Objective-Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. Methods and Results-Simvastatin, atorvastatin, and lovastatin (0.1 to 10 mol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-B) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay ( Key Words: statins Ⅲ nuclear factor-B Ⅲ activator protein-1 Ⅲ hypoxia-inducible factor-1␣ Ⅲ vascular endothelial growth factor R andomized clinical trials have clearly shown the benefit of statin therapy in the reduction of cardiovascular events and total mortality in coronary heart disease patients with either high or normal cholesterol levels. 1 In these studies, survival curves began to diverge within a relatively short period of time and before effects on plaque size were likely to occur. Demonstrated effects of HMG-CoA reductase inhibitors were not reflected by a regression in coronary stenoses as assessed by angiography. These findings have suggested that mechanisms of statins beyond lipid lowering are likely to be involved in the reduction of coronary events. 2 Both in vivo and in vitro studies support the notion that statins counteract the chronic subclinical vascular inflammatory state associated with atherosclerosis. 3,4 Statins inhibit leukocyte-endothelium interaction 5-7 and decrease inflammation in carotid lesions in humans. 8 Many of the vasculoprotective effects of HMG-CoA reductase inhibitors seem to be mediated by enhanced availability of nitric oxide. 9 There is increasing evidence that statins may act on the transcriptional level as well, eg, simvastatin inhibited endothelial secretion of PAI-1, which was correlated with reduced mRNA transcription and activity of the promoter. 10 Despite extensive research on molecular mechanisms of statins, little is known about the interactions of these drugs with transcription factors. The aim of this study was to characterize the effects of simvastatin, atorvastatin, and lovastatin on the activation of nuclear factor (NF)-B, activator protein (AP)-1, and hypoxia-inducible factor (HIF)-1␣ in endothelial and arterial smooth muscle cells. Because these factors regulate the transcription of many genes, including cytokines, chemokines, adhesion molecules, and growth factors, such interactions of statins on vascular cell signaling and gene expression may explain atheroprotective effects not directly related to cholesterol lowering. MethodsSimvastatin (MSD) and lovastatin (Calbiochem) prodrugs were activated from their inactive lactone proforms to their active dihy-

Background-Endothelial function is impaired by hyperhomocyst(e)inemia. We have previously shown that homocyst(e)ine (Hcy) inhibits NO production by cultured endothelial cells by causing the accumulation of asymmetric dimethylarginine (ADMA). The present study was designed to determine if the same mechanism is operative in humans. Methods and Results-We studied 9 patients with documented peripheral arterial disease (6 men; 3 women; age, 64Ϯ3 years), 9 age-matched individuals at risk for atherosclerosis (older adults; 9 men; age, 65Ϯ1 years), and 5 young control subjects (younger adults; 5 men; age, 31Ϯ1 years) without evidence of or risk factors for atherosclerosis. Endothelial function was measured by flow-mediated vasodilatation of the brachial artery before and 4 hours after a methionineloading test (100 mg/kg body weight, administered orally). In addition, blood was drawn at both time points for measurements of Hcy and ADMA concentrations. Plasma Hcy increased after the methionine-loading test in each group (all, PϽ0.001). Plasma ADMA levels rose in all subjects, from 0.9Ϯ0.2 to 1.6Ϯ0.2 mol/L in younger adults, from 1.5Ϯ0.2 to 3.0Ϯ0.4 mol/L in older adults, and from 1.8Ϯ0.1 to 3.9Ϯ0.3 mol/L in peripheral arterial disease patients (all, PϽ0.001). Flow-mediated vasodilatation was reduced from 13Ϯ2% to 10Ϯ1% in younger adults, from 6Ϯ1% to 5Ϯ1% in older adults, and from 7Ϯ1% to 3Ϯ1% in peripheral arterial disease patients (all, PϽ0.001

Hypoxia, cytokines, and nitric oxide (NO) stimulate the generation of vascular endothelial growth factor (VEGF) and induce heme oxygenase-1 (HO-1) expression in vascular tissue. HO-1 degrades heme to carbon monoxide (CO), iron, and biliverdin, the latter being reduced to bilirubin by biliverdin reductase. In the present study, we investigated the role of HO-1 in the modulation of VEGF synthesis in rat vascular smooth muscle cells (VSMC). In VSMC stimulated with cytokines, inhibition of NO production significantly, but not completely, reduced VEGF release. In contrast, inhibition of HO activity by tin protoporphyrin IX (SnPPIX) totally prevented cytokine-induced increase in VEGF, despite an augmented synthesis of intracellular NO. Stimulation of HO-1 activity by hemin enhanced VEGF production; this effect was abrogated by blockade of the HO pathway. Similarly, VEGF synthesis induced by hypoxia was down-regulated by SnPPIX, but not by inhibitors of NO synthase. To elucidate further a direct involvement of HO-1 in the observed effects, we generated transfected cells that overexpressed the HO-1 gene. Notably, these cells synthesized significantly more VEGF protein than cells transfected with a control gene. Among the products of HO-1, biliverdin and bilirubin showed no effect, whereas iron ions inhibited VEGF synthesis. Exposure of cells to 1% CO resulted in a marked accumulation of VEGF (20-fold increase) over the basal level. Our data indicate that HO-1 activity influences the generation of VEGF in VSMC in both normoxic and hypoxic conditions. As CO and iron, respectively the inducer and the inhibitor of VEGF synthesis, are concomitantly produced during the degradation of heme, these data indicate that HO by-products may differentially modulate VEGF production.