Rapid, direct effects of statin treatment on arterial redox state and nitric oxide bioavailability in human atherosclerosis via tetrahydrobiopterin-mediated endothelial nitric oxide synthase coupling

Abstract

Background: Treatment with statins improves clinical outcome, but the exact mechanisms of pleiotropic statin effects on vascular function in human atherosclerosis remain unclear. We examined the direct effects of atorvastatin on tetrahydrobiopterin-mediated endothelial nitric oxide (NO) synthase coupling in patients with coronary artery disease.

Methods and results: We first examined the association of statin treatment with vascular NO bioavailability and arterial superoxide (O(2)(·-)) in 492 patients undergoing coronary artery bypass graft surgery. Then, 42 statin-naïve patients undergoing elective coronary artery bypass graft surgery were randomized to atorvastatin 40 mg/d or placebo for 3 days before surgery to examine the impact of atorvastatin on endothelial function and O(2)(·-) generation in internal mammary arteries. Finally, segments of internal mammary arteries from 26 patients were used in ex vivo experiments to evaluate the statin-dependent mechanisms regulating the vascular redox state. Statin treatment was associated with improved vascular NO bioavailability and reduced O(2)(·-) generation in internal mammary arteries. Oral atorvastatin increased vascular tetrahydrobiopterin bioavailability and reduced basal and N-nitro-l-arginine methyl ester-inhibitable O(2)(·-) in internal mammary arteries independently of low-density lipoprotein lowering. In ex vivo experiments, atorvastatin rapidly improved vascular tetrahydrobiopterin bioavailability by upregulating GTP-cyclohydrolase I gene expression and activity, resulting in improved endothelial NO synthase coupling and reduced vascular O(2)(·-). These effects were reversed by mevalonate, indicating a direct effect of vascular hydroxymethylglutaryl-coenzyme A reductase inhibition.

Conclusions: This study demonstrates for the first time in humans the direct effects of statin treatment on the vascular wall, supporting the notion that this effect is independent of low-density lipoprotein lowering. Atorvastatin directly improves vascular NO bioavailability and reduces vascular O(2)(·-) through tetrahydrobiopterin-mediated endothelial NO synthase coupling. These findings provide new insights into the mechanisms mediating the beneficial vascular effects of statins in humans.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01013103.

Figure 1

In study 1 (n=492), patients receiving regular statin treatment, had significantly better flow-mediated dilation (FMD) compared with those not receiving statins (A). In saphenous veins obtained from 256 of these patients during coronary artery bypass graft surgery, ex vivo vasorelaxations in response to acetylcholine (ACh) were significantly greater in subjects receiving statins (n=212) compared with those not receiving statins (n=44; B). In internal mammary artery samples obtained from these patients, vascular O₂^·− was significantly lower in those receiving statin treatment compared with those not receiving statins (C). The statin treatments in this population were simvastatin (63.1%), atorvastatin (26.8%), rosuvastatin (4.3%), pravastatin (3%), lovastatin (1.6%), and fluvastatin (1%). Values expressed as median (25th to 75th percentile; A and C) or mean±SEM (B). *P<0.01 vs no statin treatment (P values were derived from unpaired t test of the log-transformed values for A and C and by 2-way ANOVA for repeated measures for B).

Figure 2

In study 2, there was no significant difference in the change in low-density lipoprotein (LDL) levels between patients who received atorvastatin (n=21) and those who received placebo (n=21; P=0.704; A). However, flow-mediated dilation (FMD) was improved in the atorvastatin-treated group compared with placebo (B). There was no significant difference (P=0.774) in the changes in endothelium-independent dilatation of the brachial artery in response to nitroglycerine (NTG) between the 2 groups (C). P values were derived by using 2-way ANOVA for repeated measures with time-by-treatment interaction on the log-transformed values. Values expressed as median (25th to 75th percentile).

Figure 3

In study 2, vascular superoxide (O₂^·−) generation in internal mammary arteries obtained from patients who received atorvastatin (n=18) was significantly lower compared with patients who received placebo (n=20; A). In the same vessels, O₂^·− generation was decreased by N-nitro-L-arginine methyl ester (L-NAME) in mammary arteries obtained from patients who received placebo (B), whereas this effect was reversed in vessels from atorvastatin-treated patients. Values expressed as median (25th to 75th percentile). *P<0.01 vs placebo.

Figure 4

In study 2, plasma tetrahydrobiopterin (BH4; A) and total biopterins (tBio; B) were significantly reduced in patients who received atorvastatin (n=18) compared with placebo (n=20). However, vascular BH4 (C) and tBio (D) were significantly greater in mammary arteries (IMA) obtained from patients who received atorvastatin compared with placebo. Values are expressed as median (25th to 75th percentile). P values in A and B were derived from 2-way ANOVA with time-by-group interaction and in C and D from unpaired t test between the 2 groups on the log-transformed values.

Figure 5

In study 3, ex vivo incubation of human mammary arteries with atorvastatin (5 μmol/L) induced a significant reduction of vascular superoxide (O₂^·− ; A) and reversed N-nitro-L-arginine methyl ester (L-NAME)–inhibitable O₂^·− (B) in these vessels (n=10 patients). In a second set of experiments (C; n=5 patients), the reduction of vascular O₂^·− by atorvastatin was reversed in the presence of mevalonate (P=0.650 vs control after Bonferroni correction). In this experiment (C), L-NAME reduced vascular O₂^·− in the absence of atorvastatin and increased vascular O₂^·− in vessels preincubated with atorvastatin. Coincubation of these vessels with atorvastatin and mevalonate prevented the effect of atorvastatin on L-NAME–induced changes in vascular O₂^·− (P=0.08 for L-NAME plus atorvastatin plus mevalonate vs L-NAME only). The direct effect of atorvastatin on vascular wall biology was also confirmed by demonstrating a change in vascular Rac1 activation in these vessels (C). − Indicates no atorvastatin or mevalonate; +, atorvastatin 5μmol/L or mevalonate 200μmol/L. Values are expressed as median (25th to 75th percentile; A and B) or mean±SEM (C). One-way repeated measures ANOVA in C revealed a significant effect of treatment in all readouts (P_ANOVA<0.001). *P<0.01 for individual comparisons vs control (no atorvastatin, no mevalonate and no L-NAME) after Bonferroni correction; †P<0.01 for individual comparisons vs L-NAME alone (no atorvastatin, no mevalonate) after Bonferroni correction.

Figure 6

Dihydroethidium staining in cryosections of serial internal mammary artery rings from the same vessel incubated for 6 hours with atorvastatin 0 μmol/L (A and B), atorvastatin 5 μmol/L (C and D), and atorvastatin 5 μmol/L plus mevalonate 200 μmol/L (E and F). Endothelium-derived dihydroethidium staining was reduced after atorvastatin, whereas this effect was reversed by mevalonate. N-nitro-L-arginine methyl ester (L-NAME) induced a reduction of endothelium-derived fluorescence in control (B) and atorvastatin plus mevalonate vessels (F), whereas an opposite effect was observed in the atorvastatin-treated vessels (D). This observation suggests an improvement of endothelial nitric oxide synthase coupling by atorvastatin, an effect that is prevented by mevalonate. Images are representative of 8 sets of internal mammary arteries.

Figure 7

In study 3, ex vivo incubation of human internal mammary arteries (from 10 patients) with atorvastatin induced a significant increase of vascular tetrahydrobiopterin (BH4; A) and total biopterins (tBio; B) compared with control. Atorvastatin also significantly increased GCH1 mRNA (relative to GAPDH) in these vessels (C). Values are expressed as median (25th to 75th percentile). − Indicates no atorvastatin; +, atorvastatin 5 μmol/L. *P<0.05, **P<0.01 vs control (no atorvastatin).

Figure 8

In study 3, 6 hours of incubation of serial internal mammary artery rings (from 8 patients) with atorvastatin increased both vascular tetrahydrobiopterin (BH4; A) and total biopterins (tBio; B). However, in the presence of the GTP cyclohydrolase I inhibitor DAHP, vascular BH4 (A) and tBio (B) were both significantly reduced and atorvastatin could not prevent this reduction, suggesting that the impact of atorvastatin on vascular biopterins is mediated through an effect on GTP cyclohydrolase I enzymatic activity. − Indicates no atorvastatin or DAHP; +, atorvastatin 5 μmol/L or DAHP 1 mmol/L. Values are expressed as mean±SEM. One-way repeated measures ANOVA revealed a significant effect of treatment on all readouts (P_ANOVA<0.001). *P<0.05, **P<0.01 for individual comparisons vs control (no atorvastatin, no DAHP) after Bonferroni correction.