Alleviating ischemia-reperfusion injury in aged rat liver by induction of heme oxygenase-1

Alleviating Ischemia-Reperfusion Injury in Aged Rat Liver by Induction of Heme Oxygenase-1 X.H. Wang, K. Wang, F. Zhang, X.C. Li, X.-F. Qian, F. Cheng, G.Q. Li, and Y. Fan ABSTRACT Background. Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic ischemia-reperfusion (I/R) injury, a critical factor in the dysfunction of the aged liver after transplantation. Methods. We used hemin to overexpress HO-1 and analyze its effects in a model of I/R in aged livers used for orthotopic transplantation. Results. The SGOT levels in the hemin group were significantly lower than those of the saline treatment group. Hemin liver grafts showed markedly fewer apoptotic (TUNEL⫹) liver cells after reperfusion compared with the controls. The plasma nitric oxide levels in the hemin group were significantly lower than those in the control group. Unlike untreated or hemin ⫹ Znpp–treated orthotopic liver transplant controls, iNOS expression in the hemin group was almost absent at 12 and 24 hours, after reperfusion. In contrast, eNOS was comparable in hemin and saline orthotopic liver transplants. The increased levels of Bcl-2 expression compared with saline controls were most pronounced at 12 hours after transplantation. In contrast, caspase 3 was lower at 24 hours among the hemin-pretreated group compared with saline-treated liver transplant controls. Conclusions. HO-1 alleviated the I/R injury in the aged liver by suppressing local expression of inducible nitric oxide synthase and by modulating pro- and antiapoptotic pathways.

A

DVANCES IN SURGICAL procedures and immunosuppression protocols have considerably improved patient survival after orthotopic liver transplantation (OLT). One limiting problem is the disparity between the increasing number of potential recipients and the modest numbers of eligible deceased donors. The need to expand the donor population has attracted attention to the possible use of aged donor livers, which are frequently discarded because of fear of primary nonfunction. One of the most critical events leading to nonfunction or early dysfunction of aged liver grafts is the ischemia-reperfusion (I/R) injury, an antigen-independent component of organ “harvesting”.1,2 Reversible liver impairment or severe injury that results in cell death and ultimate liver failure depend on the extent of liver damage caused by the I/R injury. Heme oxygenases (HOs) are ubiquitous enzymes that catalyze the initial and rate-limiting steps in the oxidative degradation of heme to bilirubin. HOs cleave a mesocarbon of the heme molecule, producing equimolar quantities of biliverdin, iron, and carbon monoxide (CO).3 Biliverdin is reduced to bilirubin by bilirubin reductase; the free iron is

used in intracellular metabolism or sequestered in ferritin. Three HO isoforms have been identified: HO-1, an inducible heat shock protein 32, is highly induced and confers protective effects on the oxidative stress response both in vivo and in vitro. The mechanism(s) by which HO-1 confers protection against oxidative stress have not yet been defined. It is believed that the by-products derived from the catalysis of heme by HO, namely biliverdin, ferritin accumulated from released iron, and CO, mediate the physiological effects of HO-1. Both biliverdin and bilirubin possess antioxidant properties,4 whereas iron released during heme catabolism can stimulate ferritin synthesis.5 Attention has been centered on the potential antioxidant, anti-inflammatory, antiapoptotic, and possible immune modulatory functions From the Department of Hepatic Surgery, Liver Transplantation Center of Jiangsu Province, Nanjing, Jiangsu, China. Address reprint requests to Dr Xue Hao Wang and Dr Ke Wang, the Department of Hepatic Surgery, Liver Transplantation Center of Jiangsu Province, Guangzhou Road 300, Nanjing, Jiangsu 210029, China. E-mail: [email protected]

© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.10.066

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of the reaction product(s).6 –11 However, putative mechanisms by which HO-1 induction may lead to cytoprotection during I/R insult before transplantation remain unclear. We hypothesized that one possible pathway by which HO-1 confers protection against oxidant injury was via its ability to impart antiapoptotic activity. Thus, the present study was designed to examine whether the attenuation of the I/R injury by prior treatment with hemin, an HO-1 inducer, involved the antiapoptotic pathway in rat liver undergoing a cold I/R insult. MATERIALS AND METHODS Animals Aged 16-month-old and 3-month-old male Wistar rats were obtained from the Laboratory Animal Center of Jiangsu Province. The animals were fed a standard rodent diet and water and cared for according to guidelines approved by the China Association of Laboratory Animal care.

Study Design Syngeneic liver transplantation was performed using livers that had been harvested from aged rats and stored for 6 hours at 4°C in University of Wisconsin solution. The transplantation technique achieved revascularization without hepatic artery reconstruction.12,13 There were three treatment groups. In group 1, prospective liver donors (n ⫽ 25) were given 9 g/L saline (5 mL/kg SC) 24 hours before liver harvest. Group 2 donors (n ⫽ 25) received hemin (40 umol/kg SC), an HO-1 inducer, 24 hours before procurement. After 6 hours of storage at 4°C in University of Wisconsin solution, livers in both groups were then transplanted into rats. Group 3 donors (n ⫽ 10) were treated with hemin 24 hours before harvest. After 6 hours of cold storage, livers were transplanted into rats, which were then infused with ZnPP (1.5 mg/kg IV), an HO-1 inhibitor, at the end of surgery after unclamping the vessel. Separate groups of rats (n ⫽ 5/group) were killed at 3, 6, 12, 24, and 48 hours after unclamping.

Assay of Serum Glutamic Oxaloacetic Transaminase, and NO2⫺ ⫹ NO3⫺ Levels Blood samples were collected for measurement of SGOT at 3, 6, 12, 24, and 48 hours after reperfusion. Blood samples was immediately centrifuged to obtain plasma, which was stored at ⫺80°C. Plasma AST were measured using an automated chemistry analyzer, plasma NO2⫺ ⫹ NO3⫺ (NOx) levels were measured using an automated NO detector in a high-performance liquid chromatography system.

Enzymatic Assay for HO-1 Activity To determine HO-1 activity, liver isografts were removed and homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 5 ml/L Triton X-100 and protease inhibitors. Samples were frozen in small aliquots until use. Graft homogenates were mixed with 0.8 mmol/L NADPH, 0.8 mmol/L glucose-6-phosphate, 1.0 U of G-60P dehydrogenase, 1 mmol/L MgCl2, and 10 mL of purified rat liver biliverdin reductase at 4°C. The reaction was initiated by the addition of hemin (final concentration 0.25 mmol/L). The reaction mixture was incubated at 37°C in the dark for 15 minutes. At the end of the incubation period, all insoluble material was removed by centrifugation; supernates were analyzed for bilirubin concentra-

WANG, WANG, ZHANG ET AL tions based upon the extinction coefficient of 40 mmol/L⫺1 cm⫺1 at A 460 to 530 nm. Controls included naïve samples in the absence of the NADPH generating system on all the ingredients of the reaction mixture in the absence of graft homogenates. Biliverdin reductase was purified from rat liver as previously described.14

Histology Liver specimens fixed in a 10% buffered formalin solution were embedded in paraffin. Sections were cut at 4-␮m and stained with hematoxylin and eosin.

Electron Microscopic Analysis Livers fixed with 10% buffered formaldehyde were cut into 1-mm cubes and stored at 4°C overnight. After three rinses for 30 minutes in 0.1 mol/L phosphate buffer (pH 7.2), liver samples were 1-hour postfixed in phosphate-buffered 1% OsO4. After rinsing in three changes of double-distilled water for 5 minutes, the tissue, dehydrated in progressive concentrations of ethanol and propylene oxide, was embedded in epoxy resin. Thin (90-nm) sections were cut, placed on 200-mesh copper grids, and stained with 2% uranyl acetate and lead citrate. Photomicrographs were taken on a Hitachi H500 (Tokyo, Japan).

Detection of Apoptosis For these studies, part of the specimens was immediately fixed in 4°C buffered formalin and embedded in paraffin. In all cases, conventional histological examination was performed on 4-␮m thick sections of paraffin-embedded tissue. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed essentially according to the instructions of the commercial kit (ApopTag, Intergen Co, NY; USA). Nuclear counterstaining was performed with methyl green. Negative and positive control reactions were performed for each reaction step. Negative controls were obtained by omission of terminal deoxynucleotidyl transferase. All the negative controls showed no positive signal. Positive controls included sections that were pretreated by DNAase I. All the positive controls were positive. Morphometric analysis of the cells in tissue stained by the TUNEL method was performed under high-power magnification (⫻400) in a blinded fashion. Thirty random fields were counted for each TUNELstained tissue sample. The numbers of hepatocytes were expressed as the percentages of the total number of the respective populations of liver cells. Although hepatocytes were clearly identified as a specific cell population, sinusoidal endothelial cells (SLCs) were a mixed population including endothelial cells, Kupffer cells, and possibly adherent neutrophils. All nucleated cells lining the sinusoids were evaluated.

Western Blot Analysis of Bcl-2, Caspase 3, and NOS Expressions Protein was extracted from liver tissue samples with PBSTDS buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 g/L sodium dodecyl sulfate [SDS], 10 g/L sodium deoxycholate, and 10 mL/L Triton X-100, pH 7.2). Proteins (30 g/sample) in SDS loading buffer (50 mmol/L Tris, pH 7.6, 100 g/L glycerol, 10 g/L SDS) were subjected to 120 g/L SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif, USA). The gel was then stained with Coomassie blue to document equal protein loading. The membrane was blocked with 3% dry milk and 1 mL/L Tween 20 (USB, Cleveland, Ohio, USA) in PBS and

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Fig 1. Serum SGOT levels in the hemin group are decreased compared with that in saline and hemin ⫹ znpp groups (P ⬍ .05). There is no difference in saline group and hemin ⫹ znpp groups. After 6-hour reperfusion, the serum SGOT levels is highest. incubated with primary antibody against Bcl-2, caspase-3, eNOS, iNOS, and actin. After washing the filters were incubated with horseradish peroxidase and donkey anti-rabbit antibody (Amersham, Arlington Heights, Ill, USA). Relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY, USA).

Statistics Results are expressed as mean values ⫾ SEM. Statistical comparisons between the groups were performed using an unpaired two-tailed Student t test. P values of less than .05 were considered statistically significant.

RESULTS Alleviation of I/R Injury by the Induction of HO-1

SGOT release, a well-established marker of hepatocellular injury after I/R, was measured at 3, 6, 12, 24, and 48 hours of reperfusion. The SGOT levels in the hemin group were significantly decreased compared with the saline and Hemin ⫹ ZnPP group at all time points after reperfusion (P ⬍ .05, Fig 1). Histology and Electron Microscopic Analysis

The extent of hepatic I/R injury was also assessed by hematoxylin and eosin staining of liver specimens (Fig 2). Compared with the hemin group (Fig 2A) livers pretreated with saline showed severe disruption of their lobular architecture with significant periportal edema, and vacuole-type degeneration (Fig 2B) at 6 hours after reperfusion. Six hours after reperfusion, specimens from recipients the control group were evaluated by electron microscopy, revealing a great variation in vacuoles with hepatic cytoplasm in saline group (Fig 2D) compared treatment with hemin (Fig 2C).

Changes in Plasma NO2⫺ ⫹ NO3⫺ Levels

The plasma NOx level was expressed as a percent of the preischemic level. The preischemic levels were used to normalize the data obtained after anesthesia as well as at 3, 6, or 12 hours after reperfusion. Plasma NOx levels in the hemin group were significantly lower than those in the saline or hemin ⫹ Znpp group (P ⬍ .05, Fig 3). At 24 or 48 hours after reperfusion, the plasma NOx level in the control group was almost the same as that in the hemin group. Hemin Treatment Enhances the HO-1 Enzyme Activity

Pretreatment with hemin significantly increased HO-1 liver activity in aged rat liver and isograft samples [nmol of bilirubin/(mg protein min)] (Table 1) at 3 hours, 2.13 ⫾ 0.04 versus 1.09 ⫾ 0.03 in controls; and 6 hours, 2.17 ⫾ 0.04 versus 1.20 ⫾ 0.03 in controls; at 12 hours 2.22 ⫾ 0.05 versus 1.27 ⫾ 0.04 in controls; at 24 hours 2.03 ⫾ 0.04 versus 1.15 ⫾ 0.03 in controls; and at 48 hours 1.94 ⫾ 0.03 versus 1.05 ⫾ 0.03, (all P ⬍ .05). Adjunctive ZnPP given at reperfusion inhibited HO-1 activity in the liver isografts. HO-1 Overexpression Depresses Liver Apoptotic Cell Death

To detect apoptotic cells, we performed TUNEL labeling of liver isografts that had undergone a cold I/R insult before transplantation. Classic TUNEL positivity was characterized by focal nuclear staining, with nuclear and cell membrane integrity in intact apoptotic cells. Table 1 shows the results of TUNEL staining of liver isografts subjected to 3, 6, 12, 24, and 48 hours of reperfusion after 24 hours of cold preservation. The frequency of TUNEL ⫹ liver cells was reduced in sections from rats pretreated with Hemin (Fig 4A), as compared with saline-treated controls (Fig 4B) or those after adjunctive

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Fig 2. (A) No evidence of any degeneration. (B) The central vein is dilated in the saline group, with vacuole-type degeneration. (Magnification ⫻200.) (C) Both the cell nucleus and cellular organ had no significant breakdown in the hemin group. (D) There are many inequal-sized vacuoles in hepatic cytoplasm in the saline group.

Hemin ⫹ ZnPP therapy. Consequently, an apoptotic index, calculated as the percentage of TUNEL-nuclei divided by the counter stained nuclei, was significantly diminished in the group treated with hemin, compared with the saline or Hemin ⫹ ZnPP–treated groups. HO-1 Inhibits iNOS Expression in Aged Orthotopic Liver Transplants

We used Western blots to determine whether an increased HO enzymatic activity modulates iNOS expression. Interestingly, at both 12 and 24 hours after transplantation, the iNOS expression was almost absent in livers pretreated with

hemin (Fig 5), contrasting with enhanced levels in saline and hemin ⫹ Znpp group. In contrast, no differences in eNOS expression were detected between the three orthotopic liver transplant groups. HO-1 Overexpression Enhances Bcl-2 and Depresses the Caspase 3 Expression

To evaluate whether hemin therapy affected intragraft apoptotic networks, we assessed the expression of antiapoptotic Bcl-2 and proapoptotic (caspase-3) gene products in orthotopic liver transplants by Western blots (Fig 6). Indeed, in agreement with increased HO enzymatic activity in

Fig 3. Changes in plasma NO2 ⫹ NO3 (NOx) levels in the control, hemin, and hemin ⫹ Znpp groups. Values are expressed as a percent of the preischemic level. The plasma NOx levels in the hemin group were significantly lower than those in the control group (P ⬍ .05) 3 to 12 hours after reperfusion. At 24 hours after reperfusion, the plasma NOx level in the hemin group was almost the same as that in the control group.

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Table 1. Apoptotic Index After Reperfusion

Saline Hemin Hemin ⫹ Znpp

3 hours (%)

6 hours (%)

12 hours (%)

24 hours (%)

48 hours (%)

7.82 ⫾ 1.05 5.16 ⫾ 0.73 7.8 ⫾ 0.83

15.94 ⫾ 1.82 10.2 ⫾ 0.67 15.94 ⫾ 1.58

11.67 ⫾ 1.59 9.28 ⫾ 0.78 11.68 ⫾ 1.59

8.28 ⫾ 1.09 7.14 ⫾ 1.12 8.76 ⫾ 0.88

6.36 ⫾ 0.67 4.78 ⫾ 0.65 6.48 ⫾ 0.70

the liver treated with hemin, we detected increased levels of Bcl-2 (1.5-fold) expression compared with saline controls. These differences were most pronounced at 12 hours after transplantation. In contrast, at 24 hours the active form of the proapoptotic caspase 3 (p20) protein was 2.9-fold lower in hemin-pretreated group compared with the saline liver transplant controls. DISCUSSION

We reported herein the results of our studies on the effects of HO-1 overexpression to mitigate the I/R injury in aged rat livers. The principle findings of this work were: (1) hemin prevented the I/R insult following cold ischemia and reperfusion; (2) hemin-induced HO-1 overexpression, as determined by enzymatic assays, was associated with decreased hepatocellular apoptosis; (3) treatment with ZnPP, an HO-1 inhibitor, abolished these beneficial effects, documenting the direct involvement of HO-1 in protection against the I/R injury in aged rat livers. HO-1 activity, up-regulated after various stimuli, is considered one of the most sensitive indicators of cellular stress.15,16 Analogous to heat shock regulation, HO-1 overexpression may represent an endogenous adaptive mechanism protecting cells from stress after radiation, heat shock, inflammation, and ischemia. Indeed in our present study, hemin-induced HO-1 overexpression prevented or significantly decreased injury to aged livers in a stringent, clinically relevant model of 6-hour cold ischemia followed by syngeneic transplantation. Unlike saline-pretreated controls, which showed high serum levels of SGOT, aged livers harvested from hemin-pretreated donors displayed low SGOT levels. The results of the studies in which ZnPPmediated inhibition of HO-1 activity negated the beneficial effects after hemin treatment support the hypothesis that in HO-1 induction rather than modulation of other biochemical pathways protected the liver from oxidative injury.

The effects of HO-1 overexpression have been recently studied in organ transplantation. Strikingly, studies have shown that up-regulated HO-1 expression exerts important adaptive antioxidant and anti-inflammatory functions in cellular protection from pathophysiological conditions, including graft rejection and I/R injury.17–22 But, the mechanisms underlying the beneficial effects of HO-1 against I/R injury have yet to be elucidated. Emerging evidence suggests that NO has an important role in the ischemia injury. iNOS is triggered in many cell types by cytokines, such as tumor necrosis factor-␣ or interferon-␥, generating large amounts of NO,23 the role of which in liver pathophysiology remains controversial. Evidence suggests that moderate levels of NO may be beneficial, whereas high levels are associated with pathological liver conditions. In vivo NO interact with superoxide (O2⫺) to produce peroxynitrite (ONOO⫺), a potent oxidant associated with NO-mediated tissue damage.24 Indeed overexpression of iNOS has been correlated with several acute and chronic diseases.25 We evaluated the dynamic interplay between HO-1 and NOS expression. Saline or saline ⫹ Znpp livers were characterized by high iNOS levels but no appreciable difference in eNOS expression. This finding suggests that eNOS may be sufficient to generate most, if not all, of the necessary NO associated with its protective functions. We also investigated the time courses of changes in plasma NOx (the final products of NO oxidation) as markers of NO synthesis in vivo. At 3 to 12 hours after reperfusion plasma NOx levels in the hemin group were significantly lower than those in the control group, suggesting that HO-1 attenuated NO production mainly from iNOS. The finding that increased HO activity inhibited iNOS expression supports the concept that modulation of iNOS activity/expression may be the mechanism of HO-1 action. The exact mechanisms whereby free radicals, calcium entry, or inflammation might result in fulminant. I/R insult are currently unknown, although cellular apoptosis has been sug-

Fig 4. TUNEL-positive cells in saline-pretreated group and hemin-pretreated group in 6 hours after reperfusion (⫻200). (A) Hemin-pretreated group; (B) saline-pretreated group.

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Fig 5. (A) Hemin group; (B) saline group. There is no difference in eNOS expression, but the iNOS expression in hemin group at 6 and 12 hours after reperfusion was significantly less than that in the saline group.

gested as a key early event.26 –28 Thus, inhibition of the apoptotic pathway seems to be a rational therapeutic strategy to reduce the risk of preservation injury in transplanted organs. Up-regulation of HO-1 inhibited apoptosis both in vitro and in vivo,10,29 consistent with our present TUNELbased findings of a decreased frequency of apoptotic cells among hemin-pretreated liver isografts compared with controls. The cellular and physiological mechanisms by which HO-1 exerts cytoprotective functions against I/R injury might involve expression of antipoptotic proteins. Indeed in this study adjunctive treatment with ZnPP, an HO-1 enzyme activity inhibitor, prevented expression of Bcl-2 and promoted activation of caspase 3. Caspase 3 activation is a key step in apoptosis. Bcl-2 prevents the release from mitochondria into the cytosol of apoptogenic factors including cytochrome c and apoptosis-inducing factor.30,31 Moreover, Bcl-2 interacts with apoptosis-related family members, such as Bax, and several non–family member proteins including Raf-1 and Bag-1, the

latter factor cooperating with Bcl-2 to suppress apoptosis.32 In this study, the up-regulation of antipoptotic Bcl-2 occurred 3 hours after unclamping the vessels. Bcl-2 overexpression has been shown to block cell death in a caspase-independent manner, preserving mitochondria integrity during hypoxia and promoting ATP generation even in the absence of glucose by utilizing respiratory substrates during reoxygenation.33 The mechanism through which HO-1 influences the production of antipoptotic proteins remains to be elucidated. Others have shown that HO-1-induced antipoptotic effects might be mediated via CO or through the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway.29 Indeed, CO generation prevents xenograft rejection via its ability to suppress endothelial cell apoptosis in vivo.34 Low CO concentrations have been identified as key factors in HO-1–induced protection against tumor necrosis factor–induced apoptosis in cultured fibroblasts10 and endothelial cells29 in vitro. Moreover,

Fig 6. Western blot analyses of Bcl-2 and caspase-3 in orthotopic liver transplants at 12 hours (lanes A and C) and 24 hours (lanes B and D) after transplantation. At 12 and 24 hours, antiapoptotic Bcl-2 was found in higher levels in hemin-treated livers (lane A, lane B) as compared with saline controls (lane C, lane D). Proapoptotic caspase 3 was found to be decreased in hemin-treated livers (lane A, lane B) as compared with saline controls (lane C, lane D).

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animals exposed to CO in vivo exhibited significant attenuation of hyperoxia-induced lung apoptosis, at least in part via the anti-inflammatory mitogen-activated protein kinase kinase 3 (MKK3)/p38 mitogen-activated protein kinase (p38 MAPK) pathway.35 Three major MAPKs in cardiac myocytes subjected to I/R and the endoplasmic reticulum kinase (ERK) pathway may be critical for the survival of cells by protecting them from the programmed cell death caused by stress-induced activation of p38 and c-jun Nterminal kinase (JNK).36 In summary, our data demonstrate that HO-1 up-regulation provides potent protection against cold ischemia and reperfusion injury in the aged rat liver. This beneficial effect depends, at least in part, on HO-1 modulation of the antipoptotic pathway. This study documents the potential utility of HO-1– inducing agents to prevent I/R injury in marginal donors. Further studies are needed to determine whether iNOS mediates apoptosis after HO-1 overexpression and whether HO-1 or its downstream mediator(s) directly affects apoptotic networks in aged orthotopic liver transplants.

14. Kutty RK, Maines MD: Purification and characterization of biliverdin reductase from rat liver. J Biol Chem 256:3956, 1981 15. Bonnell MR, Visner GA, Zander DS, et al: Heme-oxygenase-1 expression correlates with severity of acute cellular rejection in lung transplantation. J Am Coll Surg 198:945, 2004 16. Lakkisto P, Palojoki E, Backlund T, et al: Expression of heme oxygenase-1 in response to myocardial infarction in rats. J Mol Cell Cardiol 34:1357, 2002 17. Attuwaybi BO, Kozar RA, Moore-Olufemi SD, et al: Heme oxygenase-1 induction by hemin protects against gut ischemia/ reperfusion injury. J Surg Res 118:53, 2004 18. Ito K, Ozasa H, Kojima N, et al: Pharmacological preconditioning protects lung injury induced by intestinal ischemia/reperfusion in rat. Shock 19:462, 2003 19. Braudeau C, Bouchet D, Tesson L, et al: Induction of long-term cardiac allograft survival by heme oxygenase-1 gene transfer. Gene Ther 11:701, 2004 20. Amersi F, Buelow R, Kato H, et al: Upregulation of heme oxygenase-I protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 104:1631, 1999 21. Hangaishi M, Ishizaka N, Aizawa T, et al: Induction of heme oxygenase-1 can act protectively against cardiac ischemia/reperfusion in vivo. Biochem Biophys Res Commun 279:582, 2000 22. Squiers EC, Bruch D, Buelow R, et al: Pretreatment of small bowel isograft donors with cobalt-protoporphyrin decreases preservation injury. Transplant Proc 31:585, 1999 23. Nathan C: Inducible nitric oxide synthase: what difference does it make? J Clin Invest 100:2417, 1997 24. Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271:C1424, 1996 25. Sass G, Koerber K, Bang R, et al: Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice. J Clin Invest 107:439, 2001 26. Liu X, Drognitz O, Neeff H, et al: Apoptosis is caused by prolonged organ preservation and blocked by apoptosis inhibitor in experimental rat pancreatic grafts. Transplant Proc 36:1209, 2004 27. Huet PM, Nagaoka MR, Desbiens G. et al: Sinusoidal endothelial cell and hepatocyte death following cold ischemiawarm reperfusion of the rat liver. Hepatology 39:1110, 2004 28. Zhao ZQ, Nakamura M, Wang NP, et al: Reperfusion induces myocardial apoptotic cell death. Cardiovasc Res 43:651, 2000 29. Brouard S, Otterbein LE, Anrather J, et al: Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. J Exp Med 192:1015, 2000 30. Susin SA, Zamzami N, Castedo M, et al: Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 184:1331, 1996 31. Kluck RM, Bossy-Wetzel E, Green DR, et al: The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132, 1997 32. Wang HG, Takayama S, Rapp UR, et al: Bcl-2 interacting protein Bag-1, binds and activates the kinase Raf-1. Proc Natl Acad Sci USA 93:7063, 1996 33. Saikumar P, Dong Z, Patel Y, et al: Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17:3401, 1998 34. Sato K, Balla J, Otterbein L, et al: Carbon monoxide generated by hemeoxygenase-1 suppresses the rejection of mouseto-rat cardiac transplants. J Immunol 166:4185, 2001 35. Otterbein LE, Bach FH, Alam J, et al: Carbon monoxide mediates anti-inflammatory effects via the mitogen activated protein kinase pathway. Nat Med 6:422, 2000 36. Yue TL, Wang C, Gu JL, et al: Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perused heart. Circ Res 86:692, 2000

REFERENCES 1. Deschenes M, Forbes C, Tchervenkov J, et al: Use of older donor livers is associated with more extensive ischemia damage on intraoperative biopsies during transplantation. Liver Transplant Surg 5:357, 1999 2. Busquets J, Xiol X, Figueras J, et al: The impact of donor age on liver transplantation: influence of donor age on early liver function and on subsequent patient and graft survival. Transplantation 71:1765, 2001 3. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37:517, 1997 4. Baranano DE, Rao M, Ferris CD, et al: Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A 99:16093, 2002 5. Balla G, Jacob HS, Balla J, et al: Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 267:18148, 1992 6. Oztezcan S, Kirgiz B, Unlucerci Y, et al: Heme oxygenase induction protects liver against oxidative stress in x-irradiated aged rats. Biogerontology 5:99, 2004 7. Vicente AM, Guillen MI, Habib A, et al: Beneficial effects of heme oxygenase-1 up-regulation in the development of experimental inflammation induced by zymosan. J Pharmacol Exp Ther 307:1030, 2003 8. Soares MP, Lin Y, Anrather J, et al: Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med 4:1073, 1998 9. Katori M, Busuttil RW, Kupiec-Weglinski JW: Heme oxygenase-1 system in organ transplantation. Transplantation 74:905, 2002 10. Petrache I, Otterbein LE, Alam J, et al: Heme oxygenase-1 inhibits TNF-induced apoptosis in cultured fibroblasts. Am J Physiol Lung Cell Mol Physiol 278:L312, 2000 11. Thom SR, Fisher D, Xu YA, et al: Adaptive responses and apoptosis in endothelial cells exposed to carbon monoxide. Proc Natl Acad Sci USA 97:1305, 2000 12. Amersi F, Buelow R, Kato H, et al: Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 104:1631, 1999 13. Kato H, Amersi F, Buelow R, et al: Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation. Am J Transplant 1:121, 2001