Effects of selective inhibitors of nitric oxide synthase-2 dimerization on acute cardiac allograft rejection.

BACKGROUND: Nitric oxide synthase-2 (NOS2) is expressed during acute cardiac allograft rejection in association with myocardial inflammation, contractile dysfunction, and death of cardiomyocytes by necrosis and apoptosis. Recently, allosteric inhibitors of NOS2 monomer dimerization that block NOS2 activity have been developed. METHODS AND RESULTS: To investigate effects of selective NOS2 blockade, 15 mg/kg of BBS-1 or BBS-2 was administered twice daily subcutaneously to rats starting the day of heterotopic heart transplantation. Cardiac allograft survival was increased significantly, from 6.8 days in controls to 13.3 and to 14.2 days in NOS2-inhibited allografts. At day 5 after heart transplantation, synthesis of NOx was reduced by 53%. There were significantly fewer T lymphocytes and macrophages in the inflammatory infiltrate, as well as less edema and cardiomyocyte damage, and the International Society of Heart and Lung Transplantation score fell from 5 to 4 and 3.5. NOS2 and nitrotyrosine immunostaining and the mean numbers of apoptotic cells and of apoptotic cardiomyocytes were significantly diminished in the treated allografts. CONCLUSIONS: The data indicate that selective inhibition of NOS2 dimerization prolongs survival and reduces myocardial inflammation and cardiomyocyte damage in acute cardiac allograft rejection.

Metabolic and functional protection by selective inhibition of nitric oxide synthase 2 during ischemia-reperfusion in isolated perfused hearts.

BACKGROUND: Drugs that selectively block nitric oxide synthase (NOS) 2 enzyme activity by inhibiting dimerization of NOS2 monomers have recently been developed. METHODS AND RESULTS: To investigate whether selective inhibition of NOS2 is cardioprotective, rats were pretreated for 2 days with BBS2, an inhibitor of NOS2 dimerization, at 15 mg/kg SC. Isolated buffer-perfused hearts from treated (n=9) and control (n=7) hearts were subjected to 20 minutes of ischemia followed by 60 minutes of reperfusion. NOS2 protein was upregulated in all hearts at the end of ischemia and of reperfusion; NOS2 enzyme activity was 60% lower in hearts from the treated animals. In the treated hearts, the increase in end-diastolic pressure was significantly attenuated at the end of ischemia, and the return of developed pressure at reperfusion was greater (P<0.05). Creatine kinase release at reperfusion was lower in treated hearts than in controls (P=0.02). At the end of ischemia and of reperfusion, myocardial ATP levels were significantly higher in the treated hearts than in controls (P<0.05). In the treated hearts under ischemic conditions, lactate content was higher and the lactate/pyruvate ratio was lower than in controls (P<0.05); GAPDH activity was higher; and G-3-P and aldose reductase activity were lower. At reperfusion, in the treated hearts, there was less histological damage and less apoptosis of cardiac muscle cells. CONCLUSIONS: Pretreatment with BBS2, a selective inhibitor of NOS2, improves contractile performance, preserves myocardial ATP, and reduces damage and death of cardiac myocytes during ischemia and reperfusion of isolated buffer-perfused rat hearts.

The effect of selective inhibition of cyclooxygenase (COX)-2 on acute cardiac allograft rejection.

BACKGROUND: Using a rat (Lewis-Wistar Furth) abdominal heterotopic transplantation model, we reported previously that the expression of cyclooxygenase (COX)-2 is increased in parallel with that of nitric oxide synthase (NOS)-2 during cardiac allograft rejection. METHODS: To investigate effects of COX-2 inhibition in this model, allograft recipients were treated orally (PO) with 5 mg/kg per day of the tetra substituted furanone selective COX-2 inhibitor 5,5-dimethyl-3-(3 fluorophenyl)-4-(4 methylsulfonal) phenyl-2 (5H)-furanone (DFU) in 1% methyl cellulose solution. RESULTS: In the treated animals, allograft survival was increased from 6.3+/-0.5 to 12.6+/-2.6 days (P = .001). At days 3 and 5 posttransplantation, there were reductions in the extent of the inflammatory infiltrate, endovasculitis, myocardial edema, and cardiomyocyte damage in rejecting allografts. The mean numbers of apoptotic cardiomyocytes determined with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) technique were significantly reduced in DFU-treated grafts compared with untreated controls (P < 0.05). At day 3 posttransplantation, prostaglandin E2 synthesis by myocardial slices incubated with 100 microM bradykinin was reduced from 1,097+/-156 to 153+/-63 pg/mg of protein in the treated allografts (P < .005). At day 5, COX-2 protein and mRNA together with COX-2, NOS-2, and nitrotyrosine immunostaining in damaged cardiomyocytes were diminished in treated versus control grafts. CONCLUSION: The data indicate that the inhibition of COX-2 prolongs allograft survival and reduces myocardial damage and inflammation during acute cardiac allograft rejection.

Effects of inhibition of poly(adenosine diphosphate-ribose) synthase on acute cardiac allograft rejection.

BACKGROUND: Nitric oxide synthase (NOS)-2 is expressed during acute cardiac allograft rejection in association with death of heart muscle cells. The nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) synthase (PARS) is activated by agonists such as NO and peroxynitrite, which cause single-strand DNA breaks; PARS, in turn can promote both necrosis and apoptosis. To investigate the hypothesis that NO produced by NOS-2 in cardiomyocytes activates PARS and contributes to heart muscle cell death by apoptosis, experiments were performed using a heterotopic rat abdominal heart transplant model and cytokine-stimulated heart muscle cells in tissue culture. METHODS: Cardiac allografts were treated after transplantation with either the PARS inhibitor 5-aminoisoquinolinone at 3 mg/kg subcutaneously daily or with vehicle. Isolated purified adult rat cardiomyocytes incubated with cytokines to induce NOS-2 were treated in vitro with another PARS inhibitor, 3-aminobenzamide (3AB). RESULTS: PARS inhibition increased cardiac-allograft survival from 6 +/- 2 to 10 +/- 3 days (n=6, n=6, P<0.05). The inflammatory infiltrate, NOS-2-positive macrophages, myocyte apoptosis, and myocyte content of nitrotyrosine and poly(ADP-ribose) were significantly decreased in PARS inhibited allografts at day 5 posttransplantation. Similarly, apoptosis and PARS activity were diminished in cytokine-stimulated adult rat cardiomyocytes when either 3AB or L-NMMA were applied. CONCLUSIONS: The data indicate that PARS activation occurs during acute cardiac-allograft rejection and contributes significantly to the inflammatory response and to the death of cardiac muscle cells by apoptosis. They suggest that PARS inhibition combined with immunosuppression might enhance salvage of heart-muscle cells during acute cardiac-allograft rejection.