cl⁻]_i was examined using an MEQ fluorescence probe. c The correlation between [Cl⁻]_i and cell viability was analyzed. d and e – The expression of ClC-2 in the cells treated as described in (a) was examined using western blotting (d) and quantitative real-time PCR (e). f Cells were treated with ClC-2 siRNA (20 nM) or negative siRNA for 48 h before AngII incubation (10^− 7 M) for a further 48 h. [Cl⁻]_i was examined. *p < 0.05, **p < 0.01 vs. control, ^##p < 0.01 vs. AngII alone, n = 6

Fig. 2

Lack of ClC-2 reduced AngII-induced HBVSMC proliferation. a and b Cells were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) for 48 h in prior to angiotensin II (AngII) treatment (10^− 7 M) for another 48 h. Cell proliferation was determined using the CCK-8 assay (a) and BrdU incorporation (b). c and d The protein expressions of PCNA (c) and Ki67 (d) were detected using western blotting. **p < 0.01 vs. control, ^##p < 0.01 vs. AngII alone, n = 5

Fig. 3

ClC-2 downregulation prevented AngII-induced HBVSMC migration and invasion. a HBVSMCs transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) were subjected to angiotensin II (AngII) treatment (10^− 7 M). The wound healing assay was performed. Representative images are shown (× 100). b The quantification results for the wound closure. c HBVSMC migration was examined via transwell analysis. Representative images are shown (× 100). d The columns represent the relative numbers of invasive cells. **p < 0.01 vs. control, ^##p < 0.01 vs. AngII alone, n = 6

Fig. 4

ClC-2 inhibition attenuated the AngII-induced activation of Wnt/β-catenin signaling. a through f HBVSMCs were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) and then stimulated with angiotensin II (AngII; 10^− 7 M) for 48 h. Shown are the western blotting results for β-catenin phosphorylation (a), β-catenin cytosol (b) and nuclear protein (c) levels, GSK-3β phosphorylation (d), and survivin (e) and cyclin D1 (f) protein expression. g Quantitative real-time PCR analysis of Wnt3a and Wnt5a mRNA expression. h The cells were treated with recombinant Wnt3a (100 ng/ml) for 48 h. Wnt3a expression was examined using quantitative real-time. i Viability of HBVSMCs transfected with ClC-2 siRNA followed by co-incubation with recombinant Wnt3a and AngII. **p < 0.01 vs. control, ^##p < 0.01 vs. AngII alone, ^$$p < 0.01 vs. AngII+siClC-2, n = 4

Fig. 5

ClC-2 deficiency blocked cerebrovascular remodeling. a C57BL/6 mice were injected with ClC-2-shRNA adenovirus (sh-ClC-2) or Lacz adenovirus before AngII infusion. Representative images of hematoxylin and eosin staining of basilar arteries. b Vascular remodeling was evaluated based on the cross-sectional area (CSA). c Representative images of immunofluorescence staining for α-SMA expression. d Bar graph showing the the relative fluorescence density values for α-SMA expression. e through h β-catenin (e) and GSK-3β (f) phosphorylation, and survivin (g) and cyclin D1 (h) protein expression were determined via western blotting. *p < 0.01, **p < 0.01 vs. Lacz, ^##p < 0.01 vs. AngII+Lacz, n = 8 mice in each group