Cytochrome b558-dependent NAD(P)H oxidase-phox units in smooth muscle and macrophages of atherosclerotic lesions.

Abstract

Despite studies implicating superoxide anion-producing oxidases in atherosclerosis, their characteristics, expression, and regulation in cells of lesions are poorly understood. We examined the following: (1) whether cytochrome b558-dependent NAD(P)H oxidase-phox peptides are expressed by intimal smooth muscle cells (iSMCs) and macrophages of human aortic atherosclerotic lesions and their regulation and (2) whether cytochrome b558-dependent NAD(P)H oxidase represents a major NAD(P)H oxidase in iSMCs. Using a combination of immunochemical and reverse transcription-polymerase chain reaction procedures, we demonstrate that p22(phox) and gp91(phox) (cytochrome b558) expression in normal intima was restricted to a quarter of the iSMCs. In fatty streaks, a similar fraction of iSMCs expressed cytochrome b558, whereas macrophages also expressed low levels of p47(phox) and p67(phox). In fibrofatty lesions, the majority of iSMCs expressed the cytochrome b558 subunits; p67(phox) was also detected. Macrophages and macrophage-derived foam cells expressed the 4 phox subunits that constitute superoxide-producing cytochrome b558-dependent NAD(P)H oxidase. These were upregulated by transforming growth factor-beta1 and interferon-gamma. Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91(phox). In human aortic fibrofatty lesions, a cytochrome b558-dependent NAD(P)H oxidase appears to be a major iSMC and macrophage oxidase whose expression is upregulated by cytokines.