Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
Circulation. 2000;101:430-438

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, X.
Right arrow Articles by Cannon, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, X.
Right arrow Articles by Cannon, P. J.
Related Collections
Right arrow Biochemistry and metabolism
Right arrow Animal models of human disease
Right arrow Apoptosis
Right arrow CV surgery: transplantation, ventricular assistance, cardiomyopathy

(Circulation. 2000;101:430.)
© 2000 American Heart Association, Inc.


Basic Science Reports

Upregulation of COX-2 During Cardiac Allograft Rejection

Xiaochun Yang, MD; Ninsheng Ma, MD; Matthias J. Szabolcs, MD; Jing Zhong, MD; Eleni Athan, PhD; Robert R. Sciacca, ScD; Robert E. Michler, MD; Gary D. Anderson, PhD; Joseph F. Wiese, PhD; Kathleen M. Leahy, PhD; Susan Gregory, PhD; Paul J. Cannon, MD

From the Departments of Medicine (X.Y., E.A., R.R.S., P.J.C.), Surgery (R.E.M.) and Pathology (M.J.S.), Columbia University College of Physicians and Surgeons, New York, and G.D. Searle/Montsanto Co (G.D.A., J.F.W., K.M.L., S.G.), St. Louis, Mo.

Correspondence to Paul J. Cannon, MD, Department of Medicine, Division of Cardiology, Columbia University, 630 West 168th Street, New York, NY 10032. E-mail pjc4{at}columbia.edu


*    Abstract
up arrowTop
*Abstract
down arrowIntroduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background—The hypothesis that cyclooxygenase-2 (COX-2) is involved in the myocardial inflammatory response during cardiac allograft rejection was investigated using a rat heterotopic abdominal cardiac transplantation model.

Methods and Results—COX-2 mRNA and protein in the myocardium of rejecting cardiac allografts were significantly elevated 3 to 5 days after transplantation compared with syngeneic controls (n=3, P<0.05). COX-2 upregulation paralleled in time and extent the upregulation of iNOS mRNA, protein, and enzyme activity in this model. COX-2 immunostaining was prominent in macrophages infiltrating the rejecting allografts and in damaged cardiac myocytes. Prostaglandin (PG) levels in rejecting allografts were also higher than in native hearts. Because NO has been reported to modulate PG synthesis by COX-2, additional transplants were performed using animals treated with a selective COX-2 inhibitor (SC-58125) and a selective inhibitor of the inducible nitric oxide synthase (iNOS) N-aminomethyl-L-lysine. At posttransplant day 5, inhibitor administration resulted in a significant reduction of COX-2 mRNA expression (3764±337 versus 5110±141 arbitrary units, n=3, P<0.05) and iNOS enzymatic activity (1.7±0.4 versus 22.8±14.4 nmol/mg protein, n=3, P<0.01) compared with vehicle-treated allogeneic transplants. Allograft survival in treated animals was increased modestly from 5.4 to 6.4 days (P<0.05). However, apoptosis of cardiac myocytes (TUNNEL method) was only marginally reduced relative to vehicle controls in treated graft recipients. The intensity of allograft rejection was also similar in the treated and untreated allografts.

Conclusions—The data indicates that COX-2 expression is enhanced in parallel with iNOS in the myocardium during cardiac allograft rejection.


Key Words: prostaglandins • nitric oxide • rejection


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Myocardial inflammation constitutes a major component of the pathologic changes observed during cardiac allograft rejection. Prostaglandins (PGs), along with leukotrienes and lipoxins, are lipid mediators which contribute to the vasodilation, edema, and plasma protein leakage which occur during the inflammatory response. Arachidonic acid released from cellular phospholipids by phospholipase A2 is converted by the bis-oxygenase activity of cyclooxygenase to the prostaglandin endoperoxide PGG2 and then to PGH2. Prostaglandin H2 is metabolized to biologically active products such as PGE2, prostacyclin, and thromboxane A2.1 It is known that cyclooxygenase exists in at least 2 isoforms.1 2 The constitutive isoform, COX-1, is believed to be responsible for the constitutive production of PGs such as PGI2 by endothelial cells and the gastric mucosa and thromboxane A2 by platelets. The other isoform, COX-2, is not constitutively expressed in most tissues but can be induced in endothelial cells, fibroblasts, smooth muscle cells, and macrophages by proinflammatory cytokines, endotoxin, PGs, tumor promoters, mitogens, and hypoxia.2 3 It is believed that COX-2 is induced during both acute and chronic inflammatory responses and is primarily responsible for the PG synthesis that ensues. This has led to the development of drugs that selectively inhibit PG production by COX-2, avoiding the adverse consequences, such as gastric ulcers, that may result from inhibition of COX-1.4

In different inflammatory settings, the expression of COX-2 can be deleterious or protective.5 It has been reported that the activity of COX-1 and COX-2 can be enhanced by NO, augmenting the inflammatory response.6 7 Conversely, PGE2 produced by the inducible isoform of COX can inhibit expression of inducible nitric oxide synthase (iNOS).7 The induction of COX-2 in endothelial cells increases the synthesis of PGI2 thereby augmenting its protective actions to inhibit platelet aggregation, promote vasodilation, and reduce monocyte adhesion and activation on endothelial surfaces.8

In previous studies we demonstrated that during cardiac allograft rejection in rats, iNOS mRNA, protein, and enzyme activity are induced.9 Using immunostaining, iNOS protein was demonstrated in infiltrating macrophages and lymphocytes, endothelial cells, vascular smooth muscle cells, and cardiac myocytes within the rejecting cardiac allografts. This work was confirmed by other groups who found that iNOS is expressed in macrophages and smooth muscle cells in the vasculopathic coronary arteries of allografts undergoing chronic rejection.10 11 12 iNOS mRNA and positive immunostaining have also been demonstrated in human cardiac allografts during rejection.13 NO produced by iNOS in this setting may impair contractile properties of the ventricle, reduce cardiomyocyte viability, and modulate the development of transplant vasculopathy.9 10 11 14 15 It may also modulate PG synthesis.3 6

Accordingly, the present study was designed to investigate in a rat heterotopic heart transplantation model whether COX-2 is expressed and contributes to myocardial inflammation during cardiac allograft rejection.


*    Methods
up arrowTop
up arrowAbstract
up arrowIntroduction
*Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Cardiac Transplantation and Drug Treatment
Male Lewis (TR-11) and Wistar-Furth (WF, RT-lu) rats weighing 180 to 200 g each were purchased from Harlan Sprague-Dawley Inc (Indianapolis, Ind). Syngeneic (Lewis-Lewis) and allogeneic (Lewis-Wistar-Furth) heterotopic abdominal cardiac transplantation was performed as previously described.9 16 Heart grafts in the abdomen were palpated daily. Rejection was determined by the lack of a heart beat and confirmed by inspection at laparotomy and by histological examination. Drug administration was begun on day 1 posttransplantation and continued until each assessment day (days 1, 3, and 5). SC-58125, {1-[(4-methylsulfonyl) phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyrazole} (G.D. Searle/Montsanto Co., St. Louis, Mo), which selectively inhibits mouse COX-2 enzyme (IC50=0.07 µM) without inhibiting COX-1 (IC50 >100 µM) was given at a dosage of 10 mg · kg-1 · wk-1 (1 dose per week, i.p.) in a suspension in 0.5% aqueous methylcellulose and 0.025% Tween-20. The selective iNOS inhibitor, N-aminomethyl-L-lysine (L-NIL) (G.D. Searle/Montsanto Co, St. Louis, Mo), was given at a dose of 10 mg · kg-1 · d-1 (BID, i.p.). Animals were randomly grouped into drug and vehicle (0.9% saline) treatments.

Histology and Labeling of Apoptotic Cells
Hearts were fixed in 10% phosphate buffered formalin, embedded in paraffin, and 4 µm thick sections were cut and mounted on sialine-coated slides. For routine histologic examination, sections were stained with hematoxylin and eosin to determine the extent and severity of rejection according to the International Society of Heart and Lung Transplantation classification (ISHLT).17 Apoptotic cells were detected by in situ end-labeling, which detects the abundant DNA fragments in apoptotic nuclei using biotinylated deoxyuridine 5-triphosphate as described previously.18 19 Sections were also labeled for muscle actin with monoclonal antibody HHF-35 (Dako, Carpinteria, CA) using an immunoperoxidase technique which stained the cytoplasm of cardiac myocytes brown. The same immunoperoxidase technique was used to characterize the inflammatory infiltrate by labeling for T cell markers (anti-CD3) and macrophages (ED1).

COX-2 Immunohistochemistry
The RR6 monoclonal anti-mouse-COX-2 antibody was obtained from Dr Peter Isakson (G.D. Searle/Montsanto Co., St. Louis, Mo).20 The monoclonal anti-rat COX-1 antibody was obtained from Accurate Chemical Co (Westbury, NY). Immunohistochemistry was performed as previously described.20 Sections were then incubated with the anti-COX-2 Ab (clone RR6) diluted 1:150 in 5% horse serum PBS overnight at 4°C. Binding of the primary Ab to COX-2 was detected with the avidin-biotin-peroxidase technique labeling the site of the target antigen (COX-2) brown. For the COX-1, the primary antibody was diluted with 3% horse serum in PBS up to 1:150 and incubated at 4°C overnight. Horse anti-mouse secondary antibody was diluted 1:200 (Vector Laboratories, Burlingame, CA) and incubated for 30 minutes. Normal mouse serum was used as a negative control.

COX-2 and iNOS mRNA Ribonuclease Protection Assay
Specific mRNAs for COX-2 and iNOS were quantified by ribonuclease protection assay (RPA). Assay reagents and the procedures used were from an Ambion RPAIITM kit (Ambion Inc, Austin, Tex). The plasmid DNA used as a template for the rat iNOS probe was generously provided by Charles Rodi, Searle/Montsanto, St. Louis, Mo.

For the COX-2 RPAs, frozen graft or native hearts were thawed in guanidine isothiocyanate. Total RNA was isolated using the Ambion To-TallyTM RNA kit. Samples of total RNA were hybridized, digested, and separated. After electrophoresis, the gel was fixed and dried. Band intensities were quantified by electronic autoradiography using a Packard Instant Imager. The plasmid DNA used as a template for the rat COX-2 probe was graciously provided by P. Worley, Johns Hopkins School of Medicine, Baltimore, Md.

COX-2 Enzyme Protein Assay
The excised hearts were rinsed and flushed via the aorta with ice-cold saline to completely remove blood, then immediately frozen at -70°C. The frozen ventricular tissue was homogenized at 4°C in RIPA-lysis buffer supplemented with 10 µg/mL antipain, leupeptin and trypsin-inhibitor, and 0.1 mg/mL phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 16 000g for 30 minutes at 4°C. The protein concentration of supernatants were determined by BCA protein assay (Pierce) with BSA as standard. The total protein equivalents (40 µg per lane) for each sample were separated by 8% SDS-PAGE and electrotransferred to nitrocellulose membrane. After blocking nonspecific binding with TBS buffer containing 8% nonfat dried milk and 2% BSA, the membranes were immunoblotted with a mouse COX-2–specific (clone RR6) monoclonal antibody at a dilution of 1:2000 (>1000-fold selectivity for mouse COX-2 compared with mouse COX-121 ). The blots were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody and detected by the enhanced chemiluminescence method (DuPont NEM). The level of COX-2 protein was quantified using densitometric analysis (NIH image 1.60 software).

iNOS Enzyme Protein and Activity Assay
iNOS enzyme protein was measured as described above for the COX-2 protein assay. The anti-mouse iNOS polyclonal antiserum was a gift from Dr Mark Currie, G.D. Searle/Montsanto Co. iNOS enzyme activity was measured as previously described.9 18

Prostaglandin Assay
Both transplanted and native hearts were removed, cut into 2 slices, and immediately immersed in a HEPES-buffered Krebs solution. One slice was incubated in oxygenated Krebs solution only, the other was incubated in oxygenated Krebs solution containing bradykinin (100 µM) at 37°C, for 30 minutes. The supernatant was collected, mixed with water and methanol (10%), centrifuged at 4°C for 15 minutes at 375g and loaded on a C18 cartridge (Millipore, Bedford, MA). This was followed by serial washings with deionized water, 10% methanol, petroleum ethyl, and elution with redistilled ethyl acetate, followed by evaporation of the organic phase under nitrogen. After suspending the pellets in phosphate buffer, prostaglandin E2 in the samples was measured with the TiterZyme PGE2 kit (PerSeptive Biosystems, Framingham, Mass).

Statistical Analysis
The time course of changes in COX-2 mRNA and protein and iNOS mRNA levels and enzyme activity were analyzed by ANOVA. PGE2 values were analyzed by ANOVA after log transformation due to the markedly nonnormal distribution of the values. The degree of apoptosis at each of the time points was analyzed using the nonparametric Kruskal-Wallis procedure. Survival rates were compared using the Mann-Whitney test.


*    Results
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
The expression of COX-2 mRNA was significantly increased in the rejecting cardiac allografts in comparison to syngeneic grafts on days 3 to 5 following transplantation (Figure 1Down). The COX-2 protein was also significantly upregulated in the rejecting allografts on days 4 to 5 posttransplantation (Figure 2Down). COX-1 mRNA was also increased in rejecting allografts on days 3 to 5 (Figure 3Down). To assess PG production in the rejecting graft tissue, myocardial slices from day 5 cardiac allografts and from the native hearts of the same rats were incubated in oxygenated buffer alone and in oxygenated buffer containing bradykinin (100 µmol/L). The mean PGE2 concentration per milligrams protein in the incubation media was significantly higher (P<0.05) in rejecting allografts in comparison to nonrejecting native hearts under control conditions, 474±678 versus 135±156 pg/mg of tissue and after bradykinin treatment, 1434±2451 versus 321±309 pg/mg of tissue (n=10).



View larger version (21K):
[in this window]
[in a new window]
 
Figure 1. Time course of upregulation of COX-2 mRNA in syngeneic cardiac grafts and in rejecting cardiac allografts. Values are based on densitometric readings and are expressed as percent of the mean for the 5-day syngeneic grafts. Significantly higher levels of COX-2 mRNA were observed at days 3 to 5 in the rejecting allografts (n=3).



View larger version (46K):
[in this window]
[in a new window]
 
Figure 2. Time course of the upregulation of COX-2 protein in syngeneic cardiac grafts and in rejecting cardiac allografts. A, Western blot; B, COX-2 protein in Western blot is quantified from densitometric readings and expressed as a percent of the mean for the 5-day syngeneic grafts (n=3).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 3. Time course of the upregulation of COX-1 mRNA in syngeneic cardiac grafts and rejecting cardiac allografts. Values are derived from densitometric readings and are expressed as percent of the mean for the 5-day syngeneic grafts. Significant increases in COX-1 mRNA were observed at days 3 to 5 following transplantation (n=3).

Positive immunostaining for COX-2 was not observed in native hearts and syngeneic hearts other than a slight staining of rare endothelial cells (Figure 4CDown). COX-2 immunostaining in rejecting allografts was markedly increased in macrophages, damaged cardiomyocytes and in endothelial cells and smooth muscle cells especially in myocardial regions with an inflammatory infiltrate (Figure 4DDown). The increased expression of COX-2 mRNA (Figure 1Up) was similar in time and extent to the expression of iNOS mRNA in the allografts (Figure 5Down). Positive immunostaining for COX-1 was apparent in endothelial and endocardial cells of the native hearts (Figure 4ADown). In rejecting allografts, COX-1 immunostaining was increased in endothelial cells. It was not observed in macrophages but was present in damaged cardiomyocytes (Figure 4BDown).



View larger version (148K):
[in this window]
[in a new window]
 
Figure 4. Native rat heart (A and C) and rat heterotopic abdominal allograft (B and D) 5 days posttransplantation labeled for COX-1 (A and B) and COX-2 (C and D) represented by brown reaction product. The native hearts show COX-1 positive endothelial and endocardial cells (A). Cox-2 staining is weak in native hearts, where it is present in rare capillaries (C). Myocytes of native hearts are consistently COX-1– and COX-2–negative. During cardiac allograft rejection, COX-1 is also expressed in damaged cardiac myocytes in addition to endothelial cells (B) but not in infiltrating inflammatory cells (arrow). COX-2 is also upregulated in damaged cardiac myocytes (circle) and vascular smooth muscle cells (not shown). Magnification x400.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 5. Time course of the expression of iNOS mRNA in rejecting cardiac allografts. Values are derived from densitometric readings and are expressed as percent of the mean for the 5-day syngeneic grafts. Significant increases in iNOS mRNA were observed at days 4 to 5 following transplantation (n=3).

Treatment of the allograft recipients with L-NIL had no significant effect on COX-2 protein levels in the rejecting allografts but was associated with a reduction in iNOS enzyme activity (Figures 6BDown and 7Down). Treatment with the combination of SC-58125 and L-NIL was associated with a reduction of COX-2 protein in the 5-day rejecting allografts of 10% to 15% (Figure 6BDown). Survival of the cardiac allografts treated with the 2 inhibitors was increased slightly but significantly from 5.4±0.5 to 6.4±0.5 days (n=8, P<0.05). However, both the allografts treated with vehicle and the allografts treated with the inhibitors (Figure 8Down) showed severe inflammation and multiple foci of myocyte damage at day 5. In the SC-58125 plus L-NIL–treated animals, COX-2 immunostaining was most apparent in the infiltrating macrophages, with decreased immunostaining of cardiac myocytes (Figure 9Down). In both treated and untreated cardiac allografts, the number of apoptotic cardiac myocytes and the total number of apoptotic nuclei increased exponentially during rejection (Figure 10Down). There was no significant difference in the mean numbers of apoptotic nuclei in treated (n=6) versus untreated (n=3) allografts (16.1±9.6 versus 7.5±4.5) at day 5. Similarly, there was no significant reduction in the rejection grade of the treated cardiac allografts (TableDown).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 6. A, Effect of treatments with SC-58125 and L-NIL or L-NIL alone on COX-2 protein expression by Western blot. B, Results of quantification of COX-2 protein in Western blot. Values are expressed as percent of control. COX-2 expression was decreased significantly by treatment with both SC-58125 and L-NIL but not by the L-NIL alone in comparison with vehicle-control (n=3).



View larger version (12K):
[in this window]
[in a new window]
 
Figure 7. Comparison of iNOS activity in grafts from animals on day 5 posttransplantation. Treatments with L-NIL alone or SC-58125 and L-NIL significantly reduced iNOS activity (n=3).



View larger version (166K):
[in this window]
[in a new window]
 
Figure 8. Cardiac allografts 5 days posttransplantation treated with vehicle (a) or with SC-58125 and L-NIL (b). Both allografts show severe inflammation and multiple foci of myocyte damage. Magnification x250.



View larger version (162K):
[in this window]
[in a new window]
 
Figure 9. COX-2 immunolabeling of vehicle (a) and SC-58125– and L-NIL–treated (b) 5-day cardiac allografts. Immunoreactivity for COX-2 in vehicle-treated grafts is observed in damaged cardiac myocytes (large arrow), vascular smooth muscle cells and endothelial cells (asterisk), and infiltrating macrophages (small arrow). In SC-58125 and L-NIL–treated grafts, COX-2 immunoreactivity was mostly present in macrophages (arrows). Magnification x400.



View larger version (166K):
[in this window]
[in a new window]
 
Figure 10. Cardiac allografts 5 days posttransplantation from a vehicle-treated (a) and SC-58125– and L-NIL–treated (b) rat labeled for apoptotic nuclei (blue) and cardiac myocytes (brown). The number of apoptotic cardiac myocytes (arrow) increased exponentially in allografts from both treated and untreated animals. Many apoptotic nuclei are also found within the inflammatory infiltrate. Magnification x400.


View this table:
[in this window]
[in a new window]
 
Table 1. Grades of Rejection in Control and Treated Rat Cardiac Allografts


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The data in this study indicate that during cardiac allograft rejection COX-2 mRNA and protein are upregulated significantly in the rejecting allografts at days 3 to 5 following transplantation, in comparison to the levels observed in syngeneic grafts at the same time points. Prostaglandin synthesis was also significantly higher in myocardial tissue from the rejecting allografts 5 days after transplantation in comparison to that observed in native hearts. Immunostaining indicated that COX-2 protein was markedly expressed in endothelial cells, vascular smooth muscle cells, macrophages infiltrating the myocardium, and focally in cardiac myocytes that appeared damaged. The upregulation of COX-2 during cardiac allograft rejection in this rat model paralleled in time and extent the upregulation of iNOS mRNA, protein, and enzyme activity.9 18 COX-1 protein was also significantly increased at days 3 to 5, probably due to increased expression of COX-1 in endothelial cells.

The expression of COX-2 together with that of iNOS has previously been noted in a model of ureteral obstruction in rats which leads to hydronephrosis and renal inflammation,22 in rat adjuvant arthritis,21 in carrageenin-induced pleurisy,23 in a model of subcutaneous inflammation produced by air injection24 25 in human osteoarthritis-affected cartilage,26 and in rat hearts following treatment with lipopolysaccharide.27 The co-induction of COX-2 and iNOS has also been observed in studies in vitro of rat vascular smooth muscle cells,28 29 glomerular mesangial cells,7 murine macrophages,30 rat islets of Langerhans,31 human endothelial cells,32 articular chondrocytes,33 and rabbit hepatocytes34 incubated with endotoxin and/or cytokines but not in similar studies of human fetal cell fibroblasts6 or bovine aortic endothelial cells.35 Proinflammatory cytokines known to be synthesized and released by T lymphocytes and macrophages during cardiac allograft rejection are probably responsible for induction of COX-2 in this situation.36 In studies of porcine endothelial cells, exposed to xenoreactive antibodies and complement, IL-1ß mediated the upregulation of COX-2 and synthesis of PGE2 and TXA2.37 In studies of neonatal ventricular myocytes, IL-1ß induced iNOS, COX-2 mRNA, and protein along with a 200-fold increase in PGE2.38

The relationship between the cyclooxygenase and NO pathways varies depending on the circumstances. In cultured bovine endothelial cells, NO or NO donor drugs have been shown to inhibit PGI2 release by bradykinin (COX-1)39 and to inhibit COX-2 induction and activity in rat Kupffer cells.34 In contrast, however, NO and NO donor drugs have been shown to stimulate COX-1 and COX-2 activity in endotoxin-activated murine macrophages6 and in vascular smooth muscle cells28 and human endothelial cells.39 40 Similarly, in the hydronephrotic model of renal inflammation,22 in air-pouch–induced inflammation,24 there is evidence that NO augments the activity of COX-1 and COX-2, leading to enhanced synthesis of prostaglandins. The mechanism responsible for the effect of NO on COX activity is unclear but may involve nitrosylation of a cysteine residue in the active site of the COX enzymes,41 leading to the formation of nitrosothiols; these can produce structural changes in the enzyme, leading to increased COX catalytic efficiency.42 In other studies, it was demonstrated that NO enhanced the IL-1ß–induced expression of the COX-2 mRNA and protein.43 The cytokine and endotoxin upregulation of COX-2 and iNOS in various cells and tissues is suppressed by dexamethazone and by other immunosuppressive drugs such as cyclosporin A and FK506.4 10 22 25 44 As mentioned previously, endothelial cell expression of COX-2 may be vasculoprotective by augmenting PGI2 synthesis.8 The finding of increased myocardial fibrosis in COX-2 knockout mice suggests that endocardial endothelial cell COX-2 may also be protective by augmenting PGI2 production.45

In the present study, potent selective inhibitors of COX-2 and of iNOS were administered to rats undergoing cardiac transplantation.21 46 The administration of SC-58125 together with L-NIL resulted in the downregulation of the expression of COX-2 protein in the treated allografts, a finding that has been reported previously using an arthritis model in rats.21 The explanation for this is unclear but may reflect a role for PGs in the enhancement of COX-2 expression. The administration of L-NIL was associated with a marked reduction of iNOS enzyme activity in the rejecting allografts. However, there was only a slight increase in the survival of the cardiac allografts treated with both inhibitors.

In previous studies using cardiac allograft experimental models in rats, the expression of iNOS in the rejecting allografts was associated with increased myocardial inflammation. In the studies of Worrel et al,10 44 animals treated with aminoguanidine, a potent iNOS inhibitor (which also has antioxidant and other effects), was associated with a reduction in the intensity of the pathological changes of rejection in the cardiac allografts. In a report from Koglin et al47 and in unpublished experiments in our laboratory, there was also reduced myocardial inflammation in cardiac allografts transplanted into iNOS-deficient mice in comparison to that seen in wild type control recipients. In the present study, there were slight but not significant reductions in the myocardial inflammation in the animals treated with the selective COX-2 and iNOS inhibitor drugs. Modest reductions in the degree of inflammation and the magnitude of the inflammatory infiltrate have also been reported following administration of SC-58125 to rats with adjuvant arthritis21 and to rats with carrageenin-induced pleurisy.23

Apoptosis of cardiac myocytes and of infiltrating macrophages has also been observed in parallel with the upregulation of iNOS mRNA, protein, and enzyme activity in rejecting rat cardiac allografts and in human endomyocardial biopsies from hearts undergoing class 3 (ISHLT) rejection.18 48 These associations, along with in vitro studies of NO-mediated cardiomyocyte apoptosis, have suggested that NO may be an apoptotic trigger in this situation.49 50 The slight reductions observed in apoptotic cell numbers in the animals treated with the COX-2 and iNOS inhibitors were not statistically significant. Recently, however, Koglin et al reported that during cardiac allograft rejection in iNOS knockout mice, the number of apoptotic cells was significantly reduced.47 It is of interest that von Knethen and Brune, in studies of NO-mediated apoptosis of RAW 264.7 macrophages in vitro, developed strong evidence that COX-2 is an essential regulator of apoptosis.51

In summary, COX-2 mRNA and enzyme protein are upregulated in parallel with iNOS during cardiac allograft rejection. Although in this experimental model allograft survival was prolonged slightly, myocardial inflammation and cardiomyocyte apoptosis were not significantly reduced by treatment with a combination of inhibitors of COX-2 and iNOS.


*    Acknowledgments
 
This work was supported in part by NIH grants HL 54764 and HL 56984.

Received April 8, 1999; revision received August 5, 1999; accepted August 5, 1999.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMethods
up arrowResults
up arrowDiscussion
*References
 
1. Smith WL. Prostanoid biosynthesis and mechanisms of action. Am J Physiol. 1992;263(2 pt 2):F181–F191.

2. Mitchell JA, Larkin S, Williams TJ. Cyclooxygenase-2: regulation and relevance in inflammation. Biochem Pharmacol. 1995;50:1535–1542.[Medline] [Order article via Infotrieve]

3. Wu KK. Inducible cyclooxygenase and nitric oxide synthase. Adv Pharmacol. 1995;33:179–207.

4. Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, Isakson PC, Siebert K. Selective inhibition of inducible cyclooxygenase-2 in vivo is anti-inflammatory and nonulcerogenic. Proc Natl Acad Sci U S A. 1994;91:3228–3232.[Abstract/Free Full Text]

5. Wu, KK. Cyclooxygenase-2 induction in congestive heart failure Friend or Foe Circulation. 1998;98:95–96. Editorial.[Free Full Text]

6. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A. 1993;90:7240–7244.[Abstract/Free Full Text]

7. Tetsuka T, Caphna-Iken D, Srivastava SK, Baier LD, DuMaine J, Morrison AR. Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1. Proc Natl Acad Sci U S A. 1994;91:12168–12172.[Abstract/Free Full Text]

8. Wu KK. Injury-coupled induction of endothelial eNOS and COX-2 genes: a paradigm for thrombo resistant gene therapy. Proc Assoc Am Physicians. 1998;110:163–170.[Medline] [Order article via Infotrieve]

9. Yang X, Chowdhury N, Cai B, Brett J, Marboe C, Sciacca RR, Michler RE, Cannon PJ. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest. 1994;94:714–721.

10. Worrall NK, Lazenby WD, Misco TP, Lin T-S, Rodi CP, Manning PT, Gilton RG, Williamson JR, Ferguson TB Jr. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med. 1995;181:63–70.[Abstract/Free Full Text]

11. Russell ME, Wallace AF, Wyner LR, Newell JB, Karnovsky MJ. Upregulation and modulation of inducible nitric oxide synthase in rat cardiac allografts with chronic rejection and transplant arteriosclerosis. Circulation. 1995;92:457–464.[Abstract/Free Full Text]

12. Akyurek LM, Fellstrom BC, Yan Z, Hansson GK, Funa K, Larsson E. Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic allografts. Am J Pathol. 1996;149:1981–1990.[Abstract]

13. Lewis NP, Tsao PS, Rickenbacher PR, Xue C, Johns RA, Haywood GA, von der Leyen H, Trindade PT, Cooke JP, Hunt SA, Billingham ME, Valantine HA, Fowler MB. Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation. 1996;93:720–729.[Abstract/Free Full Text]

14. Worrall NK, Pyo RT, Botney MD, Misko TP, Sullivan PM, Alexander DG, Lazenby WD, Fergusen TB Jr. Inflammatory cell-derived NO modulates cardiac allograft contractile and electrophysiological function. Am J Physiol. 1997;(1 pt 2):H28–H36.

15. Pinsky DJ, Cai B, Yang X, Rodriguez C, Sciacca RR, Cannon PJ. The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor ß. J Clin Invest. 1995;95:677–685.

16. Ono K, Lindsay ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg. 1969;7:225–229.

17. Billingham ME, Cary NRB, Hamond ME, Kemnitz J, Marboe C, McAllister HA, Snovar DC, Winters GL, Zerbe A. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. J Heart Lung Transplant. 1990;71:219–225.

18. Szabolcs M, Michler RE, Yang X, Aji W, Roy D, Athan E, Sciacca RR, Minanov OP, Cannon PJ. Apoptosis of cardiac myocytes during cardiac allograft rejection. Relation to induction of nitric oxide synthase. Circulation. 1996;94:1665–1673.[Abstract/Free Full Text]

19. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl and nick translation assays. Cancer Res. 1993;53:1945–1954.[Abstract/Free Full Text]

20. Kaufmann WE, Worley PJ, Pegg J, Bremer M, Isakson P. COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex. Proc Natl Acad Sci U S A. 1996;93:2317–2321.[Abstract/Free Full Text]

21. Anderson GD, Hauser SC, McGarity KL, Bremer ME, Isakson PC, Gregory SA, Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J Clin Invest. 1996;97:2672–2679.[Medline] [Order article via Infotrieve]

22. Salvemini D, Seibert K, Masferrer JL, Misko TP, Currie MG, Needleman P. Endogenous nitric oxide enhances prostaglandin production in a model of renal inflammation. J Clin Invest. 1994;93:1940–1947.

23. Tomlinson A, Appleton I, Moore AR, Gilroy DW, Willis D, Mitchell JA, Willoughby DA. Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy. Br J Pharmacol. 1994;113:693–698.[Medline] [Order article via Infotrieve]

24. Appleton I, Tomlinson A, Colville-Nash PR, Willoughby DA. Temporal and spatial immunolocalization of cytokines in murine chronic granulomatous tissue. Implications for their role in tissue development and repair processes. Lab Invest. 1993;69:405–414.[Medline] [Order article via Infotrieve]

25. Vane JR, Mitchell JA, Appleton I, Tomlinson A, Bishop-Bailey D, Croxtall J, Willoughby DA. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc Natl Acad Sci U S A. 1994;91:2046–2050.[Abstract/Free Full Text]

26. Amin AR, Attur M, Patel RN, Thakker GD, Marshall PJ, Rediske J, Stuchin SA, Patel IR, Abramson SB. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage: influence of nitric oxide. J Clin Invest. 1997;99:1231–1237.[Medline] [Order article via Infotrieve]

27. Liu SF, Newton R, Evans TW, Barnes PJ. Differential regulation of cyclo-oxygenase-1 and cyclo-oxygenase-2 gene expression by lipopolysaccharide treatment in the rat. Clin Sci (Colch). 1996;90:301–306.[Medline] [Order article via Infotrieve]

28. Inoue T, Fukuo K, Marimoto S, Koh E, Ogihara T. Nitric oxide mediates interleukin-1 induced prostaglandin E2 production by vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;194:420–424.[Medline] [Order article via Infotrieve]

29. Rimarachin JA, Jacobson JA, Szabo P, Maclouf J, Creminon C, Weksler BB. Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells. Arterioscler Thromb. 1994;14:1021–1031.[Abstract/Free Full Text]

30. Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem. 1992;267:25934–25938.[Abstract/Free Full Text]

31. Corbett JA, Kwon G, Turk J, McDaniel ML. IL-1 beta induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. Biochemistry. 1993;32:13767–13770.[Medline] [Order article via Infotrieve]

32. Mitchell JA, Swierkosc T, Warner TD, Gross S, Thiemermann C, Vane JR. Regulation of prostaglandin synthesis by the release of endogenous nitric oxide in response to bacterial lipopolysaccharide. Br J Pharmacol. 1993;109:4P. Abstract.

33. Stadler J, Stefanovic-Racic M, Billiar TR, Curren RD, McIntyre LA, Geogescu H, Simmons RL, Evans CH. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J Immunol. 1991;147:3915–3920.[Abstract]

34. Stadler J, Harbrecht BG, DiSilvio M, Curran RD, Jordan ML, Simmons RL, Billier TR. Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. J Leukoc Biol. 1993;53:165–172.[Abstract]

35. Akarasereenont P, Mitchel JA, Appleton I, Thiemermann C, Vane JR. Involvement of protein tyrosine phosphorylation in the induction of cyclooxygenase and nitric oxide synthase by endotoxin in cultured cells. Br J Pharmacol. 1994;113:1522–1528.[Medline] [Order article via Infotrieve]

36. Russell ME, Wallace AF, Hancock WW, Sayegh MH, Adams DH, Siginga NES, Wyner LR, Karnovsky MJ. Upregulation of cytokines associated with macrophage activation in the Lewis to F344 rat chronic cardiac rejection model. Transplantation. 1995;59:572–578.[Medline] [Order article via Infotrieve]

37. Buston M, Coffman TM, Saadi S, Platt JL. Modulation of eicosanoid metabolism in endothelial cells in a xenograft model: role of cyclooxygenase-2. J Clin Invest. 1997;100:1150–1158.[Medline] [Order article via Infotrieve]

38. LaPointe MC, Sitkins JR. Phospholipase A2 metabolites regulate inducible nitric oxide synthase in myocytes. Hypertension. 1998;31(1 pt 2):218–224.

39. Doni MG, Writtle BJR, Palmer RMJ, Moncada S. Actions of nitric oxide on the release of prostacyclin from bovine aortic endothelial cells in culture. Eur J Pharamcol. 1988;151:19–25.[Medline] [Order article via Infotrieve]

40. Davidge ST, Baker PN, MacLaughlin MK, Roberts JM. Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. Circ Res. 1995;47:274–283.

41. Hajjar DP, Lander HM, Pearce FS, Upmacis RK, Pomerantz KB. Nitric oxide enhances prostaglandin-H synthase activity by a heme-independent mechanism: evidence implicating nitrosothiols. J Am Chem Soc. 1995;117:3340–3346.

42. Kroncke K-D, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities. Biol Chem Hoppe Seyler. 1995;376:327–343. Review.[Medline] [Order article via Infotrieve]

43. Tetsuka T, Daphna-Iken D, Miller BW, Guan Z, Baier LD, Morrison AR. Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells. J Clin Invest. 1996;97:2051–2056.[Medline] [Order article via Infotrieve]

44. Worrall NK, Misko TP, Sullivan PM, Hui Jia-J, Ferguson TB Jr. Inhibition of inducible nitric oxide synthase attenuates established acute cardiac allograft rejection. Ann Thorac Surg. 1996;62:378–385.[Abstract/Free Full Text]

45. Dinchuk JE, Car BD, Focht RJ, Johnson JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM, Gorry SA, Trzaskos JM. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature. 1995;378:406–409.[Medline] [Order article via Infotrieve]

46. Mitchell JA, Akarasereenont P, Thiemermann C, Flower RJ, Vane JR. Selectivity of nonsteroidal anti-inflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci U S A. 1994;90:11693–11697.[Abstract/Free Full Text]

47. Koglin J, Glysing-Jensen T, Mudgett JS, Russell ME. NOS-2 mediates opposing effects in models of acute and chronic rejection-Insights from NOS2 knockout mice. Am J Pathol. 1998;153:1371–1376.[Abstract/Free Full Text]

48. Szabolcs MJ, Ravalli S, Minanov O, Sciacca RR, Michler RE, Cannon PJ. Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection. Transplantation. 1998;65:804–812.[Medline] [Order article via Infotrieve]

49. Pinsky DJ, Aji W, Szabolcs M, Athan ES, Liu Y, Yang, Y-M, Kline, RP, Olson KE, Cannon PJ. Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture. Am J Physiol. 1999;277:H1189–H1199.

50. Ing DJ, Zang J, Dzau VJ, Webster KA, Bishopric NH. Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, bak, and bcl-x. Circ Res. 1999;84:21–23.[Abstract/Free Full Text]

51. Von Knethen A, Brune B. Cyclooxygenase-2: an essential regulator of NO-mediated apoptosis. FASEB J. 1997;11:887–895.[Abstract]




This article has been cited by other articles:


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Guo, Z. Xia, J. Jiang, and J. H. McNeill
Downregulation of NADPH oxidase, antioxidant enzymes, and inflammatory markers in the heart of streptozotocin-induced diabetic rats by N-acetyl-L-cysteine
Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1728 - H1736.
[Abstract] [Full Text] [PDF]


Home page
Asian Cardiovasc. Thorac. Ann.Home page
S. G Raja and G. D Dreyfus
Modulation of Systemic Inflammatory Response after Cardiac Surgery
Asian Cardiovasc Thorac Ann, December 1, 2005; 13(4): 382 - 395.
[Abstract] [Full Text] [PDF]


Home page
J. Thorac. Cardiovasc. Surg.Home page
T. Yokoyama, O. Aramaki, T. Takayama, S. Takano, Q. Zhang, M. Shimazu, M. Kitajima, Y. Ikeda, N. Shirasugi, and M. Niimi
Selective cyclooxygenase 2 inhibitor induces indefinite survival of fully allogeneic cardiac grafts and generates CD4+ regulatory cells
J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1167 - 1174.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
M. C. LaPointe, M. Mendez, A. Leung, Z. Tao, and X.-P. Yang
Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse
Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1416 - H1424.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
G. M. Pieper, V. Nilakantan, G. Hilton, X. Zhou, A. K. Khanna, N. L. N. Halligan, C. C. Felix, B. Kampalath, O. W. Griffith, M. A. Hayward, et al.
Variable efficacy of N6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection
Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H525 - H534.
[Abstract] [Full Text] [PDF]


Home page
Cardiovasc ResHome page
R. Bolli, K. Shinmura, X.-L. Tang, E. Kodani, Y.-T. Xuan, Y. Guo, and B. Dawn
Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning
Cardiovasc Res, August 15, 2002; 55(3): 506 - 519.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, X.
Right arrow Articles by Cannon, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, X.
Right arrow Articles by Cannon, P. J.
Related Collections
Right arrow Biochemistry and metabolism
Right arrow Animal models of human disease
Right arrow Apoptosis
Right arrow CV surgery: transplantation, ventricular assistance, cardiomyopathy