Uncovering a Novel Functional Interaction Between Adult Hepatic Progenitor Cells, Inflammation and EGFR Signaling During Bile Acids-Induced Injury.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-ß regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

Behavioral Problems and Family Impact in Congenital Cytomegalovirus Infection.

We examined behavior (Child Behavior Checklist) and family functioning (Family Impact Questionnaire) in 65 children with congenital cytomegalovirus. Behavioral problems were present in 30.8%. Parents of children with moderate/severe outcomes reported strain on all areas of family functioning. Behavioral problems were associated with negative impact on parental feelings and marital/partnership relationship. Our findings inform planning support services.

New and Old Key Players in Liver Cancer.

Liver cancer represents a major health problem worldwide with growing incidence and high mortality, hepatocellular carcinoma (HCC) being the most frequent. Hepatocytes are likely the cellular origin of most HCCs through the accumulation of genetic alterations, although hepatic progenitor cells (HPCs) might also be candidates in specific cases, as discussed here. HCC usually develops in a context of chronic inflammation, fibrosis, and cirrhosis, although the role of fibrosis is controversial. The interplay between hepatocytes, immune cells and hepatic stellate cells is a key issue. This review summarizes critical aspects of the liver tumor microenvironment paying special attention to platelets as new key players, which exert both pro- and anti-tumor effects, determined by specific contexts and a tight regulation of platelet signaling. Additionally, the relevance of specific signaling pathways, mainly HGF/MET, EGFR and TGF-ß is discussed. HGF and TGF-ß are produced by different liver cells and platelets and regulate not only tumor cell fate but also HPCs, inflammation and fibrosis, these being key players in these processes. The role of C3G/RAPGEF1, required for the proper function of HGF/MET signaling in HCC and HPCs, is highlighted, due to its ability to promote HCC growth and, regulate HPC fate and platelet-mediated actions on liver cancer.

Intestinal BMP-9 locally upregulates FGF19 and is down-regulated in obese patients with diabetes.

Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.

C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process.

Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-ß signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL-6 trans-signaling.

BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice.

Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs-Oval cells (OCs)-are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in different conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of KrasG12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the KrasG12D mutation was detected in hepatic culture conditions of undifferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with KrasG12D mutation in an undifferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic process.

BMP9 Promotes an Epithelial Phenotype and a Hepatocyte-like Gene Expression Profile in Adult Hepatic Progenitor Cells.

Bone morphogenetic protein 9 (BMP9), a member of the TGF-ß superfamily, has emerged as a new player in chronic liver diseases (CLDs). Its levels increase in the fibrotic liver where it promotes fibrogenesis. It also regulates hepatic progenitor cells (oval cells in rodents), a cell population that contributes to repopulate the liver and recover functionality upon severe damage, but it can also be pro-fibrogenic, depending upon the hepatic microenvironment. Here we analyze the effect of chronic exposure to BMP9 in oval cells. We show that cells chronically treated with BMP9 (B9T-OC) display a more epithelial and hepatocyte-like phenotype while acquiring proliferative and survival advantages. Since our previous studies had revealed a functional crosstalk between BMP9 and the HGF/c-Met signaling pathways in oval cells, we analyzed a possible role for HGF/c-Met in BMP9-induced long-term effects. Data evidence that active c-Met signaling is necessary to obtain maximum effects in terms of BMP9-triggered hepatocytic differentiation potential, further supporting functionally relevant cooperation between these pathways. In conclusion, our work reveals a novel action of BMP9 in liver cells and helps elucidate the mechanisms that serve to increase oval cell regenerative potential, which could be therapeutically modulated in CLD.

Sensitization to benzyl salicylate and other allergens in patients with frontal fibrosing alopecia.

BACKGROUND: Contact sensitization is frequent among patients with frontal fibrosing alopecia (FFA) (52%-76%). OBJECTIVE: To evaluate the frequency of sensitization/photosensitization in an FFA population. METHODS: A population of FFA patients were patch tested (Spanish Contact Dermatitis Research Group [GEIDAC] baseline; cosmetic and fragrance series), and photopatch tested (sunscreen series). RESULTS: Thirty-six patients (mean age: 64.6 years; 35/36: women) were studied. A history of dermatitis was recorded in 69.4% (frequently involving the face). Overall, 80.5% patients showed positive patch-test reactions. The most frequently positive allergens were nickel sulfate (25%), benzyl salicylate (22%), gallates (16.6%), propolis (16.6%), and limonene hydroperoxides (13.8%). Benzyl salicylate was likely relevant to the dermatitis (labeled on personal care products and most patients reporting clinical improvement with allergen avoidance). Patch tests with sunscreens showed positive reactions to 11 materials (five patients). Photopatch tests were positive in one case. CONCLUSION: We speculate a possible relationship between sensitization to benzyl salicylate and FFA. Hypothetically, the most likely explanation is that sensitization to benzyl salicylate involving FFA patients is a consequence of increased exposure to it. It is unclear whether allergen avoidance may impact the prognosis of alopecia. However, it seems to significantly improve the patients´ quality of life by lessening dermatitis and pruritus.

Editorial Special Issue TGF-beta/BMP Signaling Pathway.

The transforming growth factor ß (TGF-ß) superfamily plays key roles in development and tissue homeostasis, controlling the maintenance and regeneration of mature tissues. Cytokines belonging to this family can be multifunctional (TGF-ß and bone morphogenetic proteins, BMPs) or develop highly specialized functions (anti-Müllerian hormone, AMH, or growth differentiation factor 8, myostatin, GDF8) and they control a variety of cellular processes such as proliferation, differentiation, cell death, adhesion and movement, metabolism, pluripotency and stemness. (...).

C3G Is Upregulated in Hepatocarcinoma, Contributing to Tumor Growth and Progression and to HGF/MET Pathway Activation.

The complexity of hepatocellular carcinoma (HCC) challenges the identification of disease-relevant signals. C3G, a guanine nucleotide exchange factor for Rap and other Ras proteins, plays a dual role in cancer acting as either a tumor suppressor or promoter depending on tumor type and stage. The potential relevance of C3G upregulation in HCC patients suggested by database analysis remains unknown. We have explored C3G function in HCC and the underlying mechanisms using public patient data and in vitro and in vivo human and mouse HCC models. We found that C3G is highly expressed in progenitor cells and neonatal hepatocytes, whilst being down-regulated in adult hepatocytes and re-expressed in human HCC patients, mouse HCC models and HCC cell lines. Moreover, high C3G mRNA levels correlate with tumor progression and a lower patient survival rate. C3G expression appears to be tightly modulated within the HCC program, influencing distinct cell biological properties. Hence, high C3G expression levels are necessary for cell tumorigenic properties, as illustrated by reduced colony formation in anchorage-dependent and -independent growth assays induced by permanent C3G silencing using shRNAs. Additionally, we demonstrate that C3G down-regulation interferes with primary HCC tumor formation in xenograft assays, increasing apoptosis and decreasing proliferation. In vitro assays also revealed that C3G down-regulation enhances the pro-migratory, invasive and metastatic properties of HCC cells through an epithelial-mesenchymal switch that favors the acquisition of a more mesenchymal phenotype. Consistently, a low C3G expression in HCC cells correlates with lung metastasis formation in mice. However, the subsequent restoration of C3G levels is associated with metastatic growth. Mechanistically, C3G down-regulation severely impairs HGF/MET signaling activation in HCC cells. Collectively, our results indicate that C3G is a key player in HCC. C3G promotes tumor growth and progression, and the modulation of its levels is essential to ensure distinct biological features of HCC cells throughout the oncogenic program. Furthermore, C3G requirement for HGF/MET signaling full activation provides mechanistic data on how it works, pointing out the relevance of assessing whether high C3G levels could identify HCC responders to MET inhibitors.

A Signaling Crosstalk between BMP9 and HGF/c-Met Regulates Mouse Adult Liver Progenitor Cell Survival.

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-ß type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response.

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR.

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.

Syringocystadenoma papilliferum associated with verrucous carcinoma of the skin in the same lesion: Report of four cases.

The association of syringocystadenoma papilliferum (SCAP) with verrucous carcinoma (VC) of the skin in the same lesion is a rare, but well-documented event. Although human papillomaviruses (HPV) have been proposed to have an etiologic role in the development of the verrucous proliferations associated with SCAP, most of the immunohistochemical and molecular studies have failed to show the presence of their genomic material in these lesions. We report a series of four cases of SCAP associated with VC in anogenital lesions. In two of the cases, we demonstrated the presence of the BRAF V600E mutation by polymerase chain reaction and immunohistochemistry, both in the glandular and in the squamous component. No HPV-related histopathologic changes were found, nor could the presence of viral DNA be showed.

Clathrin switches transforming growth factor-ß role to pro-tumorigenic in liver cancer.

BACKGROUND & AIMS: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-ß (TGF-ß) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-ß-induced signalling in liver cells and its relevance in liver cancer. METHODS: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-ß and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). RESULTS: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-ß phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-ß-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-ß signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CONCLUSIONS: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-ß pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-ß-targeted therapy. LAY SUMMARY: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-ß in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-ß.

c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

BACKGROUND & AIMS: Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. METHODS: WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. RESULTS: Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. CONCLUSIONS: We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases.

Dual role of protein tyrosine phosphatase 1B in the progression and reversion of non-alcoholic steatohepatitis.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH). METHODS: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2-7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice. RESULTS: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated. CONCLUSIONS: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD.