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42 ROLE OF THE NADPH OXIDASE SUBUNIT NOX4 IN GENERATION OF REACTIVE OXYGEN SPECIES AND GROWTH ARREST OF VASCULAR SMOOTH MUSCLE CELLS IN ATHEROSCLEROSIS
  1. S. P. Xu,
  2. F. J. Miller
  1. Iowa City, IA

Abstract

NADPH oxidase-derived reactive oxygen species (ROS) contribute to atherosclerosis. We have previously shown that smooth muscle cells isolated from atherosclerotic lesions of murine aorta (plaque-derived SMC) exhibit increased expression of the NADPH oxidase subunit Nox4, increased ROS levels, and decreased rate of growth, as compared to medial-derived SMC. In this study, we tested the hypothesis that increased expression of Nox4 contributes to growth arrest in plaque-derived SMC. Sequence-specific gene silencing of Nox4 by RNA interference (siNox4) reduced mRNA expression of Nox4 by 80% and decreased superoxide levels by 36% in plaque-derived SMC, as compared to cells treated with control siRNA. Cell growth of plaque-derived SMC, however, was not improved following siNox4 treatment. To further investigate the effect of Nox4 on SMC growth, human aortic SMC were transfected with pCMVNox4 (Nox-SMC) or pCMVscript (control-SMC). Real-time PCR confirmed a 6-fold increase of Nox4 expression, whereas chemiluminescence indicated a 2-fold increase in NAPDH oxidase activity, in Nox-SMC compared to control-SMC. In 10% serum, Nox-SMC exhibited increased percentage of cells in G0/G1 phase. As detected by Western blotting, phosphorylation of ERK and Akt were increased in plaque-derived SMC compared to medial SMC. siNox4 failed to reduce phosphorylation of Akt or ERK in plaque-derived SMC. Forty-eight hour treatment of plaque-derived SMC, but not medial SMC, with wortmannin (10 μM), an inhibitor of PI3k/Akt activation, or PD98059 (20 μM), an inhibitor of ERK activation, increased the percentage of cells entering S phase. In summary, (1) reduction of Nox4 expression by siRNA lowers ROS levels but does not improve growth of plaque-derived SMC. (2) Expression of Nox4 increases ROS levels and decreases proliferation of human SMC. (3) Activation of Akt and ERK is increased in plaque-derived SMC, and pharmacologic inhibition of activation induces proliferation. In conclusion, reduction in Nox4-derived ROS is not sufficient to improve growth of plaque-derived SMC.

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