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Cyclooxygenase-2 (COX-2) in acute myocardial infarction: cellular expression and use of selective COX-2 inhibitor1,2

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Takayuki Saito, Ian W. Rodger, Hani Shennib, Fu Hu, Lara Tayara, and Adel Giaid 0

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Abstract: Our previous work has shown strong expression of COX-2 in the myocardium of patients with end-stage ischemic heart failure. The purpose of this study was to determine the cellular expression of this enzyme in the setting of acute myocardial infarction (AMI) and determine the role of COX-2 in experimental animals using a selective COX2 inhibitor. Experimental AMI was induced in rats by ligating the left coronary artery. Animals were either treated with a selective COX-2 inhibitor (5 mg·kg–1·day–1) or vehicle. Three days after ligation, cardiac function was assessed and infarct size was determined. Myocardial specimens were immunostained with antiserum to COX-2. Plasma concentration of prostanoids was measured by enzyme immunoassay. There was strong expression of COX-2 in the myocytes, endocardium, vascular endothelial cells, and macrophages in the infarcted zone of the myocardium. In contrast, little expression was seen in the myocardium of control rats. Animals treated with the COX-2 inhibitor showed a significant improvement in left ventricular (LV) end-diastolic pressure (P < 0.05) and LV systolic pressure (P < 0.01), and a reduction in infarct size (P < 0.05). Inhibition of COX-2 significantly decreased plasma concentration of thromboxane B2 (P < 0.05); however, it did not affect 6-keto-prostaglandin F1α. Induction of COX-2 during AMI appears to contribute to myocardial injury, and treatment with the specific inhibitor of the enzyme ameliorated the course of the disease. Key words: cyclooxygenase-2, inhibitor, acute myocardial infarction. Résumé : Nos travaux antérieurs ont montré une forte expression de COX-2 dans le myocarde de patients atteints d’insuffisance cardiaque terminale. La présente étude a eu pour but de déterminer l’expression cellulaire de cette enzyme dans l’évolution de l’infarctus myocardique aigu (IMA) et d’évaluer son rôle chez des animaux expérimentaux en utilisant un inhibiteur sélectif. L’IMA expérimental a été induit chez les rats en ligaturant l’artère coronaire gauche. Les animaux ont été traités avec un inhibiteur sélectif de COX-2 (5 mg·kg–1·jour–1) ou avec un véhicule. Trois jours après la ligature, la fonction cardiaque a été évaluée et la taille de l’infarctus déterminée. Les spécimens myocardiques ont été immunomarquées avec un antisérum anti-COX-2. La concentration plasmatique des prostanoïdes a été mesurée par dosage immunoenzymatique. L’expression de COX-2 a été élevée dans les myocytes, le myocarde, les cellules endothéliales vasculaires et les macrophages dans la zone infarcie du myocarde. À l’inverse, l’expression a été faible dans le myocarde des rats témoins. Les animaux traités avec l’inhibiteur de COX-2 ont montré une amélioration significative de la pression télédiastolique ventriculaire gauche (VG) (P < 0,05) et de la pression systolique VG (P < 0,01), et une réduction de la taille de l’infarctus (P < 0,05). L’inhibition de COX-2 a diminué significativement la concentration plasmatique de thromboxane B2 (P < 0,05); toutefois, elle n’a pas eu d’effet sur la 6-kéto-prostaglandine F1α. L’induction de COX-2 durant un IMA semble contribuer à la lésion myocardique, et un traitement avec l’inhibiteur spécifique de l’enzyme a ralenti l’évolution de la maladie. Mots clés : cyclooxygénase-2, inhibiteur, infarctus myocardique aigu. [Traduit par la Rédaction]

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Received 14 May 2002. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 24 February 2003. 100

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T. Saito, F. Hu, L. Tayara, and A. Giaid.3,4 Department of Pathology, The Montreal General Hospital and McGill University, Montreal, QC, Canada. H. Shennib. Department of Surgery, The Montreal General Hospital and McGill University, Montreal, QC, Canada. I.W. Rodger. Merck Frosst Inc., White Lane, NJ.

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This paper has undergone the Journal’s usual peer review process. 2 Presented at the Satellite Meeting of the XVII World Congress of the International Society for Heart Research in the Cardiovascular System and Inflammatory Mediators, Montréal, Que., 12–14 July 2001. 3 Present address: The Montreal General Hospital and McGill University, 1650 Cedar Ave, Suite L3–314, Montreal, QC H3G 1A4, Canada. 4 Corresponding author (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 81: 114–119 (2003)

I:\cjpp\Cjpp-8102\Y03-023.vp Wednesday, February 19, 2003 9:55:36 AM

doi: 10.1139/Y03-023

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Introduction 75

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Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2, which serves as a substrate for cell specific isomerases and synthase to produce the various prostaglandins, prostacyclin, and thromboxanes (Smith 1992). COX exists in two isoforms (Funk et al. 1991; O’Banion et al. 1992; Xie et al. 1991), the constitutive isoform (COX-1) and the inducible isoform (COX-2). It was generally believed that COX-2 is stimulated by inflammatory and immunogenic mediators and is responsible for the pathophysiological effects of prostaglandins while COX-1 is mainly responsible for physiological effects of prostaglandins; however, recent data have shown that both enzymes are constitutively expressed in a large number of cells and both participate in inflammation (Wu 1995). Myocardial infarction (MI) is associated with local inflammatory response that accompanies the progression of the ischemic myocardial injury. There is cellular infiltration by platelets, leukocytes, and other inflammatory cells, which assist in phagocytosis but have the potential to damage non-necrotic myocardial tissue as well. Edema and loss of contractile function also occur. There are elements of MI that are known to be a potent stimulus for COX-2 expression (Schrör et al. 1998) and local prostaglandins generation and release (Levine et al. 1990). Indeed, we have recently demonstrated induction of COX-2 in the myocardium of patients with end-stage congestive heart failure (Wong et al. 1998), particularly in those with MI. To date, expression of this enzyme in the setting of AMI (acute MI) has not been addressed. We therefore sought to identify the expression of COX-2 in the heart of rats with AMI by immunohistochemistry. We hypothesize that induction of COX-2 plays an important role in the development of myocardial injury and dysfunction associated with AMI, and intervention in COX pathway by selective inhibition of COX-2 may ameliorate the course of the disease. In the present study, strong immunoreactivity for COX-2 was seen in the myocytes, macrophages, and microvascular endothelial cells in the infarcted myocardium. Animals treated with a COX-2 inhibitor showed a significant reduction in infarct size and an improvement of cardiac function.

Methods

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Experimental MI Lewis male rats weighing 270–320 g were used for this study. All animal work was performed in accordance with institutional guidelines, and in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication 85-23, revised 1996). Left ventricular free-wall MI was induced as described previously (Saito et al. 2000). In brief, each rat was anesthetized with isoflurane, intubated with a 16-gauge intravenous catheter, and mechanically ventilated with room air by use of a small rodent ventilator at a rate of 80 cycles per minute and a tidal volume of 1 mL/100 g body weight. A left thoracotomy was performed in the fourth intercostal space. After the pericardium was incised, the proximal portion of the left coronary artery was ligated with one suture of 5-0 silk. Apart from the coronary artery ligation, shamoperated rats underwent an identical procedure. Subse-

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quently, the chest was closed with three layers of 3–0 Vicryl (Ethicon, Somerville, N.J.) and the rats were allowed to recover. Administration of selective COX-2 inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-fluranone (DFU), a gift from Merck Frosst Canada Inc., which is an orally active and highly selective COX-2 inhibitor (Riendeau et al. 1997), was dissolved in 1% methylcellulose solution and administered to rats by oral gavage. Rats were divided into three groups: sham-operated rats (sham group, n = 7); rats with MI and receiving DFU (5 mg·kg–1·day–1) 30 min prior ligation and continued for three days after operation (DFU group, n = 12); and rats with MI and receiving methylcellulose solution in the same manner (vehicle group, n = 13). A recent pharmacological study has shown that DFU inhibited the arachidonic aciddependent production of PGE2 with at least 1000-fold selectivity for COX-2 (IC50 = 41 ± 14 nM) over COX-1 (IC50 > 50 µM) (Riendeau et al. 1997). It also showed that at least up to 10 mg/kg of DFU was effective in inhibiting the carrageenan-induced rat paw edema (ED50 of 1.1 mg/kg) and reversing LPS-induced pyrexia in rats (ED50 of 0.76 mg/kg).

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Hemodynamic measurements Three days following MI or sham surgery, animals were anesthetized as described above. A fluid catheter connected to a transducer was inserted into the right carotid artery. After measuring arterial blood pressure and heart rate, the catheter was advanced into the left ventricle (LV) and LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and first derivative of LV (LV ± dP/dt) were determined. The catheter was then re-inserted into the right jugular vein to measure central venous pressure (CVP) and right ventricular systolic pressure (RVSP). Mean arterial pressure (MAP) was calculated according to the following formula: diastolic arterial pressure + pulse pressure/3. Morphometric analysis After hemodynamic measurements, rats were sacrificed and hearts were excised. Coronary vascular beds and cardiac cavities were flushed with 4% phosphate buffered paraformaldehyde, and the ventricles were weighed. Then the ventricles were cut into three transverse sections of approximately identical thickness. Sections were dehydrated and embedded in paraffin. From these sections, histological slices 5 µm thick were obtained and stained with hematoxylin and eosin. Slides were examined by light microscope coupled to a computerized morphometric system (Image Pro Plus, Media Cybernetics, Md.). Infarcted myocardium was defined by loss of normal myocyte appearance and homogeneity and accumulation of inflammatory cells (Jennings et al. 1994). Infarct size (IS), infarct wall thickness (IWT), left ventricular diameter (LVD), and septal wall thickness (SWT) were morphologically measured as described elsewhere (Sandmann et al. 1998). Excised lung tissues from all animals were dried at 37°C for two weeks, followed by determination of lung water content using the following formula: (wet weight – dry weight)/wet weight × 100 (%). © 2003 NRC Canada


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Immunohistochemistry Heart sections of both human and animals were immunostained with antiserum to COX-2 (Merck Frosst, Que.) by a modification of the avidin-biotinylated horseradish peroxidase method as previously described (Fukuchi et al. 1998; Wong et al. 1998). Briefly, sections were incubated serially with the following solutions: (1) 2% hydrogen peroxide for 30 min to block endogenous peroxide activity; (2) 0.3% Triton-X 100 for 15 min to permeabilize the cell membrane; (3) 10% normal goat serum for 60 min to reduce nonspecific binding of the antiserum; (4) primary antisera for 16 h at 4°C; (5) biotinylated goat anti-mouse or goat antirabbit IgG for 45 min; and (6) avidin-biotinylated horseradish peroxidase complex (Vectastain, Vector Laboratories, Burlingame, Calif.) for 45 min. Immunoreactive sites were visualized by incubation with 0.025% 3,3-diaminobenzidine and 0.01% hydrogen peroxide for 3 min. PBS, pH 7.4, was used to dilute each solution and to wash the sections 3 times between each step. Assay of plasma prostanoids After completion of hemodynamic measurements, whole blood sample was collected from the right atrium. Blood samples were centrifuged at 1500 rpm (151.2 × g) for 15 min at 4°C. Then the supernatant was collected and preserved at –80°C. After the plasma was purified using C-18 reverse phase Sep-Pak cartridge (Water Co., Milford, Mass.), the stable metabolites of PGI2 and TXA2, 6-keto-PGF1α, and TXB2 were determined (Anderson et al. 1996) in the extract using competitive enzyme immunoassay (Cayman Chemical, Ann Arbor, Mich.). Statistical analysis All results are presented as means ± SE. One-way ANOVA followed by Fisher’s test was used for comparing the differences among groups. An unpaired t test was used for comparing the values obtained from infarct size and infarct wall thickness. Significant differences among the groups were defined by a value of P < 0.05.

Results

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Myocardial localization of COX-2 In AMI rats, localization of COX-2 immunoreactivity was seen in the myocytes, endocardium, and endothelial cells of intramyocardial vessels (Figs. 1A to 1D). Macrophages surrounding necrotic cardiomyocytes showed strong immunoreactivity for COX-2 (Fig. 1B). Surviving cardiomyocytes at the border line between infarcted and noninfarcted myocardium also showed strong immunoreactivity for this enzyme (Fig. 1C). There was a large number of microvessels in the infarcted myocardium, endothelial cells of these vessels showed strong immunoreactivity for COX-2 (Fig. 1D). Noninfarcted myocardium of MI hearts had weak to moderate immunoreactivity for COX-2. In contrast, the myocardium of sham rats showed weak immunoreactivity for COX2 over the cardiomyocytes (Fig. 1E). COX-2 immunoreactivity in all cell types was similar between DFU and vehicle groups. No immunostaining was seen in the negative control sections (Fig. 1F).

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Effect of the selective COX-2 inhibitor DFU on cardiac function and structure Significantly depressed cardiac function was seen in vehicle-treated rats with AMI compared with those in the sham group. Administration of DFU 30 min prior to ligation and continued for three days significantly improved left ventricular function (Table 1). LVSP was significantly decreased in the vehicle group compared to both sham and DFU groups (P < 0.01). Cardiac compliance represented by +dP/dt and –dP/dt was significantly lower in the vehicle group than sham and DFU groups (P < 0.01 and P < 0.05; respectively). The vehicle group showed higher value in LVEDP compared to the DFU group (P < 0.05). DFU treatment suppressed systemic congestion as was evident by a significant drop in CVP compared to vehicle treatment (P < 0.01). Heart weight – body weight ratio was significantly increased in the vehicle group compared to the sham group (P < 0.01). DFU treatment significantly reduced this ratio when compared to the vehicle group (P < 0.05). Lung water content was significantly increased in the vehicle group compared to the sham group (P < 0.01), and treatment with DFU significantly reduced this ratio to the level of the sham group (P < 0.01; DFU vs. vehicle group). Infarct size was significantly lower in DFU compared to vehicle group (P < 0.05). There was no significant difference in the infarct wall thickness between groups. LVD was significantly greater in the vehicle group compared to sham (P < 0.01), and was slightly reduced in the DFU group. Septal wall thickness, a parameter for noninfarcted wall thickness, was not significantly different among the three groups.

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Plasma concentration of prostanoids The vehicle group showed significantly increased plasma level of TXB2 compared to the sham group (vehicle, 859 ± 185 pg/mL; sham, 493 ± 72 pg/mL; P < 0.05). Treatment with DFU significantly decreased the concentration of TXB2 (432 ± 60 pg/mL, P < 0.05) compared to the vehicle group. Similarly, the vehicle group showed significantly increased plasma level of 6-keto-PGF1α compared to the sham group (vehicle, 1668 ± 359 pg/mL; sham, 704 ± 260 pg/mL; P < 0.05). However, there was no significant difference between the vehicle and DFU groups (DFU, 1288 ± 239 pg/mL).

Discussion Although it is well known that several factors that contribute to the initiation and (or) progression of AMI induce COX-2 expression in inflammatory and endothelial cells, and myocytes in vitro, it was not known till now whether this enzyme is increased in AMI. In the present study, we demonstrate abundant expression of COX-2 in inflammatory and vascular endothelial cells, and cardiac myocytes in the infarcted myocardium of AMI rats. Conversely, specimens from hearts of sham animals displayed weak expression of COX-2. Treatment with the selective COX-2 inhibitor, DFU, resulted in a better cardiac performance and reduced myocardial damage. The generation and release of PGs is known to induce expression of other inflammatory and growth mediators. Thus, inhibiting their formation via COX-2 would lead to a reduction in the extent of inflammation (Seibert et al. 1994), thereby reducing release of inflammatory media© 2003 NRC Canada


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Fig. 1. COX-2 immunoreactivity in the hearts of AMI and sham rats. Immunostaining for COX-2 in the hearts of rats with AMI following left coronary artery ligation (panels A to D, and F), and sham-operated rats (panel E). Panel A shows an infarct lesion in the left ventricle of AMI rat (arrow indicates endocardium). Panel B is a higher magnification of the heart specimen shown in panel A showing strong expression of COX-2 in the macrophages (arrows) surrounding necrotic myocardium. Panel C shows surviving myocytes (arrow) located at the border line between infarcted and noninfarcted myocardium, exhibiting strong immunoreactivity for COX-2. Panel D is taken from a mid-portion of the infarcted myocardium where no myocytes can be seen. Note several microvessels exhibiting COX-2 immunoreactivity in endothelial cells (arrows). Panel E shows noninfarcted myocardium of sham rat with little COX2 immunoreactivity. Panel F shows a negative control section for immunostaining. Magnifications: A (×100); B to F (×400).

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Table 1. Effect of selective COX-2 inhibition on cardiac function and structure in rat model of AMI. Group Sham

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HR (bpm) MAP (mmHg) LVSP (mmHg) LVEDP (mmHg) LV + dP/dt (mmHg/s) LV – dP/dt (mmHg/s) CVP (mmHg) RVSP (mmHg) IS (%) IWT (mm) LVD (mm) SWT (mm) HW–BW ratio (%) Lung W.C. (%)

355±10 104±4 122±5 6±1 5058±144 4779±129 5.0±0.4 25±2 — — 4.64±0.13 1.73±0.11 0.30±0.01 78.5±1.9

Vehicle ††

328±7 80±4†† 83±5†† 17±2†† 3279±363†† 3198±339†† 11.7±0.9†† 29±2† 57.9±2.5 0.97±0.03 6.37±0.37†† 1.79±0.05 0.35±0.02†† 81.3±0.5††

DFU 333±6 82±3 100±3** 11±1* 4077±195* 4020±177* 7.4±0.6** 28±1 52.8±1.9* 1.01±0.02 6.06±0.38 1.80±0.10 0.31±0.01* 77.2±0.5**

Note: HR indicates heart rate; MAP, mean arterial pressure; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LV ± dP/dt, positive and negative first derivatives of left ventricular pressure; CVP, central venous pressure; RVSP, right ventricular systolic pressure; IS, infarct size; IWT, infarct wall thickness; LVD, left ventricular diameter; SWT, septal wall thickness; HW–BW ratio, heart weight – body weight ratio; lung W.C., lung water content. *, P < 0.05, **, P < 0.01 DFU compared to vehicle group; †, P < 0.05, ††, P < 0.01 vehicle compared to sham.

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tors known to cause exudation and injury (Bishop et al. 1998). Similarly, inhibition of COX-2 leads to a reduction in oxidant formation (Agha et al. 1999). Oxidant injury has been shown to induce negative effects on the function and structure of the myocardium (Ranaut et al. 1993). Therefore, it is reasonable to suggest that selective COX-2 inhibition suppresses the production of proinflammatory prostanoids in AMI, thereby holding the promise for therapeutic application in this disease. Our data have shown that selective inhibition of COX-2 suppressed the increase in heart weight seen in the vehicle treated animals. This may be partially explained by direct effect of DFU on myocardial edema, since fluid exudation is one of the most important pathological events seen in inflammation. Evidently, the selective COX-2 inhibitor used in this study has previously been shown to reduce edema in a rat arthritis model (Anderson et al. 1996). An increase in the water content of lungs of vehicle treated animals, which may result from systemic response to the focal inflammation and (or) increased filling pressure of the left ventricle, was also seen in our study. The increase in plasma levels of TXB2 and 6-keto-PGF1α shown in our study was consistent with other previously published reports in ischemia hearts of dog (Sakai et al. 1982), human (Friedrich et al. 1985; Chlewicka and Ignatowska-Switalska 1992) and rat (Swies et al. 1990; Giannessi et al. 1992; Feng et al. 1999). Interestingly, plasma concentration of TXB2 was significantly reduced by the treatment with the COX-2 inhibitor DFU although there was no significant difference in the concentration of 6-ketoPGF1α. Enhanced thromboxane biosynthesis in the setting of AMI is likely to reflect episodes of platelet activation, since previous studies have reported the suppression of

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TXA2 generation by low-dose aspirin (Vejar et al. 1990). Moreover, Adderley and Fitzgerald (1999) has shown the primary production of TXB2 in cultured rat cardiomyocytes. Therefore, it may be reasonable to suggest that one of the possible mechanisms by which selective inhibition improves cardiac function and structure is through inhibition of myocardial thromboxane synthesis. Anti-inflammatory agents such as nonsteroidal antiinflammatory drugs have been shown to ameliorate early ischemia-induced damage to the myocardium. For example, it is well recognized that the non-selective COX inhibitor acetylsalicylic acids (ASA) improves prognosis and reduces the likelihood of re-infarction after MI. Furthermore, chronic treatment of MI rats with ASA has been reported to reduce collagen deposition in the non-infarcted myocardium (Kalkman et al. 1995). Despite these beneficial effects, inhibition of prostaglandin production in organs such as the stomach and kidney can result in gastric lesions, nephrotoxicity, and increased bleeding mainly due to inhibition of COX-1. Therefore, it is not always desirable to administer ASA, or other non-steroidal anti-inflammatory drugs. In contrast, long-term administration of selective COX-2 inhibitor prevented myocardial dysfunction and improved cardiomyocytes survival in MI rats without affecting plasma level of PGI2. A molecule structurally related to the one we used in this study, rofecoxib, is now commercially available for use in rheumatoid arthritis, osteoarthritis, and pain without known gastric or renal toxicity. As such, in light of our current observation, it would be reasonable to examine the effect of rofecoxib on the course of AMI.

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Conclusion We have demonstrated induction of COX-2 in the heart of rats with AMI. We have also shown that induction of this enzyme contributes to myocardial injury, supported by the findings that selective inhibition of COX-2 resulted in a significant improvement in cardiac function and reduced tissue injury.

Acknowledgements This work was supported by the Canadian Institutes for Health Research. Dr. Adel Giaid is supported by the Fonds de Recherché en Santé du Quebec.

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