Objective Examine the contribution of Nox1 NADPH oxidase to atherogenesis. Methods and Results Male apolipoprotein E deficient mice (ApoE−/−) and male mice deficient in both apolipoprotein E and Nox1 (ApoE−/− Nox1−/y) received an atherogenic diet for 18 weeks. Mean blood pressures, body weights, and serum cholesterol levels were similar between the two groups of mice. Deficiency of Nox1 decreased superoxide levels and reduced lesion area in the aortic arch from 43% (ApoE−/−) to 28% (ApoE−/− Nox1−/y). The reduction in lesion size at the level of the aortic valve in ApoE−/−/Nox1−/y was accompanied by a decrease in macrophage infiltration as compared to ApoE−/− mice. Carotid artery ligation in ApoE−/− mice induced accelerated intimal hyperplasia with decreased cellular proliferation and increased collagen content in the neointima of vessels deficient in Nox1. Conclusions Nox1-derived ROS modify lesion composition and contribute to lesion size in a murine model of atherosclerosis.

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-␣ (TNF-␣) 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-␣ 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. Conclusion-ClC-3 is necessary for the activation of SMCs by TNF-␣ 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.

NADPH oxidase-derived reactive oxygen species (ROS) contribute to the pathobiology of vascular disease. However, studies investigating NADPH oxidases in atherosclerosis have been limited in their ability to distinguish between the role of vascular cell and inflammatory cell -derived ROS. In this study, we examined the contribution of Nox1-derived ROS in a mouse model of atherosclerosis. Nox1 is a primary catalytic subunit of vascular cell, but not inflammatory cell, NADPH oxidase. At weaning, male apolipoprotein E deficient mice (AS, n=12) and male mice deficient in both apolipoprotein E and Nox1 (AS/Nox, n=16) received an atherogenic diet for 18 weeks. Mean blood pressures (116±3 vs. 110 ±3 mmHg; AS vs. AS/Nox, n=6), weights, and serum cholesterol levels (1578±150 vs. 1631±80 mg/dl; AS vs. AS/Nox) were similar between the AS and AS/Nox mice. As measured by lucigenin-enhanced chemiluminescence, superoxide levels were increased in segments of thoracic aorta from AS mice as compared to aorta from control mice (22±2 vs. 14±2 RLU/sec/mm 2 ; AS vs. C57BL/6; p<0.05, n=6). In contrast, superoxide levels in segments of thoracic aorta from AS/Nox mice were significantly lower (9±1 RLU/sec/mm 2 , n=6) than both AS and C57BL/6 mice. Dihydroethidium staining confirmed decreased superoxide levels in aorta of AS/Nox mice. Atherosclerotic lesion area was measured by staining the aorta en face with Oil Red O. Although atherosclerotic lesion area was reduced over the entire length of aorta in AS/Nox mice as compared to AS mice (10±1% vs. 14±1%, p<0.01), the reduction in lesion was primarily limited to the aortic arch (28±3% vs. 43±2%, p<0.001). In summary, Nox1 contributes to generation of ROS and lesion formation in atherosclerosis. These data confirm a role for vascular cell NADPH oxidases, and in particular Nox1, in vascular disease.