LRRC8A is essential for volume‐regulated anion channel in smooth muscle cells contributing to cerebrovascular remodeling during hypertension

Abstract Objectives Recent studies revealed LRRC8A to be an essential component of volume‐regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A‐dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension. Materials and Methods siRNA‐mediated knockdown and adenovirus‐mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle–specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling. Results LRRC8A is essential for volume‐regulated chloride current (I Cl, Vol) in BASMCs. Overexpression of LRRC8A induced a voltage‐dependent Cl− current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K‐p85/AKT axis. Smooth muscle‐specific upregulation of LRRC8A aggravated Angiotensin II‐induced cerebrovascular remodeling in mice. Conclusions LRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension.


| INTRODUC TI ON
Cell volume homeostasis is essential for many biological processes involved in vertebrate heath and survival. 1,2 The decreased extracellular or increased intracellular osmolarity under hypotonic stress will evoke the process of regulatory volume decrease (RVD), which involves the activation of the channel-mediated Cl − and K + transport as well as taurine efflux. 3,4 Volume-regulated anion channel (VRAC) is ubiquitously expressed in eukaryotic cells and is involved in many physiological and pathophysiological functions. [5][6][7][8][9] Although the physiological and pharmacological properties of VRAC have been described in detail over the past 30 years, the precise molecular identity is still controversial. Previous studies have identified some candidates such as P-glycoprotein, ClC-3, and Bestrophin-1 as key components required for VRAC activity and the RVD process. [10][11][12] However, the sufficiency of these candidates for VRAC activity was not fully verified in all cell types by the subsequent studies. LRRC8A, a leucine-rich repeat-containing protein, was recently identified as an essential component of VRAC using an unbiased Genomewide RNAi Screen in many cell types including HEK293T and Tlymphocytes. 13,14 However, Andrea Milenkovic et al. found that Bestrophin 1, but not LRCC8A, was indispensable for cell volume regulation in retinal pigment epithelium (RPE) cells differentiated from human-induced pluripotent stem cells. 15 Another study also demonstrated that LRRC8A was not essential for VRAC activation in HeLa cells, 16 suggesting that the contribution of LRRC8A to cell volume regulation and the molecular identity of VRAC are variable in different cell types or tissues.
Vascular remodeling is the alteration of vascular structure, and it is characterized by the thinning of the lumen diameter inside and outside the blood vessels and increased tube wall area during the development of hypertension. Vascular remodeling is an active process in response to the development of hypertension and subsequently contributes to the pathophysiology of cardiovascular diseases such as stroke and myocardial infarction. [17][18][19] Our previous study found that the activity of VRAC enhanced cell proliferation in hypertensive rat basilar smooth muscle cells (BASMCs) and cerebrovascular remodeling, suggesting that VRAC is involved in vascular remodeling process during chronic hypertension. 20,21 Moreover, we also demonstrated that Simvastatin could attenuate rat cerebrovascular remodeling via inhibition of VRAC, 22 suggesting that VRAC is a promising therapeutic target for vascular remodeling. Particularly, we previously demonstrated ClC-3 as a potential molecular identity of VRAC, which is at least partially contributing to cell volume regulation in smooth muscle cells and involves in the process of cerebrovascular remodeling during hypertension. 23,24 Given the complexity and controversy of VRAC subunit composition in different tissues and based on our previous studies, we hypothesize that LRRC8A is an essential component of VRAC in smooth muscle cells, and that LRRC8A-mediated BASMCs proliferation is required for cerebrovascular remodeling during hypertension. Here, we found that LRRC8A is indispensable for volume regulation in BASMCs. We also provided evidence that LRRC8A regulates the process of BASMCs proliferation through phosphorylation of lysine-deficient protein kinase 1 (WNK1), and that the smooth muscle-specific overexpression of LRRC8A aggravates Angiotensin II-induced cerebrovascular remodeling in mice.

| MATERIAL S AND ME THODS
The sources of reagents are given in Table S1. All experimental procedures were approved by the Sun Yat-sen University Animal Care and Use Committee (SYSU-IACUC-2020-000446) and were in accordance with the "Guide for the Care and Use of Laboratory Animals" issued by the Ministry of Science and Technology of China and the current NIH guidelines.

| Cell culture
Four-week-old healthy male Sprague-Dawley rats (80-100 g) were used for BASMCs isolation. Rat BASMCs were isolated and cultured from rat basilar arteries using the method as described previously. 22 BASMCs were incubated in Dulbecco's Modified Essential Medium (DMEM)/F-12 supplemented with 20% fetal bovine serum (FBS).
Passages 8-12 of BASMCs were used for experiments. A10 vascular smooth muscle cells (American Type Culture Collection) were cultured in DMEM/F-12 containing 10% Newborn Calf Serum (NCS) as described before. 25

| siRNA transfection
Gene silencing was performed using gene-specific small interfering RNA (siRNA) as described before. 26 The sequences of siRNA against rat LRRC8A and WNK1 were synthesized by InvitrogenTM (Life Technologies). The sequences were Rat LRRC8A, 5′GCCUGCAUUGGUUUGCCAATT 3′ and Rat WNK1, 5′GGUGUCGGCAAAUCCUUAATT3′. BASMCs were transfected with negative siRNA (scrambled siRNA), LRRC8A siRNA, or WNK1 siRNA in serum-free-medium using Hiperfect Transfection Reagent (Qiagen). Following 4-6 h of incubation, fresh medium containing 20% FBS was added to BASMCs for another 48 h. to the protocol as previously described. 27 BASMCs were transduced with adenovirus when the cells reached 50% confluence.

| Western blot
Western blot was performed as described before. 28 Briefly, tissues or cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail (Merk). Proteins were separated by 8-12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% milk at room temperature for 1 h, blots were incubated overnight with primary antibodies at 4°C and then incubated for 1 h with secondary antibodies at room temperature. Bands were detected with Pierce ECL western blotting substrate and quantified with the computeraided 1-D gel imaging system (Bio-Rad).

| Cell viability assay
Cell viability was measured by a Cell Counting Assay Kit-8 (CCK-8; Dojindo Molecular Technologies) according to our established method. 30 Briefly, 2 × 10 3 BASMCs were seeded in a 96-well plate and preincubated in a humidity chamber for 24 h. Ten microliters of CCK-8 solution was added to each well and incubated for 2 h, after which the absorbance at 450 nm was measured using a microplate reader.

| LRRC8A smooth muscle-specific transgenic mice
The tissue-specific LRRC8A transgenic LoxP line was produced by Cyagen as described before. 34 Briefly, the fertilized eggs of C57BL/6 mice were transfected with the targeting carrier containing the  Table S2.

| Animal models of hypertension
The Ang II-induced hypertensive Animal model was implemented with an osmotic pump (Alzet model, 1004) filled with Ang II in saline solution as described previously. 34 Mice in control group were implanted with an osmotic pump with normal saline only. The 2k2c hypertensive rats were prepared as described previously. 35

| Immunostaining
Mice were perfusion-fixed at a constant pressure (100 mmHg) via the left ventricle with Krebs solution, followed by 4% freshly depolymerized paraformaldehyde for 10 min. The brains were carefully removed, and sections (8 µm) of freshly frozen basilar arteries were prepared as described before. 26 Immunofluorescence staining was carried out using specific primary and secondary antibodies as described before. 36 All the images were scanned by a Zeiss microscope (Axio Imager Z1).

| Electron microscopy
The mice were perfusion-fixed as described before. 24 Brains were removed, and tissue blocks containing the basilar artery at midpoint were cut into cubes of 1 × 1 × 3 mm. Ultrathin sections at 80-100 nm were prepared and stained with uranyl acetate and lead citrate, which were then viewed under a transmission electron microscope (FEI TECNAI spirit G2).

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software). Data were expressed as means ± SE. Statistical significance was determined by Student's t test or ANOVA followed by the Bonferroni multiple comparison test. N represented the number of independent experiments performed with different batches of cells or different mice. A value of p < 0.05 was considered statistically significant.

| LRRC8A is essential for VRAC channel activity in BASMCs
To investigate the association between LRRC8A and VRAC activity in VSMCs, we first examined the endogenous expression and localization of LRRC8A in A10 vascular smooth muscle cell line and freshly isolated BASMCs. Western blot results showed that LRRC8A was widely expressed in both A10 cells and BASMCs ( Figure 1A).
Immunostaining using FITC-labeled LRRC8A antibody showed that LRRC8A was predominantly expressed on cell surface in BASMCs ( Figure 1B).
To determine whether LRRC8A is essential for VRAC activity in BASMCs, we directly recorded hypotonic solution-induced VRAC currents by whole-cell patch-clamp recording in BASMCs.
We effectively silenced the endogenous expression of LRRC8A in BASMCs by a specific siRNA ( Figure S1). The results showed that knockdown of LRRC8A significantly decreased hypotonic solutioninduced Cl − current in BASMCs ( Figure 1C). The current density reduced from −6.80 ± 0.90 pA/pF to −3.84 ± 0.61 pA/pF at −80 mV and 19.04 ± 3.40 pA/pF to 6.63 ± 0.87 pA/pF at +80 mV, respectively ( Figure 1D,E). It has been reported that overexpression of LRRC8A in HEK293T and HeLa cells did not enhance the I Cl, Vol current density. 13 Consistent with that previous study, we also found that adenovirus-mediated overexpression of LRRC8A in BASMCs could not further increase the density of hypotonic solutioninduced current ( Figure 1F-H, Figure S2). However, we unexpectedly found that the overexpression of LRRC8A induced an enlarged outwardly rectifying current in isotonic solution; the current density was increased from −3.09 ± 0.67 pA/pF to −6.18 ± 1.20 pA/pF at −80 mV and 5.39 ± 1.10 pA/pF to 20.19 ± 2.39 pA/pF at 80 mV, respectively, in the isotonic bath solutions.

| Overexpression of LRRC8A induced a voltage-dependent Cl − current independently of hypotonicity stimulation
To better understand the electrophysiological properties of

LRRC8A-activated Cl − current in isotonic solution
LRRC8A is a protein consisting of four transmembrane helices in its N-terminal region, followed by leucine-rich repeats domain (LRRD) in the middle and its C-terminal regions. It has been demonstrated that N-terminal transmembrane pore domain is essential for channel activity. 13,37 However, the function of LRRD remains unclear.
We constructed a truncated LRRC8A, in which the C-terminal LRRD was deleted, and transiently expressed this construct in BASMCs ( Figure 3A). Whole-cell patch-clamp recording showed that transient expression of LRRD-truncated LRRC8A did not induce larger VRAC current compared to the control group during hypotonic stimulation.
Interestingly, unlike LRRC8A full length protein, the Cl − current triggered by LRRC8A overexpression in the absence of hypotonic stimulation was not observed in LRRD-truncated mutant transfected cells ( Figure 3B,C). Consistent with the current recording data, chloride concentration was not altered in LRRD-truncated mutant transfected cells ( Figure 3D,E). These data suggest that LRRD was indispensable for this voltage-dependent Cl − current in the absence of hypotonicity stimulation.

| LRRC8A promotes proliferation in BASMCs
It has been demonstrated that VRAC and volume-regulated Cl − movement are strongly associated with cerebrovascular remodeling by affecting BASMCs proliferation in our previous studies. 20 We next wanted to examine the effect of LRRC8A on BASMCs proliferation.
Our results showed that siRNA-mediated knockdown of LRRC8A significantly reduced the proliferation rate, whereas overexpression of LRRC8A increased the proliferation rate of BASMCs ( Figure 4A).

| LRRC8A promotes BASMCs proliferation through WNK1/PI3K-p85/AKT signaling pathway
Our previous studies have demonstrated that WNK1 is sensitive to hypotonic stimulation-dependent Cl − transport from inside the cell to outside the cell, which leads to the activation of WNK1 through phosphorylation and PI3K-p85/AKT signaling axis. 31 Figure S5). LRRC8A SOE mice were implanted with Angiotensin II infusion pumps for 4 weeks starting from 8 weeks after birth. Our results indicated that Angiotensin II infusion significantly increased cerebrovascular vascular remodeling; the remodeling of basilar arteries in LRRC8A SOE mice was much more severe than control mice after Angiotensin II infusion, leading to a significant morphological change, including increased media thickness and media/lumen ratio ( Figure 6G). Meanwhile, the cerebrovascular vascular remodeling was further validated by electron microscopy analyses. As shown in Figure 6H

| DISCUSS ION
The molecular identity of VRAC is complicated. Although some candidates, such as P-glycoprotein, ClC-3, and Bestrophin-1, [10][11][12] have been reported as components of VRAC, none of them have been demonstrated to be sufficient for VRAC activity in all cell types. In 2014, the studies from two laboratories identified LRRC8A as an essential component of VRAC. 13,14 It has been demonstrated that LRRC8A homohexamers poorly recapitulated VRAC electrophysiological properties. 38 Accumulating evidence from the structure of LRRC8A and other electrophysiological studies indicated that heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog are crucial for VRAC activity. [37][38][39][40] However, some other studies show that LRRC8A is dispensable for VRAC activity, 15,16 suggesting that LRRC8A alone is not sufficient for volume regulation in response to hypotonic stimulation and VRAC activity in all cell types. The controversy of these studies led us to in-

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request.