A critical role for chloride channel-3 (CIC-3) in smooth muscle cell activation and neointima formation

Abstract

Objective: We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs. In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury.

Methods and results: Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-α (TNF-α) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-α were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury.

Conclusions: ClC-3 is necessary for the activation of SMCs by TNF-α but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.

Figure 1. ClC-3 is necessary for TNF-α-mediated SMC proliferation

³H-thymidine incorporation was measured in WT and ClC-3 null SMCs in response to (A) thrombin (2 U/ml) or (B) TNF-α (10 ng/ml). * p<0.05 vs. WT control; # p<0.05 vs. WT + TNF-α. (C) WT and ClC-3 null SMCs were treated with adenovirus for the expression of ClC-3 (AdClC-3) or control vector (AdGFP). * p<0.05 vs. WT + AdGFP; # p<0.05 WT + AdGFP + TNF-α. Data are normalized to WT control, n=4 to 6.

Figure 2. ClC-3 is necessary for TNF-α-mediated activation of MMP-9

Top panel shows gelatin zymography for MMP-9 of the culture media from WT and ClC-3 null SMCs after (A) thrombin (2 U/ml) or (B) TNF-α (10 ng/ml). Summary densitometry data is shown in the lower panel and includes ClC-3 null SMCs infected with AdClC-3 or AdGFP. (C) Summary data of SMCs MMP-9 protein expression normalized to GAPDH. All summary data are normalized to WT control, n=4, * p<0.05 vs. WT control, # p<0.05 vs. WT + TNF-α.

Figure 3. TNF-α activation of ERK1/2 requires ClC-3

Representative Western blots showing phospho-ERK1/2 and total ERK1/2 in WT and ClC-3 null SMCs collected five minutes after stimulation with (A) thrombin or (B) TNF-α. Treatments for each lane correspond to the summary data shown in the bottom panel. Densitometry data were normalized to WT control, n=4, * p<0.05 vs. WT control; # p<0.05 vs. WT + TNF-α. TNF-α-mediated (C) ³H-thymidine uptake and (D) MMP-9 activation are inhibited when WT SMCs are pre-treated with the MEK inhibitor U0126 (10 μM). * p<0.05 vs. WT control, # p<0.05 vs. WT + TNF-α, n=3.

Figure 4. ClC-3 expression increases in response to TNF-α and carotid injury

ClC-3 mRNA levels were measured by real time PCR in (A) WT SMCs treated with TNF-α for 6, 24, or 48 hours (* p<0.05 vs. control, n=3) and (B) carotid arteries collected 10 days following ligation (* p<0.05 vs. non-injured, n=3).

Figure 5. Deficiency of ClC-3 protects from neointimal formation

Twenty-eight days following common carotid ligation, right (non-injured) and left (injured) carotid arteries were collected, sectioned 0.5 mm proximal to the ligation, and stained. (A) Representative photomicrographs, original magnification 10x, scale bar represents 50 microns. (B) Intimal area of injured carotid artery. Each symbol represents the mean intimal area of three sections 0.5 mm proximal to the ligation from one animal. Mean ± SEM is shown by bar to right of group data, * p<0.05 vs. WT. (C) Summary data of the ratio of intimal area to medial area of injured carotid in WT and ClC-3 null mice. * p<0.05 vs. WT, n=6.

Figure 6. Proposed role for ClC-3 in the activation of SMCs

TNF-α causes Nox1 generation of ROS into endosomes whereas thrombin increases intracellular ROS via EGFR activation. ClC-3 is necessary for ERK1/2 phosphorylation, MMP-9 activation, and cell proliferation in response to TNF-α but not thrombin. This observation is consistent with a Cl⁻/H⁺ antiporter function of ClC-3 to provide charge neutralization for Nox1 generation of endosomal ROS , .