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》

Extended preservation of rat liver graft by induction of heme oxygenase-1.

Livers can be preserved only for a short period without jeopardizing the transplantation outcome. Heat shock proteins (HSPs) protect against ischemia and reperfusion injury. We studied whether their induction and, in particular, the induction of heme oxygenase 1 (HO-1), improves transplantation survival after an extended time of cold storage. Rats were subjected to heat preconditioning (42 degrees C for 20 minutes). Livers were harvested 24 hours later, preserved in cold University of Wisconsin solution for 44 hours, and transplanted in isogeneic rats (arterialized transplantation). HO-1 was specifically induced and inhibited by cobalt protoporphyrin and tin protoporphyrin, respectively. All animals receiving a graft without preconditioning and subjected to 44 hours of cold preservation died within 3 days, whereas 89% of rats who received a graft exposed to heat survived for 3 weeks (P =.0004). Preconditioning reduced serum aspartate transaminase (AST) and lactate dehydrogenase activities after reperfusion, improved bile flow, and decreased the histologic lesions of reperfusion injury. These significant effects of heat preconditioning were prevented by administration of tin protoporphyrin and could be reproduced by administration of cobalt protoporphyrin. In grafts without preconditioning, only a small fraction (<5%) of hepatocytes were positive with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay, and even less expressed activated caspase 3. Preconditioning tended to reduce the number of positive cells and to stimulate the expression of antiapoptotic Bcl-X(L). In conclusion, heat preconditioning and, specifically, overexpression of HO-1 improve posttransplantation survival and graft function after prolonged cold ischemia preservation. The mechanism underlying these beneficial effects does not appear to be prevention of apoptosis.

Redaelli CA ，Tian YH ，Schaffner T ，Ledermann M ，Baer HU ，Dufour JF ... -  《HEPATOLOGY》

Heme oxygenase 1 mediates the immunomodulatory and antiapoptotic effects of interleukin 13 gene therapy in vivo and in vitro.

This study analyzes mechanisms by which interleukin 13 (IL-13) affects "infectious tolerance" in rat recipients of cardiac allografts, with emphasis on interactions between intragraft Ad-IL-13 gene transfer and systemic infusion of regulatory cells. Although exogenous viral IL-13 was modestly effective on its own, adjunctive Ad-IL-13 gene therapy and adoptive transfer of suboptimal dose of regulatory T cells exerted synergistic effects, as evidenced by long-term cardiac allograft survival in test recipients. Local IL-13 induction (determined by enzyme-linked immunosorbent assay and immunohistology) diminished intragraft apoptosis, and upregulated antiapoptotic A20 and antioxidant heme oxygenase 1 (HO-1). Ad-IL-13 plus regulatory cells synergistically diminished the frequency of cells positive by TUNEL (TdT [terminal deoxynucleotidyltransferase]-mediated dUTP nick-end labeling) assay, and enhanced cytoprotective gene expression. These findings correlated with in vitro studies in which Ad-IL-13 decreased tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity, conferred resistance to apoptosis, and increased HO-1/A20 expression in human umbilical vein endothelial cell (HUVEC) cultures. However, inhibition of HO-1 after treatment with tin protoporphyrin reversed the immunomodulatory/antiapoptotic effects of Ad-IL-13 both in vivo (infectious transplantation tolerance), and in vitro (HUVECs). Thus, by decreasing apoptosis/TNF-alpha-mediated cytotoxicity, and by facilitating induction of antiapoptotic/antioxidant molecules in HUVECs, this study documents the cytoprotective function of Ad-IL-13 in vitro, and points toward in vivo synergy between Ad-IL-13 and regulatory cells in the infectious transplantation tolerance pathway. Results of HO-1 neutralization studies suggest that HO-1 represents one of the putative IL-13 downstream effectors.

Ke B ，Shen XD ，Zhai Y ，Gao F ，Busuttil RW ，Volk HD ，Kupiec-Weglinski JW ... -  《HUMAN GENE THERAPY》

Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation.

This study analyzes the effects and mechanisms of heme oxygenase-1 (HO-1)-mediated cytoprotection in rat livers exposed to cold preservation. In the first series, rats were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h, and then perfused ex vivo for 2 h. Livers pretreated with CoPP had significantly higher portal venous blood flow and increased total bile production, as compared with the ZnPP group. This correlated with histologic (Banff) criteria of hepatocyte injury/liver function. In the second series, rat livers were stored at 4 degrees C for 24 h or 40 h, and then transplanted into syngeneic recipients. After 24 h of preservation, 80% of rats bearing CoPP-pretreated liver grafts survived 21 days (vs. 50% in controls). After 40h of cold preservation, liver transplant survival at day 1, 7 and 21 for the CoPP group was: 100%, 71% and 57%, respectively (vs. 50%, 50% and 33% in controls). This correlated with improved hepatic function/histologic (Suzuki) criteria of hepatocyte injury after HO-1 overexpression (immunohistology/Western blots) by infiltrating macrophages. This study documents the potential utility of HO-1-inducing agents in preventing ischemia/reperfusion injury resulting from prolonged storage of liver transplants.

Kato H ，Amersi F ，Buelow R ，Melinek J ，Coito AJ ，Ke B ，Busuttil RW ，Kupiec-Weglinski JW ... -  《AMERICAN JOURNAL OF TRANSPLANTATION》

Heme oxygenase-1 attenuates ischemia/reperfusion-induced apoptosis and improves survival in rat renal allografts.

Kidneys can be preserved only for a limited time without jeopardizing graft function and survival. Induction of heat shock proteins (HSPs) can protect against ischemia/reperfusion (I/R) injury. Therefore, we investigated whether the induction of the HSP, heme oxygenase-1 (HO-1), improves outcome following isotransplantation after an extended period of cold storage. Rats were subjected to heat preconditioning (HP; 42 degrees C for 20 minutes). Kidneys harvested after 24 hours, were preserved in cold University of Wisconsin (UW) solution at 4 degrees C for 45 hours and transplanted into bilateral nephrectomized rats. Cobalt protoporphyrin (CoPP) was administered in another group of animals in order to induce HO-1 pharmacologically, while other groups of animals received the HO-1 inhibitor, tin protophorphyrine (SnPP), following HP or CoPP. Cold ischemia caused a complete attenuation of graft function within 3 days following transplantation and subsequent death of all animals, whereas HP protected graft function and five of nine rats survived for 3 weeks. HP inhibited the induction of osteopontin and induced the expression of HO-1, HSP 70 and 90, and the antiapoptotic factor Bcl-XL. Grafts exposed to HP were protected against structural I/R injuries as revealed by histologic assessment using a semiquantitative score. Furthermore, induction of apoptosis was attenuated and activation of caspase-3 was inhibited. Comparable results were observed after administration of CoPP, whereas SnPP inhibited the effects of HP and CoPP. HP or administration of CoPP induced both HO-1, preserved kidney graft function, and prevented postreperfusion apoptosis after cold preservation.

Wagner M ，Cadetg P ，Ruf R ，Mazzucchelli L ，Ferrari P ，Redaelli CA ... -  《KIDNEY INTERNATIONAL》