This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Silver, M. J. Articles by Nicolini, F. A. Search for Related Content PubMed PubMed Citation Articles by Silver, M. J. Articles by Nicolini, F. A.

(Circulation. 1995;92:492-499.)
© 1995 American Heart Association, Inc.

Articles

Adjunctive Selectin Blockade Successfully Reduces Infarct Size Beyond Thrombolysis in the Electrolytic Canine Coronary Artery Model

Presented in part at the 44th Scientific Sessions of the American College of Cardiology, New Orleans, La.

Mitchell J. Silver, DO; Joseph M. Sutton, MD; Sharon Hook, DO; Philmo Lee, BS; Janis L. Malycky, BS; M. Laurie Phillips, PhD; Stephen G. Ellis, MD; Eric J. Topol, MD; Francesca A. Nicolini, MD, PhD

From the Experimental Thrombosis Laboratory, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio; and Cytel Corporation (M.L.P.), San Diego, Calif.

Correspondence to Joseph M. Sutton, MD, Department of Cardiology F-15, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background An adjunctive pharmacological strategy to thrombolytic therapy that is tailored to limit reperfusion injury after thrombolysis could further maximize the unquestioned benefit of restoring flow to ischemic myocardium. Ischemia–reperfusion injury exhibits features characteristic of an acute inflammatory response, including the rapid activation and infiltration of neutrophils. The initial process of neutrophil migration from the circulation to injured tissue is modulated by a group of adhesion molecules called selectins. The purpose of the present study was to assess the efficacy of a selectin blocker (CY 1503) given as an adjunct to thrombolytic therapy to interfere with the inflammatory response after ischemia–reperfusion and subsequently reduce myocardial infarct size in the electrolytic canine model.

Methods and Results A fully occlusive thrombus was formed in the left circumflex coronary artery by electrolytic injury in 20 anesthetized open-chest dogs. After occlusion, an infusion of 1 mg/kg recombinant tissue-type plasminogen activator (rTPA) was administered over 20 minutes with either a bolus of placebo or the selectin blocker CY 1503 (40 mg/kg). At the onset of reperfusion, 20 µg/kg per minute rTPA was administered for 1 hour to prevent reocclusion. After 1 hour of reperfusion, infarct size, myocardial myeloperoxidase activity, and reperfusion arrhythmias were measured. In CY 1503–treated dogs, there was a significant 69% reduction in infarct size when expressed as a percentage of the area at risk (6.7±8.4% versus 21.8±13.6%; P=.008) and a marked reduction in myeloperoxidase activity (0.014±0.009 versus 0.0370±0.025 U/min per gram; P=.02) compared with the placebo group. There was no difference between the groups in the occurrence of reperfusion arrhythmias.

Conclusions Selectin blockade as an adjunct to rTPA-mediated thrombolysis significantly reduces infarct size and myocardial neutrophil infiltration well beyond thrombolysis alone in the electrolytic canine model. These data suggest that selectin blockade is extremely effective at reducing ischemia–reperfusion injury and myocardial infarct size in this model and that the neutrophil is a potent mediator of ischemia–reperfusion injury.

Key Words: adhesion molecules • neutrophils • thrombolysis • reperfusion • selectin

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
In recent years, it has been proposed that reversible and irreversible manifestations of myocyte injury after a transient episode of ischemia may not be caused directly by the cellular milieu existing immediately before the moment of reperfusion but rather may result from some deleterious aspect of reperfusion per se.^{1 2 3} With the advent of thrombolytic therapy for acute myocardial infarction, the clinical importance of reperfusion injury has been the focus of intense investigation. It follows that, in theory, an adjunctive pharmacological strategy to limit reperfusion injury developed for concomitant use with thrombolytic agents could reduce the extent of endothelial and myocyte injury after thrombolysis for acute myocardial infarction.

The presence of a typical inflammatory reaction associated with reperfusion of previously ischemic myocardium has been well described.⁴ The neutrophil, a component of this inflammatory reaction, plays a critical role in the genesis of reperfusion injury.^{2 3 4 5} Potentially a source of oxygen free radicals, peroxides, and caustic enzymes, neutrophils may also contribute to the microvascular injury and "no reflow" seen after experimental and clinical reperfusion by collectively plugging the vascular lumen.^{6 7} In support of the role of the neutrophil as a mediator of reperfusion injury, neutrophil depletion before induction of regional ischemia^{8 9} or pharmacological suppression of neutrophil activation^{8 9 10 11 12 13 14 15} results in a limitation of myocardial infarct size in experimental preparations.

Neutrophils must adhere to endothelium before migration from circulation into tissue, where they can cause cellular damage. The circulating neutrophil initially comes into brief contact with the vessel wall, slows its movement, and rolls on the endothelium—a process known as "tethering." Tethering is mediated by a family of three lectinlike carbohydrate-binding molecules called selectins—E (endothelial), P (platelet), and L (leukocyte).^{16 17} Thus, if an agent could inhibit this process of neutrophil tethering, the initial process of neutrophil migration from the circulation would be impeded, and the resultant inflammatory reaction that contributes to reperfusion injury may in turn be limited.

Several promising compounds are under development as an analogue of the carbohydrate structure sialyl–Lewis X, which is expressed on the surface glycoproteins of neutrophils and serves as the ligand for the selectin adhesion molecules.¹⁸ By blocking neutrophil interaction with E- and P-selectin, one such agent, CY 1503 (Cytel Inc), may limit the recruitment of neutrophils to myocardial tissue after ischemia–reperfusion and therefore limit reperfusion injury.

The purpose of the present study was to test the hypothesis that a selectin blocker (CY 1503) administered as an adjunct to thrombolytic therapy would limit reperfusion injury, expressed as a reduction in myocardial infarct size, in the electrolytic canine coronary artery model. Furthermore, we sought to more precisely define the role of the neutrophil as a mediator of reperfusion injury.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Induction of Coronary Artery Thrombosis
The method of inducing an occlusive coronary thrombus has been described previously.^{19 20 21} Briefly, 27 mongrel dogs of either sex (weight, 20±0.5 kg) were anesthetized with pentobarbital sodium (25 mg/kg), intubated, and placed on assisted mechanical ventilation with a respirator (Bear Medical Systems). Serial arterial blood gas determinations were performed to maintain a physiological level of oxygenation and ventilation. A left thoracotomy was performed in the fifth intercostal space, and the heart was suspended in a pericardial cradle. The circumflex coronary artery (Cx) was isolated distal to the first marginal branch for a length of 2 cm. An ultrasonic Doppler flow probe (Crystal Biotech) was placed on the Cx to measure the coronary blood flow (CBF). Thrombosis was induced with the electrolytic injury technique.²² The endothelium of the Cx was damaged by gentle rubbing of the artery distal to the flow probe. A coronary electrode, consisting of a silver-coated copper wire with a 26-gauge needle tip, was inserted into the Cx, ensuring its contact with the intraluminal surface of the vessel. The electrode was then connected in series with a 250 000- variable resistor to the positive terminal of a 9-V nickel cadmium battery. The circuit was closed, with the negative terminal secured to the subcutaneous tissue of the dog. Distal to the flow probe and the electrode, a vascular occluder was placed on the vessel and then adjusted to totally abolish the peak reactive hyperemia after a 15-second period of total occlusion (to an 80% stenosis) without affecting the resting flow. Formation of thrombus was initiated by delivery of 100 µA continuous anodal current to the tip of the coronary electrode. When the CBF was zero, the occluder was removed gradually, and electrical stimulation was suspended. Mean aortic blood pressure was continuously monitored with a pressure transducer (SpectraMed Inc) connected to a catheter placed in the ascending aorta via the carotid artery. The heart rate was monitored through lead II of the ECG. All hemodynamic parameters were continuously recorded on a multichannel recorder (EFM). Catheters were inserted in both femoral veins and advanced into the inferior vena cava for infusion of the different therapeutic regimens.

Administration of Drug Regimens
An aqueous solution of CY 1503 in 500-mg vials (mixed in sodium acetate and water for injection, USP) was provided by Cytel Corporation. Recombinant tissue-type plasminogen activator (rTPA) was produced through recombinant DNA technology and supplied by Genentech, Inc in vials containing 50 mg of rTPA. Dilutions of the agents were prepared in sterile water, according to the dog's body weight, just before use.

Formation of a fully occlusive thrombus was indicated by zero CBF. After the vascular occluder was removed and electric stimulation was discontinued, the dogs received intravenous saline for 30 minutes to confirm the stability of the thrombus. Dogs then were randomized to one of two groups: (1) administration of rTPA (1 mg/kg) over 20 minutes plus a bolus of 20 mL of placebo (saline) or (2) administration of rTPA (1 mg/kg) over 20 minutes plus a bolus of CY 1503 (40 mg/kg). The investigators were blinded to the drug regimen received.

Because the principal aim of the present study was to evaluate the ability of CY 1503 to limit the extent of myocardial infarction attributable to reperfusion injury, a continuous infusion of rTPA was initiated at the onset of reflow administered at a rate of 20 µg/kg per minute for a period of 1 hour to prevent reocclusion (Fig 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Timeline depicting the sequence of experimental events, including induction of a stable thrombus, administration of drug regimens, and onset of reperfusion. Solid bar indicates an occluded coronary artery; open bar, an open artery. There was a slight variation in the time of occlusion among dogs (81±33 minutes); however, the period of reperfusion was held constant for 1 hour in all dogs. Time (in minutes) is indicated at the bottom of the bar. rTPA indicates recombinant tissue-type plasminogen activator.

None of the animals received aspirin or heparin. Thrombolysis was defined as restoration of CBF to at least 30% of the baseline flow value, occurring at any time after the onset of rTPA infusion. Dogs were observed for a total of 1 hour beyond thrombolysis and then were killed with a rapid injection of 80 mEq potassium chloride.

Infarct Size Determination
Infarct size determination was performed using the ex vivo dual perfusion histochemical method.¹² This method delineates viable and nonviable myocardium by perfusing the cannulated Cx with a 1.5% triphenyltetrazolium chloride (TTC) solution buffered with 20 µmol/L potassium phosphate (pH 7.4), while the cannulated left anterior descending coronary artery (LAD) is simultaneously perfused with Evans blue dye (0.25%). Both cannulas were introduced via the left main coronary ostium. Both perfusates (TTC and Evans blue dye) were delivered to the respective vascular territory under a constant pressure of 150 mm Hg at a temperature of 37°C for 10 minutes. The cannulated Cx and LAD were then perfused for 2 hours under a constant pressure of 150 mm Hg with a fixative solution (HistoChoice, Amresco).

After removal of the right ventricle and atria, the hearts were cut into five transverse sections, each approximately 1 cm thick; cumulative infarct size and the region at risk were then determined by a blinded investigator with computer-assisted direct planimetry of each section. The region at risk was defined as the cumulative area of myocardium from each section that was not stained blue, ie, myocardium subtended by the Evans blue–perfused LAD. The ability to distinguish viable from nonviable myocardial tissue via the exposure of myocardial dehydrogenase enzymes to TTC has been demonstrated by others as an accurate method of quantifying infarct size.^{23 24 25 26}

Histology
Extensive histological samples were taken from each transverse section, processed by conventional methods, and stained with hematoxylin and eosin. The histological sections were examined by a pathologist, who was blinded with respect to the treatment regimen, for the extent of myocardial tissue injury, the intensity of neutrophil infiltration (the mean of the absolute number of neutrophils from five random high-power fields), and the extent grade (the number of high-power fields, among five randomly sampled, that contained any number of neutrophils), with a grade of 1 equal to one high-power field; two, two high-power fields; through five, five high-power fields.

Neutrophil Accumulation: Myeloperoxidase Activity
Additional samples of myocardial tissue were taken from the left ventricle within the area at risk and from normal, noninfarcted sections of myocardium and were frozen at -70°C. The tissue samples were assayed for myeloperoxidase (MPO) activity as described by Schierwagen and coworkers.²⁷ The samples were thawed at room temperature and homogenized in 0.05 mol/L potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyl-trimethylammonium bromide (HTAB; Sigma Chemical Co) at 4°C for 15 seconds. After centrifugation (1700g for 30 minutes at 4°C), 1 mL of the supernatant was removed and heated to 60°C for 2 hours on a waterbath controlled with a thermostat. The supernatant was again centrifuged (10 000g for 5 minutes at 4°C) and then assayed for enzyme activity.

MPO activity was determined by measuring the hydrogen peroxide–dependent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB).²⁸ In its oxidized form, TMB is blue, which was measured spectrophotometrically at a wavelength of 650 nm. The reaction mixture for analysis consisted of a 25-µL tissue sample, 25 µL TMB (final concentration, 0.16 mmol/L; Sigma) dissolved in dimethylsulfoxide (Sigma), and 200 µL of hydrogen peroxide (final concentration, 0.24 mmol/L; York Pharmacal) diluted in 0.08 mol/L phosphate buffer (pH 5.4).

The reaction was performed in a 96-well microtiter plate. The mixture was incubated for 5 minutes at 37°C and stopped with 25 µL bovine catalase (final concentration, 13.6 µg/mL; Boehringer Mannheim). To ensure linearity of the reaction during this time period, MPO standards (human leukocyte MPO, 0.001 to 0.5 U/mL; Sigma) were included in each assay. One unit of MPO activity was defined as the amount of enzyme reducing 1 µmol of peroxide per minute. Histological evidence of neutrophil infiltration has been correlated to the content of myocardial MPO activity by several other investigators.^{12 13 14 29 30}

Evaluation of Reperfusion Arrhythmias
All dogs were monitored with the use of a two-channel Holter monitor (Del Mar Avionics) during the period of reperfusion. A blinded investigator analyzed all Holter full disclosures. A reperfusion arrhythmia event was defined as one of the following: (1) accelerated idioventricular rhythm, (2) nonsustained ventricular tachycardia (more than three consecutive premature ventricular contractions of less than 30 seconds' duration), and (3) ventricular fibrillation. The absolute number of events per dog were recorded after analysis of the Holter full disclosure report.

Statistical Analysis
Sample size calculations were performed to detect a conservative 30% reduction in infarct size,³¹ a .05 level of significance, and an 80% power. Based on these results, a total of 20 dogs were used in the final data analysis.

All data were analyzed by applying an unpaired two-tailed Student's t test with the use of the SPSS statistical package (SPSS Inc). A linear regression analysis was performed on the variable area of infarct versus area at risk to generate Fig 5.

View larger version (26K): [in this window] [in a new window]   Figure 5. Plot of influence of presenting area at risk as a covariate determinant of final infarct size for the two treatment strategies. Reference line (y=x, or infarct area equals risk area) indicates the maximum infarct size that may result from complete necrosis due to ineffective therapy. Expected reduction in final infarct size for any given area at risk afforded by the two interventional strategies is shown by linear regression of the plotted data. Infarct size reduction derived from thrombolysis alone (recombinant tissue-type plasminogen activator [rTPA; tPA in figure] plus placebo; r=.89) is considerable; however, further diminution in final infarct size was afforded by adjunctive selectin blockade therapy (r=.65; P=.008 CY 1503 versus placebo).

	Results

Top Abstract Introduction Methods Results Discussion References

 
A total of 20 dogs were randomized and included in the final data analysis. Seven dogs were not included in the final data analysis because of lethal arrhythmic events occurring during the thrombus induction phase before randomization. All investigators were blinded with respect to the treatment regimen. All values are given as mean±SD.

Hemodynamic Measurements
No significant differences were found in baseline mean arterial blood pressure, heart rate, or CBF (Table 1).

View this table: [in this window] [in a new window]   Table 1. Baseline Characteristics of Experimental Animals

Effect on Infarct Size
There was no significant difference in the time of ischemia between the treatment and placebo groups (80±20.4 versus 81±43.0 minutes; P=NS, respectively) (Table 1).

The area at risk proved to be similar in the treated and control animals, as indicated in Table 2. In dogs treated with CY 1503, there was a statistically significant reduction in infarct size when expressed either as a percentage of the area at risk (CY 1503, 6.7±8.4%; placebo, 21.8±13.6%; P=.008) or as absolute infarct size (CY 1503, 2.5±3.2 mm²; placebo, 8.8±5.4 mm²; P=.006) (Fig 2, Table 2). This translates into a 69% relative reduction in infarct size in dogs receiving CY 1503. These results strongly suggest that the inhibition of neutrophil–endothelial cell adhesion by CY 1503 markedly attenuates reperfusion injury as demonstrated by a significant reduction in infarct size after rTPA-mediated thrombolysis.

View this table: [in this window] [in a new window]   Table 2. Myocardial Area at Risk and the Effects of Placebo and CY 1503 on Infarct Size During Thrombolysis

View larger version (14K): [in this window] [in a new window]   Figure 2. Pie charts illustrating the amount of reduction in infarct size seen in CY 1503–treated animals expressed as a percentage of the area at risk. LCx indicates left circumflex artery.

Histology
Histological sections were analyzed for the extent of myocardial injury and both the intensity of neutrophil infiltration and extent grade. The myocardium in ischemic–reperfused Cx regions in dogs administered rTPA plus placebo showed evidence of extensive myocardial injury, ie, wavy fibers, cell separation, tissue edema, and frequent myocardial contraction bands (Fig 3A). On the other hand, in ischemic–reperfused Cx regions in dogs treated with rTPA plus CY 1503, the evidence of myocardial injury was minimal (Fig 3B). Of note, there was less intense neutrophil infiltration in the ischemic–reperfused Cx territory in CY 1503–treated dogs (36.1±21.1 neutrophils per high-power field) than in dogs that received placebo (52.8±43.6 neutrophils per high-power field), but this trend was not statistically significant (Fig 3). A similar trend was observed in the neutrophil extent grade within the ischemic–reperfused Cx territory. The mean extent grade in the CY 1503–treated dogs was 1.8±1.13 compared with 2.88±1.45 in the placebo group. Again, this substantial trend approached but did not achieve statistical significance (P=.08).

View larger version (0K): [in this window] [in a new window]   Figure 3. A, Hematoxylin and eosin–stained section of myocardium examined under light microscopy from the peri-infarcted zone of the ischemic–reperfused circumflex artery region from a dog treated with recombinant tissue-type plasminogen activator (rTPA) plus placebo. B, Hematoxylin and eosin–stained section of myocardium examined under light microscopy from the peri-infarcted zone of the ischemic-reperfused circumflex artery region from a dog treated with rTPA plus CY 1503.

Myocardial MPO Activity
Tissues from both the ischemic–reperfused Cx territory and nonischemic myocardium were assayed for MPO activity as an indicator of neutrophil accumulation. There was significantly less MPO activity in the ischemic–reperfused Cx regions in dogs receiving CY 1503 (0.014±0.009 U/min per gram) than in the placebo group (0.037±0.025 U/min per gram) (P=.02) (Fig 4). Likewise, there was less MPO activity within the nonischemic myocardial territory in dogs receiving CY 1503 (0.009±0.006 U/min per gram) compared with the placebo group (0.02±0.011 U/min per gram) (P=.02). These data in combination with the findings on light microscopy and reduction in infarct size provide cumulative evidence for the role of the neutrophil as a major contributor to reperfusion injury after thrombolytic therapy in this model.

View larger version (31K): [in this window] [in a new window]   Figure 4. Bar graph illustrating the significant reduction in myeloperoxidase activity found in the ischemic–reperfused LCx (left circumflex) and the normal left anterior descending coronary artery (LAD) territories in dogs treated with recombinant tissue-type plasminogen activator plus CY 1503.

Reperfusion Arrhythmias
There was no difference in the frequency of reperfusion arrhythmia events between dogs treated with CY 1503 (9±2 events) and dogs receiving placebo (9±2 events). Of note, there was no adverse, proarrhythmic effect in the CY 1503–treated dogs. The lack of effect of CY 1503 on reperfusion arrhythmias despite a significant reduction in infarct size suggests that the etiology of reperfusion arrhythmias may not be dependent on myocardial neutrophil infiltration or that some of the arrhythmic events noted in our study population occurred independent of reperfusion, since these events are common during the acute phase of myocardial infarction.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that the administration of a selectin blocker as an adjunct to rTPA-mediated thrombolysis significantly reduces myocardial infarct size attributable to reperfusion injury in the electrolytic canine coronary artery model compared with thrombolytic monotherapy. This was manifested by a marked reduction in both infarct size expressed as a percentage of the area at risk (P=.008) and absolute infarct size (P=.006) (Fig 2, Table 2). The results also support the hypothesis that neutrophils are an important component of myocardial reperfusion injury, since dogs treated with the selectin blocker had significantly less MPO activity (P=.02), a marker of neutrophil infiltration, in myocardium from the ischemic–reperfused Cx territory (Fig 4) and less tissue injury as assessed by myocardial histology (Fig 3A and 3B).

Effect on Infarct Size
The most direct evidence for contribution of the neutrophil to myocardial ischemia–reperfusion injury is provided by independent studies that used different methods to deplete neutrophils and demonstrated that neutrophil depletion was associated with a reduction in infarct size.^{8 9 32} The study by Litt and coworkers³² is of particular interest because neutrophil depletion achieved with filters was associated with decreased tissue injury, even when the depletion was affected only at the time of reperfusion. This observation confirms the idea that rapid neutrophil influx into the reperfused myocardium exacerbates the development of tissue injury.

CY 1503 is an analogue of the carbohydrate structure sialyl–Lewis X, which is abundantly expressed on the surface glycoproteins of neutrophils and serves as the ligand for the two adhesion molecules E- and P-selectin. Neither E- nor P-selectin is found on resting vascular endothelial cells. They are expressed in response to different inflammatory mediators that are present in conditions of inflammation and ischemia–reperfusion. Direct evidence that selectin blockade effectively reduces neutrophil-mediated tissue damage has been obtained in animal models of disease with the use of monoclonal antibodies to E- and P-selectin. E-selectin antibodies have been effective in reducing lung injury in models of immune complex–induced lung injury in the rat as well as in a primate model of asthma.³³ P-selectin antibodies have also been found to prevent cobra venom–induced lung injury (complement mediated),³⁴ tissue damage after ischemia–reperfusion of the cat heart in a direct coronary ligature model,³¹ and inhibition of "no reflow" in the dog gracilis muscle model.³⁵ It is of interest that E-selectin is expressed in response to inflammatory cytokines (interleukin-1_ß and tumor necrosis factor) and endotoxin and is notably expressed in diseases of inflammation where these factors are present. In contrast, P-selectin is rapidly expressed in response to agents such as oxygen free radicals and thrombin, which are present in conditions of ischemia–reperfusion.^{31 36} Unlike E-selectin, which is synthesized de novo, P-selectin is prefabricated in storage granules and thus is rapidly expressed after cell stimulation. It is likely, therefore, that the attenuation of reperfusion injury in the present study was more from the ability of CY 1503 to block the P-selectin receptor than the E-selectin receptor, since a prolonged infusion of CY 1503 was not used to coincide with delayed E-selectin expression. Weyrich and colleagues³¹ elegantly demonstrated that neutrophil adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerted a significant reduction of myocardial necrosis after ischemia–reperfusion in a feline ligature model.³¹

It follows that the electrolytic canine coronary artery model may be well suited for the study of the effects of a selectin blocker on reperfusion injury, since the occlusive thrombus generated in this model more closely parallels the acute closure experience of a human coronary artery at the time of acute myocardial infarction compared with the direct coronary ligature models. In contrast to the direct coronary ligature schema, this model is a representative physiological analogue of clinical myocardial infarction—a thrombotic event. Indeed, it is of considerable theoretical importance to observe the efficacy of CY 1503 when administered with a thrombolytic as opposed to mechanical reperfusion, since pharmacological lysis of an occlusive thrombus generates a thrombin-rich environment that may cause significant upregulation of the P-selectin receptor.

Effect on Neutrophil Accumulation
Because no standard technique currently exists for quantifying neutrophil infiltration by direct microscopic visualization of myocardium after ischemia–reperfusion, we both performed light microscopy on hematoxylin and eosin–stained sections of myocardium and measured MPO activity in both ischemic and nonischemic regions of myocardium. Measurement of MPO activity in tissue after ischemia–reperfusion has been accepted as a reliable method for quantifying neutrophil infiltration.^{12 13 14 29 30} Ischemia–reperfusion exhibits features characteristic of an acute inflammatory response that are manifested by a maximum neutrophil infiltration in the infarct-related artery territory where the greatest amount of cellular injury and necrosis occurs. However, a nonspecific and less intense neutrophil infiltration in the noninfarct- related territory also occurs, most likely due to a systemic release of chemoattractant factors from the necrotic area. As such, any systemic therapy that modulates the intensity of the inflammatory response after ischemia–reperfusion injury may cause a relative reduction in the inflammatory response in both the infarct- and noninfarct-related territories. Accordingly, our data on MPO showed a significant reduction of MPO activity (Fig 4) in both Cx- and LAD-related myocardial territories (both P=.02) after CY 1503 treatment. These observations in combination with the trend toward minimal myocardial tissue injury and less neutrophil intensity and extent grade observed with light microscopy from the ischemic–reperfused Cx regions of CY 1503–treated dogs support the hypothesis that CY 1503–mediated blockade of E- and P-selectin attenuates neutrophil migration from the circulation after ischemia–reperfusion. This reduction in neutrophil infiltration, in concert with a significant reduction in infarct size (P=.006), strongly suggests a cause-and-effect relation between the ability of CY 1503 to block neutrophil–endothelial cell adhesion and the subsequent reduction in reperfusion injury.

In the present short-term study, the length of reperfusion for only 1 hour could be seen as a limitation to extrapolate our findings to a clinical situation. However, an early accelerated neutrophil recruitment into myocardium after ischemia–reperfusion injury has been previously validated.^{9 10 30 37 38 39 40 41} Furthermore, it has recently been shown⁴¹ that the rate of neutrophil accumulation is greatest within the first hour of reperfusion, where it can increase sixfold to sevenfold, and declines thereafter, although the cumulative influx of neutrophils proceeds for 18 to 24 hours. In fact, the early neutrophil sequestration matches the appearance of chemotactic factors in the cardiac lymph draining the reperfused myocardium.⁴² Therefore, on a quantitative basis, the early moments of reperfusion are critical for neutrophil invasion. Because we postulated a reduction in infarct size after P- and E-selectin inhibition due to a reduction in myocardial neutrophil accumulation, we did not envision a reperfusion time of 1 hour as a significant limitation in this animal model.

Effect on Reperfusion Arrhythmias
There is evidence that oxygen free radicals are involved in the electrophysiological disturbances that are associated with reperfusion arrhythmias.^{43 44} In the isolated rat heart subjected to a short period of coronary occlusion, reperfusion arrhythmias were reduced by the administration of different anti–free radical agents, including the xanthine oxidase inhibitor allopurinol,⁴⁵ the enzymes superoxide dismutase and catalase,^{46 47} and the free radical scavengers mannitol, glutathione, and methionine.⁴⁶

It is of interest in the present study that despite the marked reduction in infarct size observed in CY 1503–treated dogs, no difference was found in the frequency of arrhythmias observed after reperfusion. The one feature that all of the antioxidant agents cited above have in common is the ability to prevent the formation, or enhance the elimination, of reactive oxygen intermediates. The fact that CY 1503 has no direct antioxidant properties, and therefore may not be able to entirely modify a large oxidant stress, may explain why no difference was found in the incidence of arrhythmias after ischemia–reperfusion. CY 1503 may indirectly decrease local superoxide production by diminishing the number of neutrophils contributing to the oxidative burst and diminish available sources of peroxide release, but unlike the other direct antioxidants, it does not enhance clearance of these reperfusion injury mediators. Furthermore, our sample size had the power to detect infarct size reduction, not reperfusion arrhythmias, as a primary end point. Because arrhythmic events are common during acute myocardial infarction, the possibility of a type II error exists.

Clinical Implications
Allowing for the influence of the important covariate myocardial area at risk on subsequent infarct size, Fig 5 illustrates the additional benefit afforded by the adjunctive antiselectin therapy. The jeopardy reference line (y=x, or infarct area equals risk area) indicates the maximum infarct size that may result from complete necrosis of any given area at risk; larger ischemic territory directly equates with a larger infarct zone if no salvage is afforded by the experimental interventional strategy. The effect of rTPA alone (versus rTPA plus placebo) is striking, yielding a substantial rightward shift in the relation between area at risk and final infarct size. The linearity of the control cohort data is excellent (r=.89). Adjunctive selectin blockade (rTPA plus CY 1503) yields a further reduction in expected final infarct size for any given area at risk (r=.65, P=.008 placebo versus CY 1503). The clinical manifestations of such a reduction in infarct size beyond that achieved with thrombolysis alone will be tested in future clinical trials. Historically, it is interesting that other attempts to transfer results from experimental models of reperfusion injury to humans with the use of agents such as superoxide dismutase and catalase,³ N-acetylcysteine,³ adenosine,⁴⁸ and perfluorochemicals⁴⁹ have yet to show any benefit.

Phase I clinical data with CY 1503 have been accrued, and a phase II pilot trial will soon be under way to determine whether the effect on infarct size found in the present study can be achieved in humans. The Cylexin as Adjunct to Lytic Therapy to Prevent SuperOxide Reflow Injury (CALYPSO) pilot trial will use serial tomography sestamibi imaging in a randomized, placebo-controlled study of patients undergoing primary angioplasty for acute myocardial infarction. This clinical setting facilitates the precise timing of reflow and allows direct assessment of the effectiveness of reperfusion therapy analogous to the direct feline and canine coronary ligature models. If effective, CY 1503 will be tested as an adjunct to thrombolysis in the CALYPSO thrombolytic trial.

Conclusions
It is apparent from these data that inhibition of neutrophil–endothelial cell adhesion by CY 1503 reduces myocardial infarct size well beyond thrombolytic-mediated reperfusion alone in the electrolytic canine coronary artery model. Given the sobering limited effects of currently available adjuncts to thrombolytic therapy,⁵⁰ the addition of CY 1503 to existing reperfusion strategies is a highly attractive strategy for further clinical investigation.

Received November 28, 1994; revision received January 19, 1995; accepted January 30, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Reimer K, Jennings R. Myocardial ischemia, hypoxia and infarction. In: Fozzurd HA Jr, Harber E, Katz AM, Morgan HB, eds. The Heart and the Cardiovascular System, Vol 2, 2nd ed. New York, NY: Raven Press; 1991:1927-1935.
   
2. Opie I. Reperfusion injury and its pharmacologic modification. Circulation. 1989;80:1049-1062. [Abstract/Free Full Text]
   
3. Kloner R, Przyklenk K, Whittaker P. Deleterious effects of oxygen radicals in ischemia/reperfusion: resolved and unresolved issues. Circulation. 1989;80:1115-1127. [Abstract/Free Full Text]
   
4. Lucchesi B, Mullane K. Leukocytes and ischemia-induced myocardial injury. Annu Rev Pharmacol Toxicol. 1986;26:201-224. [Medline] [Order article via Infotrieve]
   
5. Engler R, Covell J. Granulocytes cause reperfusion ventricular dysfunction after 15-minute ischemia in the dog. Circ Res. 1987;61:20-28. [Abstract/Free Full Text]
   
6. Engler R, Dahlgren M, Morris D, Peterson M, Schmid-Schonbein G. Role of leukocytes in response to acute myocardial ischemia and reflow in dog. Am J Physiol. 1986;251:H314-H323. [Abstract/Free Full Text]
   
7. Werns S, Brinker J, Gruber J, Rothbaum D, Henser R, George B, Burwell L, Kereiakas D, Mancini G, Flaherty J. A randomized, double-blind trial of recombinant human superoxide dismutase (SOD) in patients undergoing PTCA for acute MI. Circulation. 1989;80(suppl II):II-113. Abstract.
   
8. Romson J, Hook B, Kunel S, Abrams G, Schork M, Lucchesi B. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation. 1983;67:1016-1023. [Abstract/Free Full Text]
   
9. Mullane K, Read N, Salmon J, Moncada S. Role of leukocytes in acute myocardial infarction in anesthetized dogs: relationship to myocardial salvage by anti-inflammatory drugs. J Pharmacol Exp Ther. 1984;228:510-522. [Abstract/Free Full Text]
   
10. Romson J, Hook B, Rigot V, Schork M, Swanson D, Lucchesi B. The effect of ibuprofen on accumulation of 111-indium-labeled platelet and leukocytes in experimental myocardial infarction. Circulation. 1982;66:1002-1011. [Abstract/Free Full Text]
    
11. Simpson P, Mitsos S, Ventura A, Gallagher K, Fantone J, Lucchesi B. Prostacyclin protects ischemic reperfused myocardium in the dog by inhibition of neutrophil activation. Am Heart J. 1987;113:129-137. [Medline] [Order article via Infotrieve]
    
12. Simpson P, Mickelson J, Fantone J, Gallagher K, Lucchesi B. Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in the canine heart. Circ Res. 1987;60:666-673. [Abstract/Free Full Text]
    
13. Simpson P, Mickelson J, Fantone J, Gallagher K, Lucchesi B. Reduction of experimental myocardial infarct size with prostaglandin E₁-inhibition of neutrophil migration and activation. J Pharmacol Exp Ther. 1988;244:619-624. [Abstract/Free Full Text]
    
14. Bednar M, Smith B, Pinto A, Mullane K. Nafazatrom-induced salvage of ischemic myocardium in anesthetized dogs is mediated through inhibition of neutrophil function. Circ Res. 1985;57:131-141. [Abstract/Free Full Text]
    
15. Nicolini F, Mehta J, Nichols W, Donnelly W, Luostarinen R, Saldeen T. Leukocyte elastase inhibition and t-PA-induced coronary artery thrombolysis in dogs: beneficial effects on myocardial histology. Am Heart J. 1991;122:1245-1251. [Medline] [Order article via Infotrieve]
    
16. Lawrence M, Springer T. Leukocytes roll on a selectin at physiologic flow rates: distinction from prerequisite from adhesion through integrins. Cell. 1991;65:859-873. [Medline] [Order article via Infotrieve]
    
17. Lasky L. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science. 1992;258:964-969. [Abstract/Free Full Text]
    
18. Phillips M, Nudelman E, Gaeta F, Perez M, Sighal A, Hakomori S, Paulson J. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lewis X. Science. 1990;250:1130-1132. [Abstract/Free Full Text]
    
19. Nicolini F, Mehta J, Nichols W, Saldeen T, Grant M. Prostacyclin analogue iloprost decreases the thrombolytic potential of t-PA in canine coronary thrombosis. Circulation. 1990;81:1115-1122. [Abstract/Free Full Text]
    
20. Nicolini FA, Nichols WW, Mehta JL, Saldeen TG, Schofield R, Ross M, Player DW, Pohl GB, Mattsson C. Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue-plasminogen activator. J Am Coll Cardiol. 1992;20:228-235. [Abstract]
    
21. Nicolini FA, Lee P, Rios G, Kottke-Marchant K, Topol EJ. Combination of platelet fibrinogen receptor antagonist and direct thrombin inhibitor at low doses markedly improves thrombolysis. Circulation. 1994;89:1802-1809. [Abstract/Free Full Text]
    
22. Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine, a model for in vivo evaluation of antithrombotic agents. Thromb Res. 1980;17:841-853. [Medline] [Order article via Infotrieve]
    
23. Roberts AJ, Cipriano PR, Alonso DR, Jacobstein JG, Combes JR, Gay WA. Evaluation of methods for the quantification of experimental myocardial infarction. Circulation. 1978;57:35-41. [Abstract/Free Full Text]
    
24. Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, Ganz W. Early phase of acute myocardial infarct size quantification: validation of the triphenyltetrazolium chloride technique. Am Heart J. 1981;101:593-600.[Medline] [Order article via Infotrieve]
    
25. Vivaldi MY, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol. 1985;121:522-530. [Abstract]
    
26. Romaschin AD, Rebeyka I, Wilson GJ, Mickle DAG. Conjugated dienes in ischemic and reperfused myocardium: an in vivo chemical signature of oxygen free radical mediated injury. J Mol Cell Cardiol. 1987;19:289-302. [Medline] [Order article via Infotrieve]
    
27. Schierwagen C, Bylund-Fellenius AC, Lundberg C. Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity. J Pharm Methods. 1990;23:179-186.
    
28. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem. 1983;132:345-352. [Medline] [Order article via Infotrieve]
    
29. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol. 1982;78:206-209. [Medline] [Order article via Infotrieve]
    
30. Mullane KM, Kramer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharm Methods. 1985;14:157-167.
    
31. Weyrich AS, Ma XY, Lefer DJ, Albertine KH, Lefer AM. In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invest. 1993;91:2620-2629.
    
32. Litt MR, Jeremy RW, Weisman HF. Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Circulation. 1989;80:1816-1827. [Abstract/Free Full Text]
    
33. Forrest M, Paulson JC. Leukocyte adhesion. In: Granger DN, Schmid-Schonbein G, eds. Physiology and Pathophysiology of Leukocyte Adhesion. In press.
    
34. Mulligan MS, Polley MJ, Bayer RJ, Nunn MF, Paulson JC, Ward PA. Neutrophil-dependent acute lung injury. J Clin Invest. 1992;90:1600-1607.
    
35. Jerome SN, Dore M, Paulson JC. P-selectin and ICAM-1 dependent adherence reactions: role in the genesis of post-ischemic no-reflow. Am J Physiol. 1994;216:H1316-H1321.
    
36. Winn RK, Liggitt D, Vedder NB. Anti P-selectin monoclonal antibody attenuates reperfusion injury to the rabbit ear. J Clin Invest. 1993;92:2042-2047.
    
37. Engler R, Schmid-Schonbein GW, Pavalec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol. 1983;111:98-111. [Abstract]
    
38. Smith EF III, Egan JW, Bugelski PJ. Temporal relation between neutrophil accumulation and myocardial reperfusion injury. Am J Physiol. 1988;255:H1060-H1068. [Abstract/Free Full Text]
    
39. Go LO, Murry CE, Richard VJ. Myocardial neutrophil accumulation during reperfusion after reversible or irreversible ischemic injury. Am J Physiol. 1988;255:H1188-H1198. [Abstract/Free Full Text]
    
40. Richard VJ, Brooks SE, Jennings RB. Effect of a critical coronary stenosis on myocardial neutrophil accumulation during ischemic and early reperfusion in dogs. Circulation. 1989;80:1805-1815. [Abstract/Free Full Text]
    
41. Dreyer WJ, Michael LH, West MS. Neutrophil accumulation in ischemic canine myocardium: insights into the time course, distribution, and mechanism of localization during early reperfusion. Circulation. 1991;84:400-411. [Abstract/Free Full Text]
    
42. Dreyer WJ, Smith CW, Michael LH. Canine neutrophil activation by cardiac lymph obtained during reperfusion of ischemic myocardium. Circ Res. 1989;65:1751-1762. [Abstract/Free Full Text]
    
43. Manning AS, Hearse DJ. Reperfusion-induced arrhythmias: mechanisms and preventions. J Mol Cell Cardiol. 1984;16:497-518. [Medline] [Order article via Infotrieve]
    
44. Richard VJ, Murray CE, Jennings RB, Reimer KA. Oxygen-derived free radicals and postischemic myocardial reperfusion: therapeutic implications. Fundam Clin Pharmacol. 1990;4:85-103. [Medline] [Order article via Infotrieve]
    
45. Manning AS, Coltart DJ, Hearse DJ. Ischemia and reperfusion–induced arrhythmias in the rat: effect of xanthine oxidase inhibition with allopurinol. Circ Res. 1984;55:545-548. [Abstract/Free Full Text]
    
46. Bernier M, Hearse DJ, Manning AS. Reperfusion-induced arrhythmias and oxygen free radicals: studies with anti–free radical interventions and free radical–generating system in the isolated perfused rat heart. Circ Res. 1986;58:331-340. [Abstract/Free Full Text]
    
47. Woodward B, Zakaria M. Effect of some free radical scavengers on reperfusion-induced arrhythmias in the isolated rat heart. J Mol Cell Cardiol. 1985;17:485-493. [Medline] [Order article via Infotrieve]
    
48. Babbitt DG, Virmani R, Forman MB. Intracoronary adenosine administered after reperfusion limits vascular injury after prolonged ischemia in the canine model. Circulation. 1989;80:1388-1399. [Abstract/Free Full Text]
    
49. Bajaj AK, Cobb MA, Virmani R, Gay JC, Light RT, Forman MB. Limitation of myocardial reperfusion injury by intravenous perfluorochemicals: role of neutrophil activation. Circulation. 1989;79:645-656. [Abstract/Free Full Text]
    
50. Popma JJ, Topol EJ. Adjuncts to thrombolysis for myocardial reperfusion. Ann Intern Med. 1991;115:34-44.
    

This article has been cited by other articles:

				C. Duilio, G. Ambrosio, P. Kuppusamy, A. DiPaula, L. C. Becker, and J. L. Zweier Neutrophils are primary source of O2 radicals during reperfusion after prolonged myocardial ischemia Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2649 - H2657. [Abstract] [Full Text] [PDF]

				M. L. Schermerhorn, D. P. Nelson, E. D. Blume, L. Phillips, and J. E. Mayer Jr Sialyl LewisX oligosaccharide preserves myocardial and endothelial function during cardioplegic ischemia Ann. Thorac. Surg., September 1, 2000; 70(3): 890 - 894. [Abstract] [Full Text] [PDF]

				M. L. Schermerhorn, M. Tofukuji, P. R. Khoury, L. Phillips, P. R. Hickey, F. W. Sellke, J. E. Mayer Jr, and D. P. Nelson Sialyl LewisX oligosaccharide preserves cardiopulmonary and endothelial function after hypothermic circulatory arrest in lambs J. Thorac. Cardiovasc. Surg., August 1, 2000; 120(2): 230 - 237. [Abstract] [Full Text] [PDF]

				K. Jaakkola, S. Jalkanen, K. Kaunismaki, E. Vanttinen, P. Saukko, K. Alanen, M. Kallajoki, L.-M. Voipio-Pulkki, and M. Salmi Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-Selectin mediate leukocyte binding to ischemic heart in humans J. Am. Coll. Cardiol., July 1, 2000; 36(1): 122 - 129. [Abstract] [Full Text] [PDF]

				K. M. KERR, W. R. AUGER, J. J. MARSH, R. M. COMITO, R. L. FEDULLO, G. J. SMITS, D. P. KAPELANSKI, P. F. FEDULLO, R. N. CHANNICK, S. W. JAMIESON, et al. The Use of Cylexin (CY-1503) in Prevention of Reperfusion Lung Injury in Patients Undergoing Pulmonary Thromboendarterectomy Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 14 - 20. [Abstract] [Full Text]

				T. Shin'oka, M. Nagashima, G. Nollert, D. Shum-Tim, P. C. Laussen, H. G. W. Lidov, A. d. Plessis, and R. A. Jonas A NOVEL SIALYL LEWIS X ANALOG ATTENUATES CEREBRAL INJURY AFTER DEEP HYPOTHERMIC CIRCULATORY ARREST J. Thorac. Cardiovasc. Surg., June 1, 1999; 117(6): 1204 - 1211. [Abstract] [Full Text] [PDF]

				M. J. Claeys, J. Bosmans, L. Veenstra, P. Jorens, Herbert De Raedt, and C. J. Vrints Determinants and Prognostic Implications of Persistent ST-Segment Elevation After Primary Angioplasty for Acute Myocardial Infarction : Importance of Microvascular Reperfusion Injury on Clinical Outcome Circulation, April 20, 1999; 99(15): 1972 - 1977. [Abstract] [Full Text] [PDF]

				A. Kumar, M. P. Villani, U. K. Patel, J. C. Keith Jr, and R. G. Schaub Recombinant Soluble Form of PSGL-1 Accelerates Thrombolysis and Prevents Reocclusion in a Porcine Model Circulation, March 16, 1999; 99(10): 1363 - 1369. [Abstract] [Full Text] [PDF]

				Y. Merhi, P. Provost, P. Chauvet, J.-F. Theoret, M. L. Phillips, and J.-G. Latour Selectin Blockade Reduces Neutrophil Interaction With Platelets at the Site of Deep Arterial Injury by Angioplasty in Pigs Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 372 - 377. [Abstract] [Full Text] [PDF]

				A. J. Palazzo, S. P. Jones, D. C. Anderson, D. N. Granger, and D. J. Lefer Coronary endothelial P-selectin in pathogenesis of myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1865 - H1872. [Abstract] [Full Text] [PDF]

				A. Kumar, J. L. Hoover, C. A. Simmons, V. Lindner, and R. J. Shebuski Remodeling and Neointimal Formation in the Carotid Artery of Normal and P-Selectin–Deficient Mice Circulation, December 16, 1997; 96(12): 4333 - 4342. [Abstract] [Full Text]

				C. Cobbaert, W. Th. Hermens, P. P. Kint, P. J. Klootwijk, F. Van de Werf, and M. L. Simoons Thrombolysis-induced coronary reperfusion causes acute and massive interstitial release of cardiac muscle cell proteins Cardiovasc Res, January 1, 1997; 33(1): 147 - 155. [Abstract] [Full Text] [PDF]

				T. Miura, D. P. Nelson, M. L. Schermerhorn, T. Shin'oka, G. Zund, P. R. Hickey, E. J. Neufeld, and J. E. Mayer Jr Blockade of Selectin-Mediated Leukocyte Adhesion Improves Postischemic Function in Lamb Hearts Ann. Thorac. Surg., November 1, 1996; 62(5): 1295 - 1300. [Abstract] [Full Text]

				M. C.G. Horrigan, A. I. MacIsaac, F. A. Nicolini, D. G. Vince, P. Lee, S. G. Ellis, and E. J. Topol Reduction in Myocardial Infarct Size by Basic Fibroblast Growth Factor After Temporary Coronary Occlusion in a Canine Model Circulation, October 15, 1996; 94(8): 1927 - 1933. [Abstract] [Full Text]