Abstract-Atherosclerosis is associated with an impairment of endothelium-dependent relaxations, which represents the reduced bioavailability of nitric oxide (NO) produced from endothelial NO synthase (eNOS). Among various mechanisms implicated in the impaired EDR in atherosclerosis, superoxide generated from dysfunctional eNOS has attracted attention. Under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a cofactor for NOS, are deficient or lacking, eNOS becomes dysfunctional and produces superoxide rather than NO. Experimental studies in vitro have revealed that NO from eNOS constitutes an anti-atherogenic molecule. A deficiency of eNOS was demonstrated to accelerate atherosclerotic lesion formation in eNOS knockout mice. In contrast, eNOS overexpression with hypercholesterolemia may promote atherogenesis via increased superoxide generation from dysfunctional eNOS. Thus, eNOS may have 2 faces in the pathophysiology of atherosclerosis depending on tissue BH4 metabolisms. An improved understanding of tissue BH4 metabolisms in atherosclerotic vessels is needed, which would help in developing new strategies for the inhibition and treatment of atherosclerosis. Key Words: endothelial nitric oxide synthase Ⅲ atherosclerosis Ⅲ tetrahydrobiopterin Ⅲ superoxide Ⅲ nitric oxide N itric oxide (NO) is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS), which requires Ca 2ϩ /calmodulin, FAD, FMN, and tetrahydrobiopterin (BH4) as the cofactors. [1][2][3][4] In the vessels, NO is produced from the endothelium by constitutive expression of the endothelial isoform of NOS (eNOS), which is activated by mechanical stress such as blood shear-stress and stimulation with agonists such as bradykinin and acetylcholine. NO has a variety of functions, but its action as the endothelium-derived relaxing factor (EDRF) is the most important for the maintenance of vascular homeostasis. 5 An impairment of endotheliumdependent relaxations (EDR) is present in atherosclerotic vessels even before vascular structural changes occur and represents the reduced eNOS-derived NO bioavailability. Endothelial dysfunction as characterized by an impairment of EDR, and thereby reduced eNOS-derived NO bioactivity, is the critical step for atherogenesis. Among various mechanisms responsible for the impaired EDR, the increased NO breakdown by superoxide is important, and there is augmented production of superoxide in atherosclerotic vessels. Recently, it was revealed that under certain circumstances, eNOS becomes dysfunctional and produces superoxide rather than NO. The pathophysiological role of dysfunctional eNOS has attracted attentions in vascular disorders, including atherosclerosis. This review focuses on the role of dysfunctional eNOS on atherosclerotic vessels and refers to the possible role of dysfunctional eNOS on atherogenesis. Impaired EDR in AtherosclerosisAll major risk factors for atherosclerosis such as hyperlipidemia, diabetes, hypertension, and smoki...

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone. We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter. In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L -arginine/citrulline conversion. Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung. Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release (estimated by N -nitro-L -arginine-induced facilitation of the contraction by prostaglandin F 2 ␣ ) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas.

The mutation of the potential heme-binding site of the p22 phox gene may reduce susceptibility to CAD. Our observations suggest that the C242T polymorphism of the p22 phox gene is a novel genetic marker that has a protective effect on coronary risk.

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.

As atherosclerosis progressed, the expression of p22phox increased through the vessel wall. p22phox might participate in the pathogenesis and pathophysiology of atherosclerotic coronary disease.