REDUCTION OF RAT MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY BY A SYNTHETIC SELECTIN OLIGOPEPTIDE

JOURNAL OF PATHOLOGY, VOL. 178: 335-342 (1996)

REDUCTION OF RAT MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY BY A SYNTHETIC

SELECTIN OLIGOPEPTIDE YOSHINOR! SEKO*~$., YOSHIFUMI ENOKAWA*, TOHRU NAKAO~, HIDEO YAGITA~, KO OKUMURA? AND YOSHIO YAZAKI*

*Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; ?Department of Immunology, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan; $Institute for Adult Diseases, Asahi Life

Foundation, Shinjuku-ku, Tokyo, Japun STaisho PharmacruticaL Co., Ltd., Research Center, Omiya, Saitama, Japan

SUMMARY Neutrophils infiltrate into myocardial tissue subjected to ischaemia followed by reperfusion and play a major role in myocardial

reperfusion injury. The infiltration of neutrophils begins within 2 h after reperfusion, indicating the engagement of rapidly inducible adhesion molecules, such as P-selectin, on vascular endothelial cells of myocardial tissue. To investigate the essential role of P-selectin in myocardial reperfusion injury, this study examined the expression of P-selectin in rat hearts subjected to 30 min of ischaemia followed by reperfusion. The induction of P-selectin was also evaluated on the surface of cultured rat vascular endothelial cells subjected to 60 min of hypoxia, followed by reoxygenation in vituo. Finally, the effects of in vivo administration of a synthetic selectin oligopeptide on myocardial necrosis were analysed. Reperfusion of ischaemic myocardial tissue resulted in enhanced expression of P-selectin on the luminal surface of vascular endothelium and surface expression of P-selectin was induced on cultured vascular endothelial cells by hypoxialreoxygenation in vitro. The in vivo administration of a synthetic selectin oligopeptide significantly reduced the area of myocardial infarction produced by 30 min of ischaemia, followed by 48 h of reperfusion. These data offer therapeutic possibilities for acute myocardial infarction.

KEY WORDS-myocardial ischaemialreperfusion injury; selectins; oligopeptides; vascular endothelial cells; hypoxialreoxygenation

INTRODUCTION

I t is well known that neutrophils infiltrate into ischaemic, reperfused myocardial tissue and play a critical role in the development of reperfusion injury. ‘p6 Pre- vious studies7p9 from this and other laboratories showed that administration of antibodies against p2 integrins (CD 1 1, CD 18) or their ligand intercellular adhesion molecule- 1 (ICAM- 1) significantly reduced myocardial reperfusion injury. Because the infiltration of neutrophils begins within 2 h after r e p e r f u ~ i o n , ~ ~ ’ ~ it is strongly suggested that not only ICAM-1, which was shown to be induced on vascular endothelial cells (VECs) by about 8 h after reperfu~ion,~ but also rapidly inducible adhesion molecules, such as P-~e lec t in ,” -~~ are involved.

Lawrence et a1.I6 showed that neutrophils adhere to and roll on rapidly induced P-selectin on endothelial cells in the early stages of inflammation; this adhesion is then later strengthened through interaction of integrins on neutrophils with ICAM-1 on endothelial cells. Several studies17- l9 have shown that in vivo administration of anti-P-selectin monoclonal antibody (MAb) significantly reduces neutrophil-dependent tissue injury, indicating that the initial rolling attachment is a pre- requisite for firm adhesion and accumulation of neutrophils. There is, however, a risk of inducing serum sickness by the administration of animal immunoglobulin in human trials. Geng et aL20 demonstrated that several oligopeptides corresponding to the lectin domain

Addressee for correspondence: Yoshinori Seko, MD, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

0 1996 by John Wiley & Sons, Ltd. CCC 0022-341 7/96/030335-08

of human P-, E-, and L-selectin, which may represent contact sites for carbohydrate structures on target cells, inhibited neutrophil adhesion to immobilized P-selectin in vitro.

The purpose of the present study was to investigate in more detail the role of P-selectin in myocardial ischaemiah-eperfusion injury, using an in vivo rat model of ischaemialreperfusion, with corresponding in vitro studies.

MATERIALS AND METHODS Animals

Wistar rats (male, 250-280 g) and (five-day-old) were purchased from Charles River Japan Inc. (Tokyo, Japan).

Monoclonal antibody (MAb)

A mouse anti-human P-selectin MAb (clone PB1.3), which was shown to cross-react with rat P-selectin, was a generous gift from Sumitomo Pharmaceuticals Co., Ltd. (Osaka, Japan) and Cytel Corporation (San Diego, CA, U.S.A.).

Selectin oligopeptide

Recently, Geng et aLZ0 investigated whether or not selectin oligopeptides corresponding to residues 23-30, 54-63, and 70-79 of the N-terminal lectin domain of human P-, E-, and L-selectin, which may represent contact sites for carbohydrate structures on target cells, can inhibit neutrophil adhesion to P-selectin in vitro. The

Received 26 January 1995 Accepted 17 July I995

336 Y. SEKO ET AL.

23-30 peptide of E-selectin and the 54-63 peptide of E- and L-selectin were shown effectively to inhibit neutrophil adhesion to immobilized P-selectin. They were also shown effectively to inhibit adhesion of HL-60 promyelocytic cells to E-selectin-transfected COS-7 cells in vitro.*O The 23-30 peptide of E-selectin (YTHLVAIQ) was used in this study because its inhibitory effect is the most efficient and it dissolves easily in saline. The potency of the 23-30 peptide of P- and L-selectin (YTDLVAIQ; the sequences of both peptides are identical and differ from the sequence of the 23-30 peptide of E-selectin in only one amino acid) to inhibit neutrophil adhesion to immobilized P-selectin was less than that of the 23-30 peptide of E-selectin.20 The peptide was synthesized and purified by high performance liquid chromatography and its composition was confirmed by amino acid analysis. A control peptide was prepared, identical in amino acid composition to the authentic peptide, but with the sequence altered so that no residue was located in the same position or adjacent to the same residues as in the authentic sequence. The sequence of the control peptide was LQTAYHVI. A stock solution of the peptide was prepared in sterile saline at a concentration of 1 or 5 mg/nil.

Surgical pvepavation

Rats (male, 250-280 g) were subjected to coronary artery ligation by previously described techniques.2’ Briefly, rats were anaesthetized with sodium pento- barbital (40 mg/kg, intraperitoneally), intubated, and ventilated with room air (tidal volume: 20mVkg at rate of 60lmin) with a respirator (SN-480-7, Shinano Manufacturing Co., Ltd., Tokyo, Japan). After lateral thoracotomy and pericardiectomy, a 6-0 silk stitch was placed near the intramyocardial location of the left coronary artery beneath the left atrial appendage. We performed coronary artery occlusion by pressing a short length of tube over the ends of the suture and clamping it firmly against the heart. We achieved reperfusion by removing the clamp. The standard limb lead I1 electrocardiogram was monitored continuously. We confirmed the ischaemia and reperfusion of the regional myocardium by following the changes of the ST segment level on the electrocardiogram and observing the change in colour of the myocardium. After removing the clamp, the thoracotomy incision was closed and the rats were allowed to recover from surgical anaesthesia.

Pf*eparation of cultured vat cardiac V E G Rat cardiac VECs were isolated according to the

method of Issekutz and Wykretowicz.22 Briefly, hearts were removed from 5-day-old rats and chopped into small pieces, then repeatedly treated with trypsin and DNAse. After each incubation the tissue was allowed to settle and the cells in the supernatant were removed and suspended in Dulbecco’s modified Eagle’s medium (DME) containing 20 per cent fetal calf serum (FCS). Cells were cultured for 2 h in a tissue culture flask and the non-adherent cells, containing virtually all of the contaminating cardiac myocytes. were removed and

fresh medium was added. The identity of the cells was confirmed by their characteristic cobblestone morpho- logical appearance, as well as by immunofluorescence staining for Factor VIII-related antigen. Rat cardiac VECs were cultured in DME containing 10 per cent FCS in tissue culture chamber slides (Miles Inc., Diagnostics Div., Kankakee, IL, U.S.A.) or 98-well tissue culture plates for several days until they were 70-80 per cent confluent. After culturing in a new medium without FCS for 24 h, the cells were used for immunocytochemical study or cell enzyme-linked immunosorbent assay (ELISA).

Immunohistochemistry

Rat hearts were excised after 30 min of ischaemia followed by reperfusion for the time periods indicated. Cryostat sections of 6 pm thickness were prepared from freshly frozen left ventricles and fixed in 100 per cent acetone for 5 min. The sections were incubated in 0.3 per cent H,02 for 30 min and then blocked with 3 per cent goat serum. They were incubated sequentially with anti- P-selectin MAb, biotinylated anti-mouse IgC (Caltag Laboratories Inc., South San Francisco, CA, U.S.A.) and avidin-biotinylated peroxidase complex (ABC- immunoperoxidase kit, Vector Laboratories Inc., Burlingame, CA, U.S.A., according to the manufac- turer’s instructions). The sections were reacted with diaminobenzidine tetrahydrochloride (0.2 mg/ml) and then counterstained with haematoxylin.

Hypaxialveoxygenation

Hypoxia was achieved by using an anaerobic jar, AnaeroPack Series (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan), equipped with an AnaeroPack dispos- able 0, absorbing and CO, generating agent and an indicator to monitor oxygen depletion. The AnaeroPack Series jar is capable of depleting the concentration of O2 down to less than 0.1 per cent in 2 h and providing a 21 per cent CO, atmosphere. By placing a dish which contained serum-free medium, in an AnaeroPack jar overnight, the medium was balanced with the hypoxic atmosphere. Cultured rat cardiac VECs were subjected to hypoxic conditions by immediately replacing the medium with the hypoxic medium in an AnaeroPack jar. To maintain the hypoxic conditions, all the procedures were performed in a glove bag filled with 95 per cent N2 and 5 per cent CO,. After incubating in hypoxic conditions for 60 min, the cells were reoxygenated by imnie- diately replacing the hypoxic medium with a normoxic serum-free medium.

Immunocytochemistvy

Non-treated VECs and VECs subjected to 60 min of hypoxia followed by 60 rnin of reoxygenation were incubated with anti-P-selectin MAb (PB1.3) at 37°C for 30 min, washed in PBS, and then fixed in 95 per cent EtOH for 5 min. For the VECs subjected to hypoxia/ reoxygenation, the incubation with anti-P-selectin MAb was performed during the last 30 min of reoxygenation.

SELECTINS IN MYOCARDIAL REPERFUSION INJURY 337

The cells were incubated sequentially with biotinylated anti-mouse IgG (Caltag Laboratories Inc., South San Francisco, CA, U.S.A.) and avidin-biotinylated peroxidase complex (ABC-immunoperoxidase kit, Vector Laboratories), and then reacted with diaminobenzidine tetrahydrochloride. The cells were counterstained with haematoxylin.

Cell ELISA VECs on 98-well tissue culture plates were subjected

to 60 min of hypoxia followed by reoxygenation for 0, 30, 60, 90, 120, or 150 min. Cells of each group were incubated with anti-P-selectin MAb (PBl.3) for 30 rnin during the last 30 min of hypoxialreoxygenation, washed in 0.1 per cent Tween 20-PBS (T-PBS), and then fixed in 95 per cent EtOH for 5 min. The cells were incubated sequentially with biotinylated anti-mouse IgG (Caltag Laboratories Inc.) and avidin-biotinylated peroxidase complex (ABC-immunoperoxidase kit, Vector Labora- tories), and then reacted with ABTS (2,2'-azino-di-[3- ethylbenzthiazoline sulphonate]). The absorbance at 405 nm was determined.

In vivo selectin oligopeptide treatment study Forty-seven rats were assigned to the selectin oligo-

peptide treatment in a low dose or a high dose group, the control oligopeptide treatment in a low dose group, and the saline control group. The synthetic oligopeptide of E-selectin (2 mg/kg or 10 mg/kg of body weight) or the control oligopeptide (2 mg/kg) was administered via the left femoral vein 5 min before coronary artery occlusion. The same volume (50Opl) of saline was administered in the saline control group. After 30 rnin of ischaemia followed by 48 h of reperfusion, the left ventricle of each heart was excised and washed with phosphate-buffered saline (PBS) and cut transversely into six sections from the apex to the base. The tissue samples were then incubated in a 1 per cent triphenyl tetrazolium chloride (TTC) solution buffered in 0 . 2 ~ Tris buffer to pH 8.0 for 4 rnin at 25°C. With TTC, viable myocardium is stained brick red and infarcted myocardium fails to stain. The basal side of each slice was then photo- graphed with colour-positive film. From these photographs, we determined the area of infarction in each slice by computerized planimetry. The infarct size was then determined by adding all of the areas of infarction for each left ventricle. Infarct size =expressed as a percentage of the left ventricle.

Statistical analyses All data were expressed as meansf standard error

(SE). Statistical comparison was made with Wilcoxon's two-sample rank-sum test (Mann-Whitney test), with P values corrected by the Bonferroni method. Values of P<0.05 were considered significant.

RESULTS Expression of P-selectin on vascular endothelium

There was almost undetectable expression of P-selectin on the luminal surface of vascular endothe-

lium in the left ventricle of sham-operated rats (Fig. 1A). Reperfusion for 2 h after 30 rnin of ischaemia significantly increased the expression of P-selectin on the luminal surface of vascular endothelium (Fig. lB, arrow-heads), resulting in the attachment of several neutrophils (Fig. lB, arrows). There was no staining without the first antibody on the luminal surface of vascular endothelium in a serial section, indicating the specificity of the reaction (Fig. 1C). The expression of P-selectin on the luminal surface of vascular endothelium was rather weak and partial, compared with that of ICAM-1 ,7 the reaction products being patchy rather than circumferential and distributed focally in the myocardium. We could observe the expression of P-selectin up to 10 h after the start of reperfusion.

Induction of P-selectin on the surface of cultured rat cardiac VECs by hypoxialreoxygenation in vitro

The induction of P-selectin was verified by immunoperoxidase, as shown in Fig. 2. There was almost no expression of P-selectin on the surface of untreated VECs (Fig. 2A), but significant expression was induced on reoxygenation for 60 min after 60 rnin of hypoxia (Fig. 2B). As shown in Fig. 3 , after the start of reoxygenation, the intensity of P-selectin expression increased rapidly to a maximum of 2.8-fold at 120 rnin of reoxygenation, then decreased rapidly within 150 min.

Reduction of myocardial infarct size by treatment with a synthetic selectin oligopeptide

Myocardial infarct size expressed as a percentage of the left ventricle was 38 per cent in the control oligopeptide-treated group and 17 per cent in the selectin oligopeptide-treated (10 mg/kg) group (Fig. 4). The mean ( f SE) infarct sizes in the selectin oligopeptide- treated groups were significantly smaller (2 mg/kg: 21.1 f 2.8 per cent, n= 15; 10 mg/kg: 17.6 f 2.8 per cent, n = 10) compared with the control oligopeptide-treated group (35.6 i~ 1.9 per cent, n= 10) (P<0.005; P<0.002). The mean ( f SE) infarct size in the saline control group was 36.3 k 1.3 per cent (n=12) (Fig. 5). Although the mean percentage area of myocardial infarction was smaller in the high-dose group, the differences between the two selectin oligopeptide-treated groups were not significant.

Figure 6 shows representative myocardial tissue around the necrosis in rats subjected to myocardial ischaemiaheperfusion. The density of neutrophil infiltration seemed to be higher in the saline control (Fig. 6A) than in the selectin oligopeptide-treated group (Fig. 6B). Because the area of myocardial necrosis was significantly lower in the selectin oligopeptide-treated groups, the total area of neutrophil infiltration was also significantly lower.

DISCUSSION

The present study, in keeping with our previous result^,^ showed that myocardial ischaemia/reperfusion

338 Y. SEKO ET AL.

Fig. l~lmmunohistochemical study of P-selectin expression. (A) Left ventricular myocardium of a sham-operated rat. There was almost no expression of P-selectin on the luminal surface of vascular endothelium. (B, C ) Serial sections of left ventricular myocardium subjected to 30 min of ischaemia followed by 2 h of reperfusion. Note the clear expression of P-selectin on the luminal surface of some parts of the vascular endothelium (B, arrow-heads), associated with the attachment of several neutrophils (B, arrows). Immunoperoxidase staining using anti-P-selectin MAb (PBl.3) (A, B) or without the first antibody (C). (All magnifications x 400)

SELECTINS IN MYOCARDIAL REPERFUSION INJURY 339

Fig. 2-Immunocytochemical study of P-selectin expression on the surface of cultured rat cardiac vascular endothelial cells (VECs). Untreated VECs (A) and VECs subjected to 60 min of hypoxia followed by 60 min of reoxygenation (B) were stained with anti-P-selectin MAb (PB1.3) by immunoperoxidase. There was almost no expression of P-selectin on the surface of untreated VECs (A). Note that clear expression was induced on the surface of VECs by hypoxia-reoxygenation. ( X 200)

induced a rapid expression of P-selectin on the surface of VECs, followed by a gradual expression of ICAM-1. Surface expression of P-selectin was also induced on cultured VECs by hypoxialreoxygenation in vitro. We have also shown that myocardial ischaemidreperfusion injury can be significantly reduced by the in vivo

L?A (mean * SE)

0 0 0 30 60 90 120 150 (min)

Fig. 3-The time course of P-selectin expression on the surface of cultured rat cardiac vascular endothelial cells (VECs) subjected to 60 min of hypoxia followed by reoxygenation for 0, 30, 60,90, 120, or 150 min. The intensity was determined by ELISA. One representative result is shown here from three independent experiments. Mean ( f SE) from six samples

administration of a synthetic selectin oligopeptide, as well as by MAbs against cell-adhesion molecules of leukocyte integrin or immunoglobulin fa mi lie^.^

Rapid infiltration of neutrophils into reperfused tissue is known to play a pivotal role in reperfusion injury, but the mechanisms for the expression of P-selectin and ICAM-I are still unknown. There are at least two possibilities. First, the stimuli of hypoxia and reoxygenation may directly induce VECs to express these antigens. Second, cardiac myocytes or VECs subjected to ischaemiaheperfusion may produce chemotactic factors or cytokines such as interleukin-8 (IL-S), which induce neutrophil activation and infiltration; the cytokines released from the infiltrating neutrophils could then enhance the expression of P-selectin or ICAM-1 on VECs around the infiltrating neutrophils, in turn accel- erating neutrophil adhesion and accumulation. Recent s t ~ d i e s ~ ~ , ~ ~ have indeed demonstrated that neutrophil chemotactic factor (IL-8) locally produced by reperfused tissues plays a causal role in neutrophil infiltration and therefore in reperfusion injury, As for ICAM-1 expression, the second mechanism proposed above is thought to be consistent with the results of our previous s t ~ d y , ~ because neutrophil infiltration began in the very early stage after reperfusion, whereas ICAM- 1 expression on

340 Y. SEKO ET AL.

Fig. LMacroscopic view of sliced left ventricles after 30 min of ischaemia followed by 48 h of reperfusion. Normal myocardium is stained red by the triphenyl tetrazolium chloride (TTC) method and infarcted myocardium is not stained and therefore appears pale. The ‘Control’ is a case from the control oligopeplide-treated group (2 mg/kg) and the ‘Treated’ is a case from the selectin oligopeptide-treated (10 mg/kg) group. Myocardial infarct size is 38 and 17 per cent, respectively. ( x 3.1)

P < 0.002 - p c 0.005 40

Saline Control Selectin Selectin ZmgiKg 2mgKg 10mgKg

(n=12) (n=lO) (n=15) (n=lO)

Fig. 5-EEects of selectin oligopeptide treatment on myocardial reperfusion injury. The mean ( f SE) infarct sizes in the selectin oligopeptide-treated groups were significantly smaller (2 mg/kg: 21.1 + 2.8 per cent, n=15; 10 mg/kg: 17.6 f 2.8 per cent, n=10) compared with the control oligopeptide-treated group (35.6 i: 1.9 per cent, n= 10) (P<0.005; P<0.002, respectively). There was no significant difference between the two selectin oligopeptide-treated groups (2 mg/ kg vs. 10 mg/kg). The mean ( & SE) infarct size in the saline control group was 36.3 f 1.3 per cent (n=12)

VECs was enhanced later. Furthermore, in vivo administration of MAbs against cell-adhesion molecules reduced the area in which ICAM-1 expression was up-regulated. Recently, we have demonstrated enhanced expression of ICAM-1 in murine hearts with acute viral myoca rd i t i~ ,~~ which was considered to have been induced by the cytokines released from infiltrating natural killer cells.

P-selectin is expressed in Weibel-Palade bodies of VECs, is rapidly mobilized to the cell surface after activation, and mediates the binding between VECs and neutrophils at the site of in f lamrnat i~n . ’~- ’~ In the present study, using an in vitro model system of ischaemialreperfusion, we have shown that ischaemial reperfusion can directly induce the surface expression of P-selectin on VECs within I h. This contrasts sharply with the mechanism of the enhancement of ICAM-I expression and is also supported by a previous study26 showing that oxidants directly induce the expression of P-selectin on the surface of VECs, which results in enhanced neutrophil adhesion. These data strongly suggest that the early infiltration of neutrophils into reperfused myocardium is mainly mediated by the rapid expression of P-selectin. This adhesion is later strengthened through interaction of integrins on neutrophils

SELECTINS IN MYOCARDIAL REPERFUSION INJURY 34 1

Fig. &-Effects of selectin oligopeptide treatment on neutrophil infiltration. Left ventricular myocardium of a case from the saline control group (A) and from the selectin oligopeptide-treated (10 mg/kg) group (B). Haematoxylin and eosin

with ICAM-1 on VECs, which may lead to further neutrophil accumulation. The in vitro study of Lawrence and Springeri6 also supports this concept.

We have also demonstrated for the first time that the in vivo administration of a synthetic selectin oligopeptide significantly reduces reperfusion injury. Previous studies27 29 showed that the soluble form of P-selectin inhibits not only neutrophil adhesion to endothelium, but also superoxide anion release, serving as a regulator for the inadvertent activation of neutrophils in the circulation. The effects of in vivo selectin oligopeptide therapy thus seem to be due to the inhibition of neutrophil activation as well as accumulation. It is known that the lectin domains of the three members of the selectin family (P-, E-, and L-selectin) recognize carbohydrate ligands such as LewisX and sialyl Lewisx. Recently, Mulligan et uL30 and Buerke et ~ 1 . ~ ’ reported that in vivo administration of oligosaccharides coii- taining sialyl Lewisx, which is one of the ligands for P-~electin,’~-?~ significantly reduced P-selectin- dependent lung injury as well as myocardial reperfusion injury.

The selectin oligopeptides seem to inhibit neutrophil interaction with the target cells by binding to their carbohydrate ligands on neutrophils in the circulation. In the in vitro study,*O the 23-30 peptide of E-selectin was shown effectively to inhibit neutrophil adhesion to E-selectin as well as P-selectin. However, E-selectin seems to be less important in neutrophil infiltration and

accumulation in the early stage of reperfusion, because it requires several hours to be expressed on the vascular endothelial cell^.^^,'^ This was supported by the finding that anti-E-selectin MAb treatment did not protect against myocardial ischaemiaheperfusion injury in vivo.” Anti-L-selectin MAb treatment does, however, protect.3s Because the sequences of the 23-30 peptide of P- and L-selectin are identical, the 23-30 peptide of E-selectin may also inhibit neutrophil adhesion to VECs by binding to the L-selectin ligands on VECs in vivo. L-selectin, which is constitutively expressed on the surface of neutrophils, may also thus be involved in the in vivo effects of the 23-30 peptide of E-selectin.

Species-specific oligopeptide therapy against cell- adhesion molecules, unlike monoclonal antibodies, carries no risk of serum sickness. In combination with reperfusion therapy, such as percutaneous transluminal coronary angioplasty (PTCA) or percutaneous transluminal coronary recanalization (PTCR), selectin oligopeptide therapy for reperfusion injury may help to improve the course of acute myocardial infarction.

ACKNOWLEDGEMENTS We thank Mr Y. Iigo, Daiichi Seiyaku Co., Ltd.,

for preparing cultured rat cardiac VECs and Mr N. Machiyama, University of Tokyo Hospital, for taking photographs. We also thank Sumitomo Pharmaceuticals Co., Ltd. (Osaka, Japan) and Cytel Corporation (San

342 Y. SEKO ET AL.

17. Mulligan MS, Polley MJ, Bayer RJ, Nunn MF, Paulson JC, Ward PA. Neutrophil-dependent acute lung injury: requirement for P-selectin (GMP-

Diego, CA, U.S.A.) for anti-P-selectin MAb (PB1.3). 140). J Clin Invest 1992; 90: 1600 1607.

and a grant for intractable vasculitis from the Ministry 18. Weyrich AS, Ma XL, Lefer DJ. Albertine KH, Lefer AM. In v i v o neutral- ization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. J Clin Invesr 1993; 91: 2620-2629, of Health and Welfare, Japan; a grant from the Kowa

Life Science Foundation; a grant from the Ichiro 19. Winn RK, Liggitt D, Vedder NB, Paulson JC, Harlan J M . Anti-P-selectin Kanehara Foundation; a grant from the Kanae Foun- monoclonal antibody attenuates reperfusion injury to the rabbit ear. J Clin

Inicst 1993: 9 2 2042-2047. dation Of Research for New Medicine; and a grant from 20. Geng JG, Heavner GA, McEver RP. Lectin domain peptides from selectins the Japan Foundation of Cardiovascular Research. interact with both cell surface ligands and Ca2+ ions. J Eiol Chrni 1992; 267:

This work was supported by a grant for cardiomyopathy

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REDUCTION OF RAT MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY BY A SYNTHETIC SELECTIN OLIGOPEPTIDE