Volume Matters: Novel Roles of the Volume-regulated ClC-3 Channels in Hypertension-induced Cerebrovascular Remodeling

The adaptive remodeling of cerebral arteries, including hypertrophy (medial thickening) and eutrophy (luminal narrowing), is a prominent feature of hypertension and a major risk factor for stroke.^1–3 Multiple mechanisms, such as arterial smooth muscle cell proliferation, migration and apoptosis, endothelial cell dysfunction, inflammation and fibrosis, have been implicated in structural remodeling in response to chronic hypertension.⁴ Although the hypertrophic increase in cell volume of arterial smooth muscle cells (SMCs) is the primary mechanism for the thickening of mesenteric arterial media in spontaneously hypertensive rats¹ and of cerebral arteries in the 2-kidney, 2-clip (2-k,2-c) renal hypertensive rats,³ the impact of the hypertension-induced changes in cell volume of the arterial SMCs and its clinical significance are still unknown.

Alterations in cell volume are common adaptive mechanisms of most mammalian cells, including arterial SMCs, in response to metabolic, osmotic and/or static pressure perturbations.⁵ Cells are able to precisely maintain their size through the regulated loss or gain of intracellular ions or other osmolytes to avoid excessive alterations of cell volume that may jeopardize structural integrity and a variety of cellular functions.⁵ Acute increase in cell volume will initiate the regulatory volume decrease (RVD) process in order to bring the cells back to their initial volume, which is achieved by the opening of volume-regulated Cl⁻ channels (VRCCs) and other channels and transporters mediating Cl⁻, K⁺, and taurine efflux.⁵ As one of the most important mechanisms for cell volume homeostasis activation of VRCCs has been implicated in a number of vital cellular functions involved in hypertension-induced vascular remodeling, including the regulation of membrane potentials, vascular myogenic tome, cell proliferation, migration and apoptosis (Figure 1).^6–8 For example, high blood pressure-induced depolarization and contraction of cerebral artery smooth muscle may be partially mediated by VRCCs.⁷ There is evidence that the magnitude of VRCC currents in actively growing vascular SMCs is higher than in growth-arrested or differentiated SMCs, suggesting that VRCCs may be important for SMC proliferation.⁷ Therefore, hypertension-induced increase in cell volume and activation of VRCCs may contribute to the structural and functional remodeling through an integrated regulation of multiple cellular functions.

Figure 1. Schematic representation of ClC-3 Cl⁻ channels in vascular smooth muscle cells.

ClC-3, a member of voltage-gated ClC Cl⁻ channel family, encodes Cl⁻ channels in vascular smooth muscle cells that are volume regulated (I_Cl,vol) and can be activated by cell swelling (I_Cl,swell) induced by exposure to hypotonic extracellular solutions or possibly membrane stretch. I_Cl,b is a basally activated ClC-3 Cl⁻ current. α-helices of ClC-3 are shown as a–r (see review by Duan¹). ClC-3 proteins are expressed on both sarcolemmal membrane and intracellular organelles including mitochondria (mito) and endosomes. The proposed model of endosome ion flux and function of Nox1 and ClC-3 in the signaling endosome is adapted from Miller Jr. et al.⁸ Binding of IL-1β or TNF -α to the cell membrane initiates endocytosis and formation of an early endosome (EEA1 and Rab5), which also contains NADPH oxidase subunits Nox1 and p22phox, in addition to ClC-3. Nox1 is electrogenic, moving electrons from intracellular NADPH through a redox chain within the enzyme into the endosome to reduce oxygen to superoxide. ClC-3 functions as a chloride proton exchanger, required for charge neutralization of the electron flow generated by Nox1. The ROS generated by Nox1 result in NF-κB activation. Both ClC-3 and Nox1 are necessary for generation of endosomal ROS and subsequent NF-κB activation by IL-1β or TNF – α in VSMCs. Statins block ClC-3 channels, which causes hyperpolarization of the cell membrane, closure of Ca²⁺ channels and vasorelaxation, and inhibition of cell proliferation. PKC, protein kinase C; PP, serine threonine protein phosphatases; α − AR, α-adrenergic receptor; Gi, heterodimeric inhibitory G protein. Nox: NADPH oxidase.

The short isoform of ClC-3 (sClC-3), a member of the ClC superfamily of voltage-dependent Cl⁻ channels, has been proposed to be the molecular correlate of a key component of the native VRCCs in cardiac myocytes and vascular SMCs. ^{7, 9} A series of recent independent studies from many laboratories further strongly corroborated this hypothesis (please see recent reviews by Duan⁶ and Hume et al.⁷). It has been demonstrated that ClC-3 is expressed in aortic, pulmonary, and cerebral artery SMCs of many species including human. Knockdown of ClC-3 by siRNA, shRNA, and antisense and intracellular dialysis of anti-ClC-3 antibody (Ab) all consistently eliminated VRCC currents in many types of cells. Recent accumulating evidence suggests an important role of ClC-3 and VRCCs in the regulation of cell proliferation induced by hypertrophic alternations in cell volume. A recent study found that static pressure increased VRCCs and ClC-3 expression and promoted rat aortic VSMC proliferation and cell cycle progression. Inhibition of VRCCs with pharmacological blockers (such as diphenyleneiodonium, DPI) or knockdown of ClC-3 with ClC-3 antisense oligonucleotide dramatically inhibited pressure evoked cell proliferation and cell cycle progression of rat aortic SMCs. These data suggest that ClC-3 and VRCCs may play a critical role in static pressure induced cell proliferation and cell cycle progression. Since arterial SMC proliferation is a key event in the development of hypertension associated vascular disease, ClC-3 and VRCCs may be of unique therapeutic importance for treatment of hypertension attendant vascular complications.

Recent studies have demonstrated that statins are effective in attenuating vascular remodeling although the underlying mechanisms are still not determined. In this issue of Hypertension Liu et al. ¹⁰ used integrated, multiple approaches and performed a thorough investigation on the effects of simvastatin on the hypertension induced cerebrovascular remodeling and VRCCs in basilar smooth muscle cells (BASMCs). They first demonstrated that simvastatin improved the hypertension-caused cerebrovascular remodeling in 2-k,2-c renal hypertensive rats. Then they used cultured rat BASMCs to further study the effects of simvastatin on cell proliferation and the whole-cell VRCC current and volume-regulated Cl⁻ movement. They found that simvastatin inhibited cell proliferation and also the volume-regulated chloride movement and VRCCs which could be abolished by pretreatment of the cells with mevolonate (MVA) or geranylgeranyl pyrophosphate (GGPP). In addition, they found that both Rho A inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 reduced the cell proliferation and inhibited the volume-regulated chloride channel. Then the authors went on to examine the expression of ClC-3 gene in vascular smooth muscles and many other cell types in the basilar arteries; they found the expression of ClC-3 was increased during hypertension and simvastatin treatment reduced the upregualtion of ClC-3 expression. Finally, the authors used a gain-of-function approach to examine whether ClC-3 overexpression would antagonize the inhibitory effect of simvastatin on cell proliferation. Indeed they found that increased ClC-3 activity diminished the inhibitory effect of simvastatin on cell proliferation. A positive correlation between cell proliferation and activation of the ClC-3 channels was revealed.

Therefore, this study provided novel and convincing experimental evidence that simvastatin improves cerebrovascular remodeling in 2-k,2-c hypertensive rat through inhibition of the vascular SMC proliferation by suppression of volume-regulated ClC-3 channels. These results provided novel mechanistic insight into the beneficial effects of statins in the treatment of hypertension and stroke.

In addition to its important role in cell volume regulation, ClC-3 may also regulate the redox signaling pathway through interaction with NADPH oxidase (Nox) and/or transport of superoxide to improve myocyte viability against oxidative damage.⁸ It has been reported that activation of ClC-3 may improve the resistance of vascular SMCs to reactive oxygen species (ROS) in an environment of elevated inflammatory cytokines in hypertensive pulmonary arteries (please see recent reviews by Hume et al.⁷). ROS has been implicated in cellular signaling processes as well as a cause of oxidative stress-induced cell proliferation.⁴ One of the major sources of ROS in the vasculature is through one or more isoforms of the phagocytic enzyme NADPH oxidase, a membrane-localized protein which generates the superoxide (O₂^•−) anion on the extracellular surface of the plasma membrane (Figure 1).⁸

As a charged and short lived anion, it is believed that O₂^•− flux is insufficient to initiate intracellular signaling due to the combination of poor permeability through the phospholipid bilayer and a rapid dismutation to its uncharged and more stable derivative, hydrogen peroxide. Recent studies have also shown that ClC-3 may also function as an anti-apoptotic mechanism through regulation of cell volume and intracellular pH; and as a regulator of other transport functions involved in the etiology of hypertension (Figure 1).

Whether statins’ beneficial effects could be attributed also to their effects on these cellular functions of ClC-3 in cerebrovascular SMCs during hypertension is still an unanswered question. Nevertheless, regulation of ClC-3 functions in the cardiovascular system is emerging as a novel and important mechanism for the structural remodeling of the vasculature and may provide a novel therapeutic approach for the treatment of many vascular diseases such as hypertension and stroke.