Preclinical validation of a customized circuit for ex situ uninterrupted cold-to-warm prolonged perfusion of the liver.

CONTEXT: Clinical adoption of ex situ liver perfusion is growing. While hypothermic perfusion protects against ischemia-reperfusion injury in marginal grafts, normothermic perfusion enables organ viability assessment and therefore selection of borderline grafts. The combination of hypothermic and normothermic perfusion, known as "cold-to-warm," may be the optimal sequence for organ preservation, but is difficult to achieve with most commercial perfusion systems. We developed an adaptable customized circuit allowing uninterrupted "cold-to-warm" perfusion and conducted preclinical studies on healthy porcine livers and discarded human livers to demonstrate the circuit's efficacy. METHODS: In collaboration with bioengineers, we developed a customized circuit that adapts to extracorporeal circulation consoles used in cardiovascular surgery and includes a proprietary reservoir enabling easy perfusate change without interrupting perfusion. This preclinical study was conducted on porcine and human livers. Perfusion parameters (pressures, flows, oxygenation) and organ viability were monitored. RESULTS: The customized circuit was adapted to a LivaNova S5® console, and the perfusions were flow-driven with real-time pressure monitoring. Ten porcine liver and 12 discarded human liver perfusions were performed during 14 to 18 h and 7 to 25 h, respectively. No hyperpressure was observed (porcine and human portal pressure 2-6 and 2-8 mm Hg; arterial pressure 10-65 and 20-65 mm Hg, respectively). No severe histological tissue injury was observed (Suzuki score ≤ 3 at the end of perfusion). Seven (70%) porcine livers and five (42%) human livers met the UK viability criteria. CONCLUSION: The customized circuit and system design enables smooth uninterrupted "cold-to-warm" perfusion not present in current commercial perfusion systems.

Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases.

In vitro studies on liver diseases, such as non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma, are traditionally performed in two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines. However, this approach has limitations, as 2D cultures inadequately replicate the cell-cell and cell-extracellular matrix interactions found in three-dimensional (3D) environments. To overcome this limitation, various 3D models, such as spheroids, have been developed. These spheroids serve as simplified biomimetic in vitro models for studying liver diseases. They can be generated using a variety of cells from healthy and pathological tissues, including liver cancer. Here, we present a comprehensive protocol for performing immunofluorescent staining and confocal imaging on whole human hepatic multicellular spheroids, utilizing primary cells or cell lines. The immunofluorescence technique is a potent tool to understand the spatial distribution of different cell types within the spheroids and define the interactions that occur among these cells.

An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

Therapeutic potentials of mesenchymal stromal cells-derived extracellular vesicles in liver failure and marginal liver graft rehabilitation: a scoping review.

Liver failure includes distinct subgroups of diseases: Acute liver failure (ALF) without preexisting cirrhosis, acute-on-chronic liver failure (ACLF) (severe form of cirrhosis associated with organ failures and excess mortality), and liver fibrosis (LF). Inflammation plays a key role in ALF, LF, and more specifically in ACLF for which we have currently no treatment other than liver transplantation (LT). The increasing incidence of marginal liver grafts and the shortage of liver grafts require us to consider strategies to increase the quantity and quality of available liver grafts. Mesenchymal stromal cells (MSCs) have shown beneficial pleiotropic properties with limited translational potential due to the pitfalls associated with their cellular nature. MSC-derived extracellular vesicles (MSC-EVs) are innovative cell-free therapeutics for immunomodulation and regenerative purposes. MSC-EVs encompass further advantages: pleiotropic effects, low immunogenicity, storage stability, good safety profile, and possibility of bioengineering. Currently, no human studies explored the impact of MSC-EVs on liver disease, but several preclinical studies highlighted their beneficial effects. In ALF and ACLF, data showed that MSC-EVs attenuate hepatic stellate cells activation, exert antioxidant, anti-inflammatory, anti-apoptosis, anti-ferroptosis properties, and promote regeneration of the liver, autophagy, and improve metabolism through mitochondrial function recovery. In LF, MSC-EVs demonstrated anti-fibrotic properties associated with liver tissue regeneration. Normothermic-machine perfusion (NMP) combined with MSC-EVs represents an attractive therapy to improve liver regeneration before LT. Our review suggests a growing interest in MSC-EVs in liver failure and gives an appealing insight into their development to rehabilitate marginal liver grafts through NMP.

MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Ex-Vivo Pharmacological Defatting of the Liver: A Review.

The ongoing organ shortage has forced transplant teams to develop alternate sources of liver grafts. In this setting, ex-situ machine perfusion has rapidly developed as a promising tool to assess viability and improve the function of organs from extended criteria donors, including fatty liver grafts. In particular, normothermic machine perfusion represents a powerful tool to test a liver in full 37 °C metabolism and add pharmacological corrections whenever needed. In this context, many pharmacological agents and therapeutics have been tested to induce liver defatting on normothermic machine perfusion with promising results even on human organs. This systematic review makes a comprehensive synthesis on existing pharmacological therapies for liver defatting, with special focus on normothermic liver machine perfusion as an experimental ex-vivo translational model.

Modulatory effect of rapamycin and tacrolimus on monocyte-derived dendritic cells phenotype and function.

BACKGROUND: Immunosuppressive-drugs are needed after solid organ transplantation to prevent allograft rejection but induce severe side effects. Understanding the alloimmune response is critical to modulate it and to achieve graft operational tolerance. The role of regulatory T cells and tolerogenic dendritic cells (Tol-DCs) is undoubtedly essential in tolerance induction. Tacrolimus is considered as the cornerstone of immunosuppression in solid organ transplantation. mTOR inhibitor such as rapamycin are thought to induce tolerance and are used as anticancer drugs in several cancers. The aim of this study was to better understand the effect of these immunosuppressive drugs on the differentiation, maturation and function of human monocyte derived dendritic cells (DCs). MATERIAL AND METHODS: DCs were differentiated from monocytes of healthy donors with either rapamycin (Rapa-DCs) or tacrolimus (Tac-DCs). The phenotype was evaluated by flow cytometry analysis. The production of pro- and anti-inflammatory cytokines was assessed by ELISA. The mRNA expression level of IDO and PD-L1 was assessed by RTqPCR. Mixed leukocytes reactions were performed to analyse suppressive activity of DCs. RESULTS: Rapa-DC were characterised by a lower expression of the co-stimulatory molecules and CD83 than control-DCs (CTR-DC) (p < 0.05). In contrast, tacrolimus had no effect on the expression of surface markers compared to CTR-DCs. Rapamycin reduced both IL-12 and IL-10 secretions (p < 0.05). Rapa-DCs had a suppressive effect on CD4+ allogenic T cells compared to CTR-DCs (p < 0.05). However, neither Rapa-DCs nor Tac-DCs favoured the emergence of a CD4+CD25highFoxp3+ population compared to CTR-DCs. Surprisingly, Rapa-DCs had a reduced expression of IDO and PD-L1 compared to Tac-DCs and CTR-DCs. CONCLUSION: Rapa-DCs exhibit an incomplete phenotypic tolerogenic profile. To our knowledge this is the first paper showing a reduction of expression of pro-tolerogenic enzyme IDO in DCs. Tacrolimus does not change the phenotypical or functional characteristics of moDCs.

Influence of everolimus-based treatment on circulating regulatory T cells after liver transplantation: Comparative study with tacrolimus-based therapy.

Liver transplantation remains the only treatment for terminal liver diseases. However, immunosuppressive drugs required for allograft acceptance are toxic and may be responsible for severe side effects. Modulating the immune system to induce tolerance is a promising approach to reduce immunosuppressive regimen. More particularly, promoting natural CD4+ CD25+ FoxP3+ Tregs could be crucial in achieving tolerance. Contrary to calcineurin inhibitors, reports indicate that mTOR inhibitors may have a positive impact on Tregs. Here we present the first randomized prospective clinical study where Tregs levels from liver transplanted patients receiving either tacrolimus or everolimus were monitored for 6 months, starting from the day of transplantation. A total of 30 patients from four centers were monitored. Blood samples were obtained at day 0, day 14, one month, three months and six months post-transplantation. Flow-cytometry immunophenotyping of Tregs (CD4+ CD25+ CD127- FoxP3+) and functional assays with Tregs were performed to assess their immunosuppressive capacity. Levels of Tregs were significantly reduced after one month of standard tacrolimus-based immunosuppressive regimen (p<0.05). Four months after conversion, levels of Tregs from patients treated with everolimus was significantly higher than patients under tacrolimus (p<0.02). Functional assays demonstrated that Tregs conserved their capacity to suppress the proliferation of activated PBMC.

Cold-Atmospheric Plasma Induces Tumor Cell Death in Preclinical In Vivo and In Vitro Models of Human Cholangiocarcinoma.

Through the last decade, cold atmospheric plasma (CAP) has emerged as an innovative therapeutic option for cancer treatment. Recently, we have set up a potentially safe atmospheric pressure plasma jet device that displays antitumoral properties in a preclinical model of cholangiocarcinoma (CCA), a rare and very aggressive cancer emerging from the biliary tree with few efficient treatments. In the present study, we aimed at deciphering the molecular mechanisms underlying the antitumor effects of CAP towards CCA in both an in vivo and in vitro context. In vivo, using subcutaneous xenografts into immunocompromised mice, CAP treatment of CCA induced DNA lesions and tumor cell apoptosis, as evaluated by 8-oxoguanine and cleaved caspase-3 immunohistochemistry, respectively. The analysis of the tumor microenvironment showed changes in markers related to macrophage polarization. In vitro, the incubation of CCA cells with CAP-treated culture media (i.e., plasma-activated media, PAM) led to a dose response decrease in cell survival. At molecular level, CAP treatment induced double-strand DNA breaks, followed by an increased phosphorylation and activation of the cell cycle master regulators CHK1 and p53, leading to cell cycle arrest and cell death by apoptosis. In conclusion, CAP is a novel therapeutic option to consider for CCA in the future.

Novel defatting strategies reduce lipid accumulation in primary human culture models of liver steatosis.

Normothermic perfusion provides a means to rescue steatotic liver grafts, including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis, primary hepatocytes isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist were all combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain like pseudokinase involved in necroptosis. Within 24 h, in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an upregulation of genes involved in free fatty acid ß-oxidation and autophagy, and a downregulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models, notably PCLS, are insightful to design strategies for liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy and lipogenesis.

Cancer-associated fibroblasts in cholangiocarcinoma.

PURPOSE OF REVIEW: To give a state-of-art knowledge regarding cancer-associated fibroblasts (CAF) in cholangiocarcinoma (CCA) based both on direct evidence and studies on other desmoplastic cancers. High contingency of CAF characterizes CCA, a tumor with a biliary epithelial phenotype that can emerge anywhere in the biliary tree. Current treatments are very limited, the surgical resection being the only effective treatment but restricted to a minority of patients, whereas the remaining patients undergo palliative chemotherapy regimens. In cancer, CAF shape the tumor microenvironment, drive cancer growth and progression, and contribute to drug resistance. All these functions are accomplished through an interplay network between CAF and surrounding cells including tumor and other stromal cells, i.e. immune and endothelial cells. RECENT FINDINGS: Several studies have pointed out the existence of CAF sub-populations carrying out several and opposite functions, cancer-promoting or cancer-restraining as shown in pancreatic cancer, another prototypic desmoplastic tumor in which heterogeneity of CAF is well demonstrated. SUMMARY: New CAF functions are now emerging in pancreatic and breast cancers like the modulation of immune responses or tumor metabolism, opening new area for treatments.

Impact of hepatitis C virus and alcohol, alone and combined, on the unfolded protein response in primary human hepatocytes.

Hepatitis C virus (HCV) infection and alcohol abuse are leading causes of chronic liver disease and frequently coexist in patients. The unfolded protein response (UPR), a cellular stress response ranging along a spectrum from cytoprotection to apoptosis commitment, has emerged as a major contributor to human diseases including liver injuries. However, the literature contains conflicting reports as to whether HCV and ethanol activate the UPR and which UPR genes are involved. Here we have used primary human hepatocytes (PHH) to reassess this issue and address combined impacts. In this physiologically relevant model, either stressor activated a chronic complete UPR. However, the levels of UPR gene induction were only modest in the case of HCV infection. Moreover, when combined to the strong stressor thapsigargin, ethanol exacerbated the activation of pro-apoptotic genes whereas HCV tended to limit the induction of key UPR genes. The UPR resulting from HCV plus ethanol was comparable to that induced by ethanol alone with the notable exception of three pro-survival genes the expressions of which were selectively enhanced by HCV. Interestingly, HCV genome replication was maintained at similar levels in PHH exposed to ethanol. In conclusion, while both HCV and alcohol activate the hepatocellular UPR, only HCV manipulates UPR signalling in the direction of a cytoprotective response, which appears as a viral strategy to spare its own replication.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. METHODS: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. RESULTS: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in ß-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. CONCLUSION: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. LAY SUMMARY: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Hepatitis C Virus Improves Human Tregs Suppressive Function and Promotes Their Recruitment to the Liver.

BACKGROUND: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes. METHODS: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-ß1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber. RESULTS: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner. CONCLUSIONS: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression.

Treatment with mTOR inhibitors after liver transplantation enables a sustained increase in regulatory T-cells while preserving their suppressive capacity.

BACKGROUND: The mammalian targets of rapamycin (mTOR) inhibitors (sirolimus [SRL] and everolimus [EVR]) are used after transplantation for their immunosuppressive activity. Regulatory T-cells (Tregs) play a crucial role in immune tolerance. mTOR inhibitors appear to preserve Tregs, unlike Tacrolimus (Tac). AIM: The aim of this study was to evaluate the number and function of Tregs in liver transplant recipients before and after conversion from Tac to mTOR inhibitors. METHODS: Fifteen patients with stable graft function where converted to SRL (n=5) or EVR (n=10). Tregs (CD4+ CD25+ FoxP3+ CD127low) number and activity were analysed prospectively in blood cells using flow cytometry, and functional assay. RESULTS: Patients of both groups displayed a sustained rise in Treg levels after introduction of mTOR inhibitors (Treg levels at 3 months: 6.45±0.38% of CD4 T-cells, vs. baseline level of 3.61±0.37%, P<0.001; mean fold increase 2.04±0.73). In SRL group, 3-month Treg levels were 6.01±0.53 vs. 3.79±0.39; P=0.037, while in EVR group they were 6.63±0.67 vs. 3.54±0.51; P=0.001. By contrast, no statistical change was observed in an unconverted Tac control group. Tregs also preserved their functional ability to suppress activated T-cells. CONCLUSION: These results suggest that mTOR inhibitors induce a significant increase in Tregs while maintaining suppressive activity after LT.

Heregulin-1ß and HER3 in hepatocellular carcinoma: status and regulation by insulin.

BACKGROUND: The heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC). METHODS: Using 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines. RESULTS: HER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation. CONCLUSIONS: Autocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.

Development of an in vitro model to test antifibrotic drugs on primary human liver myofibroblasts.

We have developed a culture model to assess antifibrotic drugs using normal human liver myofibroblasts (HLMFs) obtained from 31 subjects. Activation was evaluated in terms of α-smooth muscle actin (α-SMA) and collagen 1 (Coll1) expression using RT-PCR, and proliferation as the uptake of 5-ethynil-2'-deoxyuridine. Under analysis of variance, between-subject differences accounted for 70% of all variability and inter-experiment differences for 30%. The sensitivity of the model was determined by quantifying the effects in terms of relative expression, which were 0.74±0.03 for cyclosporine A (CsA) and 2.4±0.10 for transforming growth factor-beta (TGF-ß) (P<0.0001 vs no treatment) for α-SMA expression. Inter-subject variations in α-SMA and Coll1 expression enabled the classification of subjects as potentially low or high fibrosers. Finally, we observed that pirfenidone (which has beneficial effects in vivo) significantly reduced the expressions of α-SMA and Coll1, whereas the angiotensin-converting enzyme inhibitor losartan (which has no effect in vivo) had no significant effect. Our model may thus detect the antifibrotic properties of drugs. Antifibrotic drugs with promising clinical relevance could possibly be selected using a bank of HLMFs from high fibrosers.

Infection of Human Liver Myofibroblasts by Hepatitis C Virus: A Direct Mechanism of Liver Fibrosis in Hepatitis C.

BACKGROUND: Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities. METHODS: We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction. RESULTS: We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients. CONCLUSION: These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor.

UNLABELLED: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.