The effect of end-ischaemic normothermic machine perfusion on donor hepatic artery endothelial integrity.

BACKGROUND: Ex vivo normothermic machine liver perfusion (NMLP) involves artificial cannulation of vessels and generation of flow pressures. This could lead to shear stress-induced endothelial damage, predisposing to vascular complications, or improved preservation of donor artery quality. This study aims to assess the spatial donor hepatic artery (HA) endothelial quality downstream of the cannulation site after end-ischaemic NMLP. METHODS: Remnant HA segments from the coeliac trunk up to the gastroduodenal artery branching were obtained after NMLP (n = 15) and after static cold storage (SCS) preservation (n = 15). Specimens were fixed in 10% neutral buffered formalin and sectioned at pre-determined anatomical sites downstream of the coeliac trunk. CD31 immunohistostaining was used to assess endothelial integrity by a 5-point ordinal scale (grade 0: intact endothelial lining, grade 5: complete denudation). Endothelial integrity after SCS was used as a control for the state of the endothelium at commencement of NMP. RESULTS: In the SCS specimens, regardless of the anatomical site, near complete endothelial denudation was present throughout the HA (median scores 4.5-5). After NMLP, significantly less endothelial loss in the distal HA was present compared to SCS grafts (NMLP vs. SCS: median grade 3 vs. 4.5; p = 0.042). In NMLP specimens, near complete endothelial denudation was present at the cannulation site in all cases (median grade: 5), with significantly less loss of the endothelial lining the further from the cannulation site (proximal vs. distal, median grade 5 vs. 3; p = 0.005). CONCLUSION: Loss of endothelial lining throughout the HA after SCS and at the cannulation site after NMLP suggests extensive damage related to surgical handling and preservation injury. Gradual improved endothelial lining along more distal sites of the HA after NMLP indicates potential for re-endothelialisation. The regenerative effect of NMLP on artery quality seems to occur to a greater extent further from the cannulation site. Therefore, arterial cannulation for machine perfusion of liver grafts should ideally be as proximal as possible on the coeliac trunk or aortic patch, while the site of anastomosis should preferentially be attempted distal on the common HA.

Transplantation of Declined Liver Allografts Following Normothermic Ex-Situ Evaluation.

The demand for liver transplantation (LT) exceeds supply, with rising waiting list mortality. Utilization of high-risk organs is low and a substantial number of procured livers are discarded. We report the first series of five transplants with rejected livers following viability assessment by normothermic machine perfusion of the liver (NMP-L). The evaluation protocol consisted of perfusate lactate, bile production, vascular flows, and liver appearance. All livers were exposed to a variable period of static cold storage prior to commencing NMP-L. Four organs were recovered from donors after circulatory death and rejected due to prolonged donor warm ischemic times; one liver from a brain-death donor was declined for high liver function tests (LFTs). The median (range) total graft preservation time was 798 (range 724-951) min. The transplant procedure was uneventful in every recipient, with immediate function in all grafts. The median in-hospital stay was 10 (range 6-14) days. At present, all recipients are well, with normalized LFTs at median follow-up of 7 (range 6-19) months. Viability assessment of high-risk grafts using NMP-L provides specific information on liver function and can permit their transplantation while minimizing the recipient risk of primary graft nonfunction. This novel approach may increase organ availability for LT.

Biliary epithelial senescence and plasticity in acute cellular rejection.

Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-ß expression at mRNA and protein levels also showed a rapid increase in TGF-ß2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-ß receptor. These data suggest that stress induced production of TGF-ß2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

CD40 activation-induced, Fas-dependent apoptosis and NF-kappaB/AP-1 signaling in human intrahepatic biliary epithelial cells.

Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas-mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40-induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF-kappaB and AP-1 were both activated after CD40 ligation. Increased functional NF-kappaB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up-regulation of AP-1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL-dependent apoptosis of liver epithelial cells supporting the importance of cross-talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP-1 in the absence of NF-kappaB signaling may be a critical factor in determining the outcome of CD40 engagement.

Hepatocyte growth factor concentration in maternal and umbilical cord blood samples and expression in fetal liver.

OBJECTIVE: To determine whether human placenta secretes hepatocyte growth factor (HGF) and could influence fetal liver development. METHODS: Expression of HGF and c-met mRNA in paired samples of first- and second-trimester fetal liver and placenta was compared using a quantitative ribonuclease protection assay. Serum HGF concentration in 30 samples of paired umbilical and maternal blood from term pregnancies was evaluated using an enzyme-linked immunosorbent assay. RESULTS: HGF and c-met mRNA were expressed at similar levels in liver and placenta, with expression increasing from 9 to 16 weeks' gestation. Median serum HGF values were 1.4 ng/mL (maternal venous), 1.2 ng/mL (cord venous), and 1.3 ng/mL (cord arterial). The maternal venous HGF levels were significantly higher than fetal venous levels (P =.02). CONCLUSIONS: This study does not support the hypothesis that the placenta secretes HGF, because maternal serum levels were higher than fetal and there was no significant difference between umbilical arterial and venous samples. Fetal liver expresses abundant HGF mRNA during the first and second trimester and expression increases in line with receptor (c-met) expression, suggesting that hepatic growth and development are independent of placental HGF.

Hepatocyte growth factor activator (HGF-A) and its zymogen in human placenta.

HGF-activator (HGF-A) is a circulating serine protease known to be responsible for activation of hepatocyte growth factor (HGF). Active HGF is thought to be an important regulator of trophoblast growth. In vitro, HGF-A is produced via proteolytic cleavage of its zymogen by thrombin. Immunocytochemistry and Western immunoblotting were performed using human placental tissue from all three trimesters with an antibody that recognizes both HGF-A and its zymogen. Western immunoblotting revealed a 97 kDa band equivalent to the zymogen in placenta from all three trimesters. A smaller 34 kDa band equivalent to HGF-A was only seen in first and second trimester placenta. The anti-HGF-A/zymogen antibody demonstrated immunostaining in placental villi and membranes throughout gestation. Within first trimester villi immunostaining was strongest within the syncytio- and cytotrophoblast layers, but was also seen within stromal and endothelial cells. Likewise, in third trimester placenta the syncytio-cytotrophoblast layer showed the strongest immunoreactivity. In vitro, HGF can induce trophoblast DNA synthesis and the localization of HGF-A to the peri-villous trophoblast layer (which expresses c-met, the HGF receptor) suggests that it may be responsible for activation of pro-HGF at this site. This adds further weight to the hypothesis that HGF in vivo is an important regulator of trophoblast growth.

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.

CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.

Apoptosis.

Apoptosis is the genetically regulated form of cell death that permits the safe disposal of cells at the point in time when they have fulfilled their intended biological function. Examples of apoptosis can be cited throughout the whole of the animal and plant kingdoms. It is a vitally important process during normal development and the adult life of many living organisms. In humans, dysregulation of apoptosis can result in inflammatory, malignant, autoimmune, and neurodegenerative diseases. In addition, infectious agents, including viruses, exploit cellular apoptosis in the host to evade the immune system. This review gives a brief historical perspective of some of the landmark discoveries in apoptosis research. The morphological and biochemical stages of apoptosis are then covered, followed by an overview of how it can be studied in the laboratory. Finally, the implications for therapeutic intervention in disease treatment are discussed.

Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation.

We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC.

Expression and function of CXC and CC chemokines in human malignant liver tumors: a role for human monokine induced by gamma-interferon in lymphocyte recruitment to hepatocellular carcinoma.

Chemotactic cytokines (chemokines) play an important role in the recruitment of lymphocytes to tissue by regulating cellular adhesion and transendothelial migration. This study examined the expression and function of CXC (human monokine induced by gamma-interferon [HuMig], interleukin-8 [IL-8], and interferon-inducible protein-10 [IP-10]) and CC (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, regulated upon activation normal T lymphocyte expressed and secreted (RANTES), and macrophage chemoattractant protein-1 [MCP-1]) chemokines and their respective receptors on lymphocytes infiltrating human liver tumors. Chemokine and chemokine receptor expression was assessed by immunohistochemistry, flow cytometry, in situ hybridization and ribonuclease (RNAse) protection assays and function by in vitro chemotaxis of tumor-derived lymphocytes to purified chemokines and to HepG2 tumor cell culture supernatants. Tumor-derived lymphocytes showed strong chemotactic responses to both CC and CXC chemokines in vitro and expressed high levels of CXCR3 (HuMig and IP-10 receptor) and CCR5 (RANTES, MIP-1alpha, and MIP-1beta receptor). Expansion of tumor-derived lymphocytes in recombinant IL-2 increased expression of CXCR3. The corresponding chemokines were detected on vascular endothelium (HuMig, IL-8, MIP-1alpha, and MIP-1beta) and sinusoidal endothelium (HuMig, MIP-1alpha, MIP-1beta) in hepatocellular carcinoma. In vitro, HepG2 cells secreted functional chemotactic factors for tumor-derived lymphocytes that could be inhibited using anti-CCR5 or anti-CXCR3 monoclonal antibodies (MoAbs). Thus, lymphocytes infiltrating human liver tumors express receptors for and respond to both CXC and CC chemokines. The relevant chemokine ligands are expressed in hepatocellular carcinoma (HCC), particularly HuMig, which was strongly expressed by tumor endothelium, suggesting that they play a role in lymphocyte recruitment to these tumors in vivo. The ability of HepG2 cells to secrete lymphocyte chemotactic factors in vitro suggests that the tumor contributes to lymphocyte recruitment in vivo.

Fas/Fas ligand interaction in human colorectal hepatic metastases: A mechanism of hepatocyte destruction to facilitate local tumor invasion.

This study demonstrates a novel role for the Fas pathway in the promotion of local tumor growth by inducing apoptotic cell death in normal hepatocytes at the tumor margin in colorectal hepatic metastases. Our results show that >85% of lymphocytes infiltrating colorectal liver cancer express high levels of Fas-ligand (Fas-L) by flow cytometry. Using immunohistochemistry of tumor tissue we showed strong Fas expression in noninvolved hepatocytes, whereas Fas-L expression was restricted to tumor cells and infiltrating lymphocytes at the tumor margin. Apoptosis was observed in 45 +/- 13% of the Fas(high) hepatocytes at the tumor margin whereas only 7 +/- 3% tumor cells were apoptotic (n = 10). In vitro, primary human hepatocytes expressed Fas receptor and crosslinking with anti-Fas antibody induced apoptosis in 44 +/- 5% of the cells compared with 4. 6 +/- 1.0% in untreated controls (P = 0.004). Both tumor-infiltrating lymphocytes (TIL) and human metastatic colon cancer cells cells are able to induce Fas-mediated apoptosis of primary human hepatocytes in coculture cytotoxic assays. TIL induced apoptosis in 47 +/- 9% hepatocytes compared with control 4.3 +/- 1. 0% (P = 0.009) and this effect was reduced by anti-human Fas-L mAb (18.7 +/- 1.3%, P = 0.009). SW620 cells induced apoptosis in 26 +/- 2% hepatocytes compared with control 5.6 +/- 1.7% (P = 0.004) and this was reduced to 11.2 +/- 1.8% (P = 0.004) in the presence of anti-human Fas-L mAb. These data suggest that the inflammatory response at the margin of colorectal liver metastases induces Fas expression in surrounding hepatocytes, allowing them to be killed by Fas-L-bearing TIL or tumor cells and facilitating the invasion of the tumor into surrounding liver tissue.

CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

Ontogeny of hepatocyte growth factor (HGF) and its receptor (c-met) in human placenta: reduced HGF expression in intrauterine growth restriction.

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.

Effect of peritransplant FTY720 alone or in combination with post-transplant tacrolimus in a rat model of cardiac allotransplantation.

FTY720 is a recently discovered compound that is derived from the fungus Isaria sinclairii. Using a DA donor-to-LEW recipient rat combination, we assessed the efficacy of peritransplant FTY720 alone or in combination with post-transplant tacrolimus on the survival of cardiac allografts. Peritransplant FTY720 given orally at a dose of 5 mg/kg on days-1 and 0 prolonged graft survival from 5 to 13 days (P < 0.05). Combining peritransplant FTY720 with post-transplant tacrolimus resulted in a further prolongation of allograft survival. The lymphocyte count in transplanted rats decreased within 24 h to 46.6%. Analysis of lymphocyte subsets by FACS revealed that FTY720 affected the total population of CD3-bearing T cells while the ratio of CD4 to CD8 cells remained unchanged. Kidney and liver biochemistry remained elevated for 2 weeks. In conclusion, FTY720 is a powerful immunosuppressive agent when used as induction therapy and may have an additive effect--perhaps a synergistic one--with post-transplant tacrolimus.

Distinct patterns of chemokine expression are associated with leukocyte recruitment in alcoholic hepatitis and alcoholic cirrhosis.

Alcoholic liver disease is associated with three histologically distinct processes: steatosis (parenchymal fat accumulation), alcoholic hepatitis (characterized by parenchymal infiltration by neutrophil polymorphs), and alcoholic cirrhosis (in which chronic inflammation and fibrosis dominate). Chemokines are cytokines that promote subset-specific leukoycte recruitment to tissues and could therefore play a crucial role in determining which leukocyte subsets are recruited to the liver in alcoholic liver disease. This paper reports that chemokine expression is increased in the liver of patients with alcoholic liver disease and, moreover, that distinct patterns of chemokine expression are associated with the different inflammatory responses to alcohol. Interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1 alpha), and MIP-1 beta were all detected in the parenchyma at sites of inflammation in alcoholic hepatitis, whereas in alcoholic cirrhosis, chemokines were restricted to inflammatory cells and endothelium in the fibrous septa and portal tracts. In alcoholic hepatitis, chemokine transcription was localized to sinusoidal cells, leukocytes, and fibroblasts in areas of parenchymal inflammation, but hepatocytes, despite staining strongly for chemokine protein, were negative. In alcoholic cirrhosis, chemokine mRNA was detected in portal tract endothelium, leukocytes, and fibroblasts. Thus, alcoholic hepatitis and alcoholic cirrhosis are associated with distinct patterns of chemokine expression that are likely to be important factors in determining whether a patient develops acute parenchymal inflammation and alcoholic hepatitis, or chronic septal inflammation and alcoholic cirrhosis.