(Circulation. 1996;94:3327-3333.)
© 1996 American Heart Association, Inc.
Articles |
Antibody to Platelet/Endothelial Cell Adhesion Molecule-1 Reduces Myocardial Infarct Size in a Rat Model of Ischemia-Reperfusion Injury
the Medical College of Wisconsin, Departments of Cellular Biology and Anatomy (R.J.G., P.J.N.), Pharmacology (J.E.S., Z.Y., D.C.W., G.J.G, P.J.N.), Anesthesiology (D.C.W.), and Medicine, Division of Cardiology (D.K., D.C.W.), and the Blood Research Institute, the Blood Center of Southeastern Wisconsin (R.J.G., D.K., P.J.N.).
Correspondence to Garrett J. Gross, PhD, Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail gjgross@post.its.mcw.edu.
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Abstract |
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Background Antibodies to selected neutrophil or endothelial cell adhesion molecules decrease myocardial infarct size in vivo. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is an immunoglobulin gene superfamily member expressed constitutively on neutrophils and endothelium. F(ab')2 fragments of antibody against PECAM-1 inhibit transendothelial migration of neutrophils in several in vivo models of acute inflammation. Therefore, we examined the effect of F(ab')2 fragments of anti–PECAM-1 antibody in a rat model of myocardial infarction.
Methods and Results F(ab')2 fragments of the anti–PECAM-1 antibody SEW16 and control normal rabbit IgG (NRIgG) were administered at 5 mg/kg to male Wistar rats, and the rats were subjected to a 30-minute coronary artery occlusion followed by 2 hours of reperfusion. At the completion of each experiment, the area at risk, infarct size (IS), and myeloperoxidase (MPO) activity were determined. Compared with untreated (n=8; IS, 57±5%) or NRIgG-treated (n=10; IS, 62±3%) control rats, SEW16-treated rats (n=15; IS, 28.5±4%) displayed a 54% decrease in myocardial infarct size (P<.001). Hemodynamic parameters, leukocyte counts, total left ventricular weight, and area-at-risk weights did not differ significantly between the treatment groups. However, measurement of MPO activity revealed that neutrophil accumulation was reduced 83% (NRIgG, 975±55 mU/g; SEW16, 167±62 mU/g).
Conclusions These results demonstrate that blocking PECAM-1 exerts a significant protective effect in a rat model of myocardial ischemia-reperfusion injury via blockade of neutrophil accumulation in the myocardium.
Key Words: endothelium • ischemia • leukocytes • reperfusion • myocardial infarction
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Introduction |
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Platelet/endothelial cell adhesion molecule-1 (CD31) is a 130-kD member of the immunoglobulin gene superfamily1 2 whose expression is restricted exclusively to certain cells of the vasculature. On endothelial cells, PECAM-1 is expressed constitutively at
1x106 molecules per cell, making PECAM-1 the most highly expressed adhesion molecule on the endothelial cell surface.3 Furthermore, PECAM-1 is expressed constitutively on circulating platelets, monocytes, neutrophils, certain lymphocyte subsets, and megakaryocytic cell lines.3 4 In vitro studies have demonstrated that PECAM-1–mediated cellular interactions are unique among members of the immunoglobulin gene superfamily in that they can be either homophilic or heterophilic in nature.2 5 6 7 On the basis of the large concentration of PECAM-1 at the endothelial intercellular junction, its expression on leukocytes, and its adhesive properties, several groups have hypothesized that leukocytes may utilize endothelial cell PECAM-1 during the process of extravasation. Early studies revealed that certain monoclonal antibodies against PECAM-1 block both neutrophil and monocyte migration through a modified Boyden chamber membrane.8 More recently, Muller et al9 showed that monoclonal antibodies against PECAM-1, as well as recombinant soluble PECAM-1, inhibit both monocyte and neutrophil migration in an in vitro model of transendothelial cell migration. Finally, in vivo studies have demonstrated that both intact and F(ab')2 fragments of antibody specific for PECAM-1 inhibit neutrophil accumulation in several models of acute inflammation, including a peritonitis model as well as an adult respiratory distress syndrome model.10 11 Thus, both in vitro and in vivo studies have illustrated that PECAM-1 is one of a growing number of cell adhesion molecules that function in leukocyte–endothelial cell interactions and probably plays a central role in acute inflammatory processes. Leukocyte interactions with the endothelium are central to the process of acute inflammation. Numerous studies suggest that leukocyte interactions with the endothelium occur as an ordered series of adhesive events that are mediated by distinct adhesion receptor/ligand pairs12 13 14 and result in the rolling of leukocytes along the activated endothelium, a process that is regulated by selectin interactions.15 16 17 Subsequently, a tight adherence of leukocytes to the endothelium occurs, a process regulated by ß2-integrin complex–ICAM-1 interactions,12 13 17 18 19 20 21 22 which finally culminates in the migration of neutrophils between the endothelial cell-cell junction into the extravascular space,22 a process in which PECAM-1 appears to be intimately involved.9 10 11
Ischemia-reperfusion–induced myocardial injury represents an acute inflammatory response in which a growing number of leukocyte and endothelial cell adhesion receptor/ligand pairs have been implicated in regulating neutrophil-mediated damage to the myocardium.23 24 25 26 In vivo administration of antibodies specific for either P-selectin or L-selectin,27 28 29 30 CD11b,31 32 CD18,32 33 34 35 36 and ICAM-132 37 38 39 has been shown to decrease neutrophil accumulation in the myocardium or block neutrophil adherence to ischemic and reperfused vessels, decrease ischemia-reperfusion–induced endothelial cell dysfunction, and reduce myocardial infarct size.
Unlike many of the cell adhesion molecules examined in myocardial ischemia-reperfusion injury, PECAM-1 is expressed constitutively on the surfaces of both leukocytes and endothelial cells. Thus, PECAM-1 is uniquely positioned to mediate the adhesive events that take place immediately after ischemia-reperfusion–mediated myocardial injury. Furthermore, because the adhesive events mediated by PECAM-1 appear to be both heterophilic and homophilic in nature,5 6 7 it may be possible to block both sides of the PECAM-1 receptor/ligand pair with a single reagent. The effects of administration of anti–PECAM-1 antibodies in an in vivo model of myocardial infarction have not been examined to date. Therefore, on the basis of the known involvement of PECAM-1 in leukocyte–endothelial cell interactions, the purpose of the present study was to examine the effects of an anti–PECAM-1 antibody in a rat model of myocardial ischemia-reperfusion injury, expressed as myocardial infarct size. Our results demonstrate that blockade of PECAM-1–mediated cellular interactions significantly reduces myocardial infarct size by decreasing neutrophil accumulation in the myocardium.
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Preparation of F(ab')2 Fragments
F(ab')2 fragments of SEW1640 and NRIgG were prepared to prevent complement activation or activation of cells via Fc receptor in vivo. Briefly, total IgG was purified from rabbit serum via binding to protein A Sepharose. Total IgG was digested with pepsin by use of the Immunopure F(ab')2 preparation kit (Pierce Chemical Co) at an empirically determined concentration to generate F(ab')2 fragments. The F(ab')2 fragments were purified according to the manufacturer's protocol, examined by SDS-PAGE to verify removal of nondigested IgG and Fc fragments, and used in the subsequent experiments. Endotoxin levels were determined via the limulus amebocyte lysate test (Whittaker M.A. Bioproducts). Endotoxin levels in each preparation were equivalent (0.2 endotoxin unit per rat) and were below levels found to have an in vivo effect in the rat over the experimental period examined.41 42
Isolation of Rat Platelets and Leukocytes and Human Leukocytes
Rat platelets were isolated from 15 mL of whole blood as follows. Rat blood was diluted 1:2 in Phillips' buffer (in mmol/L: NaCl 96, glucose 8.5, EDTA 1, Tris 8.5). Samples were centrifuged in a Serofuge at 400g for 10 minutes to separate the blood into a lower red blood cell layer, a white blood cell interface, and upper platelet-rich plasma. The platelet-rich plasma was transferred to a new tube, PGE1 was added to a final concentration of 50 ng/mL, and the platelets were pelleted by centrifugation in a Serofuge at 800g for 10 minutes. Platelets were counted with a Coulter counter.
Rat mononuclear leukocytes were isolated from 30 mL of rat whole blood. The blood was diluted 1:1 with Phillips' buffer and layered over Fico-Lite LR (Atlanta Biologicals Inc). The sample was then centrifuged at 1000g for 20 minutes. The upper layer containing the platelets was removed, and the interface containing the mononuclear leukocytes was retained and diluted in three volumes of HBSS. The mononuclear leukocytes were isolated by centrifugation at 200g, then washed two more times to remove any residual Fico-Lite LR. The cells were counted with a hemocytometer.
Human leukocytes were isolated from 40 mL of human whole blood treated with 5% acid-citrate-dextrose anticoagulant. An equal volume of 3% dextran was added to sediment the red blood cells.43 The upper layer was then collected and centrifuged at 500g for 5 minutes at room temperature to pellet the leukocytes. Contaminating red blood cells were removed by hypotonic lysis in 0.2% saline. Leukocytes were resuspended in HBSS without calcium or magnesium and then were counted with a hemocytometer.
Flow Cytometric Analysis
For FACS analysis, platelets or leukocytes were diluted to 106 platelets/100 µL of PBS, pH 7.4, plus 0.5% BSA with 5 µg primary antibody [either intact or F(ab')2 fragments of NRIgG or SEW16 IgG] and incubated for 1 hour at 4°C. After incubation with the primary antibody, the platelets/cells were washed three times with ABB (PBS, pH 7.4/0.5% BSA/0.1% sodium azide) and then resuspended in 100 µL ABB+2 µg secondary antibody [FITC-conjugated goat anti-rabbit F(ab')2; Jackson Laboratory]. The platelets/cells were incubated with the secondary antibody at 4°C for 1 hour, washed three times with ABB, resuspended in 1 mL ABB, and analyzed on a FACScan Instrument (Becton-Dickinson, Inc).
Biotinylation of Leukocyte Cell Surface Proteins
Rat or human leukocytes were resuspended to a concentration of 1x108/mL in 0.01 mol/L PBS, pH 7.4/5 mmol/L NHS-LC-Biotin (Pierce). After incubation on ice for 30 minutes, the leukocytes were washed three times with 0.01 mol/L PBS, pH 7.4. Leukocytes were resuspended in 0.3 mL of lysis buffer (20 mmol/L Tris, pH 7.4, 2 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, 2 mmol/L PMSF, 30 µmol/L antipain, 20 µmol/L leupeptin, 1 mmol/L benzamidine HCl, 1 mmol/L PefaBloc [Boehringer Mannheim Inc], and 2 mmol/L DFP) and incubated on ice for 30 minutes with frequent vortexing. After centrifugation at 12 000g for 15 minutes, the supernatant was transferred to a fresh tube, and the protein concentration was determined by the BCA Protein Assay (Pierce, Inc). The lysate was stored at -80°C until immunoprecipitations were performed.
Immunoprecipitation of Leukocyte PECAM-1
Biotinylated leukocyte lysates (human or rat) were precleared by incubation of 50 µL lysate with 20 µL of a 50% slurry of protein A Sepharose beads and 80 µL of immunoprecipitation buffer (50 mmol/L Tris, pH 7.4, 2 mmol/L EDTA, 150 mmol/L NaCl, 1% Triton X-100, 0.2 mmol/L PMSF) (without Triton X-100) with 10 µg NRIgG at 4°C for 3 hours. After centrifugation at 10 000g for 2 minutes, the precleared lysate was transferred to a fresh Eppendorf tube, and 25 µL of a 50% slurry of SEW16-conjugated protein A Sepharose beads was added. The mixture was incubated at 4°C for 2 hours and centrifuged, and the supernatant was removed. The protein A Sepharose beads were washed five times with IPB, boiled in 30 µL of 2x reducing buffer (100 mmol/L Tris, pH 6.8, 10% ß-mercaptoethanol, 10% glycerol, 4% SDS, 0.004% bromophenol blue), and centrifuged, and the supernatant was loaded onto a 7% polyacrylamide gel. After SDS-PAGE, the proteins were transferred to a nylon membrane and detected by streptavidin-conjugated horseradish peroxidase with the ECL kit (Amersham, Inc) followed by autoradiography.
Surgical Preparation and Experimental Protocol
Male Wistar rats (340 to 450 g) were anesthetized with thiopentobarbital (Inactin, 1 mg/kg IP). Catheters were placed in the right carotid artery to monitor mean arterial blood pressure and obtain blood samples and the right jugular vein for administration of fluids and antibody. Heart rate was determined from the arterial blood pressure tracings. Rats were ventilated on a Harvard rodent respirator (model 70) at 70 breaths per minute and a tidal volume of 2.5 mL per breath. Coronary arteries were ligated by previously described techniques.44 Briefly, a thoracotomy was performed in the fifth intercostal space, and the heart was exposed. A silk suture was placed around the main coronary artery, and a snare was used for producing coronary artery occlusion and reperfusion. F(ab')2 fragments of either NRIgG or a polyclonal anti–PECAM-1 preparation were administered in a total dose of 5 mg/kg IV in a blinded fashion. In addition, untreated control animals were subjected to the same experimental protocol. The experimental protocol is shown schematically in Fig 1
. An arterial blood sample was obtained for baseline blood gas measurement and total and differential leukocyte counts. A bolus dose of 2.5 mg/kg of F(ab')2 fragments was administered 15 minutes before coronary artery occlusion. The vessel was occluded for 30 minutes, and during the last 15 minutes of occlusion, a second dose (2.5 mg/kg) of F(ab')2 fragments was administered. At the end of 30 minutes of occlusion, the snare was loosened to restore flow, and the area was reperfused for 2 hours. After 1 hour of reperfusion, another blood sample was obtained for blood gas analysis and total and differential leukocyte counts. On completion of the 2-hour reperfusion period, the coronary artery was reoccluded, and patent blue dye was injected via the jugular cannula to differentiate the AAR. The animal was killed by potassium chloride injection, and the heart was excised rapidly and rinsed in PBS.
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Determination of Myocardial Ischemic Injury
Ischemic injury was measured by TTC staining and expressed as myocardial infarct size. Briefly, the left ventricle was sliced transversely into five sections and stained with 1% TTC in 0.1 mol/L PBS, pH 7.4, at 37°C for 30 minutes. TTC stains noninfarcted myocardium a brick red color, indicating the presence of a formazin precipitate that results from the reduction of TTC by dehydrogenase enzymes present in viable tissue.45 The AAR was carefully separated from the rest of the left ventricle under a dissecting microscope. Samples used for the determination of MPO activity were processed immediately; all other samples were placed in 10% formaldehyde overnight. The total left ventricular weight, the AAR weight, and the infarcted tissue weight were determined the following day. The AAR is reported as a percentage of the total left ventricular weight and infarct size as a percentage of the AAR.
Determination of MPO Activity
Neutrophil accumulation in the myocardium was measured by determining the activity of MPO, an enzyme specific for neutrophils. This method has been commonly used to assess neutrophil accumulation46 47 and has been validated in cardiac tissue from the rat.48 Briefly, tissue was stored at -70°C until it was homogenized in 0.5% hexadecyltrimethyl ammonium bromide (Sigma). The sample was then dissolved in 50 mmol potassium phosphate buffer at pH 6.0 with a Polytron (PCU-2) homogenizer for 15 seconds at 7000 rpm two separate times. The resulting homogenate was centrifuged at 12 000g for 20 minutes at 4°C. The supernatants were transferred, and 0.167 mg/mL O-dianisodine dihydrochloride (Sigma) and 0.005% H2O2 in 50 mmol/L PBS, pH 6.0, were added. The change in absorbance at 460 nm was measured spectrophotometrically. One unit of MPO activity is defined as the quantity of enzyme degrading 1 µmol peroxide/min at 25°C. The data are expressed as MPO activity (milliunits) per gram tissue.
Statistical Analysis
All values are expressed as mean±SEM. Changes in hemodynamics, leukocyte counts, and infarct size were compared by ANOVA; a two-way ANOVA and Fisher's least significant difference were used to compare differences between treatment groups, and one-way ANOVA with repeated measures followed by a Dunnett's t test was used to compare values within individual groups with their respective baseline values. Changes in MPO activity between NRIgG- and SEW16-treated groups in the nonischemic control area and the AAR for infarction were compared by an unpaired t test. A value of P<.05 was considered statistically significant.
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Characterization of Antibodies That React With Rat PECAM-1
We have previously reported the production and characterization of rabbit polyclonal antibodies generated by immunization with purified human PECAM-1.40 Because monoclonal antibodies to rat PECAM-1 are unavailable, we took advantage of the cross-reactivity to rat PECAM-1 in SEW16. As shown in Fig 2A
, SEW16 cross-reacted with PECAM-1 expressed on rat platelets via flow cytometric analysis. To confirm the cross-reactivity of the polyclonal anti–PECAM-1 IgG, immunoprecipitation of leukocyte lysates was also conducted. As shown in Fig 2B, SEW16 reacted with rat PECAM-1. Rat PECAM-1 exhibited a slightly different mobility from that of the human platelet PECAM-1: a single 120-kD band was observed in the rat, compared with the 140- and 120-kD bands typical of human PECAM-1.2 PECAM-1 has been shown to be heavily glycosylated40 ; thus, the apparent difference in the molecular size of rat versus human PECAM-1 is most likely due to variance in glycosylation. Finally, as shown in Fig 2C, flow cytometric analysis revealed that the F(ab')2 fragments of SEW16 IgG used in our experiments cross-reacted with rat leukocytes, whereas the NRIgG exhibited no reactivity with rat leukocytes, as expected. The flow cytometric analysis revealed two populations of cells representing rat neutrophils and lymphocytes, respectively.
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Effect of Anti–PECAM-1 Antibodies on Myocardial Ischemia-Reperfusion Injury
We next examined the effect of F(ab')2 fragments of SEW16 in a rat model of ischemia-reperfusion–induced myocardial infarction. In a blinded study, animals were administered a total dose of 5 mg/kg (2.5 mg/kg per time point); 10 rats received F(ab')2 fragments of NRIgG, and 15 rats received F(ab')2 fragments of SEW16. A total of 8 rats were treated with an equivalent volume of normal saline at each time point to yield the untreated control group. Total white blood cell and differential counts were determined before administration of the first dose of F(ab')2 and 1 hour after the onset of reperfusion. No significant differences in total white blood cell and differential counts were observed between the two measurements or between the treatment groups in the animals examined (Table 1). Hemodynamic data for the untreated control rats (n=8), the NRIgG F(ab')2–treated rats (n=6), and the SEW16 F(ab')2–treated rats (n=10) are summarized in Table 2. There were no significant differences in heart rate, mean arterial blood pressure, or the rate-pressure product between the three groups. Although ventricular ectopy was common during the occlusion and reperfusion periods, only 5 rats (not included in the data) died before completion of the full experimental protocol. There were no significant differences in the total left ventricular weights or the AARs between treatment groups. The average AAR was 44.3±5.7% of the left ventricular mass for the untreated control rats, 35.5±3.0% of the left ventricular mass for the NRIgG-treated rats, and 43.5±3.0% of the left ventricular mass for the SEW16-treated rats, with no significant differences between groups (Fig 3A). However, comparison of myocardial infarct size, as shown in Fig 3B, was significantly different when the untreated control group and the NRIgG-treated control group were compared with the SEW16-treated group. Untreated control rats exhibited an infarct size (56.4±4.7% of the AAR) similar to that of rats treated with NRIgG (62±2.7% of the AAR). However, treatment with F(ab')2 fragments of SEW16 resulted in a significant decrease (P<.001) in infarct size to 28.5±3.8%, representing a 50% to 54% reduction in the extent of infarction compared with treatment with NRIgG or saline, respectively. Comparable results were obtained when the infarct size was expressed as a percentage of total left ventricular weight (data not shown). Thus, treatment with F(ab')2 fragments of an antibody to PECAM-1 appears to protect against a significant portion of ischemia-reperfusion–induced myocardial damage.
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To examine the mechanism by which F(ab')2 fragments of an antibody to PECAM-1 protect against ischemia-reperfusion–induced myocardial damage, we analyzed MPO activity, a marker enzyme commonly used to assess neutrophil accumulation.46 47 48 As shown in Fig 4, in the nonischemic control area, MPO activity was not significantly different between the NRIgG-treated (n=4) and the anti–PECAM-1–treated (n=6) animals. However, as shown in Fig 4, rats treated with F(ab')2 fragments of NRIgG exhibited MPO activity in the AAR of 995±55 mU/g, whereas animals treated with F(ab')2 fragments of an antibody to PECAM-1 exhibited an 83% reduction in MPO activity to 167±62 mU/g in the AAR (P<.001). Thus, treatment with F(ab')2 fragments of an antibody to PECAM-1 protects against ischemia-reperfusion–induced myocardial damage by inhibiting neutrophil accumulation in the myocardium.
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Discussion |
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On the basis of the large concentration of PECAM-1 found at the endothelial intercellular junction, its expression on leukocytes, and its multiple adhesive properties, PECAM-1 appears to be uniquely positioned to mediate the adhesive events that take place acutely after ischemia-reperfusion–mediated injury. In the present study, we found that administration of F(ab')2 fragments of an antibody to PECAM-1 significantly decreased myocardial infarct size in a rat model of ischemia-reperfusion injury. Treatment with F(ab')2 fragments of SEW16 preserved 50% to 54% of the ischemic and reperfused myocardium compared with the control groups. Interestingly, the reduction of myocardial infarct size produced by treatment with F(ab')2 fragments of antibody against PECAM-1 is comparable to that observed in a number of studies examining the effects of administration of antibody to various cell adhesion molecules in other models of myocardial ischemia-reperfusion injury.25 26 27 28 29 30 31 32 33 34 35 36 37 Because leukocyte counts, the size of the AARs for development of infarction, and the hemodynamic factors did not vary significantly between treatment groups, it is unlikely that the protective effect observed was due to differing intensities of ischemia during coronary occlusion or altered myocardial oxygen supply or demand between the groups or to clearing of leukocytes. Further examination revealed that blockade of PECAM-1–mediated cellular interactions significantly reduced neutrophil accumulation in the ischemic-reperfused myocardium to 17% of the control value. Thus, the mechanism by which F(ab')2 fragments of antibody against PECAM-1 exert a protective effect in myocardial ischemia-reperfusion injury is most likely the result of blocking neutrophil–endothelial cell interactions.
Although early reperfusion of ischemic myocardium leads to salvage of the jeopardized tissue, it has been demonstrated that reperfusion itself may exacerbate the injury sustained during the ischemic period.23 24 25 26 Reperfusion leads to an influx of leukocytes and the release of various inflammatory mediators such as oxygen radicals, cytokines, and chemokines.23 24 25 26 On stimulation via these mediators, the endothelium undergoes phenotypic changes, including the expression of adhesive receptors, such as P-selectin27 and ICAM-1,49 that serve to recruit leukocytes and platelets to the site of injury.12 13 14 Examination of ischemia and reperfused vessels reveals that a significant increase in leukocyte adhesion to the endothelium occurs compared with control vessels.27 28 29 30 Activated endothelial cells also release several factors, such as interleukin-8 and PAF, that act as either chemotactic or activating factors for neutrophils.50 PAF has been shown to increase after myocardial ischemia-reperfusion injury and appears to increase neutrophil adherence by upregulating CD11b/CD18 on neutrophils50 51 and to stimulate neutrophil H2O2 production.52 53 In vivo administration of inhibitors of PAF to rats has been demonstrated to decrease myocardial infarct size in a 6-hour permanent coronary artery occlusion model.54 In a feline myocardial ischemia-reperfusion model, administration of a PAF antagonist was cardioprotective.55 These studies suggest that the cardioprotective effects afforded by anti–PECAM-1 antibodies are due to either direct effects on endothelial cells or blockade of specific leukocyte–endothelial cell interactions.
The molecular interactions between leukocytes and the vascular endothelium have been well studied and shown to involve a coordinated series of adhesive events between distinct receptor/ligand pairs that ultimately leads to extravasation of neutrophils.12 13 14 25 26 27 28 29 30 31 32 33 34 35 37 38 39 Activated endothelial cells express P-selectin on their surface,56 57 which interacts with its specific ligands on the leukocyte.16 58 This interaction causes leukocytes to marginate and roll along the endothelium, which, in a poorly understood mechanism, produces leukocyte activation. Activated leukocytes upregulate ß2-integrin complexes (CD11/CD18) on their surface that, with their endothelial cell counter-receptor, ICAM-1, mediates the tight adherence of leukocytes.17 19 20 21 The tight adherence between leukocytes and endothelial cells is then followed by the migration of leukocytes between the endothelial cell-cell junction into the extravascular space.22 PECAM-1 plays an intimate role in this process, since both in vitro and in vivo studies have demonstrated that antibodies specific for PECAM-1 can inhibit leukocyte migration across activated endothelial cell monolayers9 and extravasation.10 11 Thus, PECAM-1 is one of the cell adhesion receptor/ligand pairs involved in the process of leukocyte extravasation during an acute inflammatory response.
With knowledge of the adhesion molecules and adhesive events required for leukocyte–endothelial cell interactions12 13 14 and on the basis of early studies in which depletion of circulating neutrophils from the bloodstream resulted in a significant decrease in the extent of myocardial infarction after an ischemic insult,59 60 61 it was hypothesized that decreasing neutrophil migration into the injured myocardium via blockade of specific cell adhesion receptor/ligand pairs would decrease myocardial infarct size.27 28 29 30 31 32 33 34 35 37 38 39 A number of studies have examined the effect of in vivo administration of antibodies to several neutrophil/endothelial cell adhesion receptor/ligand pairs on ischemia-reperfusion–mediated myocardial damage. Antibodies to P-selectin and L-selectin27 28 29 30 and to CD11b,31 32 CD18,32 33 34 35 and ICAM-132 37 38 39 all have been shown to reduce myocardial infarct size in a variety of species. Two specific studies have demonstrated in a rat myocardial ischemia-reperfusion model similar to the one used in the present investigation that antibodies to CD11b, CD18, and ICAM-1 decrease myocardial infarct size by decreasing neutrophil accumulation in the myocardium.32 39 These observations support the hypothesis that the cardioprotective effects afforded by anti–PECAM-1 antibodies are due to the blockade of specific leukocyte–endothelial cell interactions, which results in a decreased accumulation of neutrophils in the damaged myocardium.
In conclusion, the present investigation demonstrates that administration of F(ab')2 fragments of antibody against PECAM-1 significantly decreases myocardial damage after ischemia-reperfusion injury by blocking neutrophil accumulation in the damaged myocardium. This represents the first in vivo study to examine the role of PECAM-1 in myocardial ischemia-reperfusion injury. The results reported and the scenarios discussed provide the basis for further examination of the role of PECAM-1 in ischemia-reperfusion–mediated myocardial injury. Inhibition of PECAM-1 interactions via agents that bind to or modulate the function of PECAM-1 may represent a novel adjunctive therapeutic approach in the current treatment of acute myocardial infarction.
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Acknowledgments |
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This work was supported in part by NIH grants HL-40926 (to Dr Newman) and HL-08311 (to Dr Gross) and a predoctoral fellowship to Dr Gumina from the American Heart Association, Wisconsin Affiliate. Dr Gumina is a trainee in the Medical Scientist Training Program at the Medical College of Wisconsin. Dr Newman is an Established Investigator (92001390) of the American Heart Association. The authors thank Kim Piotrowski, Anna Hsu, and Jeannine Moore for their technical assistance during the course of these investigations.
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Presented in part at the American Federation of Clinical Research Meeting, San Diego, Calif, May 5-8, 1995, and published in abstract form (J Invest Med. 1995;43:451A).
Received April 1, 1996; revision received July 9, 1996; accepted July 30, 1996.
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