Ischemia-Reperfusion Injury at the Microvascular Level
Treatment by Endothelin A–Selective Antagonist and Evaluation by Myocardial Contrast Echocardiography
Background—The purpose of this study was to verify whether endothelin A–antagonist administration at the time of coronary reperfusion preserves postischemic microvasculature and whether myocardial contrast echo (MCE) is able to detect pharmacologically induced changes in microvascular reflow.
Methods and Results—Twenty dogs underwent 90 minutes of LAD occlusion (OCC) followed by 180 minutes of reperfusion (RP). Five minutes before LAD reopening, an intravenous bolus (5 mg/kg) of LU 135252 was given in 10 dogs and vehicle in the remaining 10. At baseline (BSL), OCC, and 90 and 180 minutes of RP, microvascular flow (BF) was assessed by microspheres, and MCE was performed with intravenous echo contrast. MCE videointensity and BF were expressed as risk area/control ratio. Myocardial thickness of the risk area was calculated by 2D echo. No differences in BF between the 2 groups were observed at BSL, OCC, and 90 minutes of RP. At 180 minutes of RP, BF was decreased in controls (70±7.4% of BSL; P<0.005 versus BSL) and preserved in LU 135252–treated animals (89±4% of BSL; P=NS versus BSL; P<0.05 versus controls). Videointensity at MCE closely followed the changes in BF observed in both groups throughout the protocol. Myocardial thickness at 180 minutes of RP increased to 138.6±9.9% of BSL in controls and remained at 108.9±7.4% of BSL in treated dogs (P<0.05).
Conclusions—Endothelin A–antagonist treatment at the time of reperfusion significantly limited the progressive decrease in postischemic microvascular reflow and the increase in myocardial thickness. MCE allowed a reliable evaluation of pharmacologically induced changes in microvascular flow.
In the early phase of acute myocardial infarction, timely reperfusion is a prerequisite to limit the extent of cellular and vascular injury. However, even after early and adequate reopening of the infarct-related artery, the full benefits of reperfusion may be attenuated by a decrease in microvascular reflow1 2 and by lethal injury of potentially viable endothelial and myocardial cells3 occurring during the restoration of flow.
Increased circulating plasma levels and myocardial tissue content of endothelin, a 21-amino-acid peptide with a powerful vasoconstrictor activity,4 have been detected during ischemia-reperfusion.5 6 Because endothelin produces direct vascular and myocardial damage,7 a possible role for this peptide in the pathophysiology of ischemia-reperfusion injury has been postulated.
Myocardial contrast echocardiography (MCE) allows coronary microvasculature evaluation in vivo.8 We have recently shown that, in a canine model of ischemia-reperfusion, MCE performed with intravenous administration of second-generation contrast agent closely follows the time course of postischemic microvascular reflow.9
This experimental model of ischemia followed by reperfusion was designed to verify whether a selective endothelin A–receptor (ETA) antagonist, administered at the time of reperfusion, is able to improve postischemic microvascular reflow and to limit the increase in reperfusion-related myocardial thickness. Moreover, the accuracy of MCE in detecting pharmacologically induced changes in microvascular flow has been tested in the same animal model.
Twenty-five open-chest mongrel dogs were subjected to 90 minutes of coronary occlusion followed by 180 minutes of reperfusion. The animal studies were performed in accordance with the “Position of the American Heart Association on Research Animal Use.” The dogs were anesthetized (0.1 to 2.5 mg/kg of propofol 1%, Zeneca Pharmaceutical), intubated, and ventilated with a respiratory pump (model 55-0715, Harvard Apparatus). Body temperature was kept within physiological limits by adjustment of a heating blanket. Two 7F catheters were placed: 1 in the right femoral artery to record arterial pressure and 1 in the left femoral vein to infuse drugs and fluids. A high-fidelity micromanometer–tipped catheter transducer (Millar Instruments Inc) was placed in the left ventricle (LV) via the left carotid artery to record LV pressure; the ECG was monitored continuously. A lateral thoracotomy was performed, and the heart was suspended in a pericardial cradle. The proximal left anterior descending coronary artery (LAD) was dissected free from surrounding tissue, and at the time of coronary occlusion, an occluder was placed around it. An ultrasonic transit-time flow probe (2.0 to 2.5 mm) was placed proximal to the occluder. The probe was connected to a flowmeter (model T108, Transonic System Inc) for digital measurement of LAD flow.
Echocardiography was performed with a phased-array broad-band 3- to 2-MHz transducer (HDI3000, ATL) operating in harmonic mode (1.67 to 3.33 MHz). The heart was imaged in the short-axis view with a dynamic range of 60 dB and a mechanical index of 0.9. ECG-triggered images were obtained at end systole every 6 heartbeats.
AF0150 (Imagent US, Alliance Pharmaceutical Corp), a perfluorochemical-stabilized contrast agent, was infused intravenously at 4 mg/min, and images were obtained during infusion after myocardial videointensity had reached a plateau.
Digital data were acquired and analyzed as previously described.9 Videointensity measurements (0 to 255 gray levels) were obtained from the myocardium of the risk area, identified as the transmural area of absent enhancement after contrast injection during coronary occlusion, and from an adjacent control myocardial zone in the perfusion territory of the left circumflex artery. The ratio of risk area to control zone videointensity was calculated.9 Myocardial thickness was measured in the LAD risk area in end diastole by the leading-edge–leading-edge method in 2D images. The average of 3 measurements was considered for the data analysis.
Myocardial Blood Flow Measurement
Myocardial blood flow (BF) was measured by standard techniques.10 Briefly, ≈3×106 15-μm fluorescent microspheres (Nuflow Spheres, Triton Technology Inc) were injected directly into the left atrium. Reference blood samples were simultaneously withdrawn from the femoral artery with a constant-rate pump (Harvard 33 syringe pump) at a withdrawal rate of 15 mL/min. The tissue and the arterial reference samples were processed with a flow cytometer to count the microspheres. The value of BF was derived for the risk area and for the control zone, and their ratio was calculated.
LU 135252 [(+)-(S)-2-(4,6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenyl-propionic acid; BASF] is a selective ETA antagonist. This drug has been characterized by our group to bind only to cloned human ET receptors, with a much higher selectivity for ETA than for ETB receptors (Ki: ETA, 1.4 nmol/L; ETB, 184 nmol/L), with no affinity to any other receptor known, up to 10 μmol/L.11 The compound was dissolved in aqueous solution containing 0.1N NaOH and later buffered by addition of HCl to a pH between 7.2 and 7.4. In pilot experiments in 3 dogs, we showed that an oral or intravenous single dose of 10 mg/kg of LU 135252 was able to totally block the ETA-mediated increase in mean arterial blood pressure produced by 0.75 nmol · L−1 · kg−1 IV endothelin given 2 hours later (Figure 1⇓). In another experimental series from our laboratories, a dose of 5 mg/kg IV of LU 135252 was maximally effective in preventing coronary artery flow reductions in a dog model of unstable angina.12
Of the total of 25 dogs instrumented, 5 died of ventricular fibrillation during LAD occlusion, before LU 135252 administration. The remaining 20 animals were randomized into 2 study groups: 10 controls and 10 LU 135252–treated. In the treated animals, a bolus of 5 mg/kg IV of LU 135252 was infused 5 minutes before reperfusion.12 Data were collected at baseline, at the end of 90 minutes of LAD occlusion, and at 90 and 180 minutes of reperfusion.
Determination of Risk Area, No-Reflow, and Infarct Size
After completion of the experimental protocol, the no-reflow area was delineated by intra-atrial injection of 1 mL/kg of the fluorescent dye thioflavin S (Sigma Chemical Co).1 Then the LAD was reoccluded, and a 1-mg/kg bolus of blue dye was injected into the left atrium to determine the in vivo risk area. The dog was then euthanized and the heart explanted. Six LV slices 1 cm thick were cut parallel to the atrioventricular groove and incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) for 30 minutes at 37°C. Regions that failed to demonstrate brick-red staining were considered to represent infarcted myocardium.13 With an ultraviolet light source (peak emission wavelength 340 nm), the areas not perfused by thioflavin S (areas of no-reflow) were identified. The outlines of the LV, risk area, no-reflow, and infarct size of the apical side of each myocardial slice were traced on transparencies, and their areas were calculated by planimetry (NIH Image). Risk area was expressed as percentage of the LV, and no-reflow and infarct size as percentage of risk area. The final values of risk area, no-reflow, and infarct size for each animal were the averages of the values for each slice.13 No-reflow was also calculated as percentage of infarct size. The cross-sectional slice of the LV corresponding to the echo short-axis image was cut into 12 wedge-shaped transmural tissue samples for BF analysis. Specimens from no-reflow areas were cut into 2- to 3-mm3 blocks, weighted, and placed in cold fixative (4% formaldehyde/1% glutaraldehyde) and then processed and analyzed under a light microscope according to established methods.2 The presence and severity of contraction-band necrosis, coagulation necrosis, endothelial injury, and neutrophil accumulation were graded by a scoring system (1 to 4) that considered the extent and severity of the damage, according to established methods.2
Continuous and normally distributed data were expressed as mean±SEM and presented as percentage of baseline values. Comparisons of repeated hemodynamics, myocardial thickness, and BF and MCE data were performed with repeated-measures ANOVA, and Student’s t test with Bonferroni correction was used to assess the statistical difference between multiple comparisons. Comparisons between groups were done with the unpaired t test. A value of P<0.05 (2-sided) was considered statistically significant.
Twenty dogs completed the study protocol; their hemodynamic data are presented in the Table⇓. In both groups, the ECG showed marked ST-segment elevation during coronary occlusion, with return to baseline and no additional shifts during reperfusion. At baseline, LAD flow was similar in controls and treated animals (18.8±1.9 versus 19.5±2.4 mL/min, respectively, P=NS). At 180 minutes of reperfusion, LAD flow was 41.7±5.3% of baseline in controls (P<0.0001 versus baseline), whereas it remained 91.5±19.9% of baseline (P=NS versus baseline) in LU 135252–treated dogs (P<0.05 versus controls).
Microvascular Flow by Microspheres and MCE
At baseline, microvascular BF within the risk area was similar in the 2 groups (0.73±0.05 mL · min−1 · g−1 in controls and 0.83±0.04 mL · min−1 · g−1 in treated dogs, P=NS). During LAD occlusion, collateral flow was detectable within the risk area to a similar extent in the 2 groups (0.19±0.04 mL·min−1 · g−1 in controls and 0.29±0.04 mL · min−1 · g−1 in treated dogs, P=NS). After 90 minutes of reperfusion, BF ratio returned to baseline in both groups, whereas at 180 minutes of reflow, BF within the risk area was reduced to 0.44±0.05 mL · min−1 · g−1 in controls (P<0.005 versus baseline) and remained at 0.80±0.15 mL · min−1 · g−1 in LU 135252–treated animals (P=NS versus baseline; P<0.05 versus controls).
Risk area/control videointensity ratio during MCE varied throughout the protocol in a pattern identical to that of BF ratio (Figure 2⇓). At baseline, it was similar in both groups (0.93±0.08 versus 0.85±0.05, respectively, P=NS); it was reduced to the same extent during coronary occlusion and returned to baseline at 90 minutes of reperfusion. At 180 minutes of reperfusion, videointensity ratio was reduced from baseline in controls (73.9±18.8% of baseline, P<0.005 versus baseline), whereas it was comparable to baseline in treated dogs (90.2±12.5% of baseline, P=NS versus baseline, P<0.05 versus controls) (Figure 3⇓).
Regional Myocardial Thickness
At baseline and during LAD occlusion, end-diastolic myocardial wall thickness of the risk area was similar in the 2 groups. In controls, it increased to 139.7±9.2% and 138.6±9.9% of baseline at 90 and 180 minutes of reperfusion, respectively (P<0.0005 versus baseline). In LU 135252, myocardial thickness did not increase over baseline at 90 and 180 minutes of reperfusion (110.3±8.2% and 108.9±7.4% of baseline, respectively, P=NS versus baseline) and remained lower than that of controls (P<0.05 versus control) (Figure 4⇓).
No-Reflow and Infarct Size
Risk area was similar for the 2 groups of animals (39.4±3.7% in the control group; 41.3±4.3% in the LU 135252–treated group; P=NS). Compared with controls, treatment with LU 135252 produced a 3-fold reduction in the extent of no-reflow (15.3±4.1% of risk area in controls versus 5±2.3% of risk area in treated animals, P<0.05) and a 2-fold reduction in infarct size (22.2±5.4% of risk area in controls versus 10.6±4.2% of risk area in treated animals, P=0.06). Furthermore, in controls, no-reflow was 60.2±12.2% of the infarct size, whereas in LU 135252–treated dogs, no-reflow was only 32±14.2% of infarct size (P<0.05).
Compared with controls, LU 135252–treated dogs showed significantly less severe ischemia-reperfusion–related tissue injury within the risk area. Myocyte injury associated with ischemia (coagulation necrosis) was scored as 3.3±1.3 in controls and 1.6±0.9 in treated dogs (P<0.01), and reperfusion-related myocyte injury (contraction band necrosis) was 2.4±1.1 in controls and 1.6±0.7 in LU 135252–treated dogs (P<0.05). Endothelial injury was also more extensive in controls than in treated dogs (2.4±0.7 versus 1±0.5, respectively, P<0.0005). Average scores of intravascular neutrophil accumulation were similar in the 2 groups (2±1.6 versus 2.7±1.4, respectively, P=NS).
In this canine model, the intravenous administration of an ETA antagonist immediately before reperfusion after 90 minutes of coronary occlusion preserved microvascular flow during reperfusion and prevented the increase in myocardial wall thickness. LU 135252 selectively protected postischemic microvascular patency, as demonstrated by the 3-fold reduction in the extent of no-reflow both in absolute values and as a percentage of infarct size. The beneficial effects of ETA–antagonist administration in this experimental model establish a potential role for adjunctive treatment at the time of coronary reperfusion.
In this same model, MCE provided a reliable in vivo evaluation of microvascular flow during ischemia-reperfusion, closely depicting pharmacologically induced changes in reflow. This is the first evidence that MCE with intravenous administration of contrast agent can be used to assess the effects of a drug intervention on coronary microcirculation noninvasively.
Endothelin A Blockade After 90 Minutes of LAD Occlusion
In our model, endothelin-antagonist treatment, administered at the time of coronary reopening, significantly improved microvascular reperfusion. This beneficial effect was very likely the result of a selective protective effect of the drug on the postischemic coronary microvasculature. In fact, in animals treated with LU 135252, microvascular reflow was mostly preserved and the spatial extent of no-reflow was drastically reduced both in absolute values and as a percentage of myocardial necrosis. Although the precise mechanism by which ETA-antagonist treatment improved postischemic microvascular patency in this model cannot be entirely elucidated from our results, several hypotheses can be postulated on the basis of the known effects of endothelin on microcirculation, such as potent constriction,4 obstruction due to neutrophil activation and accumulation,14 and increased permeability.15
After ischemia-reperfusion, coronary microvessels have been demonstrated to increase their resistance in experimental models, as stated by the so-called “injury-spasm hypothesis.”16 Because endothelin has a potent constrictor effect that is known to be selective for intramyocardial microvessels, microvascular constriction is very likely the chief effect produced by endothelin released during ischemia-reperfusion. Pilot experiments from our group have demonstrated that endothelin has a direct effect on microvascular resistance and that LU 135252 is able to prevent such an effect, as shown by the prevention of arterial pressure increase and of coronary flow reduction.12 Therefore, although we could not visualize microvessel constriction in controls and treated dogs, it is conceivable that this is the main pathophysiological mechanism of LU 135252 in protecting from reperfusion injury.
Neutrophil plugging has been clearly demonstrated to be a determinant of the no-reflow phenomenon after ischemia-reperfusion in animals.17 Because endothelin has a direct effect on neutrophil adhesion to endothelial cells,14 it is conceivable that part of the beneficial effect observed with ETA antagonist on the reduction of no-reflow is due to prevention of endothelin-mediated neutrophil plugging after ischemia-reperfusion. This hypothesis is supported by recent data on isolated rat hearts subjected to global temporary ischemia.18 In this model, LU 135252 protected from ischemia-reperfusion injury only hearts reperfused in the presence of neutrophils, thus suggesting that the cardioprotective effect of the drug is at least in part related to the inhibition of leukocyte-induced injury. Although our histological data show neutrophil plugging to the same extent in control and treated dogs, we cannot exclude a different level of leukocyte activation in the 2 groups.
Endothelin enhances microvascular permeability,15 with consequent myocardial wall swelling. In our model, wall thickness at reperfusion was significantly increased in controls but not in LU 135252–treated dogs. This is very likely the result of prevention of tissue edema and wall swelling and also of reperfusion-related contraction-band necrosis, findings less preeminent in LU 135252–treated dogs. As a result of the prevented increase in wall thickness, microvascular compression was also reduced. Consistent with our findings, Oh et al19 observed an increase in end-diastolic myocardial thickness in 22% of patients with acute reperfused myocardial infarction. Interestingly, in these thickened myocardial segments, a substantial “no-reflow” was observed at MCE.
In addition to the direct effects on the coronary microcirculation produced during ischemia-reperfusion, endothelin impairs endothelium-dependent vasodilatation, and ETA receptor–antagonist treatment prevents this effect.20 This effect was considered the result of impaired formation or enhanced degradation of endothelium-derived relaxing factor. Thus, the possibility that the observed postischemic damage was primarily endothelial in origin also has to be taken into account.
Rationale for Experimental Design
The efficacy of a variety of endothelin receptor antagonists in the treatment of ischemia-reperfusion injury has been tested in different animal models, and conflicting results have been reported.21 22 23 A number of variables may account for the observed nonuniformity of results, such as the animal species studied, the duration of coronary occlusion produced, the chemical and pharmacological characteristics of the ETA antagonist used, and the timing of drug administration. In all previous studies, endothelin antagonists were administered before the onset of ischemia and continuously during reperfusion.21 22 23 Although such administration was effective in most cases, it is not clinically applicable. We infused LU 135252 intravenously 5 minutes before the reopening of the infarct-related artery, a timing of administration potentially applicable in patients with acute myocardial infarction undergoing coronary revascularization by thrombolysis or coronary angioplasty. Most importantly, to the best of our knowledge, this is the first in vivo study looking at the protective effect of endothelin-antagonist treatment on postischemic microvascular reflow.
MCE in the Serial Assessment of Pharmacologically Induced Changes in Reflow
In patients with a first, uncomplicated anterior myocardial infarction, Taniyama et al26 showed the potential of verapamil in limiting infarct area and vascular damage and in attenuating reperfusion injury, as assessed by MCE performed with intracoronary contrast echocardiography during coronary angiography, thus establishing the potential role of this drug in the treatment of postischemic reperfusion injury and highlighting the value of intracoronary MCE in the detection of pharmacologically induced changes in microvascular reflow.
In our study, MCE was performed with intravenous administration of second-generation contrast agent, and the observed changes in videointensity closely depicted the changes in microvascular flow produced by endothelin-antagonist treatment at the time of reperfusion.
Limitations of the Study
We did not measure ET levels in the coronary sinus; however, in a similar canine experiment, Velasco et al5 showed that the progressive reduction of microvascular reflow was associated with an enhanced spillover of endothelin during reperfusion.
Perfluorochemicals have been reported to modulate neutrophil function.27 Theoretically, it is possible that perfluorochemicals used to stabilize the MCE contrast agent activated neutrophils in our model; however, this did not influence the results, because the same agent was used in both controls and treated dogs.
In this study, we used background-subtracted videointensity as an estimate of myocardial BF. Although videointensity is related primarily to myocardial blood volume, changes in volume can be reflected by changes in flow. We observed a close relationship between videointensity and flow, suggesting that the changes in flow observed were coupled with alterations of blood volume.9
The no-reflow phenomenon during acute myocardial infarction has functional and clinical relevance.24 Thus, therapeutic interventions at the time of reperfusion are desirable to protect the postischemic myocardium from reperfusion injury and to limit the extent of the no-reflow phenomenon. Our study provides evidence of the potential benefit of the systemic administration of a selective ETA antagonist at the time of reperfusion in reducing microvascular damage produced by 90 minutes of ischemia followed by 180 minutes of reperfusion. Furthermore, MCE is a valuable tool not only in the noninvasive assessment of microvascular reflow but also in the evaluation of drug intervention at the microvascular level.
Dr Galiuto was a recipient of grant 203.04.14 from the Consiglio Nazionale delle Ricerche (CNR), Italy. We are grateful to Barry Peters, MD, Jim Reynolds, and Jessica Weigenseil for their technical support.
Guest Editor for this article was Lewis C. Becker, MD, Johns Hopkins Medical Institutions, Baltimore, Md.
Presented in part at the 72nd Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7–10, 1999, and published in abstract form (Circulation. 1999;100[suppl I]:I-205). Award Winner Abstract of the 21st European Congress of Cardiology, Barcelona, Spain, August 28–September 1, 1999.
- Received April 28, 2000.
- Revision received June 26, 2000.
- Accepted July 10, 2000.
- Copyright © 2000 by American Heart Association
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