Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway

Abstract

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.

Keywords: FOXO3a; LRRC8A; WNK1; extracellular matrix; matrix metalloproteinases; vascular remodeling.

Fig. 1. Vascular smooth muscle-specific LRRC8A knockout (CKO) ameliorates angiotensin II (Ang II)-induced cerebrovascular remodeling.

Ang II upregulates the protein expression of LRRC8A in the basilar artery (BA) (a) and mouse aortic smooth muscle cells (MASMCs) (b). The expression of LRRC8a was analyzed by immunofluorescence staining (a) and Western blotting (b). The scale bar represents 50 μm. (n = 5, *P < 0.05 vs. sham in (a); n = 6, *P < 0.05 vs. control in b). c Systolic blood pressure (SBP) values in LoxP and CKO mice with or without Ang II perfusion for 4 weeks. (n = 8, *P < 0.05 vs. LoxP+sham, ^#P < 0.05 vs. LoxP+Ang II). Vascular smooth muscle was stained with HE (d) and vascular remodeling was evaluated by measuring the wall diameter (e), lumen diameter (f), media thickness (g), cross-sectional area (h), and media/lumen ratio (i). The scale bar represents 50 μm. (n = 5, *P < 0.05 vs. LoxP+sham, ^#P < 0.05 vs. LoxP+Ang II)

Fig. 2. LRRC8A knockout in vascular smooth muscle mitigates Ang II-induced extracellular matrix (ECM) deposition.

The expression of collagen I (Col I, a) and fibronectin (Fn, b) in the BAs of LoxP and CKO mice with Ang II-induced hypertension was analyzed by immunofluorescence staining. (n = 5, *P < 0.05 vs. LoxP+sham, ^#P < 0.05 vs. LoxP+Ang II) c, d. LRRC8A silencing prevented the upregulation of collagen I and fibronectin induced by Ang II in MASMCs (c), and LRRC8A overexpression accelerated it (d). The cells were transfected with LRRC8A siRNA or a LRRC8A-harboring vector for 24 h and then treated with Ang II (100 nM) for another 24 h. Neg, negative control siRNA; si, LRRC8A siRNA; vec, empty vector; 8A, LRRC8A-harboring vector. (n = 5, *P < 0.05 vs. control, ^#P < 0.05 vs. Ang II)

Fig. 3. LRRC8A knockout in vascular smooth muscle mitigates the Ang II-induced upregulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).

a GO analysis based on mRNA-sequencing of the aortas of CKO mice and their littermate LoxP mice with Ang II-induced hypertension. b The heatmap shows significantly differentially expressed mRNAs related to the ECM. c The mRNA levels of MMP2, MMP-9, and MMP-14 in the aortas of LoxP and CKO mice with Ang II-induced hypertension were examined by qPCR. (n = 5–6, *P < 0.05 vs. LoxP+sham, #P < 0.05 vs. LoxP+Ang II). d The expression of MMP-9 in the BAs of LoxP and CKO mice with Ang II-induced hypertension was analyzed by immunofluorescence staining. (n = 5, *P < 0.05 vs. LoxP+sham, #P < 0.05 vs. LoxP+Ang II) e, f. LRRC8A silencing prevented the upregulation of MMPs (MMP-2, MMP-9, and MMP-14) and TIMPs (TIMP-1 and TIMP-2) induced by Ang II in MASMCs (e), and LRRC8A overexpression accelerated it (f). The cells were transfected with LRRC8A siRNA or the LRRC8A-harboring vector for 24 h and then treated with Ang II (100 nM) for another 24 h. Neg, negative control siRNA; si, LRRC8A siRNA; vec, empty vector; 8A, LRRC8A-harboring vector. (n = 4–6, *P < 0.05 vs. control, #P < 0.05 vs. Ang II)

Fig. 4. WNK1 mediates the effect of LRRC8A on MMP expression in respond to Ang II stimulation.

The expression of p-WNK1 (a) and WNK1 (b) in the BAs of LoxP and CKO mice with Ang II-induced hypertension was analyzed by immunofluorescence staining. (n = 5, *P < 0.05 vs. LoxP+sham, #P < 0.05 vs. LoxP+Ang II) c, d. LRRC8A silencing inhibited the upregulation of p-WNK1 induced by Ang II in MASMCs(c), and LRRC8A overexpression increased it (d). The expression of WNK1 was not affected. The cells were transfected with LRRC8A siRNA or the LRRC8A-harboring vector for 24 h and then treated with Ang II (100 nM) for another 24 h. Neg, negative siRNA; si, LRRC8A control siRNA; vec, empty vector; 8A, LRRC8A-harboring vector. (n = 5, *P < 0.05 vs. control, #P < 0.05 vs. Ang II) e. WNK1 silencing abrogated the effect of LRRC8A on the expression of MMP-2 and MMP-9, as well as collagen I and fibronectin, in MASMCs treated with Ang II. The cells were transfected with the LRRC8A-harboring vector with or without WNK1 siRNA for 24 h and then treated with Ang II (100 nM) for another 24 h. 8 A, LRRC8A-harboring vector; si, WNK1 siRNA. (n = 5–6, *P < 0.05 vs. control, #P < 0.05 vs. Ang II, +P < 0.05 vs. Ang II + 8A)

Fig. 5. WNK1 interacts with FOXO3a and facilitates its phosphorylation.

a The mRNA expression of FOXOs in MASMCs. (n = 6) b. The binding of FOXO family members and WNK1. The score between FOXO3a and WNK1 was optimized. (n = 10) c. Image of the binding of FOXO3a and WNK1. d–f WNK1 interacted with FOXO3a but not FOXO1, and Ang II increased this interaction in MASMCs. Cell lysates were immunoprecipitated with an anti-FOXO1 antibody (d), an anti-FOXO3a antibody (e), or an anti-WNK1 antibody (f), and then immunoblotted with an anti-WNK1 antibody (d, e), an anti-p-WNK1 antibody (e), or an anti-FOXO3a antibody (f). (n = 3–4, *P < 0.05 vs. control) g, h. WNK1 silencing decreased the upregulation of p-FOXO3a induced by Ang II in MASMCs (g), and WNK1 overexpression potentiated it (h). The expression of FOXO3a was not affected. The cells were transfected with WNK1 siRNA or the WNK1-harboring vector for 24 h and then treated with Ang II (100 nM) for another 24 h. Neg, negative control siRNA; si, WNK1 siRNA; vec, empty vector; WNK1, WNK1-harboring vector. (n = 5, *P < 0.05 vs. control, #P < 0.05 vs. Ang II)

Fig. 6. WNK1 mediates the effect of LRRC8A on FOXO3a phosphorylation in response to stimulation with Ang II.

The expression of p-FOXO3a (a) and FOXO3a (b) in the BAs of LoxP and CKO mice with Ang II-induced hypertension was analyzed by immunofluorescence staining. (n = 5, *P < 0.05 vs. LoxP+sham, #P < 0.05 vs. LoxP+Ang II) c, d LRRC8A silencing inhibited the upregulation of p-FOXO3a induced by Ang II in MASMCs (c), and LRRC8A overexpression increased it (d). The expression of FOXO3a was not affected. The cells were transfected with LRRC8A siRNA or the LRRC8A-harboring vector for 24 h and then treated with Ang II (100 nM) for another 24 h. Neg, negative control siRNA; si, LRRC8A siRNA; vec, empty vector; 8A, LRRC8A-harboring vector. (n = 5, *P < 0.05 vs. control, #P < 0.05 vs. Ang II) e WNK1 silencing abrogated the effect of LRRC8A on the phosphorylation of FOXO3a in MASMCs treated with Ang II. The cells were transfected with the LRRC8A-harboring vector with or without WNK1 siRNA for 24 h and then treated with Ang II (100 nM) for another 24 h. 8 A, LRRC8A-harboring vector; si, WNK1 siRNA. (n = 5, *P < 0.05 vs. control, #P < 0.05 vs. Ang II, +P < 0.05 vs. Ang II + 8A) f Schematic drawing of the study conclusions. Opening of the LRRC8A Cl⁻ channel reduces intracellular Cl⁻ concentrations and phosphorylates WNK1, a “Cl⁻-sensitive kinase”. Activated WNK1 interacts with FOXO3a and increases its phosphorylation, which increases the transcription of MMPs and TIMPs, thereby resulting in ECM deposition and vascular remodeling.