Hypercholesterolemia is an important risk factor for cardiovascular disease, and several studies indicated that cholesterol reduction is associated with decreased cardiovascular morbidity and mortality under a variety of clinical conditions (1,2). Hypercholesterolemia is commonly associated with impaired endothelial function (3), and enhanced endothelial function has been proposed as an additional mechanism contributing to the cardiovascular benefits conferred by statins (4-7).
Endothelial dysfunction is considered the earliest stage of atherosclerotic disease (8) and is a predictor of future cardiovascular events in subjects with coronary heart disease (9). Flow-mediated vasodilation (FMV) of the brachial artery is considered a measure of endothelial function (10,11), and noninvasive assessment of FMV is a promising alternative to the traditional invasive and expensive techniques (3,5). Nitric oxide bioavailability is usually impaired in the presence of several established cardiovascular risk factors including age, male gender (12), hypertension (13), smoking (11), hypercholesterolemia (3), and diabetes (14). In the female gender, a physiologic decline of the endothelial function occurs in the postmenopausal age group (12). Statin treatment has been supposed to restore the endothelial function through its lipid-lowering effect, thereby increasing nitric oxide bioavailability (15). Moreover, an in vitro study demonstrated a direct effect of pravastatin on nitric oxide synthase (16). An improvement in endothelial function has been observed in hypercholesterolemic subjects after 1 month of treatment with simvastatin (4,5), and in subjects with severe hypercholesterolemia treated for 7 months with high-dose atorvastatin (6). However, no data are currently available on the short-term effects of atorvastatin on endothelial function.
The present study was designed to assess the effect of atorvastatin on endothelial function in hypercholesterolemic postmenopausal women. A second aim of the study was to evaluate the time course of drug-induced endothelial changes.
Thirty postmenopausal hypercholesterolemic women (mean age, 58 ± 6 years) were included in the present study. All subjects with concomitant cardiovascular risk factors (hypertension, cigarette smoking, diabetes, body mass index >27.3 kg/m2, overt cardiovascular disease) were excluded from the study. No subject was receiving lipid-lowering drugs, hormone-replacement therapy, or antioxidant vitamins. Average blood pressure was 131/75 ± 11/6 mm Hg, and body mass index was 23.8 ± 2 kg/m2. Twenty subjects were randomized in an open-label, parallel-group design to receive American Heart Association step 1 diet and atorvastatin, 10 mg daily at night, and the remaining 10 subjects received only American Heart Association step 1 diet. FMV of the brachial artery, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, lipoprotein (a), and LDL particle size were determined at baseline and after 1, 2, 4, and 8 weeks of treatment. The study protocol was approved by the Ethics Committee of our institution, and all patients gave their written informed consent.
Brachial artery ultrasound
FMV was assessed on the brachial artery by ultrasonography. Details of the ultrasonographic procedure have been reported elsewhere (17). The measurements were performed in supine position on the nondominant arm, after 10-20 min resting in a quiet, dark room with a temperature of 22°C. The skin was marked indelibly by ink. The brachial artery was scanned longitudinally just above the antecubital crease using a 10-MHz probe (ESAOTE AU4, Florence, Italy). Diameter of the brachial artery was measured at the R wave of the electrocardiogram, on the interface between media and adventitia of the anterior and posterior wall. Gain settings were optimized to identify the lumen and the vessel wall interfaces and were not modified during the examination. Hyperemia was induced by inflation of a pneumatic cuff (12.5 cm wide) at 230-250 mm Hg for 4 min on the most proximal portion of the upper arm. Arterial diameter measurement was repeated 45-60 s after sudden deflation of the cuff. Tracings were recorded on videotape and read by one investigator, who was unaware of the subject's clinical data and temporal sequence. The average of three measurements of basal and posthyperemia diameter was used for the analysis. FMV was expressed as the relative increase in brachial artery diameter during hyperemia, and defined as 100 (posthyperemia diameter × basal diameter)/basal diameter). Blood flow was measured as arterial cross-sectional area (xr2) times mean Doppler velocity corrected for angle. The intraobserver between-occasion reproducibility of FMV in our laboratory was assessed in 21 subjects examined 2 days apart. The mean ± SD difference between the two examinations was 1.0 ± 1.5%.
Total cholesterol, triglycerides, and HDL cholesterol were determined by enzymatic colorimetric method (Dimension Autoanalyzer; DADE Inc., Newark, NJ, U.S.A.). Triglyceride levels never exceeded 400 mg/dl; hence, LDL cholesterol was calculated from the Friedewald equation (18). Plasma lipoprotein (a) concentration was measured by enzyme-linked immunosorbent assay. An aliquot of plasma (blood anticoagulated with EDTA) was used to determine LDL size by gradient gel electrophoresis according to Rainwater et al. (19). A 2-16% polyacrylamide gel was prepared; samples for seeding were preincubated, stained with Sudan Black, and seeded with thyreoglobulin, ferritin, catalase, lacticodehydrogenase, albumin, and latex of a known size to be used as standard migration distances. The migration distances were read on densitometry (590 nm). A quadratic equation (polynomial regression of Stokes) was used to convert migration distance to particle diameter.
The Kolmogorov-Smirnov algorithm was used to determine whether each variable had a normal distribution. Parameters are expressed as mean ± SD, except for lipoprotein (a), which is expressed as median (25th-75th percentile). Analysis of variance for repeated measurements was used to assess differences among times of observation. Student's t test and Friedman's test for unpaired data were used to compare between-group differences. Pearson's correlation coefficients were used to assess the relation between treatment-induced changes in FMV and changes in lipid parameters in subjects treated with atorvastatin and in control subjects.
Serum lipid values are shown in Table 1. Entry HDL cholesterol was higher in the atorvastatin group; no other significant difference in baseline lipid parameters was found between the two groups. A significant decrease in total cholesterol, LDL cholesterol, and triglycerides was observed in patients treated with atorvastatin after 1 week of therapy, with a further reduction after 2, 4, and 8 weeks (all p < 0.05). In subjects treated with diet, total cholesterol and LDL cholesterol were slightly reduced starting from the second week, and a modest but significant reduction in serum triglycerides was found after the first week of treatment (all p < 0.05). Total cholesterol, LDL cholesterol, and triglycerides were significantly lower in the atorvastatin group at all treatment times (all p < 0.05). The other lipid parameters did not show any significant change in either group.
Brachial artery vasoactivity is reported in Table 2. In the group treated with atorvastatin, a significant increase in FMV was documented after 2 weeks, and a further improvement was observed at weeks 4 and 8 (all p < 0.05). In the group receiving diet, no significant changes in FMV were observed. FMV did not differ at baseline between the groups, but was significantly greater in the atorvastatin group after 1 week (p < 0.05) and after 2, 4, and 8 weeks (p < 0.001). Pretreatment brachial artery diameter and flow did not differ between the groups, and did not change with treatment in either group.
The relation between treatment-induced changes in FMV and in LDL cholesterol was assessed separately in the two groups (Fig. 1). A weak but significant inverse association was found in the atorvastatin group (r = −0.22; p = 0.041), as well as in control subjects (r = −0.43; p < 0.005).
Impaired endothelial function is commonly associated with hypercholesterolemia and is currently believed to play a role in explaining the relation between hypercholesterolemia and atherosclerosis (3). Several potential mechanisms for the reduced bioavailability of nitric oxide in hypercholesterolemic subjects have been proposed, including reduced transcription of nitric oxide synthase, production of a less bioactive nitric oxide, increase in asymmetric dimethylarginine levels, uncoupling of the receptor-Gi protein complex, and increased nitric oxide breakdown by superoxide radicals (20-26). The postmenopausal status also is associated with an impairment in endothelial function, which is largely attributable to changes in the hormonal status (12). Estrogens exert a protective effect on the endothelium through several mechanisms, including a reduction in the vessel wall redox potential (27).
Statin treatment improves endothelial function in coronary and peripheral arteries along with cholesterol reduction. O'Driscoll et al. found that 4-week treatment with simvastatin improves forearm dilation to acetylcholine in subjects with moderate hypercholesterolemia (5). In postmenopausal hypercholesterolemic women, simvastatin modified endothelial function after 4 weeks, in parallel with cholesterol reduction (7). Our study demonstrates that atorvastatin improves FMV within 2 weeks of treatment in postmenopausal hypercholesterolemic women. FMV increased further after 4 weeks, without major subsequent changes over an 8-week treatment. This is the first evidence of such an early FMV modification during lipid-lowering treatment. An improvement in FMV was reported by Vogel et al. (4) after 2 weeks of treatment with simvastatin in seven healthy middle-aged men with average pretreatment LDL cholesterol (133 ± 14 mg/dl). In that study, at variance with the present one, fully normal levels of LDL cholesterol were achieved after treatment (88 ± 15 mg/dl), whereas in our study, a substantial improvement in FMV was obtained after 2 weeks of treatment despite the persistence of moderately high LDL cholesterol levels (147 ± 27 mg/dl). An increase in brachial artery FMV was reported by Simons et al. (6) in 20 subjects with severe hypercholesterolemia after a 7-month treatment with high-dose atorvastatin. In that study, however, subjects had a higher pretreatment serum cholesterol (422 vs. 319 mg/dl in our study), and 80% of them had a history of coronary heart disease. Moreover, a higher dose of atorvastatin was administered (80 vs. 10 mg daily), and the time course of FMV changes was not assessed (6).
The precise mechanism responsible for the beneficial effect of statins on the endothelium is controversial. Several studies indicated that endothelial function improves with lowering of serum cholesterol (4-7). In the present study, only a weak association was found between decrease in serum cholesterol during atorvastatin treatment and improvement in FMV (Fig. 1, right). Several reasons, including the relatively small sample size, a biologic lag phase, and imprecisions in measurements, may contribute to this finding. However, the possibility cannot be excluded that the improvement in endothelial function is not due solely to the decrease in serum cholesterol but to a direct effect of the drug on nitric oxide bioavailability. Some evidence suggests that statins may exert a direct beneficial effect on the endothelium besides their lipid-lowering activity. A pravastatin-induced activation of endothelial nitric oxide synthase has been observed by Kaesemeyer et al. (16) in rat aortic rings. Moreover, an increase in nitric oxide bioavailability has been observed in hypercholesterolemic men after treatment with fluvastatin (15). To our knowledge, no available evidence supports the view that structural differences between atorvastatin and other statins may influence their effect on the endothelial function.
Reactive hyperemia is a multifactorial phenomenon, involving both vasodilator metabolites and myogenic influences (28). We did not assess vasodilation in response to exogenous nitrates; thus we cannot exclude the possibility that the treatment-induced vasodilation is by a non-endothelium-dependent mechanism. However, it has been shown that brachial artery response to hyperemia is dependent on nitric oxide bioavailability (11,29,30). In the present study, arterial occlusion was induced above the examined brachial artery segment, whereas occlusion was below the site of investigation in the experiment that demonstrated the endothelium-dependent nature of FMV (29). Despite the conceptual differences between the two approaches, a subsequent study showed that also postischemic forearm vasodilation after proximal occlusion depends on endothelium-derived nitric oxide (30). Moreover, in a direct comparison between the two methods, vasodilation induced by occlusion above the study site showed a significant direct relation with dilation induced by distal occlusion, suggesting the evaluation of the same mechanism of vasodilation (31).
In summary, atorvastatin improved endothelial function within 2 weeks of treatment in hypercholesterolemic women. The extent to which this effect is attributable to the lipid-lowering effect or to a possible direct effect of the drug on nitric oxide bioavailability remains to be established.
1. The Lipid Research Clinics Coronary Primary Prevention Trial results. II: the relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA
2. Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet
3. Chowiencyk PJ, Watts GF, Cockcroft JR, et al. Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolemia
4. Vogel RA, Corretti MC, Plotnick GD. Changes in flow mediated brachial artery vasoactivity with lowering of desirable cholesterol levels in healthy middle-aged men. Am J Cardiol
5. O'Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function
within 1 month. Circulation
6. Simons LA, Sullivan D, Simons J, et al. Effects of atorvastatin
monotherapy and simvastatin plus cholestyramine on arterial endothelial function
in patients with severe primary hypercholesterolaemia. Atherosclerosis
7. Koh KK, Cardillo C, Bui MN, et al. Vascular effects of estrogen and cholesterol-lowering therapies in hypercholesterolemic post-menopausal women. Circulation
8. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med
9. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation
10. Plotnick GD, Corretti MC, Vogel RA. Effects of antioxidant vitamins on the transient impairment of endothelium-dependent brachial artery vasoactivity following a single high fat meal. JAMA
11. Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet
12. Celermajer DS, Sorensen KE, Spiegelhalter DJ, et al. Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women. J Am Coll Cardiol
13. Treasure CB, Klein JL, Vita JA, et al. Hypertension and left ventricular hypertrophy are associated with impaired endothelium-mediated relaxation in human coronary resistance vessels. Circulation
14. Goodfellow J, Ramsey MW, Luddington LA, et al. Endothelium and inelastic arteries: an early marker of vascular dysfunction in non-insulin dependent diabetes. Br Med J
15. John S, Schlaich M, Langenfeld M, et al. Increased bioavailability of nitric oxide after lipid-lowering therapy
in hypercholesterolemic patients: a randomized, placebo-controlled, double-blind study. Circulation
16. Kaesemeyer WH, Caldwell RB, Huang J, et al. Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions. J Am Coll Cardiol
17. Marchesi S, Lupattelli G, Schillaci G, et al. Impaired flow-mediated vasoactivity during post-prandial phase in young healthy men. Atherosclerosis
2000 (in press).
18. Friedewald WT, Levy RI, Frederickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of ultracentrifuge. Clin Chem
19. Rainwater DL, Andres DW, Ford AL, et al. Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins. J Lipid Res
20. Tanner FC, Noll G, Boulanger CM, et al. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived nitric oxide. Circulation
21. Casino PR, Kilcoyne CM, Quyyumi AA, et al. Investigation of decreased availability of nitric oxide precursor as the mechanism responsible of impaired endothelium-dependent vasodilation in hypercholesterolemic patients. J Am Coll Cardiol
22. Minor RL, Myers PR, Guerra R, et al. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aortas. J Clin Invest
23. Ito A, Tsao PS, Adimoolam S, et al. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation
24. Darley-Usmar VM, Hogg N, O'Leary VJ, et al. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoproteins. Free Radic Res Commun
25. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia
increases endothelial superoxide anion production. J Clin Invest
26. John S, Schmieder RE. Impaired endothelial function
in arterial hypertension and hypercholesterolemia
: potential mechanisms and differences. J Hypertens
27. White CR, Darley Usmar V, Oparil S. No role for NO in estrogen-mediated vasoprotection? Circulation
28. Shepherd JT. Circulation to skeletal muscle. In: Handbook of physiology: the cardiovascular system: peripheral circulation and organ blood flow.
Bethesda: American Physiological Society, Sect. 2, Vol III, pt. 1, 1984:319-70.
29. Joannides R, Haefeli WE, Linder L, et al. Nitric oxide is responsible for flow-dependent dilation of human peripheral conduit arteries in vivo. Circulation
30. Meredith IT, Currie KE, Anderson TJ, et al. Postischemic vasodilation in human forearm is dependent on endothelium-derived nitric oxide. Am J Physiol
31. Mannion TC, Vita JA, Keaney JF, et al. Non-invasive assessment of brachial artery endothelial vasomotor function: the effect of cuff position on level of discomfort and vasomotor responses. Vasc Med