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-Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H 2 O 2 and O 2 Ϫ production in ECs. Next, we used ECs obtained from p47phox Ϫ/Ϫ mice (MAE-p47 Ϫ/Ϫ ), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47 Ϫ/Ϫ , but it was restored when the cells were transfected with p47 phox plasmid. Moreover, OS-induced O 2 Ϫ production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. Key words: BMP4 Ⅲ oscillatory shear Ⅲ reactive oxygen species Ⅲ monocyte adhesion Ⅲ endothelial cells Ⅲ NADPH oxidase V ascular endothelial cells (ECs) are constantly exposed to fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, the regions of branched and curved arteries exposed to disturbed flow conditions including oscillatory shear stress (OS) correspond to "lesion-prone areas" that preferentially develop atherosclerosis. 1,2 In contrast, straight arteries exposed to steady, high levels of laminar shear stress (LS) are relatively well protected from atherosclerotic plaque development. 1,2 Atherosclerosis is an inflammatory disease preferentially occurring in lesion-prone areas. 2,3 The earliest measurable markers of atherogenesis include expression of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and subsequent monocyte adhesion and recruitment into the lesion-prone areas. 2,4,5 Additional critical atherogen...

1179 in a PKA-dependent, but Aktindependent manner, whereas the NO ⅐ production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.