Anti-atherosclerotic role of smooth muscle cell NOX4 in human advanced atherosclerosis

Frieda Frank (Dresden)1, A. Hofmann (Dresden)1, B. Hamann (Dresden)1, S. Wolk (Dresden)1, D. M. Poitz (Dresden)2, H. Morawietz (Dresden)3, K. Schröder (Frankfurt am Main)4, C. Reeps (Dresden)1

1Universitätsklinikum Carl Gustav Carus an der TU Dresden Klinik und Poliklinik für Viszeral-, Thorax- und Gefäßchirurgie Dresden, Deutschland; 2Universitätsklinikum Carl Gustav Carus an der TU Dresden Institut für klinische Chemie und Laboratoriumsmedizin Dresden, Deutschland; 3Universitätsklinikum Carl Gustav Carus an der TU Dresden Med. Klinik III, Gefäßendothel/Mikrozirkulation Dresden, Deutschland; 4Universitätsklinikum Frankfurt Institut für Kardiovaskuläre Physiologie Frankfurt am Main, Deutschland


Introduction Carotid artery disease is caused by a narrowing of the internal carotid artery due to an atherosclerotic lesion that causes high-grade stenosis. Plaques may rupture or the stenosis may lead to complete occlusion of the carotid artery. Symptomatic disease manifests as ischemic stroke or transient ischemic attack. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that lead to symptomatic disease. NADPH oxidases (NOX) are the most important ROS-producing enzymes in the vascular wall. NOX4 is unique within the NOX family as it releases hydrogen peroxide (H2O2) and has anti-atherosclerotic and anti-inflammatory properties. Our previous studies showed lower NOX4 mRNA expression in plaques from patients with symptomatic disease, and low NOX4 mRNA expression was associated with an increased risk of symptomatic outcome. However, the underlying mechanisms remain to be elucidated. We hypothesized that NOX4 expression is reduced in smooth muscle cells and that this reduction could be reversed.
Vascular smooth muscle cells (VSMC) were isolated from carotid artery plaques of patients with asymptomatic or symptomatic disease. VSMC from the carotid artery of patients without arteriosclerotic lesions served as controls. First, cells were treated with TGF-β1 in the presence or absence of the NOX4 inhibitors diphenylene iodonium (DPI) and GKT137831 (GKT) and the mRNA expression of NOX4, ACTA2, SMTN and COL1A1 was analyzed.  The half-life of NOX4 mRNA was determined with actinomycin D, and cell proliferation was assessed with BrdU.
Results Stimulation with TGF-β1 led to a 13-fold increase in NOX4 mRNA in the control group (p=0.002) compared to patients with asymptomatic disease (p=0.0009), while no significant induction was observed in symptomatic disease. Treatment with DPI reversed this effect in all groups, while GKT affected NOX4 mRNA only in the symptomatic group, underlining the known non-specificity of DPI for NOX4. NOX4 mRNA stability was lower in the asymptomatic patients compared to the control group (p=0.04). Treatment of VSMC with GKT and TGF-b1 increased BrdU incorporation in all groups, suggesting a protective role of NOX4 in VSMC in preventing neointimal hyperplasia. Smoothelin (SMTN) is exclusively present in contractile SMC and was induced by TGF-b1 in the control group and only in asymptomatic disease. SMTN mRNA was decreased after NOX4 inhibition with DPI (p=0.01) and tended to be reduced by GKT (p=0.07) in both groups. COL1A1 is expressed by synthetic VSMC and was increased in patients with symptomatic disease, whereas no increase was found within the control group and asymptomatic patients. The induction in COL1A1 mRNA was reversed by DPI but not by GKT.
Conclusion Smooth muscle cell NOX4 mRNA expression could be increased by TGF-β1 in asymptomatic disease. This increase was accompanied by an increase in contractile markers, an effect mediated in part by NOX4. Mechanistically, NOX4 mRNA stability was lower in asymptomatic disease, although NOX4 could be induced by TGF-b1. Further experiments using either genetic NOX4 deletion or smooth muscle cell-specific overexpression will clarify whether induction of NOX4 is a therapeutic target for patients with advanced atherosclerosis.

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