Lipid-independent effects of statins on endothelial function and bioavailability of nitric oxide in hypercholesterolemic patients

Lipid-independent effects of statins on endothelial function and bioavailability of nitric oxide in hypercholesterolemic patients Stefan John, MD, Markus P. Schneider, MD, Christian Delles, MD, Johannes Jacobi, MD, and Roland E. Schmieder, MD, FACC Erlangen, Germany

Background Experimental evidence suggests a lipid-independent effect of statins on endothelial function and nitric oxide (NO) availability in humans. We investigated whether improvement in NO availability in hypercholesterolemia can be achieved rapidly with statins before lipid-lowering therapy is complete. Methods

We studied 41 patients (52 F 11 years) with low-density lipoprotein (LDL) cholesterol z 130 mg/dL (179 F 45 mg/dL) randomly assigned to treatment either with atorvastatin (20 mg/day) or cerivastatin (0.4 mg/day). Endotheliumdependent vasodilation of the forearm vasculature was measured by plethysmography and intra-arterial infusion of acetylcholine (ACh) after 3 days (n = 18) and 14 days (n = 39) of treatment. NO availability and oxidative stress were assessed by coinfusion of L-NMMA and vitamin C.

Results After 3 days of treatment, LDL-cholesterol decreased by 11.9% with a further decrease to 29.6% after 14 days ( P b .001). Endothelium-dependent vasodilation improved by +46.7% after 3 days of statin therapy compared with before therapy (ACh 48 Ag/min: +15.7 F 10.6 vs +10.7 F 10.8 mL/min per 100 mL, P b .05). No further improvement in endothelium-dependent vasodilation (+42.7% compared with before therapy) could be demonstrated after 14 days of treatment (ACh 48 Ag/min: +17.7 F 10.3 vs +12.4 F 9.3 mL/min per 100 mL before therapy, P b .001). Coinfusion of ACh plus vitamin C was able to improve endothelium-dependent vasodilation before but not after 3 or 14 days of statin therapy either. The improvement in endothelium-dependent vasodilation after therapy was no longer observed when the NO-synthase inhibitor L-NMMA was coinfused together with ACh. Conclusions Short-term lipid-lowering therapy with statins is able to improve endothelial function and NO availability almost completely after 3 days in hypercholesterolemic patients probably by decreasing oxidative stress. This improvement seems to be more rapid than the accompanying decline in LDL-cholesterol and not related to these lipid changes. This finding can support the concept of lipid-independent effects of statins in humans. (Am Heart J 2005;149: 473.e1-473.e10.) Prospective clinical trials have demonstrated that reductions in low-density lipoprotein (LDL) cholesterol with statins decrease morbidity and mortality rates, in particular coronary artery disease and stroke. Because serum cholesterol levels are strongly associated with the development of atherosclerotic disease, it has been initially assumed that cholesterol reduction by statins is the predominant mechanism underlying their beneficial

From the Department of Medicine IV, University of Erlangen-Nu¨rnberg, Erlangen, Germany. This study was supported by a research grant of Bayer AG, Leverkusen, Germany and of the bDeutsche Forschungs Gemeinschaft Q KFO 106 TP 3. Submitted December 3, 2003; accepted June 10, 2004. Reprint requests: PD Dr. Stefan John, MD, Department of Medicine IV / 4, University of Erlangen-Nu¨rnberg, Krankenhausstr. 12, D-91054 Erlangen, Germany. E-mail: [email protected] 0002-8703/$ - see front matter n 2005, Elsevier Inc. All rights reserved. doi:10.1016/j.ahj.2004.06.027

effects in cardiovascular disease. However, the benefit of statins occurs early in the course of statin therapy, and subgroup analysis of large clinical trials indicates that statin-treated individuals have less cardiovascular disease than placebo-controlled individuals with comparable serum cholesterol levels.1,2 Subsequently, it has been suggested that these drugs may have antiatherogenic effects in addition to their capacity to lower lipids and lipoproteins.3 In fact, intriguing experimental data have shown that statins exhibit pleiotropic effects beyond their lipid-lowering actions, including enhancement of endothelial nitric oxide (NO) production,4,5 inhibition of smooth muscle proliferation,6 and anti-inflammatory and antioxidative actions.7,8 Cholesterol lowering therapy with statins improves endothelium-dependent vasodilation in coronary9,10 and systemic11 arteries due to an increased availability of NO,12 the most important endothelium derived vasodilating substance. Apart from vasodilating effects, NO

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Table I. Comparison of baseline characteristics and lipid profiles between all included hypercholesterolemic patients (x F SD, n = 41) and the subgroup of patients which additionally participated in the assessment of endothelial function after 3 days (n = 18)

Sex (m/f) Age (y) Height (m) Weight (kg) Body mass index (kg/m2) Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Active smoker (yes/no) Hypertensives (yes/no) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Hs-CRP (mg/L)

Table II. Changes in lipid profiles and BP after the 3- (n = 18) and 14-day (n = 39) treatment period (D% F SEM) with statins from before therapy

After 3 days (n = 18)

All patients (n = 41)

P

10/8 52.6 F 9 1.72 F 0.10 78 F 12 26.4 F 2.4

22/19 52.8 F 11 1.70 F 0.09 76 F 13 25.7 F 2.7

ns ns ns ns ns

142 F 23 90 F 12 0/18 0/18 266 F 53 59 F 16 169 F 47 180 F 147 2.64 F 3.41

140 F 20 89 F 11 0/41 0/41 271 F 46 60 F 18 179 F 45 168 F 113 2.70 F 3.64

ns ns ns ns ns ns ns ns ns

has been shown to inhibit several components of the atherogenic process.13 Endothelium- derived NO mediates vascular relaxation and inhibits platelet aggregation,14 vascular smooth muscle proliferation,15 and endothelium-leukocyte interactions.16 Thus, improvement in the availability of NO seems to play a pivotal role in the regression and delayed progression of coronary atherosclerosis induced by lipid-lowering therapy.17 Rapid improvement in endothelial function18 and NO availability after lipid-lowering therapy with statins has been demonstrated even after a short time period of only 2 weeks.19 In addition, improved endothelial function has been suggested even after a single LDL apheresis within hours.20 However, these effects on endothelial function were regularly related to the accompanying decline in serum cholesterol levels. As cholesterol clamp conditions during statin therapy can be hardly achieved in humans, direct effects of statins on atherosclerotic arteries without lipid-lowering therapy have been studied in animal models.21 Moreover, statin-induced short-term improvement in flowmediated dilation of the brachial artery, as a rough measurement of global endothelium-derived vasodilation, could be demonstrated in elderly diabetic patients and in healthy volunteers.22-24 These findings appeared to be independent of changes in lipid concentration in these populations. The objective of this study was to determine whether restoration of endothelium-dependent vasodilation and availability of NO can be achieved before, and probably independent from, a complete reduction in serum cholesterol levels in patients with hypercholesterolemia.

Percent change in

After 3 days (n = 18)

Total cholesterol (%) LDL cholesterol (%) HDL cholesterol (%) Trigycerides (%) Hs-CRP (mg/L) Systolic BP (mm Hg) Diastolic BP (mm Hg)

11.7 F 2.3 11.9 F 2.8 4.5 F 2.4 18.2 F 8.6 +0.69 F 4.9 4.1 F 4.1 0.7 F 2.8

After 14 days (n = 39) 23.4 F 2.2 29.6 F 2.4 +1.6 F 3.2 21.5 F 8.3 +0.53 F 5.9 4.3 F 4.1 +0.7 F 2.8

P .001 .001 .064 .665 .142 .275 .400

P values for comparison of these changes between after 3 days and after 14 days.

Methods Study population Hypercholesterolemic patients were randomly assigned to a lipid-lowering strategy with either atorvastatin or cerivastatin for a period of up to 14 days in a double-blind study. Inclusion criteria were age between 20 and 65 years, increased serum LDL cholesterol level of z130 mg/dL, serum triglyceride level of V400 mg/dL while not receiving cholesterol-lowering medication. Exclusion criteria were patients with active smoking habits and nonsmokers b1 year of cessation, diabetes mellitus (HbA1c N7% or fasting blood glucose N120 mg/dL or antidiabetic medication), arterial hypertension (diastolic blood pressure [BP] z95 mm Hg or systolic BP z160 mm Hg) or being on any BP lowering agent, any cardiovascular or cerebrovascular event within the last 3 months, ouvert peripheral vascular disease, severe cardiac pathologies (uncontrolled arrythmias, atrial fibrillation, unstable angina, congestive heart failure NYHA II-IV, coronary artery bypass grafting surgery, or percutaneous transluminal coronary angioplasty within the previous 6 months), known intolerance or hypersensitivity to statins, severe disorders of the gastrointestinal tract (chronic diarrhea, ulcerative colitis, etc), familial mono genic hypercholesterolemia, secondary hyperlipoproteinemia, vascular abnormalities in the forearm vasculature, liver or kidney disease (aspartate aminotransferase and alanine amino transferase levels N200% of upper normal limit, alkaline phosphatase, bilirubin, and serum creatinine N150% of upper normal limit), patients regularly or occasionally practicing weightlifting or body building or working on night shifts, any lipid-lowering medication, and patients on steroids or immunosuppressive agents. Forty-one patients were included in this randomized, double-blind, placebo-controlled trial. The baseline characteristics of these patients are given in Table I. They were randomly assigned by a randomization list in a 1:1 fashion to the atorvastatin (n = 20) or the cerivastatin group (n = 21). Unfortunately, the study had to be stopped because of the withdrawal of cerivastatin from the US and European markets. Therefore, no further patients were included and 2 patients did not complete the study and were excluded from per protocol analysis. Thus, 19 patients in the atorvastatin group and 20 patients in the cerivastatin group

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Table III. Forearm blood flow (mL/min per 100 mL) at baseline and changes from baseline for different doses of intra-arterial acetylcholine, sodium nitroprusside, and acetylcholine with coinfusion of vitamin C or L-NMMA after 3 days and 14 days of statin therapy (x F SD) 3 days (n = 18) Changes (mL/min per 100 mL)

Before therapy

14 days (n = 39)

After therapy

P

Before therapy

After therapy

P

Acetylcholine Baseline 12 Ag/min 48 Ag/min + Vitamin C (18 mg/min)

3.47 F 1.16 +8.36 F 7.96 +10.7 F 10.8 +15.7 F 8.94*

3.66 +11.1 +15.7 +16.2

1.26 8.78 10.6 12.2

ns .05 .05 ns

3.22 F 0.90 +9.50 F 7.09 +12.4 F 9.29 +15.6 F 9.25**

3.43 +11.7 +17.7 +18.3

Nitroprusside Baseline 3.2 Ag/min 12.8 Ag/min

4.47 F 1.82 +9.19 F 3.48 +16.9 F 7.20

4.33 F 1.65 +9.48 F 4.24 +16.1 F 7.48

ns ns ns

4.24 F 1.47 +9.00 F 3.14 +16.1 F 6.39

4.48 F 1.66 +9.42 F 4.24 +16.6 F 7.59

ns ns ns

Acteylcholine + L -NMMA 4 Amol/min Baseline 12 Ag/min 48 Ag/min

3.56 F 1.11 +10.1 F 10.9 +15.2 F 14.3

3.55 F 1.11 +8.92 F 8.76 +15.8 F 9.68

ns ns ns

3.44 F 0.90 +8.64 F 8.69 +14.1 F 11.3

3.65 F 0.94 +8.63 F 9.41 +16.3 F 11.4

ns ns ns

F F F F

F F F F

1.00 9.19 10.3 11.9

ns .05 .001 ns

Corresponding P values for comparison between before and after the treatment period (Students t test). Asterisk, for comparison between without vitamin C and with coinfusion of vitamin C (*P b .005, **P b .001).

entered statistical analysis. The baseline characteristics of these patients were not different between the 2 treatment groups (data not shown). Before the first examination, all patients were asked to give consent for additional invasive endothelial function testing already on day 3 after the beginning of statin therapy. Eighteen patients agreed with this additional third invasive evaluation of their forearm circulation. The baseline characteristic of these 18 patients additionally examined on day 3 were not different from those of the whole study population (Table I).

Study design The study was approved by the ethics committee of the University of Erlangen-Nqrnberg and the study was performed according to bgood clinical practiceQ (GCP) guidelines. Written informed consent was obtained from all patients before study entry. After evaluation of inclusion and exclusion criteria possible concomitant use of lipid-lowering drugs was discontinued in a first washout period (days 56 to 42). During this period all patients who had not been on a stable diet for hyperlipidemia received dietary instructions according to the American Heart Association Step I dietQ from a registered dietitian. The washout period was followed by a placebo run-in period for 6 weeks (days 42 to 0). Patients were finally included according to the mean of their LDL cholesterol concentrations measured on days 14 and 7. The following active treatment phase (days 0 to 14) lasted 14 F 3 days. The primary end point of the study was the assessment of endothelium-dependent vasodilation on day 0 (baseline) and days 3 and 14 (after treatment).

Assessment of FBF Studies were performed in the morning at the same time in an undisturbed temperature-controlled environment (22-248C).

An intra-arterial catheter (20 G, 8 cm, Vygon, France) was inserted into the brachial artery of the nondominant arm by the use of Seldinger’s technique. After cannulation, the subjects rested for 60 minutes before the study started. Subjects lay supine with their left forearm supported above the level of the right atrium. The response of forearm blood flow (FBF) to vasoactive agents was assessed by strain-gauge venousocclusion plethysmography (EC 5R Plethysmograph, Hokanson, USA). Drugs were infused intra-arterially (IA) at the rate of 1.5 mL/min using syringe pumps. This flow applied to the brachial artery was kept constant also in the case of simultaneous infusion of 2 different substances by changing the single concentration of the used substance. The following substances were administered (each dose was infused IA. for 4 minutes): (1) acetycholine (ACh) to assess endothelium-dependent vasodilation at sequential dosages of 12 and 48 Ag/min. (2) Simultaneous infusion of vitamin C (18 mg/min) and acetycholine (48 Ag/min) to test whether any improvement in endothelium-dependent vasodilatation can be achieved by this substance with antioxidant capacity. (3) Sodium-nitroprusside (NP) to test endothelium-independent vascular relaxation (3.2 and 12.8 Ag/min). (4) Simultaneous infusion of l-NMMA (4 Amol/min) and acetycholine (12 and 48 Ag/min) to test whether any improvement in endotheliumdependent vasodilatation can be blocked by this NO synthase inhibitor. Before each intervention, saline was infused for 15 minutes, to let the FBF return to resting levels. The left hand was excluded from the circulation by the inflation of a wrist cuff to 220 mm Hg during each measurement period. Upper arm cuff was inflated to 40 mm Hg by a rapid cuff inflator (E20, Hokanson, Bellevue, USA) to occlude venous outflow during each measurement. Output from the strain gauges was displayed on a monitor in real time. A software program coordinated the acquisition, storage, and display of data as well as inflation and deflation of the arm cuffs. Forearm blood flow

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Figure 2

600 after 3 days before therapy

+ Vit C #

* 400

200

ANOVA * P < .05

0 baseline

12

48

Increase in forearm blood flow (∆%)

Increase in forearm blood flow (∆%)

Figure 1

600 after 14 days before therapy

+ Vit C

** ##

400

200

ANOVA * P < .005

0

+ Vit C (18mg/min)

baseline

+ Vit C (18mg/min)

48

12

i.a. acetylcholine (µg/min) i.a. acetylcholine (µg/min) Improved endothelium-dependent vasodilation after 3 days of statin therapy: increase in FBF in percent from baseline (D%) after the infusion of acetylcholine with increasing doses (n = 18; square, before therapy; filled square, after therapy) and after the infusion of ACh 48 Ag/min + coinfusion of vitamin C (circle, before therapy; filled circle, after therapy). Comparison of the changes between before and after therapy (asterisk, P b .05; ANOVA P b .05) and between with and without vitamin C (number sign, P b .005).

was obtained from an average of 8 measurements recorded for 9 seconds out of every 15 seconds during the final 2 minutes of each infusion period.

Improved endothelium-dependent vasodilation after 14 days of statin therapy: increase in FBF in percent from baseline (D%) after the infusion of acetylcholine with increasing doses (n = 18; square, before therapy; filled square, after therapy) and after the infusion of ACh 48 Ag/min + coinfusion of vitamin C (circle, before therapy; filled circle, after therapy). Comparison of the changes between before and after therapy (asterisk, P b .05; ANOVA P b .05) and between with and without vitamin C (double pound sign, P b .001).

Figure 3

LDL-Cholesterol

before

***

Routine chemical methods were used to determine serum concentrations of total cholesterol, high-density lipoprotein (HDL)- and LDL cholesterol, triglycerides, creatinine, glucose, electrolytes, and liver enzymes. High-sensitivity C-reactive protein (hs-CRP) was determined using a polystyrene-enhanced nephelometric assay (DadeBehring, Germany).

After the baseline evaluation, the patients were randomly assigned to 1 of 2 treatments: atorvastatin 20 mg or cerivastatin 0.4 mg once daily in the evening. Both substances were identical in appearance. Patients and treating physicians were blinded with regard to the chosen therapy.

Statistical analysis Differences between treatment groups in clinical characteristics, lipid profiles, other biochemical parameters, and changes in FBF were analyzed by unpaired Students t test and by paired Students t test between before (day 0) and after (day 3 or 14) treatment. In addition, ANOVA for repeated measurements was applied to test differences in dose–response curves between groups and treatment phases. Vascular reactivity data are expressed as blood flow in milliliters per

14 days

- 11.9%

(percent from before therapy)

Analytic methods

Treatment

3 days

- 29.6% *** ***

n.s.

* + 46.7%

Endothelium dependent vasodilation (∆FBF at ACh 48 µg/min

** + 42.7%

in percent from before therapy)

before

3 days

14 days

More rapid improvement in endothelial function than in LDLcholesterol: percent changes from before therapy in LDL-cholesterol and endothelium-dependent vasodilation (DFBF from baseline after ACh 48 Ag/min) after 3 and 14 days of statin therapy.

minute per 100 mL forearm tissue (x F SD) and as the percent change (x F SEM) from the corresponding baseline. Bio chemical parameters are expressed in absolute values (x F SD) and as the percent change (x F SEM) from the corresponding baseline. Linear correlation analysis (Pearson) was used to test correlations between changes in endothelium-dependent

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Increase in forearm blood flow (∆ %)

Figure 4 600

600

after 3 days before therapy

400

400

200

200

after 14 days before therapy

0

0 baseline

3.2

12.8

baseline

3.2

12.8

i.a. nitroprusside (µg/min) Unaffected endothelium-independent vasodilation after 3 (n = 18) and 14 days (n = 39) of statin therapy: increase in FBF in percent from baseline (D%) after the infusion of NP with increasing doses (square, before therapy; filled square, after therapy). Comparison of the changes between the treatment group and placebo (all ns).

vasodilation and changes in lipid profiles after therapy. Twosided P values are given throughout the text. A 2-sided P value of b0.05 was considered statistically significant.

Results Changes in lipid profiles After 3 days of lipid-lowering therapy with statins, total cholesterol levels (266 F 53 mg/dL before therapy vs 233 F 56 mg/dL after therapy, P b .001), LDL cholesterol (169 F 47 vs 149 F 50 mg/dL, P b .001), triglycerides (180 F 147 vs 116 F 37 mg/dL, ns), and HDL cholesterol (59 F 16 vs 56 F 14 mg/dL, P b .05) decreased. After 14 days of lipid-lowering therapy with statins, total cholesterol levels (274 F 45 mg/dL before therapy vs 208 F 40 mg/dL after therapy, P b .001), LDL cholesterol (181 F 45 vs 127 F 37 mg/dL, P b .001), and triglycerides (167 F 111 vs 115 F 47 mg/dL, P b .01) decreased, whereas HDL cholesterol slightly increased (61 F 18 vs 64 F 17 mg/dL, P b .05). The percent changes in lipid profiles from before therapy to therapy after 3 and 14 days are shown in Table II. A highly significant further decrease in total cholesterol and LDL cholesterol could be observed during the treatment period after 14 days in comparison to after 3 days. No differences in the changes in triglycerides, HDL cholesterol, and BP values were noted between after 3 and 14 days of statin therapy. There were no significant differences between the atorvastatin- and the cerivastatin-induced changes in lipid profiles (data not shown).

FBF responses to acetylcholine and additional vitamin C In the group of patients examined after 3 days, FBF at baseline was similar before and after treatment (Table III). Intra-arterial administration of ACh caused an increase in FBF with increasing doses before and after therapy (ANOVA all P b .001) (Table III in absolute terms, Figure 1 in percent change from baseline). After 3 days of statin therapy, however, the acetylcholine-induced increases in FBF were enhanced compared with pretreatment evaluation ( P b .05 for both doses of acetylcholine, Table III, ANOVA P b .05, Figure 1). Before therapy the ACh-induced increases in FBF could be improved by the simultaneous infusion of vitamin C (DAch 48 Ag/min: +10.7 F 10.8 mL/min per 100 mL without vitamin C, +15.7 F 8.94 mL/min per 100 mL with vitamin C, P b .005) (Table III). This effect of vitamin C was no longer observed after 3 days of therapy (DAch 48 Ag/min: +15.7 F 10.6 mL/min per 100 mL without vitamin C, +16.2 F 12.2 mL/min per 100 mL with vitamin C, ns) (Table III). In the group of patients examined after 14 days, FBF at baseline was similar before and after treatment (Table III). Intra-arterial administration of ACh caused an increase in FBF with increasing doses before and after therapy (ANOVA all P b .001) (Table III in absolute terms, Figure 2 in percent change from baseline). After 14 days of statin therapy, again the acetylcholine-induced increases in FBF were enhanced compared with pretreatment evaluation ( P b .005 for ACh 48 Ag/min, Table III, ANOVA P b .005, Figure 1),

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Figure 5

Increase in forearm blood flow (∆%)

Increase in forearm blood flow (∆%)

after 3 days

after 14 days

800

800

∆%

600

∆%

P < 0.05

n.s.

400

400

200

200

0

n.s.

ACh 12

ACH 12 + L-NMMA 4

0

ACh 12

ACh 12 + L-NMMA 4

P < .05

n.s.

800 ∆%

n.s. 600

800 ∆%

600

600

400

400

200

200

0

P < .005

n.s.

0

ACh 48

ACH 48 + L-NMMA 4

ACH 48

ACH 48 + L-NMMA 4

Improved endothelium-dependent vasodilation after 3 or 14 days of statin therapy can be blocked with L-NMMA: changes in FBF (FBF) in percent from baseline (D%) after the infusion of acetylcholine 12 Ag/min (ACH 12, upper figure) and 48 Ag/min (ACH 48, lower figure) without (left two columns) and with (right two columns) coinfusion of the NO-synthase inhibitor L-NMMA 4 Amol/min before (square) and after (filled square) lipid-lowering therapy with cerivastatin for 3 (left) or 14 days (right).

but not further enhanced compared with the evaluation after 3 days (ANOVA ns). Before therapy the ACh-induced increases in FBF could be improved by the simultaneous infusion of vitamin C (DAch 48 Ag/min: +12.4 F 9.29 mL/min per 100 mL without vitamin C, +15.6 F 9.25 mL/min per 100 mL with vitamin C, P b .001) (Table III). This effect of vitamin C was again no longer observed after 14 days of therapy (DAch 48 Ag/min: +17.7 F 10.3 mL/min per 100 mL without vitamin C, +18.3 F 11.9 mL/min per 100 mL with vitamin C, ns) (Table III). To analyze the effects in the treatment group after 3 days vs those after 14 days, we subtracted the change in FBF from baseline in response to acetylcholine after therapy from those before therapy. No difference could be detected between this improvement in vasodilator function (FBF from baseline in mL/min per 100 mL) after 3 days or 14 days of statin treatment at ACh dose 48 Ag/min (+5.5 F 11.1 after 3 days vs +5.7 F 10.4 after 14 days, ns) and at ACh dose 12 Ag/min (+3.2 F 5.7 after 3 days vs +3.9 F 7.6 after 14 days, ns). These increases in FBF (percent from baseline) indicate an increase in vasodilator function (ACh 48 Ag/min) of

+46.7% (improvement in percent from before therapy) after 3 days and of 42.7% (percent from before therapy) after 14 days (Figure 3). The corresponding decreases in LDL cholesterol in percent from before therapy were 11.9% after 3 days with a further decline to 29.6% after 14 days (Figure 3). Finally, we analyzed whether the improvement in endothelium-dependent vasodilation after lipid-lowering therapy was related to the degree of decreases in LDL cholesterol. No relations could be found between the improvement in endothelium-dependent vasodilation (D% from baseline) and the improvement in LDL cholesterol after therapy (after 3 days: r = 0.196, P = .466; after 14 days: r = 0.125, P = .480). Between the 2 treatment groups (atorvastatin vs cerivastatin) no differences could be detected in all the abovementioned changes in endothelium-dependent vasodilation in comparison to the whole study population (data not shown).

FBF responses to nitroprusside Administration of the endothelium-independent vasodilator sodium nitroprusside caused dose- dependent

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increases in FBF before and after 3 or 14 days of statin therapy (ANOVA P b .001). No differences in the observed increases after administration of sodiumnitroprusside could be found between before and after treatment, respectively (Table III; Figure 4).

Forearm blood flow responses to acetylcholine with simultaneous L-NMMA infusion Before the simultaneous intra-arterial infusion of l-NMMA (4 Amol/min) and 2 increasing doses of acetylcholine, no differences in baseline FBF were noted between before and after statin therapy after 3 or 14 days (Table III). The significant improvement in acetycholine -induced vasodilation after 3 days or 14 days of treatment with statins (see above) was no longer observed if the NO-synthase inhibitor was coinfused (Table III; Figure 5; ANOVA ns). After 3 days of treatment FBF increased from baseline at dose acetylcholine 12 Ag/min by 293% F 76% before vs 245% F 57% after treatment and at acetylcho line 48 Ag/min by 439% F 94% before vs 479% F 61% after treatment (all differences ns) (Figure 5). After 14 days of treatment FBF increased from baseline at dose acetylcholine 12 Ag/min by 262% F 44% before vs 220% F 33% after treatment and at acetylcholine 48 Ag/min by 428% F 56% before vs 482% F 61% after treatment (all differences ns) (Figure 5). Changes in hs-CRP High-sensitivity C-reactive protein serum levels changed from 2.64 F 3.41 mg/L to 3.37 F 4.89 mg/L after 3 days (ns) and from 2.70 F 3.64 mg/L to 3.23 F 5.93 mg/L (ns) after 14 days of lipid-lowering treatment (Tables I and II).

Discussion Lipid-independent improvement in NO availability This study documents that therapy with statins can rapidly improve endothelial function and the availability of NO already after 3 days of therapy, at a time when the lipid-lowering effect of statins is incomplete. Of note, no further improvement in endothelial function and NO availability was observed with ongoing therapy, in contrast to a further decline in LDL concentrations. Moreover, our data suggest that these beneficial effects of statins are mediated by the reduction of oxidative stress in the vasculature. In the current study, treatment with the statin atorvastatin or cerivastatin improved endotheliumdependent vasodilation (Figures 1 and 2) as it has been documented for lipid-lowering therapy in previous studies after 1 to 12 months of therapy in coronary9,10 and peripheral arteries.11,12,18 Moreover, the adminis tration of the NOS inhibitor l-NMMA blunted the improvement in endothelium-dependent vasodilation

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after 3 or 14 days of statin therapy (Figure 5). This finding indicates that a rapidly increased availability of NO mediates improved endothelium-dependent vasodilation found already after 3 days. To our knowledge this is the shortest period so far demonstrated for HMG-CoA-reductase inhibitors to improve pharmacologically assessed NO availability in humans. No further improvement in endothelial function could be noticed by continuing lipid-lowering therapy. In previous studies improvement in endothelial function was closely related to the accompanying decline in LDL cholesterol, suggesting that statins restore endo thelial function predominantly by lowering serum cholesterol levels. Although we observed a small decline in LDL cholesterol already after 3 days of lipid-lowering therapy, serum lipids continued to decline during the following treatment period (Figure 3). After 3 days of statin therapy, approximately one third of the maximum lipid-lowering effect was achieved. This further improvement in lipid profiles did not lead to any further improvement in NO availability, which reached its maximum response already after 3 days. The improvement in vasodilator function was not related either to the improvement in LDL cholesterol after these 3 days. Although this rapid endothelial improvement cannot be completely dissociated from the lipid-lowering effects of statins, our data suggest, at least in part, a direct, lipid-independent effect of statins on endothelial function and NO availability in patients with mild to moderate hypercholesterolemia. Laufs et al have demonstrated a disassociation from alterations of vascular function, assessed by flowmediated vasodilation, and the time course of high dose cholesterol lowering with atorvastatin in healthy young subjects.24 In addition, it has been demonstrated that a single dose of cerivastatin is able to improve vascular responsiveness measured by flow-mediated dilation of the brachial artery independent from its lipid-lowering effects again in healthy normocholesterolemic subjects.23 Another recent study has reported a direct lipidindependent effect of statins in elderly diabetic patients with or without hypercholesterolemia.22 In this study, cerivastatin was administered in a low dosage of only 0.15 mg/d also for 3 days. According to the authors this low dose of statins did not affect lipid levels. Nevertheless, an improvement in flow-mediated vasodilation of the forearm vasculature, measured by high-resolution ultrasound, could be demonstrated after this short-time therapy. Our findings can confirm this notion after direct intra-arterial stimulation with vasoactive agents directly affecting the NO-cGMP pathway in a hypercholesterolemic study population.

Potential lipid-independent mechanisms Extensive evidence accumulated over the past 2 decades has demonstrated that the endothelium- derived

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NO exerts essential vasoprotective functions and that impaired endothelial function predicts long-term atherosclerotic disease progression and cardiovascular event rates.13,25 In patients with hypercholesterolemia, the availability of endothelium-derived NO is impaired either through decreased synthesis of NO or, even more important, through increased breakdown of NO as the result of oxidative mechanisms.26,27 Superoxide directly inactivates NO and may also increase the subsequent oxidation of LDL particles by the formation of the powerful oxidant peroxynitrite.28 Although LDL cholesterol and superoxide production in particular are clearly associated with endothelial dysfunction and reduced availability of NO, therapy with statins may improve endothelial function through other mechanisms independent of their lipid-lowering effects. In fact, there is increasing experimental evidence that statins have additional beneficial antiatherosclerotic effects by acting directly on endothelial cells. In this context, statins increase NO availability by stimulating and upregulating endothelial NO synthase (eNOS) in vitro independent of their cholesterol lowering actions.5 Furthermore, statins have been shown to restore eNOS activity in the presence of hypoxia29 and oxidized LDL,30 conditions that lead to endothelial dysfunction. Statins also inhibit the expression of endothelin-1, a potent vasoconstrictor and mitogen,31 and were shown to have angiostatic effects at high concentrations.32 Another potential mechanism by which statins may favorably affect the endothelium and attenuate endo thelial dysfunction is through their antioxidant effects. Statins enhance endothelium-dependent vasodilation by inhibiting the production of reactive oxygen species, such as superoxide (O2 ) and hydroxy radicals, from aortas of cholesterol-fed rabbits, although plasma total cholesterol levels were not different from control animals.33 Although lipid-lowering therapy by itself can lower vascular oxidative stress,26,34 some of these antioxidant effects of statins appear to be cholesterol independent. For example, statins attenuate angiotensin II-induced free radical production in vascular smooth muscle cells by inhibiting NADH oxidase activity and downregulating angiotensin type 1 receptor expression.35,36 Because NO is scavenged by reactive oxidant species, these findings indicate that the antioxidant properties of statins may also contribute to their ability to improve NO availability.34 In vitro, direct measurement of diffusible NO, together with current measurements of O2 , disclosed that in the presence of statins the NOS system operates with high efficiency toward increasing NO activity by activation of NO release and by concurrent inactivation of O2 .37 In the current study impaired endothelium-dependent vasodilation before therapy could be rapidly restored by the coinfusion of high doses of the antioxidant vitamin C. This finding indicates that indeed oxidative stress has led

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to the impairment of NO availability in the forearm vasculature of these hypercholesterolemic patients. After 3 days of statin therapy, however, the effect of vitamin C was blunted (Figure 1), suggesting that the improvement in NO availability was related to simultaneous decrease of oxidative stress. As this effect occurred as early as after 3 days, before lipid-lowering effect was completely achieved, this finding supports the notion of antioxidant properties of statins independent of their lipid-lowering actions in humans. Of note, these changes were not accompanied by changes in the inflammatory parameter hs-CRP after short-term lipid-lowering therapy. Thus reduced inflammation seems not to underlie improved oxidative stress in our patients.

Possible relation to clinical trials During the last years, large clinical trials have demonstrated that statins decrease the incidence of cardiovascular diseases. Aggressive cholesterol lowering has subsequently been adopted by clinicians as part of a standard treatment regimen for patients with documented coronary artery disease.1,2,38,39 However, no apparent threshold level for LDL cholesterol could clearly be identified. Although the majority of the clinical beneficial effects obtained with statins are a direct result of their lipid-lowering properties, there appears to be an ever-growing list of actions that are attributed to statins beyond their ability to reduce serum lipid levels at least under experimental conditions.3-8,29 - 37 Our finding of a potentially cholesterol-independent improvement in NO availability can add to this growing list of evidence, but we examined patients with only mild hypercholesterolemia and applied clinically used dosages of these drugs. Recently, the Heart Protection Study40 and the ASCOT trial41 demonstrated that statin therapy at comparable dosages used in our study was associated with significant clinical benefit in a wide range of high-risk individuals irrespective of their underlying cholesterol level. As a consequence, the indication for statin therapy will grow from a lipid-lowering strategy to an overall antiatherosclerotic strategy in patients at risk for cardiovascular disease. Ongoing studies with current and new statins are likely to shed further light on the potential cholesterol-independent, antiatherosclerotic benefits of these agents. In the present study, we have shown that improved availability of NO, an effect which is highly desirable during the acute phase of coronary syndromes, can be achieved rapidly within only 3 days. Thus, our data can support the concept that lipid-lowering therapy with statins may play a role not only for prevention but also for therapy of acute coronary syndromes or among patients during percutaneous coronary intervention. Consistently, it was demonstrated recently in a large study cohort that pretreatment with statins among

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patients undergoing percutaneous coronary intervention was associated with a significant mortality advantage at early and intermediate-term follow-up.42 In addition, the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study39 found a significant risk reduction in cumulative ischemic events in those patients with acute coronary syndromes which were randomized within 24 to 96 hours of admission to highdose statin therapy. Our observations suggest that the rapid improvement in NO availability may contribute to the improved outcome after statin therapy in these patients with acute coronary syndromes.

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