Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition

Highlights

• Vascular smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling.

• TMEM16A reduces extracellular matrix deposition via suppressing MMPs expression and activity.

• TMEM16A decreases MMP-2 and MMP-9 expression via inhibiting WNK1 activation.

• TMEM16A suppresses TGF-β/Smad3 signaling pathway.

• TMEM16A suppresses JNK and ERK MAPK signaling pathways, but not p38 MAPK.

Abstract

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca²⁺-activated Cl⁻ channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl⁻-sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Introduction

Ca²⁺-activated Cl⁻ channels (CaCCs) are widely expressed in various tissues and play important roles in many physiological processes, including epithelial fluid secretion, smooth muscle contraction, neuronal and cardiac excitability, sensory transduction, and nociception [1]. The molecular identity of the CaCCs had remained unclear until TMEM16A was identified as the basic component of CaCCs by three independent laboratories in 2008 [[2], [3], [4]]. TMEM16A is the first member of “Transmembrane protein 16” family, also known as anoctamin 1 (Ano1). In the cardiovascular system, TMEM16A expression was identified in vascular smooth muscle cells (VSMCs) [5], atrial fibroblasts [6], ventricular myocytes [7], and vascular endothelial cells [8]. Previous studies showed that TMEM16A downregulation was negatively associated with vascular remodeling during hypertension [5,9]. However, the mechanism underlying the involvement of TMEM16A in hypertension-induced vascular remodeling remains largely unclear.

Cerebrovascular remodeling during hypertension is considered as the leading cause of stroke. It occurs due to chronic exposure of the arterial wall to high blood pressure, structurally and functionally modifying the vessel walls [10]. It has been well-established that the essential determinants of vascular remodeling are related to molecular disorders such as inflammation, and oxidative stress, but also to abnormalities in VSMC functions such as proliferation, apoptosis, migration, and extracellular matrix (ECM) synthesis and degradation [11]. A growing number of evidence have proven that dysregulation of various Cl⁻ channels in VSMCs is a new important mechanism underlying cerebrovascular remodeling [12]. For example, we previously demonstrated that ClC-3 Cl⁻ channel activation participated in cerebrovascular remodeling during hypertension by enhancing proliferation [13], migration [14], and ECM deposition [15] of basilar artery smooth muscle cells (BASMCs), as well as inhibiting apoptosis in these cells [16]. Two other Cl⁻ channels-cystic fibrosis transmembrane conductance regulator, and bestrophin-3-have been proven to protect BASMCs against apoptosis [17,18]. A previous study from our laboratory showed that downregulation of TMEM16A contributed to cerebrovascular remodeling during hypertension by promoting BASMC proliferation [5]. This result was further supported by another group, which showed that inhibition of TMEM16A induced aortic smooth muscle cell proliferation and vascular remodeling during Ang II-induced hypertension [9]. Recently, we have found that TMEM16A participates in BASMC apoptosis [19], which is also in line with the above results that showed an inhibitory effect of TMEM16A on proliferation. However, the effect of TMEM16A on ECM deposition is not known. Wang et al. reported that TMEM16A was upregulated, instead of being downregulated, in the arteries of spontaneous hypertensive rats, and it was positively correlated with VSMC proliferation and Ang II-induced vascular remodeling [20]. This discrepancy indicates that the potential role of TMEM16A in vascular remodeling during hypertension needs further evaluation in vivo.

Because whole-body TMEM16A knockout mice have a complex phenotype with early mortality [21], in this study, we generated vascular smooth muscle-specific TMEM16A transgenic mice. Thereby, we were able to test our hypothesis of TMEM16A protecting against cerebrovascular remodeling during hypertension via suppression of ECM deposition. Furthermore, we investigated whether the underlying mechanism was related to alterations in the expression and activity of metalloproteinases, which are the major participants in ECM degradation [11].