Cholesterol Reduction Improves Myocardial Perfusion Abnormalities in Patients With Coronary Artery Disease and Average Cholesterol Levels

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1 Journal of the American College of Cardiology Vol. 35, No. 1, by the American College of Cardiology ISSN /00/$20.00 Published by Elsevier Science Inc. PII S (99)00529-X Cholesterol Reduction Improves Myocardial Perfusion Abnormalities in Patients With Coronary Artery Disease and Average Cholesterol Levels Coronary Artery Disease Jose M. Mostaza, MD, PHD,* María V. Gomez, MD, PHD,* Felix Gallardo, MD,* María L. Salazar, MD, PHD, Raquel Martín-Jadraque, MD, PHD,* Leandro Plaza-Celemín, MD, Isidoro Gonzalez-Maqueda, MD, PHD, Luís Martín-Jadraque, MD, PHD Madrid, Spain OBJECTIVES BACKGROUND METHODS RESULTS CONCLUSIONS We sought to evaluate whether pravastatin treatment increases myocardial perfusion, as assessed by thallium-201 single-photon emission computed tomographic (SPECT) dipyridamole testing, in patients with coronary artery disease (CAD) and average cholesterol levels. Previous studies in hypercholesterolemic patients have demonstrated that cholesterol reduction restores peripheral and coronary endothelium-dependent vasodilation and increases myocardial perfusion. This was a randomized, placebo-controlled study with a cross-over design. Twenty patients with CAD were randomly assigned to receive 20 mg of pravastatin or placebo for 16 weeks and then were crossed over to the opposite medication for a further 16 weeks. Lipid and lipoprotein analysis and dipyridamole thallium-201 SPECT were performed at the end of each period. The SPECT images were visually analyzed in eight myocardial segments using a 4-point scoring system by two independent observers. A summed stress score and a summed rest score were obtained for each patient. Quantitative evaluation was performed by the Cedars-Sinai method. The magnitude of the defect was expressed as a percentage of global myocardial perfusion. Total and low density lipoprotein cholesterol levels during placebo were mg/dl and mg/dl, respectively. These levels with pravastatin were mg/dl and mg/dl, respectively. The summed stress score and summed rest score were lower with pravastatin than with placebo ( vs , p and vs , p 0.043, respectively). Quantitative analysis showed a smaller perfusion defect with pravastatin (29.2%) as compared with placebo (33.8%) (p 0.021) during dipyridamole stress. No differences were found at rest. Reducing cholesterol levels with pravastatin in patients with CAD improves myocardial perfusion during dipyridamole stress thallium-201 SPECT. (J Am Coll Cardiol 2000;35: 76 82) 1999 by the American College of Cardiology Endothelial dysfunction is an early event in the development of atherosclerosis. Subjects with a family history of coronary artery disease (CAD) (1) and those with coronary risk factors and apparently normal coronary arteries (2,3) frequently have impaired endothelial function. From the Atherosclerosis Unit, *Nuclear Medicine Service and Cardiology Service of the Centro de Investigaciones Clínicas del Instituto de Salud Carlos III and the Coronary Unit of Hospital La Paz, Madrid, Spain. This study was supported by grant 96/2128 from Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III, Spain. Manuscript received December 3, 1998; revised manuscript received July 30, 1999, accepted October 5, High cholesterol levels are associated with abnormal endothelial vasodilatory capacity (4 7). This defect is thought to be involved in the pathogenesis of myocardial ischemia. Cholesterol lowering restores endotheliumdependent vasodilation in the peripheral and coronary arteries of hypercholesterolemic patients (8 11), increases myocardial perfusion as assessed by positron emission tomography (PET) (12) and single-photon emission computed tomography (SPECT) (13) and decreases the number of ischemic episodes detected on 48-h Holter monitoring (14). Peripheral vasodilatory function can also be improved in normolipidemic patients treated with hydroxymethylglu-

2 JACC Vol. 35, No. 1, 2000 January 2000:76 82 Mostaza et al. Improve Myocardial Perfusion With Pravastatin 77 Abbreviations and Acronyms ACE angiotensin-converting enzyme ANOVA analysis of variance CAD coronary artery disease HDL high density lipoprotein HMG-CoA hydroxymethylglutaryl coenzyme A LDL low density lipoprotein PET positron emission tomography SPECT single-photon emission computed tomography taryl coenzyme A (HMG-CoA) reductase inhibitors (15). This finding could explain the rapid reduction in cardiovascular morbidity associated with pravastatin treatment in patients with CAD and average cholesterol levels (16). It is not known whether lipid reduction in patients with normal or moderately elevated cholesterol levels could improve myocardial perfusion. The aim of this study was to determine whether pravastatin treatment reduces the magnitude of the myocardial perfusion defect in patients with CAD and average cholesterol levels, as assessed by dipyridamole stress thallium-201 SPECT. METHODS Patients. We studied 20 patients with a diagnosis of CAD (history of documented myocardial infarction or stable angina pectoris with 50% diameter reduction of at least one coronary artery on angiography), fasting low density lipoprotein (LDL) cholesterol 160 mg/dl at the time of screening and reversible perfusion defects on dipyridamole stress thallium-201 SPECT performed during the preceding six months. Patients were excluded if they had taken lipid-lowering medications during the previous two months or antioxidant supplements in the preceding three months. Patients who had percutaneous transluminal coronary angioplasty or coronary artery bypass graft surgery and those with unstable angina or acute myocardial infarction during the previous three months were also excluded. None of the patients had diabetes mellitus or any other significant disease of the gastrointestinal, renal or endocrine systems. Study protocol. The study was approved by the Ethics and Clinical Trials Committee of the Instituto de Salud Carlos III, and all patients gave written, informed consent. We compared the effects of pravastatin versus placebo using a randomized, single-blinded, placebo-controlled, cross-over design. The study period was 32 weeks. All patients in the study had LDL cholesterol levels 160 mg/dl after four weeks on a 30% fat diet. Patients were randomized to receive 20 mg of pravastatin in a single nocturnal dose or placebo for 16 weeks. After completion of this period, dipyridamole stress SPECT was performed, and patients were switched to the opposite medication for another 16 weeks. Another SPECT study was carried out at the end of the second treatment phase. Fasting blood samples were taken from all patients at the end of both treatment phases for the determination of total cholesterol, LDL cholesterol, high density lipoprotein (HDL) cholesterol and triglyceride levels. SPECT study. Patients were asked to discontinue antiplatelet drugs 10 days before the study, angiotensinconverting enzyme (ACE) inhibitors three days and betablockers, calcium antagonists and nitrates 36 h before the examination. Caffeine intake was not allowed during the 24-h period preceding testing. After a 12-h fast, 0.8 mg/kg body weight of dipyridamole was injected over 6 min. Thallium-201 (2.5 mci, 92.5 MBq) was given by bolus injection immediately after dipyridamole infusion (17). Blood pressure and a 12-lead electrocardiogram were monitored and recorded before and after dipyridamole administration. Image acquisition was started 2 min after the radioisotope injection. The rest study was obtained 4 h later. Another dose of 1.5 mci (55.5 MBq) of thallium-201 was injected, and reinjection images were obtained 20 min later (18). Thallium-201 SPECT was performed using a singlehead, rotating gamma camera (7500 Orbiter, Siemens) equipped with a high resolution, parallel-hole collimator. Thirty-two projections of 20 s each were obtained over a 180 arc in a circular orbit, from the 45 right anterior oblique to the 45 posterior oblique projection. Images were reconstructed using a filtered back-projection algorithm, with a Butterworth filter (cutoff frequency 0.4 cycles/pixel). Visual assessment was performed by two independent observers (M.V.G. and F.G.) who had no knowledge of clinical data or the patients identity. Two representative short-axis slices, one from the basal region and another from the apical region, were each divided into four segments (anterior, lateral, inferior and septal). Each of the eight segments was scored from 0 to 3 (0 normal perfusion; 1 slightly diminished; 2 moderately diminished; 3 severely diminished or absence of captation). A summed stress score and a summed rest score were obtained by adding the scores of the eight segments of each patient on stress and rest-reinjection, respectively. Quantitative analysis was performed according to the Cedars-Sinai method (19). This method is highly reproducible and can be used to interpret temporal changes in myocardial perfusion (20). Three vascular territories were considered for analysis according to a polar map display of a commercially available program. The data obtained were compared with those from a normal data bank. The results were expressed as a percentage of perfusion defect in the left ventricle and in each vascular territory. A territory was considered ischemic if the magnitude of the perfusion defect exceeded 20%. A global percentage defect was calculated per patient as the average of the defects in each territory. Lipid analytical techniques. Serum concentrations of cholesterol and triglycerides were measured by enzymatic meth-

3 78 Mostaza et al. JACC Vol. 35, No. 1, 2000 Improve Myocardial Perfusion With Pravastatin January 2000:76 82 Table 1. Clinical Characteristics of Study Patients (n 18) Age (yrs) (range 48 68) Gender (M/F) 16/2 Hypertension 6 (33%) Smokers 2 (11%) Ex-smokers 10 (56%) Nonsmokers 6 (33%) Previous MI 13 (72%) Treatments Antiplatelet drugs 18 (100%) Beta-blockers 7 (39%) ACE inhibitors 1 (5.5%) Calcium antagonists 2 (11%) Nitrates 8 (44%) Estrogens 1 (5.5%) Data are presented as the mean value SD or number (%) of patients. ACE angiotensin-converting enzyme; F female; M male; MI myocardial infarction. ods. High density lipoprotein cholesterol was determined after precipitation of serum with phosphotungstic acid (21). Low density lipoprotein cholesterol was calculated using the Friedewald formula. Statistical analysis. Treatment comparisons and period effects were evaluated by two-way repeated measures analysis of variance (ANOVA), taking into account the statistical dependence of paired observations in the cross-over design. To evaluate the presence of carryover effect, we tested the statistical significance of treatment by period interactions (22). Data are expressed as the mean value SD. A two-tailed p value 0.05 was accepted as statistically significant. RESULTS Clinical characteristics. Of the 20 patients (18 men and 2 women) enrolled in the study, two were excluded from analysis. One patient refused to undergo a second SPECT study, and the other developed unstable symptoms. The clinical characteristics of the 18 patients who completed the study are presented in Table 1. Thirteen had a previous myocardial infarction. Angiographic confirmation of coronary arteriosclerosis was obtained in 12 patients. One patient was receiving treatment with ACE inhibitors, 2 with calcium antagonists, 7 with beta-blockers, 8 with nitrates and 18 with antiplatelet drugs. There were no changes in the type or doses of the medications during the study. Lipid and lipoprotein levels during placebo and pravastatin treatment are presented in Table 2. During the placebo phase, serum total cholesterol ranged from 153 to 254 mg/dl and LDL cholesterol from 112 to 169 mg/dl. Body weight remained stable throughout the study. Except for a lower systolic blood pressure after dipyridamole injection during pravastatin treatment, there were no differences in blood pressure or heart rate before and after dipyridamole during both phases of the study (data not shown). Visual assessment. Pravastatin treatment was associated with a reduction in the magnitude of the perfusion defects. Summed scores were lower with pravastatin than with placebo both during dipyridamole stress ( vs , p by ANOVA) and at rest ( vs , p by ANOVA) (Table 3, Fig. 1). Of the 144 segments evaluated (eight segments per each of the 18 patients), scores improved in 37 (26%), worsened in 16 (11%) and remained unchanged in 91 (63%) when pravastatin was compared with placebo. Figure 2 illustrates the findings in a representative patient. Quantitative evaluation. During dipyridamole stress, the magnitude of the perfusion defect was smaller with pravastatin ( %) than with placebo ( %) (p by ANOVA) (Table 3). No differences were observed between the two treatments groups (pravastatin vs. placebo) in the rest study (15 10% vs %, p 0.24 by ANOVA). Of the 54 territories evaluated (three territories per each of the 18 patients), 37 (69%) improved and 17 (31%) worsened in perfusion. Considering significant variations as those above or below 1 SD, 18 (33.3%) of 54 improved and four (7.4%) of 54 worsened in perfusion with pravastatin. Thirty of the 54 vascular territories had a stress perfusion defect 20% on placebo and were considered ischemic. Nine patients had one, 6 patients had two, and 3 patients had all 3 vascular territories that were ischemic. Perfusion improvement was restricted to these ischemic territories (magnitude of the perfusion defect % vs % [placebo vs. pravastatin], p by ANOVA). Pravastatin administration was not associated with improved perfusion in territories that had normal Table 2. Lipid Levels During Placebo and Pravastatin Treatment Placebo Pravastatin Change* Treatment p Value by ANOVA Period Carryover Total cholesterol HDL cholesterol LDL cholesterol *Mean change SEM. Other data are presented as mean value (mg/dl) SD. ANOVA analysis of variance; HDL high density lipoprotein; LDL low density lipoprotein.

4 JACC Vol. 35, No. 1, 2000 January 2000:76 82 Mostaza et al. Improve Myocardial Perfusion With Pravastatin 79 Table 3. Scintigraphic Values During Dipyridamole Stress and at Rest During the Placebo and Pravastatin Phases Placebo Pravastatin Change* Treatment p Value by ANOVA Period Carryover Visual assessment Summed stress score Summed redistribution score Quantitative assessment Dipyridamole stress Redistribution *Mean change SEM. Other data are presented as the mean value SD. ANOVA analysis of variance. perfusion during the placebo phase (magnitude of the perfusion defect 9.6 6% vs. 10 8% [placebo vs. pravastatin], p 0.86 by ANOVA). Figure 3 illustrates a polar map display of the same patient represented in Figure 2. The improved myocardial perfusion observed with pravastatin was not related to age, gender, percentage of total (or LDL) cholesterol reduction, basal or on-treatment total (or LDL) cholesterol concentrations or previous myocardial infarction (data not shown). Period and carryover effects were not significant for any of the variables analyzed (Table 3). DISCUSSION The results of this study show that myocardial perfusion defects improved after 16 weeks of pravastatin administration in patients with CAD and average cholesterol levels. The improved myocardial perfusion was mainly observed during the dipyridamole stress test. As suggested by previous reports, the improvement after a short period of treatment suggests that the benefit is related to functional rather than anatomic changes (e.g., improved endothelial function). The study does not indicate, however, whether the beneficial action of the drug is related to its hypolipidemic effects or to its direct action on the endothelial cell. Endothelial dysfunction, hypercholesterolemia and cholesterol reduction. Endothelial dysfunction is present in subjects with risk factors for CAD even before structural changes can be shown in their vessels (1 3). Hypercholesterolemic patients have blunted forearm blood flow responses to nitroprusside and metacholine (5), abnormal coronary responses to intracoronary infusion of acetylcholine (23) and reduced coronary flow reserve (24). Lipidlowering therapy can potentially restore coronary and peripheral endothelium-dependent vasodilation (8 11) and can increase myocardial perfusion as assessed by PET and Figure 1. Visual scoring of SPECT images: summed stress score and summed rest score for each of the 18 patients in the study during placebo and pravastatin treatment. Figure 2. Short-axis slices from one representative patient during dipyridamole stress testing. There is a severe defect in the anterior and septal segments with placebo (arrows, upper panels) that partially reverses with pravastatin treatment (arrows, lower panels).

5 80 Mostaza et al. JACC Vol. 35, No. 1, 2000 Improve Myocardial Perfusion With Pravastatin January 2000:76 82 SPECT (12,13). As previously discussed (12), dipyridamole increases myocardial perfusion by a direct vasodilatory effect. This action can precipitate a further increase in perfusion by a flow-mediated, endothelium-dependent arteriolar vasodilation (25). Increased blood flow primarily reflects changes in microvascular vasomotion. Hypercholesterolemia affects endothelial function in the microcirculation (26). Enhanced coronary flow after lipid-lowering therapy suggests that small resistance vessels are involved in the favorable response (10). Endothelial dysfunction in patients with average cholesterol levels. An important finding of the study is that patients with cholesterol levels in the normal to moderately elevated range can improve their myocardial perfusion with HMG-CoA reductase treatment. Cholesterol levels in the high to normal range have been associated with abnormal endothelial function in healthy subjects (27). Cholesterol reduction can improve peripheral vasomotion in this group (15). Our results support the beneficial effects of treatment with HMG-CoA reductase inhibitors in these subjects and demonstrate that atherosclerotic coronary vessels can also improve their functional properties with treatment. Enhancement of myocardial perfusion was restricted to ischemic segments, a finding reported earlier (13). Previous studies have demonstrated a progressive impairment of endothelial function in relation to different stages of atherosclerosis (4) and to the extent of local wall thickening evaluated by intracoronary ultrasound examination (28). Although the study was not powered to demonstrate changes in myocardial perfusion at rest, both qualitative and quantitative evaluations showed a tendency to improve at-rest perfusion (significant only with the former). These findings are not surprising as it has been demonstrated that endothelium-derived nitric oxide is important in the regulation of basal vascular tone (29,30). High cholesterol levels, in particular, oxidatively modified LDL, can decrease the synthesis and increase the degradation of nitric oxide (31,32). The finding that a short-term reduction of previously considered normal cholesterol levels improves myocardial perfusion suggests that even average cholesterol concentrations negatively influence the vasodilatory capacity of coronary vessels. Figure 3. Polar map display and quantitative analysis of the SPECT study obtained in the same patient represented in Figure 2. The images were obtained during dipyridamole stress and at rest, after placebo (upper panel) and pravastatin treatment (lower panel). The percentage of perfusion defect is represented for each of three vascular territories (LAD left anterior descending; RCA right coronary artery; LCX left circumflex) and for the total left ventricle. Mechanisms of improved endothelial function by HMG- CoA reductase inhibitors. The improvement in myocardial perfusion observed during HMG-CoA reductase treatment probably results from the lipid-lowering action of the drug. This is supported by the fact that other approaches, including parenteral fat-free nutrition (12), plasmapheresis (33) and cholestyramine treatment (8), can also improve the vasodilatory capacity of peripheral and coronary arteries. However, the beneficial effects observed with HMG-CoA reductase treatment could also be explained by a direct action of these drugs on the endothelium, independent of their action on blood lipids. Supporting this view is the fact that we could not find a relationship between the magnitude of cholesterol lowering and the improvement in the perfusion defects, a finding also observed by other investigators (34). Endothelial dysfunction is associated with impaired nitric oxide bioavailability (31) and increased release or activity of endothelin-1 (35); both abnormalities have been previously described in hypercholesterolemia (36). Statins can directly increase enos (endothelin nitric oxide synthase) expression and nitric oxide production and decrease endothelin expression in vascular endothelial cells (37,38). These actions could have a favorable, direct influence on endothelial function. Noninvasive evaluation of endothelial dysfunction. Most studies that have assessed endothelial coronary function in humans have been invasive. Noninvasive tests that explore the vasodilatory capacity of coronary arteries are important in clinical investigation. Positron emission tomographic studies are expensive and unavailable to many centers. Thallium-201 SPECT allows the assessment of myocardial perfusion abnormalities and is highly sensitive to detect changes in myocardial perfusion associated with cholesterol reduction.

6 JACC Vol. 35, No. 1, 2000 January 2000:76 82 Mostaza et al. Improve Myocardial Perfusion With Pravastatin 81 Study limitations. The improvement of perfusion defects during pravastatin, although significant, was modest. However, our data confirm previously reported findings that have demonstrated that HMG-CoA reductase treatment decreases the number of ischemic episodes, as assessed by 48-h Holter monitoring (14,39), and increases the tolerance to an exercise test (40). Conclusions. Administration of pravastatin in patients with CAD and average cholesterol levels improves myocardial perfusion during dipyridamole stress SPECT. This favorable action could partially explain the rapid reduction in cardiovascular morbidity associated with pravastatin treatment in patients with CAD and average cholesterol levels. Acknowledgments We appreciate the excellent technical assistance of Sandra Cañizares and Emilia Rebollo and the statistical assistance of Javier Jimenez and Eliseo Guayar. 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