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

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. We used hemin to overexpress HO-1 and analyze its effects in a model of I/R in aged livers used for orthotopic transplantation. 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. 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.

Wang XH ，Wang K ，Zhang F ，Li XC ，Qian XF ，Cheng F ，Li GQ ，Fan Y ... -  《TRANSPLANTATION PROCEEDINGS》

Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury.

Ischemia/reperfusion (I/R) injury is a critical factor in the dysfunction of steatotic orthotopic liver transplants. Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic I/R injury. We used adenovirus (Ad)-based HO-1 gene transfer to analyze the effects of HO-1 overexpression in a well-established fatty Zucker rat model of I/R followed by orthotopic liver transplantation. Ad-HO-1 gene therapy increased recipient survival (80% vs. 40-50% in controls) and significantly diminished hepatocyte injury, as compared with untreated and Ad-beta-galactosidase (Ad-beta-Gal)-treated livers. Orthotopic liver transplants in the Ad-HO-1 group exhibited less macrophage infiltration in the portal areas, as compared with controls. Unlike untreated and Ad-beta-Gal-treated orthotopic liver transplant controls, which showed elevated levels of inducible nitric oxide synthase by infiltrating macrophages, inducible nitric oxide synthase expression in the Ad-HO-1 group was almost absent. In contrast, endothelial nitric oxide synthase was comparable in Ad-HO-1- and Ad-beta-Gal-transduced fatty orthotopic liver transplants. Intragraft expression of antiapoptotic Bcl-2 and Bag-1 was increased in Ad-HO-1-treated orthotopic liver transplants, as compared with Ad-beta-Gal controls. Moreover, increased HO enzymatic activity was accompanied by inhibition of caspase-3 protein expression. HO-1 gene transfer significantly prolongs survival of steatotic orthotopic liver transplants, depresses macrophage infiltration, suppresses local expression of inducible nitric oxide synthase, and modulates pro- and antiapoptotic pathways.

Coito AJ ，Buelow R ，Shen XD ，Amersi F ，Moore C ，Volk HD ，Busuttil RW ，Kupiec-Weglinski JW ... -  《TRANSPLANTATION》

Heme oxygenase-1 alleviates ischemia/reperfusion injury in aged liver.

To investigate if ischemia/reperfusion (I/R) injury in aged liver could be alleviated by heme oxygenase-1 (HO-1). Three groups of SD rats (16 mo old) were studied. Group 1: control donors received physiological saline 24 h before their livers were harvested; group 2: donors were pretreated with hemin 24 h before their livers were harvested; and group 3: donors received hemin 24 h before their livers were harvested and zinc protoporphyrin (ZnPP, HO-1 inhibitor) was given to recipients at reperfusion. The harvested livers were stored in University of Wisconsin solution (4 degrees) for 6 h, and then transplanted to syngeneic rats. Serum glutamic oxaloacetic transaminase (SGOT), apoptotic cells, and apoptotic gene were measured 3, 6, 12, 24, 48 h after reperfusion. We measured the apoptotic index by TUNEL, determined the expression of antiapoptotic Bcl-2 and proapoptotic (caspase-3) gene products by Western blot. After 3, 6, 12, 24, and 48 h of reperfusion, the SGOT levels (584.4+/-85.8 u/L, 999.2+/-125.2 u/L, 423.4+/-161.3 u/L, 257.8+/-95.8 u/L, and 122.4+/-26.4 u/L) in hemin group were significantly (all P<0.05) lower than those in saline group (1082.2+/-101.2 u/L, 1775.2+/-328.3 u/L, 840.4+/-137.8 u/L, 448.6+/-74.3 u/L, and 306.2+/-49.3 u/L). Liver HO-1 enzymatic activity correlated with beneficial effects of hemin and deleterious effects of adjunctive ZnPP treatment. Markedly less apoptotic (TUNEL+) liver cells 3, 6, 12, 24, and 48 h after reperfusion (5.16+/-0.73, 10.2+/-0.67, 9.28+/-0.78, 7.14+/-1.12, and 4.78+/-0.65) (P<0.05) could be detected in hemin liver grafts, as compared to controls (7.82+/-1.05, 15.94+/-1.82, 11.67+/-1.59, 8.28+/-1.09, and 6.36+/-0.67). We detected the increased levels of Bcl-2 (1.5-fold) expression and compared with saline controls. These differences were most pronounced at 12 h after transplantation. In contrast, an active form of proapoptotic caspase-3 (p20) protein was found to be 2.9-fold lower at 24 h in hemin-pretreated group, as compared to saline liver transplant controls. HO-1 overexpression can provide potent protection against cold I/R injury. This effect depends, at least in part, on HO-1-mediated inhibition of antiapoptotic mechanism.

Wang XH ，Wang K ，Zhang F ，Li XC ，Li J ，De W ，Guo J ，Qian XF ，Fan Y ... -  《-》

Graft injury in relation to graft size in right lobe live donor liver transplantation: a study of hepatic sinusoidal injury in correlation with portal hemodynamics and intragraft gene expression.

To investigate the degree and mechanism of hepatic sinusoidal injury in different graft sizes in right lobe live donor liver transplantation (LDLT). Liver grafts from living donors are likely to be small-for-size for adult recipients. Graft injury after reperfusion is common, but the mechanism and degree of injury remain unclear. The hepatic sinusoidal injury in different graft sizes and its relationship with portal hemodynamics and intragraft gene response at the early phase after reperfusion have not been studied in right lobe LDLT. From May 2000 to November 2001, 40 adults receiving right lobe LDLT had portal pressure measured continuously before and after reperfusion. Liver biopsies were taken before and after reperfusion for detection of vasoregulatory genes (endothelin-1 and endothelial nitric oxide synthase) and heat shock genes (heat shock protein 70 and heme oxygenase-1), and electron microscope examination. Blood samples from the portal vein and suprahepatic inferior vena cava were taken for the measurement of plasma nitric oxide level. The recipients were grouped according to the ratio of graft weight to estimated standard liver weight: group 1 (n = 10), less than 40%; group 2 (n = 21), 40% to 60%; and group 3 (n = 9), more than 60%. The portal pressures recorded after reperfusion in group 1 were significantly higher within 30 minutes of reperfusion than those in groups 2 and 3. After reperfusion, the intragraft endothelin-1 mRNA level in group 1 increased by 161% of the basal level but decreased by 31.5% and 62% of the basal level in groups 2 and 3, respectively. The intragraft mRNA level of heme oxygenase-1 in groups 1 and 2 decreased by 75.5% and 25.3% of the basal level respectively but increased by 41% of basal level in group 3. The intragraft protein level of heat shock protein 70 decreased by 50 ng/mL after reperfusion in group 1 but increased by 12.4 ng/mL and 0.6 ng/mL in groups 2 and 3, respectively. The portal vein plasma nitric oxide level decreased more significantly after reperfusion in group 1 than in group 2. Electron microscope examination of liver biopsies in group 1 showed tremendous mitochondrial swelling as well as irregular large gaps between the sinusoidal lining cells. There were two hospital deaths in group 1 and none in the other two groups. Patients implanted with grafts less than 40% of standard liver weight suffered from transient portal hypertension early after reperfusion. The phenomenon was accompanied by intragraft upregulation of endothelin-1 and ultrastructural evidence of sinusoidal damage. The transient portal hypertension after reperfusion, subsequent endothelin-1 overexpression, and plasma nitric oxide level reduction, together with downregulation of heme oxygenase-1 and heat shock protein 70, may account for the small-for-size graft injury.

Man K ，Fan ST ，Lo CM ，Liu CL ，Fung PC ，Liang TB ，Lee TK ，Tsui SH ，Ng IO ，Zhang ZW ，Wong J ... -  《-》

Cytoprotective and antiapoptotic effects of IL-13 in hepatic cold ischemia/reperfusion injury are heme oxygenase-1 dependent.

Liver injury caused by ischemia/reperfusion (I/R) insult represents the major problem following orthotopic liver transplantation (OLT). I/R damage has been linked to Th1-like cytokine producers. This study evaluates putative cytoprotective effects/mechanisms of Th2-type IL-13 gene transfer. IL-13 overexpression prevented hepatic insult in a rat model of 24 h cold ischemia followed by OLT, as assessed: (i) profoundly decreased hepatocellular damage (sGOT levels), and ameliorated histological signs of I/R injury (Suzuki criteria), consistent with long-term OLT survival; (ii) prevented hepatic apoptosis (TUNEL stains) and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes. However, inhibition of HO-1 with tin protoporphyrin reversed cytoprotective/antiapoptotic effects of IL-13. In conclusion, cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in this clinically relevant rat hepatic cold I/R injury model was accomplished via decreased apoptosis and induction of antiapoptotic/antioxidant molecules. HO-1 neutralization studies suggest that HO-1 represents one of putative IL-13 downstream effectors. This study provides the rationale for novel approaches to maximize organ donor pool through the safer use of OLTs despite prolonged periods of cold ischemia.

Ke B ，Shen XD ，Lassman CR ，Gao F ，Busuttil RW ，Kupiec-Weglinski JW ... -  《AMERICAN JOURNAL OF TRANSPLANTATION》