Atherosclerosis 168 (2003) 367 /373 www.elsevier.com/locate/atherosclerosis
Endothelin type A receptor antagonism restores myocardial perfusion response to adenosine in experimental hypercholesterolemia Piero O. Bonetti a, Patricia J.M. Best a, Martin Rodriguez-Porcel a, David R. Holmes, Jr a, Lilach O. Lerman b, Amir Lerman a,* a
Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA b Division of Hypertension, Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, MN, USA Received 20 August 2002; received in revised form 6 March 2003; accepted 13 March 2003
Abstract Experimental hypercholesterolemia is characterized by increased endothelin-1 (ET-1) activity and is associated with an attenuated myocardial perfusion response and an inappropriate increase in coronary microvascular permeability during episodes of increased myocardial demand. This study was designed to determine the effect of chronic selective ET type A (ETA) receptor antagonism on coronary vascular response to simulated cardiac stress in experimental hypercholesterolemia. Twenty-one pigs were randomized to three groups: normal diet (N), high-cholesterol diet (HC), and HC diet plus ABT-627, a selective ETA receptor antagonist, (HC/ ABT-627). After 12 weeks, cardiac electron beam computed tomography (EBCT) was performed before and during intravenous infusion of adenosine, and myocardial perfusion (ml/min per g) and coronary microvascular permeability index (arbitrary units) were calculated. Basal myocardial perfusion was similar in all groups (N: 0.919/0.10; HC: 0.959/0.08; HC/ABT-627: 1.039/0.09; P/0.64). Adenosine infusion led to a significant increase in myocardial perfusion in the N (1.329/0.15; P B/0.001) but not in the HC (0.959/0.07) group. However, in the HC/ABT-627 group, adenosine also significantly increased myocardial perfusion (1.339/ 0.12; P/0.001). Basal permeability index did not differ between the groups (N: 1.569/0.13; HC: 1.349/0.19; HC/ABT-627: 1.629/ 0.10; P/0.38). Adenosine infusion significantly increased permeability index in HC pigs (2.299/0.22; P B/0.001) but not in N (1.719/0.21) and HC/ABT-627 (1.829/0.08) pigs. We conclude that chronic selective ETA receptor antagonism preserves myocardial perfusion response and coronary microvascular integrity during episodes of increased myocardial demand in experimental hypercholesterolemia, indicating an important role for the endogenous endothelin system in this disorder. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelin; Endothelin receptor antagonism; Hypercholesterolemia; Myocardial perfusion; Microvascular permeability
1. Introduction Endothelin-1 (ET-1), a 21-amino acid peptide that is produced by various cells including endothelial cells and vascular smooth-muscle cells (VSMCs), is a very potent vasoconstrictor [1]. In addition, ET-1 also exerts proliferative and proinflammatory effects, and its overexpression is involved in the pathogenesis of various cardiovascular disease states [2]. ET-1 exerts its biological effects via activation of two specific membrane
* Corresponding author. Tel.: /1-507-255-4152; fax: /1-507-2552550. E-mail address:
[email protected] (A. Lerman).
bound receptors. The ET type A (ETA) and ET type B (ETB) receptors on VSMCs mediate vasoconstriction. Furthermore, activation of ETA receptors on VSMCs also induces cell proliferation, whereas stimulation of endothelial ETB receptors mediates release of vasorelaxing factors, such as nitric oxide (NO) and prostacyclin [2]. Experimental hypercholesterolemia is characterized by an increase in circulating and tissue ET-1 immunoreactivity [3]. Moreover, experimental hypercholesterolemia is associated with a blunted enhancement of myocardial perfusion and an exaggerated increase in coronary microvascular permeability during episodes of cardiac stress, such as simulated by intravenous infusion of adenosine [4]. However, little is known about the role
0021-9150/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00141-2
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of the endogenous endothelin system in the pathogenesis of this disordered coronary vascular response. This study was designed to investigate the effect of chronic treatment with the selective ETA receptor antagonist ABT-627 on myocardial perfusion and coronary microvascular permeability at baseline and during intravenous infusion of adenosine in experimental hypercholesterolemia in vivo. For this purpose we used electron beam computed tomography (EBCT), a well established method to quantify myocardial perfusion and coronary microvascular permeability [4 /9].
2. Methods 2.1. Animals The Mayo Foundation Institutional Animal Care and Use Committee reviewed and approved all the study procedures, which were designed in accordance with the National Institutes of Health Guidelines. Twenty-one domestic crossbred pigs, 3 months of age, were randomized into three groups and subsequently treated for 12 weeks: Group 1 (normal group; n/7) received a normal diet. Group 2 (HC group; n /7) and group 3 (HC/ ABT-627 group; n/7) were fed an atherogenic diet of 2% cholesterol and 15% lard by weight (TD 93296, Harlan Teklad, Madison, WI). In addition to the highcholesterol diet, animals of group 3 were given ABT-627 (Abbott Laboratories, Abbott Park, IL), an orally active, non-peptide, selective ETA antagonist on a weight-adjusted scale to maintain a dose of 4 mg/kg per day. This dosage was based on our previous studies in pigs, demonstrating effective blockade of ET-1 and no undesirable side effects [10,11]. After 12 weeks, in vivo studies were performed and plasma lipid profiles (Roche, Nutley, NJ) were determined. 2.2. EBCT studies EBCT studies were performed as previously described [4 /9]. In brief, animals were anesthetized with an intramuscular bolus of ketamine (30 mg/kg) and xylazine (5 mg/kg), and anesthesia was maintained with a constant infusion of ketamine (0.3 mg/kg per min) and xylazine (0.04 mg/kg per min) in saline. After intubation, animals were ventilated with room air and the carotid artery and the jugular vein were cannulated with 8F and 7F sheaths, respectively. An intravenous bolus of 10 000 units heparin was followed by the initiation of a continuous intravenous infusion of 1000 units/h. Online blood pressure monitoring was enabled by placing a guiding catheter in the descending aorta, and a pig-tail catheter was inserted in the right atrium for the subsequent injection of contrast media.
After transfer to the EBCT gantry (Imatron C-150, Imatron Inc., South San Francisco, CA), localization scans were performed to identify cross-sectional images at two adjacent mid-left ventricular levels. Next, 40 consecutive, ECG-triggered end-diastolic scans were obtained over the preselected levels at one to three heartbeat intervals after a bolus injection (0.3 ml/kg) of non-ionic, low-osmolar contrast agent iopamidol (Isovue-370, Squibb Diagnostics, Princeton, NJ) into the right atrium. Ten minutes later an intravenous adenosine infusion (400 mg/kg per min) was initiated and EBCT flow studies were repeated after hemodynamic stabilization. After discontinuation of the adenosine infusion, an ECG-triggered cine CT of the heart was acquired during central venous injection of contrast agent at rest for the determination of left ventricular volumes and left ventricular muscle mass (LVMM). 2.3. EBCT data analysis and hemodynamic calculations Details of the methods have been described earlier [4 / 9,12]. In summary, regions of interest were traced in the anterior wall and the chamber of the left ventricle (image analysis software ANALYZETM, Biomedical Imaging Resource, Mayo Foundation, Rochester, MN), and time/density curves were generated for both regions. Next, 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 a commercially-available computer software (KALEIDAGRAPH, Synergy Software, Reading, PA) to obtain curves representing intramyocardial distribution of contrast media in both the intraand extra-vascular compartments. Mean transit time (MTT; s) and intramyocardial vascular blood volume (BV; ml/cc tissue) were calculated using the areas enclosed under the intravascular myocardial and left ventricular chamber curve. Myocardial perfusion (ml/ min per g) was calculated as: 60 /(BV/MTT)/[1.05 /(1BV)]. The factor (1-BV) served as a correction factor for dynamic changes in BV occurring in the heart in vivo. Coronary microvascular permeability index (arbitrary units) was computed as: contrast extraction rate/BV, whereas contrast extraction rate was derived from the extravascular curve as: 60 /1.05 /(slope of extravascular curve /MTT)/area under left ventricular cavity curve. By using commercially-available software (Imatron Inc., South San Francisco, CA), left ventricular enddiastolic and end-systolic frames were visually identified from each cine mode movie that spanned the cardiac cycle at the level. Left ventricular volumes were planimetered 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
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added by use of a modified Simpson’s formula to determine left ventricular end-diastolic (LVEDV) and end-systolic (LVESV) volumes. Stroke volume (SV) was then calculated as LVEDV/LVESV, cardiac output (CO) as SV /heart rate (HR), and left ventricular ejection fraction (LVEF) as (SV/LVEDV)/100. In addition, left ventricular myocardial area on each enddiastolic frame was planimetered 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). LVMM was then computed as the sum of the masses of the individual scanned sections according to a modified Simpson’s formula. Systemic vascular resistance (SVR) was calculated as mean arterial pressure (MAP)/CO and coronary vascular resistance (CVR) as MAP/(myocardial perfusion /LVMM). CO during infusion of adenosine was calculated as previously described as: injected volume of contrast (ml) /iodine content of iopamidol (mg/ml) per area under the left ventricular cavity curve [13]. 2.4. Statistical analysis Results are presented as mean9/S.E.M. Paired Student’s t-test was used for comparisons within groups. Comparisons between different groups were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni t procedure if indicated. Spearman correlation analysis was used to investigate possible relationships between measured variables. All analyses were performed using SIGMASTAT statistical software, version 2.03 (SPSS Inc., Chicago, IL). Statistical significance was accepted for P B/0.05.
3. Results
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Table 1 Plasma lipid profiles and body weight in the three experimental groups at 12 weeks
Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) Body weight (kg)
Normal (n/7)
HC (n/7)
HC/ABT-627 (n/7)
2.19/0.1* 0.99/0.1* 1.09/0.1* 0.39/0.1 60.19/2.3
10.29/1.6 7.49/1.5 2.69/0.2 0.59/0.1 60.99/2.1
12.09/1.1 9.09/1.1 2.79/0.3 0.59/0.1 61.79/1.5
Mean9/S.E.M. * P 5/0.001 vs. HC and HC/ABT-627.
ABT-627 group compared with normal and HC pigs. Further, CVR was significantly lower in the HC/ABT627 group than in the normal group. Adenosine infusion led to a significant decrease in MAP in all groups, which was associated with an increase in HR in pigs of the normal and the HC group and an increase in CO in all groups. Moreover, adenosine led to a significant decrease in CVR compared with baseline in the normal and the HC/ABT-627 group but not in HC animals. Basal LVEF, CO, and LVMM were similar in all experimental groups (Table 2). 3.3. Myocardial perfusion Anterior wall myocardial perfusion at baseline was similar in all groups (P /0.64). Adenosine infusion resulted in a significant increase in myocardial perfusion in normal animals but not in animals of the HC group. In contrast and similar to normals, HC pigs receiving ABT-627 demonstrated a normal myocardial perfusion response to adenosine. Relative change of myocardial Table 2 Systemic hemodynamics in the three experimental groups at 12 weeks
3.1. Plasma lipid profiles and body weight After 12 weeks of high-cholesterol diet total cholesterol, low density lipoprotein (LDL) cholesterol, and high density lipoprotein (HDL) cholesterol levels were significantly and similarly increased in the HC and the HC/ABT-627 group compared with normal animals, whereas triglyceride levels did not significantly differ between the three experimental groups. At the end of the 12 weeks study period there was no difference in average body weight between the animals of the three experimental groups (Table 1).
HR (bpm) MAP (mmHg)
Baseline Adenosine Baseline Adenosine Baseline
Normal
HC
HC/ABT627
729/3 879/4% 1149/5 979/4% 27.39/1.2
689/5 799/4% 1119/6 939/8% 28.19/3.6
829/5 859/4 819/4* 729/4*% 17.89/1.8*
1.39/0.2
1.19/0.1
0.79/0.1$
0.89/0.1% 4.39/0.2 5.39/0.3% 54.09/1.2 103.49/ 2.8
0.99/0.1 4.09/0.4 5.19/0.4% 57.79/1.6 109.09/ 3.2
0.59/0.1*% 4.79/0.4 6.29/0.4% 60.49/2.5 114.99/3.3
3.2. Systemic hemodynamics
SVR (mmHg/l per min) CVR Baseline (mmHg/ml per min) Adenosine CO (l/min) Baseline Adenosine LVEF (%) Baseline LVMM (g) Baseline
Basal HR was similar in all groups but basal MAP and SVR were significantly lower in pigs of the HC/
Mean9/S.E.M. * P B/0.05 vs. normal and HC; $ P B/0.05 vs. normal; % P B/0.05 vs. baseline.
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perfusion in response to adenosine was similar in the normal group and the HC/ABT-627 group (P /0.12), and significantly differed from that in the HC group (Fig. 1).
3.4. Coronary microvascular permeability index Basal coronary microvascular permeability index was similar in all experimental groups (P /0.38). In normal animals adenosine infusion was not associated with any change in coronary microvascular permeability index. In contrast, adenosine infusion led to a significant increase in this parameter in animals of the HC group. However, in HC animals treated with ABT-627 no significant increase in coronary microvascular permeability index occurred in response to adenosine. The relative change of coronary microvascular permeability observed in HC animals significantly differed from that observed in normal and HC/ABT-627 pigs (Fig. 2). Fig. 2. Upper panel: Anterior wall coronary microvascular permeability index at baseline (white dots) and during intravenous adenosine infusion (black dots) in the three experimental groups (horizontal bars denote mean values). P B/0.001. Lower panel: Relative change (percent compared with baseline) of anterior wall coronary microvascular permeability index in response to intravenous adenosine in the three experimental groups (horizontal bars denote mean values). * P B/0.001 vs. normal and HC/ABT-627. HC, hypercholesterolemic pigs; HC/ABT-627, hypercholesterolemic pigs receiving a selective ETA receptor antagonist.
3.5. Relationship between myocardial perfusion and coronary microvascular permeability index and hemodynamic variables
Fig. 1. Upper panel: Anterior wall myocardial perfusion at baseline (white dots) and during intravenous adenosine infusion (black dots) in the three experimental groups (horizontal bars denote mean values). * P 5/0.001. Lower panel: Relative change (percent compared with baseline) of anterior wall myocardial perfusion in response to intravenous adenosine in the three experimental groups (horizontal bars denote mean values). * P B/0.001 vs. normal; $ P B/0.01 vs. HC/ ABT-627. HC, hypercholesterolemic pigs; HC/ABT-627, hypercholesterolemic pigs receiving a selective ETA receptor antagonist.
No statistically significant relationships were found between myocardial perfusion values and coronary microvascular permeability index values at rest or during adenosine infusion in any of the three groups. Also, no statistically significant correlation existed between the relative changes in myocardial perfusion and coronary microvascular permeability index in response to adenosine in the groups. In normal animals a statistically significant inverse relationship was found between myocardial perfusion and CVR at baseline (r//0.96, P /0.001). However, this correlation was attenuated and became non-significant in HC animals (r //0.57, P /0.150), whereas there was a trend towards a significant relationship between these variables in HC/ABT-627 animals (r/ /0.68, P /0.074). On the other hand, no statistically significant relationships between myocardial perfusion values or coronary microvascular permeability index
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values and other hemodynamic parameters (MAP, SVR, CO) were found.
4. Discussion ET-1, aside from being a potent endogenous vasoconstrictor peptide, is involved in the pathogenesis of various forms of cardiovascular disease, such as endothelial dysfunction, atherosclerosis, and heart failure [2,14,15]. Experimental porcine hypercholesterolemia is associated with increased plasma and tissue ET-1 activity [3] and is further characterized by an attenuation of the myocardial perfusion response combined with an exaggerated increase in coronary microvascular permeability during cardiac stress [4]. However, the exact role of ET-1 for this abnormal coronary vascular response to situations of increased myocardial demand is unclear. The current study demonstrates that chronic administration of the selective ETA receptor antagonist ABT-627 prevents the attenuation of the myocardial perfusion response and the increase in coronary microvascular permeability during simulated cardiac stress associated with experimental hypercholesterolemia. These results support a role for the endogenous endothelin system as a mediator of myocardial ischemia and inadequate vascular permeability in hypercholesterolemia. Our animal model is not characterized by the development of atherosclerotic plaques [16]. Thus, the changes in myocardial perfusion observed in our study represent primarily the result of functional alterations of the coronary vasculature, especially of the small arterioles, which represent the major determinants of coronary blood flow [17]. Adenosine, when administered systemically, may affect myocardial circulation by two different mechanisms. Whereas intravenous administration of adenosine may directly dilate coronary microvessels, it also leads to indirect metabolic dilation of the coronary vasculature by increasing CO and thereby cardiac demand in order to compensate for a decrease in SVR, as is shown in the present study. ET-1 primarily leads to direct vasoconstriction by activation of ETA and ETB receptors on VSMC and selective ETA receptor antagonists partly inhibit this endothelium-independent ET-1 effect on vascular tone, thereby reducing SVR and CVR. Indeed, ABT-627 may cause a slight decrease in SVR and MAP in healthy, normotensive subjects [18]. Notably, hypercholesterolemia is not only associated with an increase in ET-1 activity [3,10] but also with an increased vasoconstrictory response to ET-1 in vivo [19], which might explain the more pronounced reduction of SVR and MAP at baseline in the HC/ABT-627 group. The concurrent
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reduction of CVR can be regarded as an autoregulative mechanism to maintain basal myocardial perfusion. However, the adenosine-induced changes of myocardial perfusion may involve additional mechanisms other than direct antagonism of ET-1-mediated vasoconstriction by ABT-627. Although adenosine is primarily considered an endothelium-independent vasodilator, its vasodilatory effect is mediated via specific A2-receptors that are located not only on VSMCs but also on endothelial cells [20,21]. Moreover, studies in both humans and pigs demonstrated that the vasorelaxant effect exerted by adenosine involves the release of NO [22 /24] and is, therefore, also somewhat dependent on endothelial function. Our animal model of hypercholesterolemia is characterized by the development of epicardial and microvascular coronary endothelial dysfunction [3,19] and, indeed, we have previously shown that therapeutic improvement in coronary endothelial function in this animal model is associated with restoration of coronary vascular response to adenosine [4]. Importantly, using the same ABT-627 protocol as was used in the present study, we have recently demonstrated that chronic selective ETA receptor antagonism preserves coronary epicardial and microvascular endothelial function in our porcine model of experimental hypercholesterolemia [10,11]. This effect was accompanied by an increase in coronary endothelial nitric oxide synthase (eNOS) immunoreactivity and a decrease in the reduction of plasma NO that is associated with hypercholesterolemia, suggesting that chronic selective ETA receptor antagonism exerts an endothelium-protecting effect which is at least in part due to upregulation of vascular eNOS expression and NO production. Finally, endothelium-derived NO was shown to play a crucial role for coronary microvascular dilation during metabolic stimulation [25]. Taken together, these findings support our presumption that improvement in coronary endothelial function may contribute to the effect of ABT-627 on myocardial perfusion response to intravenous adenosine observed in the current study. In our normal animals, relative change of myocardial perfusion in response to adenosine, though clearly significant, was lower than that observed in human studies assessing coronary flow reserve with intracoronary Doppler flow measurements [26]. In order to eliminate any adverse effect of adenosine-induced changes of intramyocardial vascular volume on absolute myocardial perfusion values, changes in this parameter were taken into account for calculation of myocardial perfusion [9], and thus, a major confounding effect by this variable is unlikely. On the other hand, we cannot exclude the possibility that the use of intravenous (rather than intraaortic) injections of contrast, which results in spread of the bolus, led to some underestimation of absolute myocardial perfusion. Impor-
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tantly, however, intravenous bolus administration of contrast did not substantially decrease myocardial perfusion 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 [9]. Thus, a certain species-variability in the response to intravenous adenosine may also have contributed to the relatively moderate, though clearly significant, increase in myocardial perfusion during adenosine infusion observed in pigs fed a normal diet. In the current study hyperlipidemia with and without concomitant treatment with a selective ETA receptor antagonist was not associated with any difference in coronary microvascular permeability compared with animals fed a normal diet at baseline. However, infusion of adenosine led to a significant increase in coronary microvascular leakage in hypercholesterolemic pigs, an effect that was abolished by simultaneous treatment with the selective ETA receptor antagonist ABT-627. Experimental studies addressing the impact of ET-1 upon vascular permeability have indicated a dual effect. It has been demonstrated that ET-1 enhances vascular permeability in the rat heart via the activation of ETA receptors [27]. In line with this finding, our results indicate that selective ETA receptor antagonism may preserve microvascular tightness in pathophysiological states associated with increased ET-1 activity, such as hypercholesterolemia [3]. On the other hand, selective ETB receptor blockade was shown to lead to an increase in vascular permeability under basal conditions, demonstrating that constitutive endothelial ET-1 release is also necessary for maintaining basal levels of permeability via the ETB receptor [28]. Notably, increased vascular permeability is also a hallmark of endothelial dysfunction [29]. Indeed, interventions that improve endothelial function in hypercholesterolemia were also associated with preservation of microvascular permeability during situations of increased cardiac demand [4]. Thus, preservation of coronary endothelial function, again, may be partly responsible for the beneficial effect of ABT627 on microvascular leakage in hypercholesterolemic pigs. Finally, given the significant blood pressure lowering associated with ABT-627 administration in the current study, a contribution of blood pressure reduction to the preservation of coronary microvascular permeability during adenosine infusion in treated hypercholesterolemic animals cannot be excluded. In summary, this study demonstrates for the first time that chronic selective ETA receptor antagonism preserves myocardial perfusion response and coronary microvascular integrity during simulated cardiac stress in experimental porcine hypercholesterolemia. These data point out an important role for the endogenous endothelin system in the regulation of myocardial perfusion and coronary vascular permeability in hypercholesterolemia.
Acknowledgements This work was supported by the Northland Affiliate of the American Heart Association (grant 9960336Z), National Institute of Health grants HL-63282 and HL63911, Miami Heart Research Institute, Margarete und Walther Lichtenstein Stiftung Basel, Switzerland, Freiwillige Akademische Gesellschaft Basel, Switzerland, and the Mayo Foundation. Additionally, we would like to thank Abbott Laboratories for providing ABT627.
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