Bibo Ke

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

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°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 °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 40 h 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.

Heme oxygenase-1 overexpression protects rat hearts from cold ischemia/reperfusion injury via an antiapoptotic pathway.

BACKGROUND Ischemia/reperfusion (I/R) injury is one of the most important causes of the early graft loss. We have shown that overexpression of heme oxygenase-1 (HO-1), an inducible heat shock protein 32, protects rat livers against I/R injury. We report on the cytoprotective effects of HO-1 in a rat cardiac I/R injury model, using cobalt protoporphyrin (CoPP) as HO-1 inducer and zinc protoporphyrin (ZnPP) as HO-1 inhibitor. METHODS Three groups of Lewis rats were studied: group 1 control donors received phosphate-buffered saline 48 hr before the harvest; group 2 donors were pretreated with CoPP at -48 hr; and in group 3, donors received CoPP at -48 hr and ZnPP was given to recipients at reperfusion. Hearts were harvested, stored in University of Wisconsin solution (4 degrees C) for 24 hr, and then transplanted to syngeneic (Lewis) rats. RESULTS Sixty percent of control grafts ceased their function in <15 min. In contrast, 80% of CoPP-pretreated grafts survived 14 days. All grafts stopped functioning within 24 hr after CoPP + ZnPP therapy. Cardiac HO-1 enzymatic activity and protein expression correlated with beneficial effects of CoPP and deleterious effects of adjunctive ZnPP treatment. Markedly less apoptotic (TUNEL+) myocyte/endothelial cells could be detected in CoPP cardiac grafts, as compared with controls. The expression of antiapoptotic (Bcl-2/Bag-1) proteins was up-regulated in the CoPP group. CONCLUSION HO-1 overexpression provides potent protection against cold I/R injury in a stringent rat cardiac model. This effect depends, at least in part, on HO-1-mediated up-regulation of a host antiapoptotic mechanism, especially in the early postreperfusion period.

Toll‐Like Receptor and Heme Oxygenase‐1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll‐like receptor 4 (TLR4) signaling is specifically required in initiating antigen‐independent IRI leading to liver inflammation, whereas local induction of anti‐oxidant heme oxygenase‐1 (HO‐1) is cytoprotective. This study analyzes in vivo interactions between HO‐1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90‐min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2‐deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzukis scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF‐α production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO‐1 expression. In contrast, tin protoporphyrin‐mediated HO‐1 inhibition restored hepatic damage in otherwise IRI‐resistant TLR4 mutant/KO mice. CoPP‐induced HO‐1 overexpression ameliorated hepatic damage in IRI‐susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO‐1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO‐1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO‐1 repressor.

Heme oxygenase 1 gene transfer prevents CD95/Fas ligand-mediated apoptosis and improves liver allograft survival via carbon monoxide signaling pathway

Apoptosis via the CD95/FasL (CD95L) pathway plays an important role in allograft rejection. Heme oxygenase 1 (HO-1), a stress-responsive cytoprotective molecule, may be essential in preventing graft rejection. We used Ad-HO-1 gene transfer to analyze HO-1-mediated effects in a rat allogeneic orthotopic liver transplantation (OLT) model. The cytotoxicity to Fas-bearing YAC-1 target cells and frequency of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells in vitro were diminished in Ad-CD95L + Ad-HO-1-transfected cells, as compared with Ad-CD95L + Ad-beta-gal controls (p < 0.001). AdHO-1 gene transfer prevented <10-day rejection of dark agouti (DA) livers in Lewis (LEW) rats (survival >32 days), and diminished apoptosis. Unlike Ad-beta-gal OLTs, which showed signs of severe acute rejection, OLTs in the Ad-HO-1 group exhibited mild to moderate rejection and improved function. These beneficial effects were abrogated after adjunctive treatment with tin protoporphyrin (SnPP), an HO-1 antagonist. Intragraft expression of HO-1 and antiapoptotic gene products (Bcl-xl/Bag-1) was enhanced in Ad-HO-1-transduced OLTs, in association with selectively depressed expression of helper T cell type 1 cytokines (interleukin 2 and interferon gamma), as compared with Ad-beta-gal controls. To deliver CO, one of the downstream HO-1 mediators, allogeneic OLT recipients were exposed to methylene chloride. Such treatment prolonged survival to >47 days, diminished apoptosis, and preserved hepatic architecture/function. Thus, Ad-HO-1 gene transfer prevents CD95/FasL-mediated apoptosis, and significantly prolongs allogeneic OLT survival via a downstream HO-1-CO signaling pathway.

Gene Transfer-Induced Local Heme Oxygenase-1 Overexpression Protects Rat Kidney Transplants From Ischemia/Reperfusion Injury

Heme oxygenase-1 (HO-1) overexpression using gene transfer protects rat livers against ischemia/reperfusion (I/R) injury. This study evaluates the effects of Ad-HO-1 gene transfer in a rat renal isograft model. Donor LEW kidneys were perfused with Ad-HO-1, Ad-beta-gal, or PBS, stored at 4 degrees C for 24 h, and transplanted orthotopically into LEW recipients, followed by contralateral native nephrectomy. Serum creatinine, urine protein/creatinine ratios, severity of histologic changes, HO-1 mRNA/protein expression, and HO enzymatic activity were analyzed. Ad-HO-1 gene transfer conferred a survival advantage when compared with PBS- and Ad-beta-gal-treated controls, with median survival of 100, 7, and 7 d, respectively (P < 0.01). Serum creatinine levels were elevated at day 7 in all groups (range, 2.2 to 5.8 mg/dl) but recovered to 1.0 mg/dl by day 14 (P < 0.01) in Ad-HO-1 group, which was sustained thereafter. Urine protein/creatinine ratio at day 7 was elevated in both PBS and Ad-beta-gal, as compared with the Ad-HO-1 group (12.0 and 9.8 versus 5.0; P < 0.005); histologically, ATN and glomerulosclerosis was more severe in Ad-beta-gal group at all time points. Reverse transcriptase-PCR-based HO-1 gene expression was significantly increased before reperfusion (P < 0.001) and remained increased in the Ad-HO-1-treated group for 3 d after transplantation. Concomitantly, HO enzymatic activity was increased at transplantation and at 3 d posttransplant in the Ad-HO-1 group, compared with Ad-beta-gal controls (P < 0.05); tubular HO-1 expression was discernible early posttransplant in the Ad-HO-1 group alone. These findings are consistent with protective effects of HO-1 overexpression using a gene transfer approach against severe renal I/R injury, with reduced mortality and attenuation of tissue injury.

Fas ligand gene transfer to renal allografts in rats : Effects on allograft survival

BACKGROUND Fas ligand (FasL) induces apoptosis of cells bearing its receptor Fas, and has been shown to be important in T-cell development and regulation and in immune privilege. We hypothesized that FasL expression by renal allografts might provide protection from rejection. METHODS The murine FasL cDNA was cloned into a replication-defective adenovirus (AdV-FasL). Protein expression was confirmed by immunostaining of AdV-FasL-transduced HeLa cells. Allogeneic kidney transplants were performed between WF (RT1u) donors and Lewis (RT1) recipients. Donor kidneys were perfused in situ with saline alone (control), or 9 x 10(9) plaque-forming units of AdV-FasL. One native kidney was removed at the time of transplant and the other at 6 or 7 days. Uremic death was the endpoint, and deaths within 7 days of transplant were excluded. Transduced allografts were stained for FasL expression using a monoclonal antibody and tested for FasL mRNA production by reverse transcriptase-polymerase chain reaction and Northern blotting. RESULTS Immunostaining of AdV-FasL-transduced allografts demonstrated efficient gene transfer lasting approximately 2 weeks, and FasL mRNA production in the AdV-FasL-transduced allografts was confirmed by Northern blotting and reverse transcriptase-polymerase chain reaction. Mean survival of animals with AdV-FasL-transduced renal allografts was 27.8 days vs. 11.6 days in control animals (P < 0.05). CONCLUSIONS (1) Adenoviral vectors can successfully transduce rat kidneys with the FasL cDNA. (2) FasL gene transfer prolongs rat renal allograft survival.

CD154-CD40 T-cell costimulation pathway is required in the mechanism of hepatic ischemia/reperfusion injury, and its blockade facilitates and depends on heme oxygenase-1 mediated cytoprotection.

Background. Ischemia/reperfusion (I/R) injury remains an important clinical problem that affects both early and later allograft outcome. This study was designed to analyze the role of T cells and CD154-CD40 T- cell costimulation pathway in a mouse liver I/R model. Methods and Results. Ninety minutes of warm ischemia followed by 4 h of reperfusion in wild-type (WT) mice resulted in a significant hepatic damage, as assessed by liver function (serum alanine aminotransferase [sALT] levels), local neutrophil accumulation (myeloperoxidase activity), and histology (Suzuki’s score). In contrast, T-cell deficiency (in T-cell deficient [nu/nu] mice), disruption of the CD154 signaling (in knockout [KO] mice), or its blockade in WT recipients (after MR1 monoclonal antibody [mAb] treatment), virtually prevented hepatic I/R insult. Unlike CD154-deficient T cells, adoptive transfer of WT spleen cells fully restored hepatic I/R injury in nu/nu mice. Finally, the improved hepatic function in CD154 KO recipients, WT mice treated with CD154 mAb, or nu/nu mice infused with CD154-deficient cells resulted in consistently enhanced expression of heme oxygenase-1 (HO-1), a heat-shock protein with cytoprotective functions. Conclusion. This study confirms the importance of T cells, and documents for the first time the role of CD154 costimulation signals in the mechanism of hepatic I/R injury. We also show that CD154 blockade-mediated cytoprotection results and depends on HO-1 overexpression. Our data provide the rationale for human trials to target CD154-CD40 costimulation in hepatic I/R injury, particularly in the transplant patient.

Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model

This study analyzes how toll‐like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild‐type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4°C for 24 hours. Unlike WT OLTs, TLR4‐deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzukis grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)‐γ‐inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM‐1), as well as tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐2, and IFN‐γ, yet increased IL‐4 and IL‐10 expression. The well‐functioning OLTs from TLR4 KO donors revealed attenuated activity of capase‐3, and enhanced heme oygenase‐1 (HO‐1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI‐dependent responses. Liver Transpl 13:1435–1443, 2007.

ASC/caspase‐1/IL‐1β signaling triggers inflammatory responses by promoting HMGB1 induction in liver ischemia/reperfusion injury

Apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors, is essential for inducing caspase‐1 activation and the consequent secretion of interleukin‐1β (IL‐1β), which is associated with local inflammation during liver ischemia/reperfusion injury (IRI). However, little is known about the mechanisms by which the ASC/caspase‐1/IL‐1β axis exerts its function in hepatic IRI. This study was designed to explore the functional roles and molecular mechanisms of ASC/caspase‐1/IL‐1β signaling in the regulation of inflammatory responses in vitro and in vivo. With a partial lobar liver warm ischemia (90 minutes) model, ASC‐deficient and wild‐type mice (C57BL/6) were sacrificed at 6 hours of reperfusion. Separate animal cohorts were treated with an anti–IL‐1β antibody or control immunoglobulin G (10 mg/kg/day intraperitoneally). We found that ASC deficiency inhibited caspase‐1/IL‐1β signaling and led to protection against liver ischemia/reperfusion (IR) damage, local enhancement of antiapoptotic functions, and down‐regulation of high mobility group box 1 (HMGB1)–mediated, toll‐like receptor 4 (TLR4)–driven inflammation. Interestingly, the treatment of ASC‐deficient mice with recombinant HMGB1 re‐created liver IRI. Moreover, neutralization of IL‐1β ameliorated the hepatocellular damage by inhibiting nuclear factor kappa B (NF‐κB)/cyclooxygenase 2 signaling in IR‐stressed livers. In parallel in vitro studies, the knockout of ASC in lipopolysaccharide‐stimulated bone marrow–derived macrophages depressed HMGB1 activity via the p38 mitogen‐activated protein kinase pathway and led to the inhibition of TLR4/NF‐κB and ultimately the depression of proinflammatory cytokine programs. Conclusion: ASC‐mediated caspase‐1/IL‐1β signaling promotes HMGB1 to produce a TLR4‐dependent inflammatory phenotype and leads to hepatocellular injury. Hence, ASC/caspase‐1/IL‐1β signaling mediates the inflammatory response by triggering HMGB1 induction in hepatic IRI. Our findings provide a rationale for a novel therapeutic strategy for managing liver injury due to IR. (HEPATOLOGY 2013)