Background-Oxidized low-density lipoprotein (ox-LDL) causes endothelial dysfunction in part by decreasing the availability of endothelial nitric oxide (NO). Although HMG CoA reductase inhibitors restore endothelial function by reducing serum cholesterol levels, it is not known whether they can also directly upregulate endothelial NO synthase (ecNOS) activity. Methods and Results-Human saphenous vein endothelial cells were treated with ox-LDL (50 g/mL thiobarbituric acid reactive substances 12 to 16 nmol/mg) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin. In a time-dependent manner, ox-LDL decreased ecNOS mRNA and protein levels (91Ϯ4% and 67Ϯ8% reduction after 72 hours, respectively). Both simvastatin (1 mol/L) and lovastatin (10 mol/L) upregulated ecNOS expression by 3.8-fold and 3.6-fold, respectively, and completely prevented its downregulation by ox-LDL. These effects of simvastatin on ecNOS expression correlated with changes in ecNOS activity. Although L-mevalonate alone did not affect ecNOS expression, cotreatment with L-mevalonate completely reversed ecNOS upregulation by simvastatin. Actinomycin D studies revealed that simvastatin stabilized ecNOS mRNA ( 1/2 , 43 versus 35 hours). Nuclear run-on assays and transient transfection studies with a Ϫ1.6 kb ecNOS promoter construct showed that simvastatin did not affect ecNOS gene transcription. Conclusions-Inhibition

Hypoxia induces vasoconstriction, in part, by downregulating endothelial cell nitric oxide synthase (ecNOS) expression. Previous studies indicate that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors improve endothelium-dependent relaxation by increasing ecNOS activity. To determine whether HMG CoA reductase inhibitors can prevent hypoxia-mediated down-regulation of ecNOS function and expression, human endothelial cells were exposed to hypoxia (3% O 2 ) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin for various durations (0 -48 h). Hypoxia decreased ecNOS protein and mRNA levels in a time-dependent manner, resulting in a 4-and 9-fold reduction after 48 h, respectively. In a concentration-dependent manner, simvastatin, and to a lesser extent, lovastatin, prevented the down-regulation of ecNOS expression by hypoxia. Simvastatin-induced changes in ecNOS expression correlated with changes in endothelial NO production and were reversed by treatment with L-mevalonate. Actinomycin D studies revealed that under hypoxic conditions, simvastatin increased ecNOS mRNA half-life from 13 to 38 h. Nuclear run-on studies showed that simvastatin had no effect on repression of ecNOS gene transcription by hypoxia. These results indicate that HMG CoA reductase inhibitors regulate ecNOS function and expression through changes in ecNOS mRNA stability and suggest that treatment with HMG CoA reductase inhibitors may have beneficial effects in patients with hypoxia-mediated pulmonary hypertension.Pulmonary hypertension is a major cause of morbidity and mortality in individuals exposed to hypoxic conditions (1). Recent studies demonstrate that pulmonary arterial vessels from patients with pulmonary hypertension have impaired release of endothelium-derived relaxing factor or nitric oxide (NO) (2, 3). Indeed, individuals with pulmonary hypertension demonstrate reduced levels of endothelial cell nitric oxide synthase (ecNOS) 1 expression in their pulmonary vessels and benefit clinically from inhalation NO therapy (4, 5). Conversely, mutant mice lacking ecNOS gene or newborn lambs treated with the ecNOS inhibitor, N -monomethyl-L-arginine (LNMA), develop progressive elevation of pulmonary arterial pressures and resistance (6, 7). We and others have shown that hypoxia causes pulmonary vasoconstriction via inhibition of ecNOS expression and activity (8 -10). Hence, hypoxia-mediated downregulation of ecNOS may lead to the vasoconstrictive and structural changes associated with pulmonary hypertension.Clinical trials with 3-hydroxy-3-methylglutaryl coenzyme A-(HMG CoA) reductase inhibitors have shown that a reduction in serum cholesterol level is correlated with improved endothelium-dependent relaxation in atherosclerotic vessels (11, 12). The HMG CoA reductase inhibitors lower serum cholesterol levels by blocking the hepatic conversion of HMG CoA to Lmevalonate in the cholesterol biosynthetic pathway (13). Although the mechanism by which HMG CoA reductase inhibitors restore endothelial function has ...

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