The importance of reactive oxygen species (ROS) in vascular physiology and pathology is becoming increasingly evident. All cell types in the vascular wall produce ROS derived from superoxide-generating protein complexes similar to the leukocyte NADPH oxidase. Specific features of the vascular enzymes include constitutive and inducible activities, substrate specificity, and intracellular superoxide production. Most phagocyte enzyme subunits are found in vascular cells, including the catalytic gp91phox (aka, nox2), which was the earliest member of the newly discovered nox family. However, smooth muscle frequently expresses nox1 rather than gp91phox, and nox4 is additionally present in all cell types. In cell culture, agonists increase ROS production by activating multiple signals, including protein kinase C and Rac, and by upregulating oxidase subunits. The oxidases are also upregulated in vascular disease and are involved in the development of atherosclerosis and a significant part of angiotensin II-induced hypertension, possibly via nox1 and nox4. Likewise, enhanced vascular oxidase activity is associated with diabetes. Therefore, members of this enzyme family appear to be important in vascular biology and disease and constitute promising targets for future therapeutic interventions.

Background-NAD(P)H oxidases are important sources of superoxide in the vasculature, the activity of which is associated with risk factors for human atherosclerosis. This study was designed to investigate the localization of superoxide production and the expression of the Nox family of NAD(P)H oxidase proteins (gp91phox, Nox1, and Nox4) in nonatherosclerotic and atherosclerotic human coronary arteries. Methods and Results-In coronary artery segments from explanted human hearts, we examined intracellular superoxide production with dihydroethidium. In nonatherosclerotic coronary arteries, superoxide was present homogenously throughout the intima, media, and adventitia. In atherosclerotic arteries, there was an additional intense area of superoxide in the plaque shoulder, which is rich in macrophages and ␣-actin-positive cells. p22phox colocalized with gp91phox mainly in macrophages, whereas Nox4 was found only in nonphagocytic vascular cells. Expression of gp91phox and p22phox mRNA was associated with the severity of atherosclerosis. gp91phox correlated with the plaque macrophage content, whereas Nox4 correlated with the content of ␣-actin-positive cells. Nox1 expression was low both in human coronary arteries and isolated vascular cells. Conclusions-Several Nox proteins, including gp91phox and Nox4, may contribute to increased intracellular oxidative stress in human coronary atherosclerosis in a cell-specific manner and thus may be involved in the genesis and progression of human coronary atherosclerotic disease.

Abstract-Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-␤1 (TGF-␤1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle ␣-actin (SM ␣-actin) de novo and produce extracellular matrix. We hypothesized that TGF-␤1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-␤1 potently upregulates the contractile marker SM ␣-actin mRNA (7.5Ϯ0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-␤1, Nox4 mRNA is dramatically upregulated by 16.2Ϯ0.8-fold (nϭ3, PϽ0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80Ϯ5%, inhibits NADPH-driven superoxide production in response to TGF-␤1 by 65Ϯ7%, and reduces TGF-␤1-induced expression of SM ␣-actin by 95Ϯ2% (nϭ6, PϽ0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-␤1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-␤1 stimulation of Smad 2/3 phosphorylation by 75Ϯ5% and 68Ϯ3%, respectively (nϭ7, PϽ0.0001). We conclude that Nox 4 mediates TGF-␤1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure. (Circ Res. 2005;97:900-907.)Key Words: Nox4 Ⅲ human cardiac fibroblasts Ⅲ transforming growth factor Ⅲ reactive oxygen species Ⅲ Smad 2/3 H eart failure remains the leading cause of hospital admissions in the United States, with more than 550 000 new patients diagnosed each year. 1 Regardless of etiology, cardiac fibrosis is a major contributor to cardiac remodeling associated with cardiomyopathies. It is characterized by expansion of the interstitial compartment due to increased deposition of extracellular matrix by activated myofibroblasts. 2 Cardiac myofibroblasts are specialized contractile fibroblasts formed by irreversible acquisition of contractile proteins such as smooth muscle ␣-actin (SM ␣-actin) in response to potent fibrogenic cytokines. 3 The expression of SM ␣-actin is regulated by transforming growth factor-␤1 (TGF-␤1), a primary fibrogenic growth factor in heart failure that is downstream of many of the pro-fibrotic actions of other fibroblast growth factors, such as angiotensin II, aldosterone, and norepinephrine. 4 TGF-␤1 is upregulated in failing human hearts and various experimental models of cardiac hypertrophy, 4 and functional blockade of TGF-␤1 prevents cardiac interstitial fibrosis induced by pressure overload in...

Objective-Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91 phox , both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. Methods and Results-We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22 phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox , a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. , are important determinants of vascular function, acting not only as signaling intermediates to promote growth and differentiation 1 but also as modulators in pathological processes such as hypertension, atherosclerosis, and diabetic microvascular disease. 2 Major sources of ROS in the vascular wall are NAD(P)H oxidases, 3 multi-subunit enzymes that differ structurally and biochemically from the prototypical phagocyte NAD(P)H oxidase active in host defense. One structural difference is that vascular smooth muscle cells (VSMC) express two proteins, Nox1 and Nox4, 4 which are homologues to the gp91 phox catalytic subunit in phagocytes. 5 Biochemical differences include kinetics of activation, output, and regulation of NAD(P)H-dependent ROS production. 3 In contrast to the activated phagocyte oxidase, which produces large quantities of ROS into an extracellular (phagosomal) compartment in inducible bursts, Nox1 and Nox4 generate low-level, predominantly intracellular ROS constitutively and in response to agonists. 2 Conclusions-The See page 625 and coverAlthough Nox1 and Nox4 generate ROS, overexpression of these two homologues in the same fibroblast background suggests that Nox1 has mitogenic activity and is growthpromoting, 6 whereas Nox4 is implicated in cellular senescence. 7 In VSMC, Nox1 is inducible and upregulated by growth factors and hormones, whereas Nox4 is downregulated by these agonists. 8 Furthermore, in an animal model of restenosis in which O 2 ·Ϫ production is increased, Nox1 and Nox4 are induced at different times after carotid injury, 9 suggesting different functions of the two Nox proteins in redox-sensitive arterial remodeling.The differential expression and growth factor-related responses of multiple Nox proteins in VSMC imply distinct mechanisms of Nox1 and Nox4 regulation. This might involve specific signaling pathways such as the biphasic production of angiotensin II-mediate...