Naohisa Kuriyama

Tenascin‐C: A novel mediator of hepatic ischemia and reperfusion injury

Hepatic ischemia/reperfusion (IRI) injury remains a major challenge in clinical orthotopic liver transplantation (OLT). Tenascin‐C (Tnc) is an extracellular matrix protein (ECM) involved in various aspects of immunity and tissue injury. Using a Tnc‐deficient mouse model, we present data that suggest an active role for Tnc in liver IRI. We show that Tnc‐deficient mice have a reduction in liver damage and a significant improvement in liver regeneration after IRI. The inability of Tnc−/− mice to express Tnc significantly reduced the levels of active caspase‐3/transferase‐mediated dUTP nick end‐labeling (TUNEL) apoptotic markers and enhanced the expression of the proliferation cell nuclear antigen (PCNA) after liver IRI. The lack of Tnc expression resulted in impaired leukocyte recruitment and decreased expressions of interleukin (IL)‐1β, IL‐6, and CXCL2 after liver reperfusion. Tnc‐deficient livers were characterized by altered expression patterns of vascular adhesion molecules, such as vascular cell adhesion molecule‐1 and platelet endothelial cell adhesion molecule‐1 post‐IRI. Moreover, matrix metalloproteinase‐9 (MMP‐9) synthesis, which facilitates leukocyte transmigration across vascular barriers in liver IRI, was markedly down‐regulated in the absence of Tnc. We also show that Tnc is capable of inducing MMP‐9 expression in isolated neutrophils through Toll‐like receptor 4. Therefore, our data suggest that Tnc is a relevant mediator of the pathogenic events underlying liver IRI. The data also support the view that studies aimed at further understanding how newly synthesized ECM molecules, such as Tnc, participate in inflammatory responses are needed to improve therapeutic approaches in liver IRI. (HEPATOLOGY 2011)

TIMP-1 deficiency leads to lethal partial hepatic ischemia and reperfusion injury†‡

Hepatic ischemia and reperfusion injury (IRI) remains an important challenge in clinical orthotopic liver transplantation (OLT). Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is the major endogenous regulator of matrix metalloproteinase‐9 (MMP‐9). In this study we investigated the functional significance of TIMP‐1 expression in a well‐established mouse model of partial liver IRI. Compared to wildtype mice, TIMP‐1−/− mice showed further impaired liver function and histological preservation after IRI. Notably, TIMP‐1 deficiency led to lethal liver IRI, as over 60% of the TIMP‐1−/− mice died postreperfusion, whereas all TIMP‐1+/+ mice recovered and survived surgery. Lack of TIMP‐1 expression was accompanied by markedly high levels of MMP‐9 activity, which facilitates leukocyte transmigration across vascular barriers in hepatic IRI. Indeed, TIMP‐1−/− livers were characterized by massive leukocyte infiltration and by up‐regulation of proinflammatory mediators, including tumor necrosis factor alpha, interferon‐gamma, and inducible nitric oxide synthase post‐IRI. The inability of TIMP‐1−/− mice to express TIMP‐1 increased the levels of active caspase‐3 and depressed the expression of Bcl‐2 and the phosphorylation of Akt, emphasizing an important role for TIMP‐1 expression on hepatocyte survival. Using independent parameters of regeneration, 5‐bromodeoxyuridine incorporation, proliferating cell nuclear antigen expression, and histone H3 phosphorylation, we provide evidence that hepatocyte progression into S phase and mitosis was impaired in TIMP‐1‐deficient livers after IRI. Inhibition of the cell cycle progression by TIMP‐1 deficiency was linked to depressed levels of cyclins‐D1 and ‐E and to a disrupted c‐Met signaling pathway, as evidenced by reduced phosphorylated c‐Met expression and elevated c‐Met ectodomain shedding postliver IRI. Conclusion: These results support a critical protective function for TIMP‐1 expression on promoting survival and proliferation of liver cells and on regulating leukocyte recruitment and activation in liver IRI. (HEPATOLOGY 2012;56:1074–1085)

MMP-9 deficiency shelters endothelial PECAM-1 expression and enhances regeneration of steatotic livers after ischemia and reperfusion injury.

BACKGROUND & AIMS Organ shortage has led to the use of steatotic livers in transplantation, despite their elevated susceptibility to ischemia/reperfusion injury (IRI). Matrix metalloproteinase-9 (MMP-9), an inducible gelatinase, is emerging as a central mediator of leukocyte traffic into inflamed tissues. However, its role in steatotic hepatic IRI has yet to be demonstrated. METHODS We examined the function of MMP-9 in mice fed with a high-fat diet (HFD), which developed approximately 50% hepatic steatosis, predominantly macrovesicular, prior to partial hepatic IRI. RESULTS The inability of MMP-9(-/-) deficient steatotic mice to express MMP-9 significantly protected these mice from liver IRI. Compared to fatty controls, MMP-9(-/-) steatotic livers showed significantly reduced leukocyte infiltration, proinflammatory cytokine expression, and liver necrosis. Loss of MMP-9 activity preserved platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, a modulator of vascular integrity at the endothelial cell-cell junctions in steatotic livers after IRI. Using in vitro approaches, we show that targeted inhibition of MMP-9 sheltered the extracellular portion of PECAM-1 from proteolytic processing, and disrupted leukocyte migration across this junctional molecule. Moreover, the evaluation of distinct parameters of regeneration, proliferating cell nuclear antigen (PCNA) and histone H3 phosphorylation (pH3), provided evidence that hepatocyte progression into S phase and mitosis was notably enhanced in MMP-9(-/-) steatotic livers after IRI. CONCLUSIONS MMP-9 activity disrupts vascular integrity at least partially through a PECAM-1 dependent mechanism and interferes with regeneration of steatotic livers after IRI. Our novel findings establish MMP-9 as an important mediator of steatotic liver IRI.

The Cytoprotective Effects of Addition of Activated Protein C into Preservation Solution on Small-for-Size Grafts in Rats

Small‐for‐size liver grafts are a serious obstacle for partial orthotopic liver transplantation. Activated protein C (APC), a potent anticoagulant serine protease, is known to have cell‐protective properties due to its anti‐inflammatory and antiapoptotic activities. This study was designed to examine the cytoprotective effects of a preservation solution containing APC on small‐for‐size liver grafts, with special attention paid to ischemia‐reperfusion injury and shear stress in rats. APC exerted cytoprotective effects, as evidenced by (1) increased 7‐day graft survival; (2) decreased initial portal pressure and improved hepatic microcirculation; (3) decreased levels of aminotransferase and improved histological features of hepatic ischemia‐reperfusion injury; (4) suppressed infiltration of neutrophils and monocytes/macrophages; (5) reduced hepatic expression of tumor necrosis factor α and interleukin 6; (6) decreased serum levels of hyaluronic acid, which indicated attenuation of sinusoidal endothelial cell injury; (7) increased hepatic levels of nitric oxide via up‐regulated hepatic endothelial nitric oxide synthesis expression together with down‐regulated hepatic inducible nitric oxide synthase expression; (8) decreased hepatic levels of endothelin 1; and (9) reduced hepatocellular apoptosis by down‐regulated caspase‐8 and caspase‐3 activities. These results suggest that a preservation solution containing APC is a potential novel and safe product for small‐for‐size liver transplantation, alleviating graft injury via anti‐inflammatory and antiapoptotic effects and vasorelaxing conditions. Liver Transpl 16:1–11, 2010.

Paradoxical impact of the remnant pancreatic volume and infectious complications on the development of nonalcoholic fatty liver disease after pancreaticoduodenectomy

The aim of the present study was to evaluate perioperative risk factors for development of nonalcoholic fatty liver disease (NAFLD) after pancreaticoduodenectomy (PD), paying special attention to remnant pancreatic volume (RPV) and postoperative infection.

Dual cytoprotective effects of splenectomy for small-for-size liver transplantation in rats.

The problems associated with small‐for‐size liver grafts (ie, high mortality rates, postoperative complications, and acute rejection) remain critical issues in partial orthotopic liver transplantation (OLT). In association with partial OLT, splenectomy (SP) is a procedure used to reduce the portal pressure. However, the precise effects of SP on partial OLT have been unclear. In this study, using small‐for‐size liver grafts in rats, we examined the cytoprotective effects of SP on OLT. Liver grafts were assigned to 2 groups: a control group (OLT alone) and an SP group (OLT after SP). SP significantly increased animal survival and decreased liver damage. SP exerted the following cytoprotective effects: (1) it improved hepatic microcirculation and prevented increases in the portal pressure after OLT, (2) it suppressed the hepatic infiltration of neutrophils and macrophages through the direct elimination of splenic inflammatory cells before OLT, (3) it decreased the hepatic expression of tumor necrosis factor α and interleukin‐6, (4) it attenuated sinusoidal endothelial injury, (5) it decreased plasma endothelin 1 levels and increased hepatic heme oxygenase 1 expression, (6) it suppressed hepatocellular apoptosis through the down‐regulation of hepatic caspase‐3 and caspase‐8 activity, and (7) it increased hepatic regeneration. In conclusion, SP for small‐for‐size grafts exerts dual cytoprotective effects by preventing excessive portal vein hepatic inflow and eliminating splenic inflammatory cell recruitment into the liver; this in turn inhibits hepatocellular apoptosis and improves liver regeneration. Liver Transpl, 2012.

Activated protein C prevents hepatic ischaemia–reperfusion injury in rats

Background: Hepatic ischaemia–reperfusion injury (IRI) is a serious complication of liver surgery, especially extended hepatectomy and liver transplantation. Activated protein C (APC), a potent anticoagulant serine protease, has been shown to have cell‐protective properties by virtue of its anti‐inflammatory and anti‐apoptotic activities.

Human Equilibrative Nucleoside Transporter 1 Expression in Endoscopic Ultrasonography-Guided Fine-Needle Aspiration Biopsy Samples Is a Strong Predictor of Clinical Response and Survival in the Patients With Pancreatic Ductal Adenocarcinoma Undergoing Gemcitabine-Based Chemoradiotherapy.

ObjectivesThis study aimed to clarify whether pretreatment human equilibrative nucleoside transporter (hENT1) expressions in endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) specimens obtained from resectable, borderline resectable, and locally advanced unresectable pancreatic ductal adenocarcinoma (PDAC) are concordant with those in the resected specimen after gemcitabine-based chemoradiotherapy (Gem-CRT) and to validate the utility of hENT1 expression using EUS-FNAB samples as a prognostic marker. MethodsWe evaluated the relationship between hENT1 expressions assessed by immunohistochemical staining and clinical outcomes in 51 of 76 patients with PDAC who were diagnosed by EUS-FNAB and received preoperative Gem-CRT. ResultsThe concordance rate of hENT1 expressions was 89.2% (K = 0.681). Median survival time (month) in the 51 whole patients and 37 patients with resection was significantly longer in hENT1 positive than in hENT1 negative: 25.0 and 30.0 versus 9.0 and 9.0, respectively. A multivariate analysis confirmed that hENT1 expression was an independent prognostic factor in both whole patients and those with resection. Regardless of T3 and T4, hENT1-positive patients with resection had significantly better prognosis than hENT1-negative patients, whose prognosis was similar to those without resection. ConclusionsThe assessment of hENT1 expression using EUS-FNAB samples before Gem-CRT provides important information on patients with PDAC who can benefit from curative-intent resection.

Tissue factor expression demonstrates severe sinusoidal endothelial cell damage during rejection after living-donor liver transplantation

BACKGROUND/PURPOSE Since it is well known that endothelial cells may be important targets during rejection after living-donor liver transplantation, in this study we investigated liver sinusoidal endothelial cell (SEC) damage during rejection by focusing on thrombomodulin (TM) and hyaluronic acid (HA) as plasma markers of SEC damage. We also examined tissue factor (TF) expression in SECs, because damage to endothelial cells leads to immediate activation of the coagulation system, with the damage being triggered mainly by TF. METHODS Living-donor liver transplantation was performed at Mie University Hospital between March 2002 and December 2007; there were 8 patients with rejection (4 with acute cellular rejection and 4 with chronic rejection) and 32 patients without rejection. Liver biopsy tissue was immunostained with an anti-TF antibody, and assessed for SEC damage. In addition, total RNA was extracted from liver biopsy tissue and tested for TF mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS The plasma TM level was significantly higher in the rejection group than in the non-rejection group. TF expression was observed in SECs, in infiltrating inflammatory cells, and in the vascular endothelium in the rejection group. TF mRNA expression was significantly higher in the rejection group than in the non-rejection group. CONCLUSIONS We demonstrated that TF expression revealed severe SEC damage in grafted liver during both acute cellular rejection and chronic rejection.

Thrombomodulin Administration Attenuates Ischemia-Reperfusion Injury of the Remnant Liver After 70% Hepatectomy in Rats: Simulated Model of Small-for-size Graft in Living Donor Liver Transplantation

BACKGROUND Hepatic ischemia-reperfusion injury (IRI) is a serious complication affecting liver function and postoperative course after liver transplantation. Thrombomodulin (TM) has been known to have anticoagulant and anti-inflammatory activities exerting a cytoprotective effect. We evaluated the cytoprotective effect of recombinant human soluble TM (rhsTM) on the remnant liver exposed to IRI after 70% hepatectomy in rats, which was the simulated model of small-for-size graft in living donor liver transplantation. MATERIALS AND METHODS A Wistar rat underwent 70% hepatectomy followed by 20-minute IRI for the remnant liver. rhsTM (1 mg/kg) (TM group) or saline (control group) was intravenously administered 30 minutes before operation. RESULTS Alanine aminotransaminase levels were more significantly decreased during the 24 hours after operation in the TM group than in control group, especially at 6 hours. Intrahepatic infiltration of macrophages/monocytes (ED-1 immunohistochemical staining) at 6 hours was significantly decreased in the TM group compared to the control group. The number of proliferating cell nuclear antigen-positive cells at 12 hours (hepatocyte proliferation) was significantly higher in the TM group than in the control group; although liver weight 7 days after operation did not differ between the two groups. Hepatocyte apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, also known as TUNEL assay) at 24 hours was more significantly diminished in the TM group than in the control group. CONCLUSION These results suggest that rshTM attenuates hepatocyte injury through its anti-inflammatory effect, and promotes hepatocyte proliferation in the reduced-size liver exposed to hepatic IRI.