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J Am Coll Cardiol, 2002; 40:546-554 © 2002 by the American College of Cardiology Foundation |
* Division of Cardiovascular DiseasesRochester, Minnesota, USA
Division of Hypertension, Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, USA
Manuscript received February 27, 2002; revised manuscript received April 18, 2002, accepted May 7, 2002.
* Reprint requests and correspondence: Dr. Amir Lerman, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.
lerman.amir{at}mayo.edu
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Abstract |
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BACKGROUND: Simvastatin preserves coronary endothelial function in experimental hypercholesterolemia independent of its lipid-lowering effect. However, the functional significance of this observation is unknown.
METHODS: Pigs were randomized to three groups: normal diet (N), high-cholesterol diet (HC) and HC diet plus simvastatin (HC+S) for 12 weeks. Subsequently, cardiac electron beam computed tomography was performed before and during intravenous infusion of adenosine and dobutamine, and MP and PI were calculated.
RESULTS: Total and low density lipoprotein cholesterol levels were similarly and significantly increased in HC and HC+S animals compared with N. Basal MP was similar in all groups. Myocardial perfusion significantly increased in response to either adenosine or dobutamine in N and HC+S animals. Dobutamine also significantly increased MP in HC animals. However, the changes of MP in response to either drug were significantly lower in the HC group compared with the other two groups (p < 0.01 for adenosine and p < 0.05 for dobutamine vs. N and HC+S). Basal PI was similar in all groups and was not altered by either drug in N and HC+S animals. In contrast, PI significantly increased in HC pigs during infusion of either adenosine (p < 0.001) or dobutamine (p < 0.05).
CONCLUSIONS: These findings demonstrate that chronic administration of simvastatin preserves myocardial perfusion response and coronary microvascular integrity during cardiac stress in experimental hypercholesterolemia independent of lipid lowering.
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Hypercholesterolemia is associated with the development of coronary endothelial dysfunction (8), which is characterized by impaired endothelium-dependent vasorelaxation (9) and an increase in microvascular permeability (10). We recently demonstrated that changes in coronary vascular function associated with hypercholesterolemia in a porcine model may also lead to an attenuated increase in myocardial perfusion (MP) and an exaggerated augmentation of coronary microvascular permeability in response to an intravenous infusion of adenosine (11). In humans with coronary artery disease, altered endothelial function contributes to ischemic manifestations and also unfavorably modulates the course of acute coronary syndromes (12). Moreover, endothelial dysfunction of the coronary resistance vessels is associated with myocardial ischemia (13) and an adverse prognosis (14), even in the absence of obstructive coronary artery disease. Cholesterol lowering with statins improves endothelial function in hypercholesterolemia (15), and is also associated with an improvement of MP (16) in patients with coronary artery disease. Recently, we demonstrated that preservation of endothelial function of epicardial coronary arteries and coronary arterioles in experimental hypercholesterolemia is one of the statin effects mediated at least partly by lipid-independent mechanisms (17). However, the functional significance of this lipid-independent effect of statins on MP and coronary microvascular permeability remains unclear.
The current study was designed to test the hypothesis that chronic administration of simvastatin would preserve MP response and coronary microvascular integrity during situations of increased myocardial demand in an animal model of porcine experimental hypercholesterolemia in vivo independent of lipid lowering. For this purpose, we used electron beam computed tomography (EBCT), an ultrafast computed tomography technique that has been shown to enable accurate quantification of global and regional MP and coronary microvascular permeability in animals as well as in humans (11,18–22).
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EBCT studies
Electron beam computed tomography studies were performed as previously described (11,18–24). In brief, animals were anesthetized with ketamine (30 mg/kg) and xylazine (5 mg/kg). Anesthesia was maintained with a constant infusion of ketamine (0.3 mg/kg/min) and xylazine (0.04 mg/kg/min) in saline. Animals were intubated and ventilated with room air. The carotid artery and the jugular vein were exposed by cutdown and cannulated with 8F and 7F sheaths, respectively. After injection of 10,000 U of heparin intravenously and initiation of a continuous intravenous infusion of 1,000 U heparin per hour, a guiding catheter was placed in the descending aorta for online blood pressure measurement and a pigtail catheter was placed in the right atrium for injection of contrast media.
Subsequently, animals were positioned supine in the EBCT (Imatron C-150, Imatron Inc., South San Francisco, California) gantry and an intravenous infusion of saline (5 ml/min) was initiated. With localization scans, cross-sectional images at two adjacent mid-left ventricle levels were identified. After a right atrial bolus injection (0.3 ml/kg over 2 s) of nonionic, low-osmolar contrast media iopamidol (Isovue-370, Squibb Diagnostics, Princeton, New Jersey), 40 consecutive electrocardiogram (ECG)-triggered end-diastolic scans were obtained over the preselected levels at one to three heartbeat intervals. Ten minutes after this baseline study an intravenous infusion of adenosine (400 µg/kg/min) was started and after hemodynamic stabilization EBCT flow studies were repeated. Subsequently, adenosine infusion was discontinued and ECG-triggered cine CT images of the heart were acquired during central venous injection of contrast agent at rest for the determination of left ventricular volumes and left ventricular muscle mass (LVMM). Finally, an intravenous infusion of dobutamine (5 to 25 µg/kg/min, to achieve a heart rate of 
150 beats/min) was started and EBCT flow studies were repeated after hemodynamic stabilization.
EBCT data analysis
The methods have been previously described in detail (11,18–25). In brief, EBCT images were transferred to a Sun workstation and regions of interest were traced in the anterior wall and chamber of the left ventricle using the image analysis software package Analyze (Biomedical Imaging Resource, Mayo Foundation, Rochester, Minnesota), and time-density curves were generated for both regions. The left ventricular curve was fitted with a standard gamma-variate curve-fit, whereas the curve obtained from the anterior wall was fitted using a custom-designed extended gamma-variate curve-fitting algorithm implemented in commercially available computer software (KaleidaGraph, Synergy Software, Reading, Pennsylvania) to obtain curves representing intramyocardial distribution of contrast media in both the intra- and extravascular compartments (11,25). The areas enclosed under the intravascular myocardial and left ventricular cavity curves were subsequently used to calculate an index of intramyocardial mean transit time(s) and vascular blood volume (ml/cc tissue). Myocardial perfusion (ml/g/min) was then computed as: 60 x (blood volume/mean transit time)/[1.05 x (1 – blood volume)]. The factor (1 – blood volume) served to correct for dynamic changes in blood volume occurring in the heart in vivo (19–21,25). Coronary microvascular permeability index (PI) (arbitrary units) was calculated as contrast extraction rate/blood volume, where contrast extraction rate (analogous to permeability-surface area product) was derived from the curve depicting the extravascular compartment of the contrast agent as 60 x 1.05 x (maximal slope of ascending part of extravascular curve x mean transit time)/area under left ventricular curve (11).
By using commercially available software (Imatron Inc., South San Francisco, California), left ventricular end-diastolic and end-systolic frames were visually identified from each cine-mode movie that spanned the cardiac cycle at the level. Left ventricular volumes were measured by planimetry from the left ventricular apex to the left ventricular base and the tomographic measurements of end-diastolic and end-systolic volume at each level were added by use of a modified Simpson’s formula to determine left ventricular end-diastolic (LVEDV) and end-systolic (LVESV) volumes. Stroke volume was then calculated as LVEDV – LVESV, cardiac output as stroke volume x heart rate, and left ventricular ejection fraction (LVEF) as stroke volume/LVEDV (19,24). In addition, left ventricular myocardial area on each end-diastolic frame was measured by planimetry after tracing the outer border of the left ventricular myocardium, and the mass of each short-axis slice was calculated as the product of the myocardial area, scan slice thickness (0.8 cm) and specific gravity of the myocardium (1.05 g/cm3). Left ventricular muscle mass was then computed as the sum of the masses of the individual scanned sections according to a modified Simpson’s formula (19,23).
Statistical analysis
Results are expressed as mean ± SEM. Comparisons within groups were performed using paired Student t test. One-way analysis of variance followed by the Bonferroni t procedure, if indicated, was used for comparisons among the three different groups. All analyses were done by using SigmaStat statistical software, version 2.03 (SPSS Inc., Chicago, Illinois). Statistical significance was accepted for a value of p < 0.05.
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Discussion |
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Assessment of pleiotropic effects of statins in vivo.. Statins have been shown to significantly reduce morbidity and mortality from coronary events in patients with a wide range of cholesterol levels (1–5). It is now clear that the clinical benefit of statins cannot be attributed to cholesterol lowering alone and that these drugs exert pleiotropic effects that clearly are independent of their impact on cholesterol levels (7). However, despite growing knowledge about numerous lipid-independent properties of this class of drugs in vitro, little is known about their consequences in vivo. One of the problems in identifying lipid-independent effects of statins in vivo is differentiating them from the indirect effects related to lipid lowering. Another confounding factor is that a direct effect of a statin may complement an indirect effect (26). In accordance with one of our previous studies (17), simvastatin did not alter plasma cholesterol levels in our animal model of dietary hypercholesterolemia, which may be attributable to the pure dietary nature of hypercholesterolemia in our model and the fact that lipid-lowering potency and efficacy of statins are much lower in pigs than in humans (27). Hence, our animal model provides a unique opportunity to study lipid-independent effects of statins in vivo.
Effect of simvastatin on MP
Our animal model of hypercholesterolemia has been well characterized and shows endothelial dysfunction of epicardial coronary arteries and coronary resistance vessels (17), without development of atherosclerotic plaques (28). Hence, the observed difference of increase in MP during simulated cardiac stress between untreated hypercholesterolemic pigs and those treated with simvastatin cannot be attributed to the presence of coronary stenoses or an inhibitory effect of simvastatin on the development of such lesions.
Several mechanisms may be responsible for the observed differences of the MP response to simulated cardiac stress between the experimental groups. The hemodynamic effects of dobutamine result from a unique and complex interaction of its alpha- and betamimetic properties (29). However, although dobutamine may have a minor direct vasodilative effect on coronary vessels, the increase in myocardial blood flow in response to this compound is due mainly to enhanced myocardial metabolic demand, which leads to dilation and/or recruitment of coronary resistance vessels, including arterioles that are 
100 µm in diameter. Importantly, endothelium-derived NO has been shown to play a pivotal role for the dilative response of the coronary microvasculature associated with the infusion of dobutamine (30). Thus, the increase in MP in response to dobutamine depends to a certain degree on endothelial function. On the other hand, adenosine is primarily considered an endothelium-independent vasodilator. However, its vasodilatory effect is mediated via specific A2-receptors that are located not only on vascular smooth muscle cells but also on endothelial cells (31,32). Moreover, several studies demonstrated that the vasorelaxant effect exerted by adenosine involves the release of NO (33,34), and is, therefore, also somewhat dependent on endothelial function. Thus, changes in endothelial function may play a role in the vasodilatory response to both dobutamine and adenosine. Furthermore, we recently demonstrated that treatment with simvastatin preserves endothelial function in epicardial arteries and resistance vessels in vitro in a lipid-independent manner (17). Hence, although direct effects of dobutamine and adenosine might have contributed to our results, it is likely that the lack of increase in MP during infusion of either drug observed in hypercholesterolemic pigs might result from coronary endothelial dysfunction, and that the normalization of MP in animals treated with simvastatin is the result of preservation of endothelial function. In this context, our results are in accordance with a previous clinical study reporting a reduction of episodes of transient myocardial ischemia with pravastatin in patients with coronary artery disease and normal to moderately elevated serum cholesterol (35). These investigators speculated that the anti-ischemic effect of pravastatin in their study was mainly mediated by a normalization of coronary endothelial function, because an angiographic study in the same patient population demonstrated only less progression but not regression of preexisting coronary atherosclerosis with pravastatin therapy (36). Our current study extends these previous observations and demonstrates that the beneficial effects of simvastatin upon MP are not only independent of lipid lowering, but also occur very early in the course of atherogenesis, even before obvious morphologic alterations of the vessel wall are present (28).
Effect of simvastatin on coronary microvascular permeability
The demonstrated changes of coronary microvascular permeability among the different experimental groups further support the hypothesis that simvastatin exerts its benefits upon MP primarily by its endothelial-protective actions. Increased microvascular permeability is a characteristic feature of endothelial dysfunction and is considered a major pathogenetic mechanism in atherogenesis (10). In accordance with our previous findings (11), we found that coronary microvascular permeability, though normal under basal conditions, significantly increased during simulated cardiac stress in hypercholesterolemic pigs. On the other hand, this abnormal alteration of vascular permeability was prevented in animals fed a high-cholesterol diet and simultaneously treated with simvastatin. Again, this effect of simvastatin was independent of any change in plasma lipid levels. These results are in line with a recently published study demonstrating a normalization of increased microvascular permeability with simvastatin therapy in hypercholesterolemic men (37). Although the relationship between endothelial dysfunction and functionally increased vascular permeability is established, other factors that might influence vessel wall permeability must also be considered. In an earlier study we have demonstrated that experimental hypercholesterolemia is associated with ultrastructural changes of the internal elastic lamina in epicardial coronary arteries (38). Thus, these structural alterations of the vessel wall might contribute to changes in vascular permeability, and the underlying mechanism for the effect of statins on microvascular permeability might also be secondary to the modification of compartments of the vessel wall other than the endothelium.
Suggested mechanism
We recently demonstrated that adverse changes of MP and coronary microvascular permeability in hypercholesterolemia such as those observed in the present study might be mediated by an increase in oxidative stress (11). Moreover, we have shown that simvastatin may reduce oxidative stress (17). Therefore, it may be speculated that the beneficial effects exerted by simvastatin are at least partly mediated by its antioxidant effects.
Pharmacologic stress tests in pigs
The magnitude of the MP response to intravenous dobutamine in normal animals was similar to that observed in healthy humans and pigs (39,40). On the other hand, although clearly significant, the increase in MP in response to intravenous adenosine in normal animals was less than that observed in previous studies in humans using intracoronary Doppler flow measurements (41). Several EBCT studies employing venous injections of contrast media reported underestimation of absolute MP at high flow rates induced by coronary vasodilators such as adenosine (22). Reasons postulated for these observations included failure to correct for intramyocardial vascular volume, which increases in relation to myocardial blood flow, and the use of intravenous (rather than intraaortic) injections of contrast, which results in spread of the bolus (22). In the present study, changes in intramyocardial vascular volume during adenosine infusion were taken into account for calculation of MP, and thus a major confounding effect by this variable is unlikely. However, we cannot exclude the possibility that the duration of the arterial input curve (left ventricular cavity curve) exceeded coronary transit time in the high flow state induced by adenosine and led to some underestimation of absolute MP. This may account for the moderate, though significant, increase in MP in response to adenosine in normal animals. Interestingly, however, intravenous administration of contrast did not substantially decrease MP reserve to intravenous adenosine in humans when EBCT methodology similar to the current study and a correction for changes in intramyocardial vascular volume were used (22). Hence, and because the EBCT-derived MP reserve to dobutamine was not attenuated, species variability in the response to intravenous adenosine might have contributed to the relatively moderate MP reserve observed in normal animals.
Conclusions
In summary, this study demonstrates that simvastatin preserves MP response and coronary microvascular integrity during situations of cardiac stress in experimental hypercholesterolemia independent of lipid lowering. These results contribute to the growing evidence that statins exert their beneficial effects to a considerable part in a lipid-independent manner.
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