Abstract 619: The Chloride Transporter ClC-3 is Required for TNF-α-induced Proliferation of Vascular Smooth Muscle Cells, Activation of Matrix Metalloproteinase-9, and Neointima Formation.
Migration and proliferation of smooth muscle cells (SMC) is fundamental to the development of vascular disease. We have recently shown that ClC-3 is required for cytokine-dependent signaling by the Nox1 NADPH oxidase in SMC. We tested the hypothesis that ClC-3 is involved in cytokine-mediated cell proliferation and activation of matrix metalloproteinase-9 (MMP-9) by TNF-α, and contributes to neointimal hyperplasia following vessel injury. Studies were performed in SMC isolated from aorta of ClC-3 null and littermate control (WT) mice. Whereas TNF-α (10 ng/ml) increased proliferation of WT SMC 2.2-fold, as measured by [3H]-thymidine incorporation, proliferation of ClC3 null SMC was markedly impaired. Adenoviral gene transfer of ClC-3 (AdClC-3) normalized the proliferation of ClC-3 null SMC to TNF-α. Using gelatin zymography, TNF-α activation of MMP-9 was reduced in ClC-3 null SMC (4.2 ± 0.3 vs. 1.3 ± 0.1 fold increase in WT vs. ClC-3 null), and restored following AdClC-3 (5.9 ± 0.4 fold increase). Since the ERK1/2 pathway is known to participate in activation of MMP-9 and cell growth, we measured ERK1/2 phosphorylation by Western blotting. Following TNF-α, ERK1/2 activation was significantly lower in ClC-3 null SMC as compared to control SMC (0.97±0.03 vs 1.61±0.15 fold increase, p<0.05) and was restored by AdClC-3. Additional evidence that ClC-3 contributes to TNF- α signal transduction was provided by the finding that the chloride channel inhibitor niflumic acid reduced WT SMC generation of ROS, activation of ERK1/2 and MMP9, and proliferation, following TNF-α stimulation. Next we examined the role of ClC-3 in vascular injury by performing left carotid ligation in mice. Ten days after carotid ligation in WT mice, ClC-3 mRNA was increased two-fold in the ligated artery as compared to the noninjured artery. Four weeks following injury, the intima-media ratio of injured arteries was 1.6 ± 0.4 in WT and 0.4 ± 0.3 in ClC-3 null mice. In summary, TNF-α-mediated activation of MMP-9 and proliferation of SMC requires ClC-3. In additon, carotid injury increases vascular ClC-3 expression and the absence of ClC-3 inhibits neointimal hyperplasia. These findings identify ClC-3 as a novel target in the redox activation of vascular cells and development of vascular disease.