Expression of NADH/NADPH oxidase p22phox in human coronary arteries.

BACKGROUND: NADH/NADPH oxidase is an important source of superoxide in the vasculature. Recently, we found that polymorphism of the gene p22phox, a critical component of this oxidase, is associated with a risk of coronary artery disease. The aim of this study was to investigate the localization of p22phox in human coronary arteries and to examine its difference in expression between nonatherosclerotic and atherosclerotic coronary arteries. METHODS AND RESULTS: Using coronary artery sections from autopsied cases (n=11), the expression of p22phox was examined by immunohistochemistry and Western blotting. In nonatherosclerotic coronary arteries, p22phox was weakly expressed, mainly in the adventitia. In atherosclerotic coronary arteries, intensive immunoreactivity was detected in neointimal and medial smooth muscle cells and infiltrating macrophages in hypercellular regions and at the shoulder region. Semiquantitative analysis and Western blotting showed that the expression of p22phox in atherosclerotic coronary arteries was more pronounced than that in nonatherosclerotic arteries. Double staining revealed p22phox expression in adventitial fibroblasts, smooth muscle cells, macrophages in the neointima and media, and endothelial cells. CONCLUSIONS: As atherosclerosis progressed, the expression of p22phox increased through the vessel wall. p22phox might participate in the pathogenesis and pathophysiology of atherosclerotic coronary disease.

Endothelial NO synthase overexpression inhibits lesion formation in mouse model of vascular remodeling.

NO produced by endothelial NO synthase (eNOS) plays important roles in the regulation of vascular tone and structure. The purpose of this study was to clarify the role of eNOS-derived NO on vascular remodeling by use of eNOS-transgenic (eNOS-Tg) mice. The common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, the proximal carotid artery of the ligation site was histologically examined. In this vascular remodeling model, the endothelium remains uninjured, but neointimal and medial thickening occurs in combination with a reduction in vascular diameter at the proximal portion of the ligation. At 4 weeks after ligation, the respective neointimal and medial areas in wild-type mice were 17 200+/-1100 and 24 300+/-1500 microm(2), whereas both were reduced to 8000+/-1900 (P:<0.01) and 18 400+/-700 microm(2) (P:<0.01) in eNOS-Tg mice (n=8). Total vascular area was not different between the 2 genotypes. N:(G)-Nitro-L-arginine methyl ester treatment increased neointimal and medial areas to the same extent in both genotypes. Leukocyte infiltration was observed in the luminal side of the vessel, but the number of infiltrating cells was significantly attenuated in eNOS-Tg mice compared with wild-type mice. This reduction of leukocyte infiltration in eNOS-Tg mice was associated with reduced expressions of intracellular adhesion molecule-1 and vascular cellular adhesion molecule-1 on the endothelium. In conclusion, chronic eNOS overexpression in the endothelium reduced leukocyte infiltration and inhibited neointimal formation and medial thickening. Our data provide the evidence for the regulatory role of NO from the endothelium on vascular structure integrity.

Expression of glutaredoxin in human coronary arteries: its potential role in antioxidant protection against atherosclerosis.

Oxidative stress is considered an important factor in atherogenesis. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase. However, the mechanisms that regulate the cellular redox state in the vessel wall remain unclear. Recent study has shown that thioredoxin, a thiol-disulfide oxidoreductase, is expressed in atherosclerotic plaques of human carotid arteries. In this study, we investigated the localization and expressional change of glutaredoxin and thioredoxin, two important members of the thiol-disulfide oxidoreductases, in autopsy samples of human coronary arteries. In nonatherosclerotic coronary arteries, glutaredoxin was expressed in endothelial cells, in fibroblasts of the adventitia, and most intensely in medial smooth muscle cells. Interestingly, in atherosclerotic lesions such as hypercellular lesions, the infiltrating macrophages highly expressed glutaredoxin. The expressional pattern of thioredoxin was quite similar to that of glutaredoxin. Western blot analysis demonstrated that hydrogen peroxide stimulated the expression of glutaredoxin in a time- and dose-dependent manner in cultured human coronary artery smooth muscle cells. Fluorescence microtopography with dihydroethidium demonstrated that the generation of reactive oxygen species was associated with the expression of glutaredoxin. These results suggest the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries.

Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in cholesterol-fed rabbits.

Studies in vitro reveal that fluvastatin, an HMG-CoA reductase inhibitor, has a strong DPPH radical scavenging activity and achieves concentration-dependent inhibition of copper- and cell-induced oxidation of low-density lipoprotein (LDL). To further examine the anti-oxidative activity of fluvastatin in vivo, we elucidated the effects of chronic treatment with fluvastatin at a dose insufficient to reduce plasma cholesterol levels (2 mg/kg per day) on vasomotion and vascular oxidative stress in thoracic aortas of 0.5% cholesterol-fed rabbits. After 12 weeks of dietary treatment, aortic segments from rabbits fed cholesterol alone showed impaired endothelium-dependent relaxation responses to acetylcholine and A23187 compared to normal chow-fed rabbits in association with a significant increase in plasma total cholesterol levels. In contrast, although plasma total cholesterol levels were not different from those in control cholesterol-fed rabbits, aortic segments from fluvastatin-treated rabbits showed normal relaxation. Compared with rabbits fed cholesterol alone, fluvastatin treatment decreased susceptibility of LDL to ex vivo copper-induced oxidation, reduced vascular superoxide generation, and atheromatous plaque formation. In conclusion, the potent anti-oxidative properties of fluvastatin in addition to its cholesterol-lowering activity appear to contribute to its anti-atherosclerotic effect in vivo.

Cell death and proliferation in Nd-YAG laser, electrocautery, and scalpel wounds on mice skin.

The purpose of this study is to compare cell death and proliferation in laser, electrocautery and scalpel wounds on the mice epidermis. Wounds were examined by transmission electron microscopy, the detection of free 3'-OH DNA ends and immunohistochemistry of proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), keratinocyte growth factor (KGF) and keratinocyte growth factor receptor (KGFR). Reepithelization was first observed 5 days after scalpel and laser incisions and 7 days after electrocautery incision. Ultrastructurally, keratinocytes in both electrocautery and laser wounds showed similar post-apoptotic necrotic changes. Interestingly, dividing cells were often observed 3 days after laser incision. Apoptotic index in electrocautery wounds was higher than in laser wounds, although there was no significant difference in the PCNA expression level between them. The expression of iNOS, KGF and KGFR in laser wounds was more intense than in electrocautery wounds. In scalpel wounds, keratinocytes did not show significant changes in morphology or of markers of cell death and proliferation during the observation period. Therefore, the increase in the number of dividing cells and in the expression level of iNOS, KGF and KGFR may induce earlier and thicker reepithelization in laser wounds than in electrocautery and scalpel wounds.

Restriction of cicatricial pemphigoid antigens to the lamina densa: confirmation by indirect immunoelectron microscopy.

Circulating anti-basement membrane zone (BMZ) antibodies in a patient with cicatricial pemphigoid (CP) were examined using an indirect immunofluorescence test, indirect immunoperoxidase electron microscopy, and Western blot analysis. An indirect immunofluorescence test on salt-split skin revealed that the anti-BMZ antibodies reacted solely to the dermal side at the separating epidermal-dermal interface, and indirect immunoelectron microscopy on intact skin indicated localization of the corresponding antigens (CP antigens) over the lamina densa and within the lower half of the lamina lucida; there were no CP antigens beneath a melanocyte. Indirect immunoelectron microscopy on salt-split skin demonstrated that the CP antigens were partly dissociated from, but restricted to, the lamina densa. Western blot analysis showed no differences in molecular weight between the CP antigens and bullous pemphigoid (BP) antigens. CP antigens, as detected by this patient's serum, appear to be constituted of molecules quite similar to BP antigens, but with different epitopes. CP antigens may be shed from basal cells and locate in the area of anchoring filaments, where they play a role in connecting basal cells to the underlying lamina densa.