Poly-ADP-ribose polymerase-inhibition protects against myocardial and endothelial reperfusion injury after heart transplantation

The Journal of Heart and Lung Transplantation Volume 22, Number 1S S.M. Woolley,1 B.V. Naidu,1 A.S. Farivar,1 A.L. Salzman,2 C. Szabo,2 M.S. Mulligan,1 1Cardiothoracic Surgery, University of Washington, Seattle, WA; 2Inotek Corporation, Beverly, MA The activation of poly(ADP)ribose synthetase (PARS), in response to free radical and oxidant induced DNA strand breaks triggers an energy consuming cycle ultimately resulting in cell death. Previous studies have reported that inhibition of PARS pharmacologically or by genetic deficiency confer protection in models of endotoxic shock and ischemiareperfusion injury (heart and gut). The purpose of this study was to determine the role of PARS inhibition in lung ischemia-reperfusion injury (LIRI). Left lungs of Long Evans rats were rendered ischemic for ninety minutes and reperfused for up to four hours. Treated animals received 3mg/kg IV of a PARS inhibitor (INO-1001) 30 minutes prior to ischemia. Injury was assessed in terms of tissue neutrophil accumulation (MPO content), vascular permeability (125-I BSA extravasation) and bronchoalveolar lavage (BAL) leukocyte count. Cytokine and chemokine content in BAL fluid was determined by ELISA. Separate tissue samples were processed for nuclear transcriptional factor activation by EMSA and apoptosis by TUNEL assay. Lung vascular permeability was reduced in treated animals by 73% compared to controls (p⬍0.009). The protective effects of PARS inhibition correlated with a 46% decrease in tissue MPO content (p⬍0.008) and a 56% reduction in BAL leukocyte counts (p⬍0.01). This positively correlated with diminshed expression of pro-inflammatory mediators. TNF␣ was decreased by 96%, MIP-1␣ by 99%, MIP-2 by 75%, and CINC by 70%.The expression of nuclear transcription factors and the degree of apoptosis were also reduced. The deleterious effects of LIRI are initiated by formation of free radicals and superoxides which in turn leads to DNA strand breaks. Consequently activation of PARS causes cellular energy depletion and ultimately cell death, as well as pro-inflammatory nuclear transcription factor activation. Amelioration of these mechanisms likely contributes to the protective effects of PARS inhibition in LIRI. 214 EFFECTS OF P38 MITOGEN-ACTIVATED PROTEIN KINASE INHIBITOR AS AN ADDITIVE TO CELSIOR SOLUTION ON CANINE HEART TRANSPLANTATION FROM NON-HEARTBEATING DONORS N. Koike,1 I. Takeyoshi,1 S. Ohki,1 M. Tokumine,1 K. Matsumoto,2 Y. Morishita,1 1Second Department of Surgery, Gunma University Faculty of Medicine, Maebashi, Gunma, Japan; 2Department of Pathology, Nippon Medical School Second Hospital, Kawasaki, Kanagawa, Japan Background: The activation of p38 mitogen-activated protein kinase (MAPK) plays an important role on ischemia/reperfusion injury. FR167653 is a novel p38 MAPK inhibitor. This study evaluated the effects of p38 MAPK inhibition using FR167653 as an additive to the Celsior solution in canine heart transplantation from non-heart-beating donors (NHBDs). Methods: Donor hearts were left in situ for 20 min after cardiac arrest induced by rapid exsanguination. Twelve donor-recipient pairs of mongrel dogs were divided into two groups, the control and FR group (n ⫽ 6 each). Both group animals underwent coronary flushing and were immersed into the Celsior solution with or without FR167653 for 4 hr. Orthotopic heart transplantation was then performed. Cardiac output (CO), left ventricular pressure (LVP) and end-systolic maximal elastance (Emax) were measured at 1 and 2 hr after weaning from cardiopulmonary bypass (CPB), and then grafts were harvested for histopathological study. The activation of MAPKs including p38 MAPK, c-Jun N-terminal protein kinase (JNK) and extracellular signal-

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regulated protein kinase (ERK) were evaluated by the use of other 12 mongrel dogs during the heart transplantation procedure. Results: CO and LVP recovery rates and Emax were significantly (p ⬍ 0.05) higher in the FR group than in the control group 2 hr after weaning from CPB. Histopathological damage was more severe in the control group. The p38 MAPK, JNK and ERK were significantly (p ⬍ 0.05) activated in the control group after reperfusion. The p38 MAPK activation was significantly (p ⬍ 0.05) inhibited in the FR group than in the control group 10 min after reperfusion. Conclusions: The addition of FR167653 to the Celsior solution improved graft viability which might be due to the inhibition of p38 MAPK activation after transplantation, and may attenuate ischemia/reperfusion injury in heart transplantation from NHBDs. 215 POLY-ADP-RIBOSE POLYMERASE-INHIBITION PROTECTS AGAINST MYOCARDIAL AND ENDOTHELIAL REPERFUSION INJURY AFTER HEART TRANSPLANTATION G. Szabo,1 N. Stumpf,1 S. Baehrle,1 S. Hagl,1 1Cardiac Surgery, University of Heidelberg, Heidelberg, BW, Germany Free radical production and related cytotoxicity during ischemia/ reperfusion may lead to DNA strand-breakage which activates the nuclear enzyme poly-ADP-ribose polymerase (PARP) and initiates an energy consuming, inefficient repair cycle with transfer of the ADPribosyl moiety of NAD⫹ to protein acceptors. We investigated the effects of PARP inhibition on ischemia/reperfusion injury in a rat heart transplantation model. Intraabdominal heterotopic transplantation was performed in Lewis rats. After one hour of ischemic preservation, reperfusion was started after application of either saline vehicle (control, n⫽12), or AIQ5 (2 mg/kg) a selective PARP-inhibitor (n⫽12). Coronary blood flow (CBF), left ventricular pressure (LVP), dP/dt, end-diastolic pressure (LVEDP), endothelium-dependent vasodilatation to acetylcholine (ACH) and endothelium-independent vasodilatation to sodium nitroprusside (SNP) were measured after one and 24 hours of reperfusion. Myocardial ATP content, the expression of the adhesion molecules P-Selectine and ICAM-1 were determined. After one hour, CBF (4.01⫾0.55 vs. 2.86⫾0.35, ml/min/g, p⬍0.05), LVP (96⫾3 vs. 83⫾4 mmHg, p⬍0.05) and dP/dt (2692⫾322 vs. 1740⫾116 mmHg/s, p⬍0.05) were significantly higher in AIQ5 group in comparison to control. Vasodilatatory response to SNP was similar in both groups. ACH resulted in a significantly higher increase in CBF in the AIQ5 group (91⫾8% vs. 51⫾9%, p⬍0.05). ATP content was significantly higher (8.2⫾0.62 vs. 2.44⫾0.26 ␮mol/g, p⬍0.05) and the expression of P-Selectine and ICAM-1 were significantly reduced after treatment with AIQ5. After 24 hours, there was no difference between the groups in basal CBF, LVP, dP/dt, LVEDP and the response of CBF to SNP. However, ACH led to a still significantly higher response in the AIQ5 group (128⫾13% vs. 88⫾7%, p⬍0.05). Thus, PARP inhibition reduces reperfusion injury by restoring the energy stores and inhibiting adhesion molecule expression. 216 HEARTS TRANSFECTED WITH INDUCIBLE NITRIC OXIDE SYNTHASE HAVE ACCELERATED RESTORATION OF CARDIAC FUNCTION FOLLOWING ISCHEMIA/REPERFUSION ASSOCIATED WITH CARDIAC TRANSPLANTATION L.L. Shears,1 S. Kanno,1 T.R. Billiar,1 R. Mahidara,1 K.R. McCurry,1 R.L. Kormos,1 1Cardiothoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA