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Biochemical and Biophysical Research Communications 273, 772–775 (2000)

doi:10.1006/bbrc.2000.3010, available online at http://www.idealibrary.com on

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nhibition of Cyclooxygenase-2 Improves Cardiacunction in Myocardial Infarction

akayuki Saito,* Ian W. Rodger,† Fu Hu,* Hani Shennib,* and Adel Giaid*,1

Department of Pathology and †Department of Medicine, Montreal General Hospital and McGill University,ontreal, Quebec, Canada; ‡Center for Innovative Cardiovascular Therapy, Continuum Heart Institute,ew York, New York; and §Merck Frosst Canada Inc., New Jersey

eceived June 1, 2000

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Induction of cyclooxygenase-2 (COX-2) in ischemicyocardium is thought to increase the production of

roinflammatory prostanoids and contribute signifi-antly to the ischemic inflammation. Left ventricularyocardial infarction (MI) was created by ligating the

eft coronary artery in Lewis rats. Hemodynamic mea-urements at 4 weeks showed better cardiac functionn the group treated with a selective COX-2 inhibitorDFU; 5 mg/kg/day) for 2 weeks after induction of MIompared to the vehicle treated group. These resultsuggest that induction of COX-2 contributes to myo-ardial dysfunction, and that selective inhibition ofOX-2 could constitute an important therapeutic tar-et for the treatment of MI. © 2000 Academic Press

Key Words: cyclooxygenase-2; myocardial infarction;elective COX-2 inhibitor; rat.

Arachidonic acids released from cellular phospholip-ds by phospholipase A2 is converted by cyclooxygenaseCOX) activity to prostaglandin (PG) G2 then to PGH2.GH2 serves as a substrate for cell-specific isomerasesnd synthase to produce the PGs; PGE2, PGD2, pros-acyclin (PGI2), and thromboxaneA2 (TXA2) (1). It isnown that there are at least two related but distinctene products that possess COX activity, termedOX-1 and COX-2 (2–4). These two isoforms have dif-

erent physiologic and pathologic roles. The constitu-ive isoform, COX-1, presents in almost all tissues andells (5), and plays an important role in “house-eeping” functions, such as the maintenance vascularomeostasis and gastrointestinal integrity (6). In con-rast, the inducible isoform, COX-2, is more selectivelyistributed in several cell types such as macrophages,

1 To whom correspondence should be addressed at The Montrealeneral Hospital, Suite L3-314, 1650 Cedar Avenue, Montreal, Que-ec H3G 1A4, Canada. Fax: (514) 934 8296. E-mail: mdga@musica.cgill.ca.

772006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ighly inducible by cytokines, growth factors, hor-ones, and oncogenes (10). The induction of COX-2,ith resultant production of prostanoids, can contrib-te to inflammation, pain, parturition, and certainypes of cancer (11).

We have recently demonstrated induction of COX-2n the myocardium of patients with end-stage conges-ive heart failure, particularly in those with ischemiceart disease (12). However, the contribution of COX-2o cardiac dysfunction and remodeling associated withhronic heart failure has not been elucidated. The anti-nflammatory effects of selective COX-2 inhibitors haveeen well documented, and two classes of COX-2 inhib-tor have been administered to patients with rheuma-oid arthritis and/or osteoarthritis (13). Since inflam-ation constitutes an important feature of ischemic

eart failure, we hypothesized that inhibition of proin-ammatory prostanoids through use of a selectiveOX-2 inhibitor could significantly ameliorate myocar-ial injury and hence improve myocardial function.In this study, we examined and demonstrated the

fficacy of selective COX-2 inhibitor; 5,5-dimethyl-3-(3-uorophenyl)-4-(4-methyl-sulphonyl-2(5H)-fluranone

DFU) on the cardiac function after left coronary arteryigation, as a model of experimental myocardial infarc-ion.

ATERIALS AND METHODS

Experimental myocardial infarction. Twenty-one Lewis maleats weighing 280–320 g were used for this study. All animal workas performed in accordance with institutional guidelines, which is

ompliance with the “Guide for the Care and Use of Laboratorynimals,” published by the National Institutes of Health (NIH pub-

ication 85-23, revised 1985). Left ventricular free-wall MI was in-uced as previously described (14). In brief, each rat was anesthe-ized with isoflurane, intubated with a 16-gauge intravenousatheter and mechanically ventilated with room air by use of a smallodent ventilator at a rate of 80 cycles per minute and a tidal volumef 1 mL/100 g body weight. A left thoracotomy was performed in the

fourth intercostal space. After the pericardium was incised, the prox-iorcr

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Vol. 273, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mal portion of the left coronary artery was ligated with one suturef 5-0 silk. Apart from the coronary artery ligation, sham-operatedats underwent an identical procedure. Subsequently, the chest waslosed in three layers of 3-0 Vicryl (Ethicon, Somerville, NJ) and theats were allowed to recover.

Administration of selective COX-2 inhibitor. DFU (a gift fromerck Frosst Canada Inc.), which is an orally active and highly

elective COX-2 inhibitor (15), was dissolved in 1% methylcelluloseolution and administered to rats by gavage. Rats were divided intohree groups: sham-operated rats (sham, n 5 6), rats with MI andeceiving DFU (5 mg/kg/day) 30 min prior to ligation and continuedor 2 weeks after operation (DFU, n 5 7), and rats with MI andeceiving 1% methylcellulose solution 30 min prior to ligation andontinued for 2 weeks after operation (MI, n 5 8). The dosage ofFU was determined according to our previously published datahich demonstrated the selectivity and effectiveness of this dosage

15).

Hemodynamic measurements. After 4 weeks following MI orham surgery, animals were anesthetized as described above. Afteredian sternotomy, arterial pressure was measured by direct punc-

ure of ascending aorta using 20 gauge needle then the needle wasdvanced into the left ventricle to measure left ventricular systolicressure (LVSP), left ventricular end-diastolic pressure (LVEDP),nd first derivative of left ventricular pressure (6dp/dt). Centralenous pressure (CVP) was also measured by direct puncture of theight atrium.

General measurements. After hemodynamic measurement, heartnd lung were excised and weighed. Lung was dried at 37°C for 2eeks, and the dry weight was measured. Lung water content (%)as calculated as (wet weight-dry weight) 3 100/wet weight. Bodyeight gain was calculated by the difference between weight at the

ime of primary surgery and at the time of sacrifice divided by theuration of the study, and data were expressed as g/day.

Statistical analysis. All results are presented as mean 6 SEM.ne-way ANOVA followed by Fisher’s test was used for comparing

he differences among multiple groups with a commercial programStatview). Significant differences among groups were defined by aalue of P , 0.05.

ESULTS

Four weeks after ligation, administration of DFU forweeks, starting at 30 min before coronary artery

igation significantly reduced LVEDP compared to theehicle-treated animals (DFU 5.47 6 0.79 mmHg vs.I 21.38 6 2.21 mmHg, P , 0.001) (Fig. 1A). DFU-

reatment also significantly improved cardiac compli-nce shown as higher value of 2dp/dt (DFU 914 6 105mHg/s vs. MI 574 6 127 mmHg/s, P , 0.05) (Fig.

B). Interestingly, body weight gain after primary sur-ery was most significant in rats treated with DFUompared to others including sham animals (Fig. 1C).onceivably, this is a consequence of the analgesicffect of DFU. Positive dp/dt was also markedly im-roved by DFU-treatment (868 6 99 mmHg/s vs. 594 609 mmHg/s); however, these data didn’t reach statis-ical significance. Other parameters such as HR andAP were not significantly different among three

roups. CVP was significantly lower in sham groupompared to Vehicle-treated group (P , 0.01); how-

773

ver, there was no significant difference betweenehicle-treated and DFU-treated groups (Vehicle;.06 6 0.46 mmHg vs. DFU; 2.86 6 1.01 mmHg). Lungater content and heart weight/body weight ratio wereot significantly different among groups.

ISCUSSION

In the present study, we administered the selectiveOX-2 inhibitor (DFU) 30 min prior to- and continued

or the first 2 weeks after coronary artery ligation.ncidentally, the peak expression of myocardial COX-2mmunoreactivity is observed at 2 weeks after coro-ary artery ligation (data not shown). We hereinhowed that selective COX-2 inhibition with DFU sig-

FIG. 1. The effects of selective COX-2 inhibitor (DFU) adminis-ration. Hemodynamic measurement was employed under anestheticondition at 4 weeks after primary surgery. (A) left ventricularnd-diastolic pressure (LVEDP); (B) the negative first derivative ofeft ventricular pressure (2dp/dt); (C) average daily gain of bodyeight after primly surgery (BW gain). *, P , 0.01, **, P , 0.05.

nificantly reduced the elevated LVEDP and CVP, andica(Coith

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Vol. 273, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ncreased 2dp/dt. These parameters are indicators ofardiac functions, and also known to be significantlyltered in congestive heart failure. Indeed, our controlsham) animals showed significantly lower LVEDP andVP, and better cardiac compliance (dp/dt) than thosef vehicle treated animals. These data suggest thatncreased formation of proinflammatory prostanoidshrough COX-2 pathway contribute to experimentaleart failure due ischemic heart disease.We have previously shown that DFU inhibited the

rachidonic acid-dependent production of PGE2 with ateast a 1000-fold selectivity for COX-2 (IC50 5 41 6 14M) over COX-1 (IC50 . 50 mM) (15). The generationnd release of prostaglandins is known to induce ex-ression of several inflammatory and growth media-ors known to play a role in ischemic disease. Thus,nhibiting COX-2 derived prostanoids could lead to aeduction in the extent of inflammation and/or releasef inflammatory mediators known to cause exudation,njury, and tissue scaring (16, 17). Similarly, inhibitionf COX-2 may lead to a reduction in oxidant formation18). Finally, a reduction in the level of COX-2 derivedGs through selective inhibition of the enzyme maymeliorate some of the previously described direct ef-ects of PGs on the function of the myocardium.

Another interesting result seen in this study is thatnimals treated with the COX-2 inhibitor showedreater body weight gain following primary surgeryompared to vehicle-treated animals. There may bewo ways to explain these findings, one is the evidencehat selective COX-2 inhibitor has an analgesic effect19), and the other is a consequence of improving car-iac dysfunction. It is well recognized that the non-elective COX inhibitors, e.g., acetylsalicylic acid, alsoave an analgesic effect; however, no available dataave clearly shown the effectiveness on cardiac func-ion after myocardial infarction as was shown in ourtudy. Although it is well known that acetylsalicyliccid reduces the likelihood of re-infarction after MIresumably secondary to its anti-platelet effect, inhi-ition of constitutive production of prostanoids mayesult in sever complications such as bleeding, gastriclcer, and nephrotoxicity. In contrast to these negativeffects of non-selective COX inhibitor, effects of DFUppear to be specific to the heart since we saw noffects on other organs (kidneys and lungs; unpub-ished data). These observations are supported byther published reports on the use of the similar inhib-tor in other experimental settings in both human (20)nd animals (15, 21).In summary, our data presented herein implies that

nduction of COX-2 in ischemic myocardium and sub-equent inflammation were critical for cardiac deteri-ration, and demonstrates a selective COX-2 inhibitoravorably alters the adverse sequence of events. These

774

ic target for treatment of myocardial infarction in thelinical evaluation of the effectiveness of selectiveOX-2 inhibitor.

CKNOWLEDGMENTS

This work was supported by the Medical Research Council ofanada. Dr. Adel Giaid is supported by the Fonds de Recherche enante du Quebec.

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