Arline Albala

Increased Immunoreactive Endothelin-1 in Human Transplant Coronary Artery Disease

BACKGROUND The pathogenesis of transplant coronary artery disease (TCAD) is unknown, but it is thought to derive from an interaction between immune and nonimmune factors, leading to smooth muscle cell proliferation and accumulation in the expanded neointima. Endothelin-1 (ET-1), a potent vasoconstrictor with mitogenic properties for vascular smooth muscle cells, has recently been demonstrated in native vessel atherosclerosis. The present study used immunohistochemistry to investigate the role of ET-1 in TCAD. METHODS AND RESULTS ET-1 immunoreactivity and cellular localization were assessed in human coronary arteries with TCAD (n = 13) and in normal coronary arteries (n = 10) with single- and double-label immunohistochemistry. The intensity of immunostaining was determined by a semiquantitative method. Diffuse and intense ET-1 immunoreactivity was found in 11 of 13 patients with TCAD (85%), mainly in myointimal cells and, in lesser amounts, in macrophages and endothelial cells. In contrast, normal coronary arteries had only faint immunostaining localized to the endothelial layer. Mean semiquantitative grade was significantly higher in TCAD than in normal arteries (1.8 versus 0.7; P < .05). ET-1 was more frequently present in lipid-rich, atheromatous lesions than in lipid-poor, proliferative ones. Intimal neovessels consistently immunostained for ET-1. CONCLUSIONS Immunoreactivity for ET-1 is significantly increased in TCAD, possibly as a result of stimulatory cytokines and growth factors that are upregulated in the posttransplant state. The results suggest a role for this mitogenic peptide in the pathogenesis of graft arteriosclerosis.

Inducible Nitric Oxide Synthase Expression in Smooth Muscle Cells and Macrophages of Human Transplant Coronary Artery Disease

BACKGROUND The inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide in response to cytokine stimulation. Previous investigations have demonstrated iNOS expression in the setting of acute and chronic rejection in experimental cardiac transplant models. The goal of this study was to investigate whether iNOS is upregulated in human transplant coronary artery disease (TCAD), a major cause of late mortality after cardiac transplantation. METHODS AND RESULTS We studied 15 patients with TCAD and 10 with normal coronary arteries. In situ hybridization and immunohistochemistry were used in tissue sections to localize iNOS mRNA and protein, respectively. The presence of peroxynitrite was indirectly assessed by immunostaining with an anti-nitrotyrosine antibody. Normal coronary arteries had no evidence of iNOS expression. In contrast, 30 of 36 coronary artery segments with TCAD (83%) were immunostained by the iNOS antibody. The presence of iNOS mRNA was demonstrated in these vessels by in situ hybridization. Specific cell markers identified iNOS-positive cells as neointimal macrophages and smooth muscle cells. Nitrotyrosine immunoreactivity colocalized with iNOS expression in arteries with TCAD, distributed in macrophages and smooth muscle cells. CONCLUSIONS iNOS mRNA and protein are expressed in human arteries with TCAD, where they are associated with extensive nitration of protein tyrosines. These findings indicate that the high-output nitric oxide pathway and possibly the oxidant peroxynitrite might be involved in the process leading to the development of TCAD.

Acute Cardiac Allograft Rejection in Nitric Oxide Synthase-2−/− and Nitric Oxide Synthase-2+/+ Mice Effects of Cellular Chimeras on Myocardial Inflammation and Cardiomyocyte Damage and Apoptosis

Background—The contribution of nitric oxide synthase (NOS)-2 to myocardial inflammation and cardiomyocyte necrosis and apoptosis during allograft rejection was investigated through heterotopic cardiac transplantation in mice. Methods and Results—In the first experiments, hearts from C3H donor mice were transplanted into NOS-2−/− and NOS-2+/+ C57BL/6J.129J recipients. A second series of experiments included NOS-2−/− donor hearts transplanted into NOS-2−/− recipients and wild-type NOS-2+/+ donor hearts transplanted into wild-type NOS-2+/+ recipients. (All donors were C57BL/6J and recipients were C57BL/6J.129J.) In the first series of experiments, no significant differences were observed in allograft survival, rejection score, total number of apoptotic nuclei (TUNEL), total number of apoptotic cardiomyocytes, or graft NOS-2 mRNA and protein. Positive NOS-2 immunostaining occurred in endothelial cells and cardiomyocytes in the allografts; the inflammatory infiltrate was NOS-2 positive only when recipients were NOS-2+/+. In the second series of experiments, cardiac allograft survival was significantly increased in the NOS-2−/− mice (26±13 versus 17±8 days, P <0.05), along with significant reductions in inflammatory infiltrate, rejection score, and total number of apoptotic nuclei (23.5±9.5 versus 56.4±15.3, P <0.01) and of apoptotic cardiomyocytes (2.9±1.6 versus 6.9±2.7, P <0.05). No NOS-2 or nitrotyrosine, a marker of peroxynitrite exposure, was detected in NOS-2−/− allografts transplanted into NOS-2−/− recipients. ConclusionsThe data suggest that NO derived from NOS-2 contributes to the inflammatory response and to cardiomyocyte damage and apoptosis during acute cardiac allograft rejection.

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.

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 tranferase-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 &mgr;M bradykinin was reduced from 1097±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.

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.