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(Circulation. 1996;94:542-546.)
© 1996 American Heart Association, Inc.

Articles

An Oligosaccharide Sialyl-Lewis^x Analogue Does Not Reduce Myocardial Infarct Size After Ischemia and Reperfusion in Dogs

Edward A. Gill, MD; Yinong Kong, MD; Lawrence D. Horwitz, MD

the Division of Cardiology, University of Colorado Health Sciences Center (Denver).

Correspondence to Dr Edward A. Gill, Division of Cardiology, Box B130, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail Ed.Gill@uchsc.edu.

	Abstract

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Background Polymorphonuclear leukocytes, particularly neutrophils, are important mediators of ischemia/reperfusion–induced myocardial and coronary vascular injury. The selectin family of glycoprotein receptors mediates neutrophil "rolling," a loose, transient adhesion to the coronary endothelium that precedes the firmer adhesion associated with cardiovascular injury. The oligosaccharide sialyl-Lewis^x (SLe^x) is the probable neutrophil counterligand for endothelial E- and P-selectin. Administration of analogues of SLe^x could potentially prevent neutrophil rolling by competing for the selectin-adhesion sites. We investigated the effects of treatment with an analogue of SLe^x in a chronic canine model of ischemia/reperfusion.

Methods and Results Anesthetized mongrel dogs were subjected to 90 minutes of ischemia through occlusion of the left anterior descending coronary artery and 48 hours of reperfusion. Five minutes before the onset of reperfusion, dogs received either the SLe^x analogue CY-1503 at a dose of 20 mg/kg or normal saline. Myocardial infarct size was measured through triphenyltetrazolium chloride staining, and polymorphonuclear leukocyte accumulation was evaluated through measurement of cardiac myeloperoxidase activity. After adjustment for blood flow, the mean infarct size of control dogs (44.7±4.2%) was not different from that of treated dogs (33.4±4.0%, P=.06), although there was a trend toward a slightly lower value in the treated dogs. Myeloperoxidase activity was not different in the infarcted myocardium of the treated group compared with that of the control group (2.7±0.71 treated versus 1.08±0.41 units/mg protein control, P=.06).

Conclusions We conclude that CY-1503 does not substantially or consistently reduce myocardial infarct size or neutrophil accumulation in dogs subjected to ischemia followed by a prolonged period (48 hours) of reperfusion.

Key Words: reperfusion • leukocytes • myocardial infarction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Early reperfusion of acute myocardial infarction ameliorates hypoxia but, through a complex inflammatory response, may cause further injury, resulting in suboptimal myocardial salvage.¹ Activated neutrophils have been implicated as a cause of tissue destruction during reperfusion.^{1 2} The rate of neutrophil recruitment into the ischemic myocardium is greatest within the first hour of reperfusion, although it is likely that the cumulative influx of neutrophils proceeds for several additional hours.³

Adhesion of neutrophils to the vascular endothelium is one of the important early events that leads to neutrophil influx into the myocardium and reperfusion injury.^{1 2 4} The selectin family of adhesion molecules includes two members, P- and E-selectin, that are expressed on the surface of the endothelium during ischemia/reperfusion.^{2 5 6 7} P-selectin is rapidly released from the Weibel-Palade bodies in the endothelial cells early in reperfusion and mediates "rolling," which is loose, transient adhesion of neutrophils to the endothelium.^{2 6} E-selectin, which is expressed more slowly because new protein synthesis is required, may also contribute to rolling.^{5 6} Rolling precedes a firmer adhesion of the neutrophil, which is mediated by the ß-2 (CD18) neutrophil integrins and immunoglobulin endothelial cell adhesion molecules ICAM-1 and ICAM-2.⁷ This firm adhesion appears to be an important component of reperfusion injury.² However, because adhesion through integrin/endothelium interactions is inefficient at high shear rates, it has been proposed that rolling is a necessary predecessor to integrin-mediated neutrophil adhesion or transmigration into the extravascular space in vivo.⁷ Therefore, blockade of P- and E-selectin or their ligands could reduce neutrophil infiltration into the myocardium and prevent or reduce damage during ischemia/reperfusion.^{2 5 6 7}

Endothelial selectins bind to a carbohydrate counter-receptor, SLe^x, on the neutrophil.^{8 9} In vitro studies have demonstrated that SLe^x-containing carbohydrates inhibit selectin-mediated neutrophil adherence to endothelium.^{8 9} Recently, there have been two reports that the administration of an SLe^x-containing oligosaccharide (CY-1503) attenuated myocardial necrosis in acute feline and canine models of regional cardiac ischemia/reperfusion in vivo.^{10 11} These studies examined myocardial infarct size very early—4.5 hours of reperfusion in both the feline and canine models. Because it is questionable whether all myocardial necrosis is accurately detectable within the first few hours of reperfusion¹² and because the process of neutrophil infiltration and myocardial injury may continue beyond this time,¹³ interpretation is difficult of measurements of infarct size at this early time point. To assess the usefulness of CY-1503 in a more demanding, and possibly more clinically relevant, canine preparation, we investigated its effects on infarct size after a considerably longer reperfusion period. We performed measurements after 90 minutes of regional myocardial ischemia and 48 hours of reperfusion, a model in which we have found marked reductions in infarct size with certain diffusible antioxidants.^{14 15}

	Methods

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Surgery
Mongrel dogs were anesthetized with sodium pentobarbital (30 mg/kg IV). The dogs were intubated and ventilated with a respirator (Harvard Apparatus) with a mixture of 35% oxygen and 65% nitrogen, permitting maintenance of arterial blood gases in the usual physiological range expected at sea level despite the altitude of Denver. With sterile technique, a thoracotomy was performed in the left fifth intercostal space, and the pericardium was opened. Catheters were inserted into the descending aorta and left atrial appendage. A 1-0 silk ligature was placed around the LAD just distal to the origin of the first diagonal branch. The ligature was left loosely in place, and the ends were pulled through a 4-cm length of polyethylene tubing. Arterial blood gases were measured, and ventilation was adjusted to maintain normal physiological levels. Aortic pressures were recorded with the use of Statham P23Db transducers (Gould). A lead II ECG was recorded with subcutaneous needle electrodes.

The protocol is diagrammed in Fig 1. An endotoxin-free SLe^x analogue (CY-1503) was obtained from Cytel Corp. After a baseline radioactive microsphere injection was made for regional myocardial blood flow determination, LAD occlusion was performed by tightening the ligature and clamping it for 90 minutes. Epicardial cyanosis was readily apparent in all dogs. At 70 minutes into the occlusion period, a second microsphere injection was made. Five minutes before the onset of reperfusion (at 85 minutes), CY-1503 (or normal saline in a similar volume for control dogs) at a dose of 20 mg/kg was injected intravenously as a bolus. The investigators were blinded as to whether drug or vehicle was infused until all of the data were analyzed. At the end of the 90-minute occlusion period, the ligature was gradually loosened over 5 minutes and then removed. One hour later, we externalized the left atrial and aortic lines to the dorsum of the neck by using subcutaneous tunnels and closed the chest. Appropriate doses of morphine sulfate were given intramuscularly by the investigators or the supervising veterinarian to relieve discomfort during the first 24 hours after surgery. The dogs were returned to the kennel after spontaneous respirations were resumed. During the next 2 days, they were fed a standard diet. The wounds were cleaned, and the catheters were flushed daily with heparinized saline. At 48 hours after reperfusion had begun, the dogs were returned to the laboratory and again anesthetized with pentobarbital sodium, through the left atrial catheter. The thoracotomy incision was reopened, and the heart and proximal aorta were removed.

View larger version (17K): [in this window] [in a new window]   Figure 1. Timetable demonstrating the protocol used.

Measurement of Infarct Size
The heart and proximal aorta were connected to a dual perfusion apparatus.^{14 15} The aortic root and LAD immediately distal to the site of prior occlusion were cannulated. The LAD was ligated at the site of the prior occlusion. The region subserved by the circumflex coronary artery was perfused with a 0.25% solution of Evans blue dye through the aortic root cannula, whereas the region subserved by the LAD was perfused through the LAD cannula with a 2% solution of TTC at 37°C. Perfusion pressures in both cannulae were equalized at 100 mm Hg.

After 2 minutes, the heart was removed from the perfusion apparatus. The left ventricle was isolated and weighed. Six transverse sections 1 cm thick were obtained through sectioning parallel to the atrioventricular groove. The slices were weighed and traced onto clear acetate sheets to demarcate (1) a remote normal region (blue stain); (2) an ischemic, noninfarcted region (TTC positive, red stain); and (3) an ischemic, infarcted region (TTC negative, unstained).

The traced areas on the acetate sheets were scanned with the use of an Epson 300 ESC scanner with PICTURE PUBLISHER 3.1 software by Micrografx. The areas were then measured with an off-line analysis system (IMAGE 1.44, NIH Graphics). The percent area of infarct was compared with the total area of myocardium based on the weight of the total transverse section, and the size of the infarct was determined in grams.

Regional Myocardial Blood Flow
Regional myocardial blood flow was assessed with 15-µm-diameter microspheres labeled with ⁴⁶Sc or ⁸⁵Sr (DuPont) as we described previously.^{14 15}

Reagents
Endotoxin-free CY-1503 was provided by Cytel Corp. Quality control in vivo and in vitro procedures had been run on the lots used in these experiments by Cytel. CY-1503 was stored at 4°C.

Measurement of Myeloperoxidase
Immediately after the dogs were killed by cardiac excision while under anesthesia, samples of myocardium from the normal, ischemic, and infarct zones were frozen and stored at -80° for later analysis for myeloperoxidase according to a modification of the method of Goldblum et al.¹⁶ Briefly, minced frozen samples of myocardium were placed in 0.5% hexadecyltrimethylammonium bromide, and 5 mmol/L EDTA in 50 mmol/L potassium phosphate was added before homogenization. The suspensions were centrifuged at 40 000g for 5 minutes at 4°C. Myeloperoxidase activity in the supernatant was assayed after mixing a 0.05-mL sample with 1.45 mL of 50 mmol/L potassium phosphate buffer (pH 6.0) containing 0.17 mg/mL o-diansoide dihydrochloride and 0.005% hydrogen peroxide. Absorbance at 460 nm was measured for 6 minutes and compared with a standard curve of measured myeloperoxide. Results are expressed as units of myeloperoxide per milligram of protein.

Statistical Analysis
Data are reported as mean±SEM. Collateral ischemic blood flow MI/RISK was assessed with analysis of covariance,^{17 18} with transmural flow in the ischemic region as the independent variable and MI/RISK as the dependent variable. For all analyses a value of P<.05 was considered to be significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Study Group
Seventeen dogs were included in the final analysis: eight in the control group and nine in the CY-1503 group, which received a 20 mg/kg dose. Four dogs were excluded due to inadequate development of ischemia (minimum blood flow, >30 mL·100 g^-1·min^-1). Two dogs did not survive the procedure because of fatal ventricular tachyarrhythmias during the ischemic or reperfusion periods (both were in the control group). Ventricular fibrillation requiring defibrillation developed twice in one dog in the CY-1503 group, but this dog was included in the analysis.

Regional Myocardial Blood Flow
There were no differences between the control and treated groups in transmural blood flow in the normal zones. Although there was a trend toward differences among the groups in transmural flow in the ischemic regions, the mean values were not statistically different. The mean transmural myocardial blood flow in the ischemic zones was 20.5±2.7 mL·min^-1·100 g tissue^-1 in the control group and 16.1±2.1 mL·min^-1·100 g tissue^-1 in the CY-1503 group (P=.22).

Myocardial Infarct Size
There were no differences between the two groups in the RISK/LV. The mean value for RISK/LV was 21.4±2.8% for the CY-1503–treated dogs and 19.0±2.4% for the control dogs (P=.53).

There was no significant difference in the infarct region as a function of the region at risk (MI/RISK) between the control group and the group treated with CY-1503. The mean infarct size (MI/RISK) was 39.3±9.0% for control dogs and 38.3±4.7% for CY-1503–treated dogs (P=.93). Because this measurement does not take into account differences in the level of collateral flow into the infarct zone, we also analyzed the relation between MI/RISK and flow through analysis of covariance.

The relation between MI/RISK and the transmural ischemic zone blood flow during coronary occlusion for each dog is shown in Fig 2. The relation between MI/RISK and flow was linear for both the control (r=.95) and CY-1503 (r=.62) groups. At the lower flows, there was a trend toward lower MI/RISK values in the CY-1503 group. However, no difference was apparent at the intermediate or higher flows. The mean values for MI/RISK normalized for flow with ANCOVA was 44.7±4.2 in the control group and 33.4±4.0 in the CY-1503 group. The normalized mean values were not quite different statistically (P=.06). Because differences in slopes in infarct experiments are unusual, we performed an additional analysis that assumed that the control and experimental groups had identical slopes; again, there was no significant difference (P=.08). The dog that had undergone two defibrillations resembled the other CY-1503 dogs, and its exclusion would not have altered the results. Thus, although there was a trend toward a slightly lower infarct size, normalized for both area at risk and flow, this did not attain statistical significance. Therefore, CY-1503 had either no effect or only a small effect on infarct size measured at 48 hours of reperfusion.

View larger version (18K): [in this window] [in a new window]   Figure 2. Infarct size corrected for region at risk (MI/RISK) plotted against mean blood flow (MBF). Data were compared with the use of analysis of covariance. Mean values for MI/RISK normalized for flow by analysis of covariance were 44.7±4.2 in the control group and 33.4±4.0 in the CY-1503 group. Normalized mean values were not quite different statistically (P=.06).

Myeloperoxidase
Comparisons of myeloperoxidase activity in normal, ischemic, and infarcted myocardium in eight control and six CY-1503 dogs revealed no reduction in leukocyte accumulation after 48 hours of reperfusion in the treated dogs (Fig 3). Despite a trend favoring more myeloperoxidase in the CY-1503–treated dogs, the mean myeloperoxidase level (2.7±0.71) was not different than in the control dogs (1.08±0.41) (P=.06). Therefore, administration of this SLe^x analogue did not reduce leukocyte accumulation in the myocardium during reperfusion.

View larger version (20K): [in this window] [in a new window]   Figure 3. Myeloperoxidase (MPO) levels were measured in control and treated dogs in the normal, risk (ischemic), and infarcted regions of myocardium as a marker for neutrophil infiltration. Despite a trend favoring more MPO in the CY-1503–treated dogs, the mean MPO level (2.7±0.71) was not different than that in the control dogs (1.08±0.41) (P=.06).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It is well established that with early reperfusion of acute myocardial infarction in animal models, there is an accumulation of large quantities of activated neutrophils in the reperfused region. In addition, there is evidence that these activated neutrophils are capable of causing myocardial or coronary vascular injury.^{1 2 19 20 21 22 23 24} Antibodies to leukocyte integrin binding sites or endothelial ICAM or selectin binding sites have been used with some success to reduce cardiac reperfusion injury.^{20 25} However, convincing evidence of favorable outcomes in nonacute animal preparations or humans has not been offered. In addition, the use of antibodies to block adhesion molecules in vivo is potentially limited by the deleterious effects of formation of antigen/antibody complexes at the endothelial cell surface, possibly through F_c receptor mechanisms.^{26 27 28} This could result in adverse long-term outcomes analogous to the vascular rejection process and accelerated atherosclerosis reported in human cardiac transplant recipients.^{29 30} Although these concerns are largely speculative at present, they have prompted searches for ways of preventing leukocyte effects in the coronary circulation through the use of agents that, in contrast to monoclonal antibodies, are not likely to trigger immune reactions at the endothelial surface. One such approach has been to attempt to block interactions of neutrophils with endothelial selectins through the administration of carbohydrate analogues of SLe^x, such as CY-1503.

P- and E-selectin differ from ICAM-1 and ICAM-2 in that in vitro studies have identified their principal leukocyte ligand as a carbohydrate moiety, SLe^x, rather than the glycoprotein ß-2 integrins CD11a/CD18 and CD11b/CD18.^{8 9 31 32} This makes the concept of a carbohydrate analogue functioning as a blocking agent attractive, since it is probably not antigenic. Analogues of the carbohydrate binding site SLe^x inhibit P- or E-selectin binding in in vitro models.^{8 33} In addition, there have been two recent studies^{10 11} that concluded that the SLe^x analogue CY-1503 reduces coronary vascular and myocardial injury measured early in reperfusion. Buerke et al¹⁰ reported that CY-1503 attenuated myocardial reperfusion injury in cats subjected to 90 minutes of regional ischemia and 4.5 hours of reperfusion. Lefer et al¹¹ reported myocardial infarct size reduction by CY-1503 in a dog model of 90 minutes of regional cardiac ischemia followed by 4.5 hours of reperfusion.

However, there are possible reasons why an agent may appear to protect myocardium at 4 to 5 hours of reperfusion, yet not be effective if measurements are obtained after longer reperfusion periods. First, neutrophil-mediated injury may continue well beyond the first 4 hours of cardiac reperfusion.^{3 13} Second, measurements of infarct size made during the first few hours of reperfusion may not accurately identify all irreversibly injured myocardium and therefore could underestimate infarct size. In a recent study,¹² no lethal reperfusion injury could be detected at 3 hours of reperfusion in a canine model. Early trials in which superoxide dismutase appeared to reduce myocardial infarct size after short reperfusion periods^{34 35} were refuted subsequently by studies performed at least 48 hours after reperfusion began.^{36 37 38 39} Because late cell death (after 4 to 6 hours of reperfusion) could develop in tissue still capable of metabolic activity after a few hours of injury, it is likely that measurements made 48 hours after reperfusion begins are more reliable in assessments of drug effects.

Accordingly, we examined the efficacy of CY-1503, the oligosaccharide used by Buerke et al¹⁰ and Lefer et al,¹¹ in a canine model of 90 minutes of regional myocardial ischemia followed by 48 hours of reperfusion. Although there was a trend toward smaller infarct sizes in the CY-1503 group, it did not reach statistical significance. The trend toward a difference with treatment occurred entirely in the most ischemic hearts; there was no suggestion of a difference in hearts in which flows were higher during ischemia (Fig 2). Even if the inclusion of additional dogs in the study would have resulted in a statistical reduction in infarct size, the magnitude of the effect of treatment would have been so minimal that its practical usefulness would have been in doubt. Furthermore, measurements of myeloperoxidase, an index of leukocyte accumulation in tissue,¹⁶ showed no reduction due to CY-1503 treatment in the reperfused myocardium.

There are several possible reasons why the SLe^x carbohydrate CY-1503 was not effective in our canine model of ischemia/reperfusion. Numerous cellular adhesion molecules, including the integrins, the immunoglobulin superfamily PECAM, and selectins, may be involved in neutrophil adhesion or transmigration during cardiovascular reperfusion.^{2 5 25 26 27 28 40} It is quite conceivable that some of these mechanisms are redundant. If so, blockade or inhibition of one adhesion molecule could be offset by increased activation of a complementary adhesion molecule. Another possibility is that CY-1503 effectively blocked P-selectin binding but did not block the later expression of E-selectin. P-selectin is translocated to the endothelial cell surface within 20 minutes of reperfusion both in vitro and in vivo and is undetectable after 240 to 270 minutes of reperfusion.^{41 42} In contrast, E-selectin takes 4 to 6 hours to be expressed on the endothelial cell surface and may continue to be expressed for up to 24 hours in models of reperfusion.⁴² In the previously cited study¹¹ showing that the agent reduced infarct size, there was no detectable CY-1503 at 180 minutes of reperfusion. This time period occurs before E-selectin expression, and P-selectin still should have been present on the endothelial cell surface at 180 minutes. CY-1503 may not have blocked all P-selectin sites. This is unlikely to be related to the dose that we used (20 mg/kg), since it is four times the dose used by Lefer et al¹¹ in their canine model, where it appeared to be effective early in reperfusion. Finally, we cannot exclude the possibility that SLe^x is not the sole ligand for endothelial selectins on neutrophils.

In conclusion, we demonstrated that in dogs subjected to 90 minutes of ischemia and 48 hours of reperfusion, the SLe^x analogue CY-1503 provides little or no protection against myocardial injury and does not reduce neutrophil accumulation. Despite reports by others of efficacy during shorter reperfusion periods in other animal models, we found no evidence of substantial prevention of myocardial cell death.

	Acknowledgments

 
This work was supported by NIH grant HL-48177 and by the Cytel Corporation. We gratefully acknowledge the technical assistance provided by Nancy Sherman.

	Selected Abbreviations and Acronyms

 

ICAM = immunoglobulin cell adhesion molecule LAD = left anterior descending coronary artery MI/RISK = infarct size as a percentage of risk area RISK/LV = ratio of weight of risk region to total left ventricular weight SLex = sialyl-Lewisx TTC = 2,3,5-triphenyltetrazolium chloride

Received December 11, 1995; revision received February 6, 1996; accepted February 16, 1996.

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