The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression.

Tissue inhibitor of metalloproteinase (TIMP) 3 is a naturally occurring inhibitor of a broad range of proteases, with key roles in extracellular matrix turnover and in the pathogenesis of various diseases. In this study, we investigated the response of mice lacking TIMP3 (TIMP3-/-) to hepatic ischemia/reperfusion injury (IRI). We report here that TIMP3-/- mice showed an enhanced inflammatory response, exacerbated organ damage, and further impaired liver function after IRI when compared with their wild-type littermates. Loss of TIMP3 led to the cleavage and shedding of E-cadherin during hepatic IRI; the full-length 120-kDa E-cadherin and the ratio of 38-kDa C-terminal fragment/120-kDa E-cadherin were decreased and increased, respectively, in TIMP3-/- livers after IRI. Moreover, GI254023X, a potent inhibitor of a disintegrin and metalloprotease (ADAM) 10, was capable of partially rescuing the expression of E-cadherin in the TIMP3-null hepatocytes. The proteolysis of E-cadherin in the TIMP3-/- livers was also linked to the loss of β-catenin from the hepatocyte membranes and to an increased susceptibility to apoptosis after liver IRI. In a similar fashion, depression of the E-cadherin/β-catenin complex mediated by TIMP3 deletion and knockdown of β-catenin by small interfering RNA were both capable of inducing caspase activation in isolated hepatocytes subjected to H₂ O₂ oxidative stress. Hence, these results support a protective role for TIMP3 expression in sheltering the hepatocyte E-cadherin/β-catenin complex from proteolytic processing and inhibiting apoptosis after hepatic IRI.

Hepatic ischemia/reperfusion injury (IRI), an inevitable antigen-independent inflammation response in cadaveric liver transplantation, correlates with poor early graft function, rejection episodes, and contributes to donor organ shortage. Sirtuin 1 (SIRT1) is a histone deacetylase that may regulate inflammatory cell activity and manage liver function in IRI, though its functional role and clinical relevance remains to be elucidated. We investigated the efficacy of SIRT1 activation in a murine liver IRI model and verified the concept of putative SIRT1-mediated hepatoprotection in clinical liver transplantation. In the experimental arm, mice were subjected to 90 minutes of liver partial warm ischemia followed by 6 hours of reperfusion with or without adjunctive SIRT1 activation in vivo (resveratrol [Res]). In parallel, bone marrow-derived macrophage (BMDM) or spleen lymphocyte cultures were treated with Res. In the clinical arm, liver biopsies from 21 adult primary liver transplant patients (2 hours after reperfusion) were divided into "low" (n = 11) versus "high" (n = 10) SIRT1 expression groups, assessed by Western blots. Treatment with Res attenuated murine liver IRI while up-regulating SIRT1, suppressing leukocyte infiltration, and decreasing proinflammatory cytokine programs. SIRT1 silencing (small interfering RNA) in BMDM cultures enhanced inflammatory cytokine programs, whereas addition of Res decreased proinflammatory response in a SIRT1-dependent manner. In addition, Res decreased interferon γ production in liver-infiltrating and spleen lymphocyte cultures. Human liver transplants with high SIRT1 levels showed improved hepatocellular function and superior survival (P = 0.04), accompanied by lower proinflammatory cytokine profile. In conclusion, our translational study is the first to identify SIRT1 as a regulator of hepatocellular function in human liver transplant recipients under ischemia/reperfusion stress. By targeting innate and adaptive immune activation, manipulation of SIRT1 signaling should be considered as a novel means to combat inflammation in liver transplantation. Liver Transplantation 23 1282-1293 2017 AASLD.

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

UNLABELLED: Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR-facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease-specific manner. Fifty-one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting-assisted hepatic analyses. Forty-three percent of OLTs failed to increase GR perioperatively under surgical stress. Post-/pre-GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase-3 and positively with B-cell lymphoma-extra large (Bcl-xL)/B-cell lymphoma 2 (Bcl-2) levels. In a murine OLT model with extended (18-hour) cold storage, treatment with rhRLX ameliorated ischemia-reperfusion (IR) damage and improved survival while up-regulating hepatocyte GR and Bcl-xL/Bcl-2 expression in OLT. rhRLX-induced GR suppressed hepatocyte high-mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll-like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX-mediated cytoprotection. CONCLUSION: This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

Background & aims

Hepatic ischemia-reperfusion injury (IRI), characterized by exogenous antigen-independent local inflammation and hepatocellular death, represents a risk factor for acute and chronic rejection in liver transplantation. We aimed to investigate the molecular communication involved in the mechanism of liver IRI.

Methods

We analyzed human liver transplants, primary murine macrophage cell cultures and IR-stressed livers in myeloid-specific heme oxygenase-1 (HO-1) gene mutant mice, for anti-inflammatory and cytoprotective functions of macrophage-specific HO-1/SIRT1 (sirtuin 1)/p53 (tumor suppressor protein) signaling.

Results

Decreased HO-1 expression in human post-reperfusion liver transplant biopsies correlated with a deterioration in hepatocellular function (serum ALT; p<0.05) and inferior patient survival (p<0.05). In the low HO-1 liver transplant biopsy group, SIRT1/Arf (alternative reading frame)/p53/MDM2 (murine double minute 2) expression levels decreased (p<0.05) while cleaved caspase 3 and frequency of TUNEL+cells simultaneously increased (p<0.05). Immunofluorescence showed macrophages were the principal source of HO-1 in human and mouse IR-stressed livers. In vitro macrophage cultures revealed that HO-1 induction positively regulated SIRT1 signaling, whereas SIRT1-induced Arf inhibited ubiquitinating activity of MDM2 against p53, which in turn attenuated macrophage activation. In a murine model of hepatic warm IRI, myeloid-specific HO-1 deletion lacked SIRT1/p53, exacerbated liver inflammation and IR-hepatocellular death, whereas adjunctive SIRT1 activation restored p53 signaling and rescued livers from IR-damage.

Conclusion

This bench-to-bedside study identifies a new class of macrophages activated via the HO-1-SIRT1-p53 signaling axis in the mechanism of hepatic sterile inflammation. This mechanism could be a target for novel therapeutic strategies in liver transplant recipients.

Lay summary

Post-transplant low macrophage HO-1 expression in human liver transplants correlates with reduced hepatocellular function and survival. HO-1 regulates macrophage activation via the SIRT1-p53 signaling network and regulates hepatocellular death in liver ischemia-reperfusion injury. Thus targeting this pathway in liver transplant recipients could be of therapeutic benefit.

Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR-facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease-specific manner. Fifty-one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting-assisted hepatic analyses. Forty-three percent of OLTs failed to increase GR perioperatively under surgical stress. Post-/pre-GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase-3 and positively with B-cell lymphoma-extra large (Bcl-xL)/B-cell lymphoma 2 (Bcl-2) levels. In a murine OLT model with extended (18-hour) cold storage, treatment with rhRLX ameliorated ischemia-reperfusion (IR) damage and improved survival while up-regulating hepatocyte GR and Bcl-xL/Bcl-2 expression in OLT. rhRLX-induced GR suppressed hepatocyte high-mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll-like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX-mediated cytoprotection.

Conclusion

This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

Macrophages polarize into heterogeneous proinflammatory M1 and antiinflammatory M2 subtypes. Heme oxygenase 1 (HO-1) protects against inflammatory processes such as ischemia-reperfusion injury (IRI), organ transplantation, and atherosclerosis. To test our hypothesis that HO-1 regulates macrophage polarization and protects against IRI, we generated myeloid-specific HO-1-knockout (mHO-1-KO) and -transgenic (mHO-1-Tg) mice, with deletion or overexpression of HO-1, in various macrophage populations. Bone marrow-derived macrophages (BMDMs) from mHO-1-KO mice, treated with M1-inducing LPS or M2-inducing IL-4, exhibited increased mRNA expression of M1 (CXCL10, IL-1β, MCP1) and decreased expression of M2 (Arg1 and CD163) markers as compared with controls, while BMDMs from mHO-1-Tg mice displayed the opposite. A similar pattern was observed in the hepatic M1/M2 expression profile in a mouse model of liver IRI. mHO-1-KO mice displayed increased hepatocellular damage, serum AST/ALT levels, Suzuki's histological score of liver IRI, and neutrophil and macrophage infiltration, while mHO-1-Tg mice exhibited the opposite. In human liver transplant biopsies, subjects with higher HO-1 levels showed lower expression of M1 markers together with decreased hepatocellular damage and improved outcomes. In conclusion, myeloid HO-1 expression modulates macrophage polarization, and protects against liver IRI, at least in part by favoring an M2 phenotype.