Notch-IGF1 signalling in biliary epithelial cells drives their expansion and inhibits hepatocyte differentiation

[no abstract available

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration

Liver regeneration through progenitor cell response : role of the microenvironment in a murine model of chronic liver injury

Rapidly after liver damage, unharmed hepatocytes divide en masse to compensate for the endured cell loss and regain normal function. However, in case of massive and/or chronic injury this process is insufficient due to either paucity of hepatocytes able to engage into the regenerative process or replicative inability of the remaining hepatocytes. In those conditions, a dormant compartment of progenitor cells is activated and considered as a rescue mechanism for functional liver mass regeneration. Liver progenitor cells (LPC) are described as bipotential being able to differentiate into biliary or hepatocytic lineages, depending on the injurious process. The accompanying microenvironment, consisting of hepatic stellate cells (HSC) /myofibroblasts (MF), Kupffer cells (KC), extracellular matrix (ECM) and soluble factors is believed to have a major role in the regulation and modulation of the LPC response. The aim of the present PhD thesis was to evaluate the contribution of this microenvironment to the liver progenitor cell response induced in mice after administration of a choline deficient diet supplemented in ethionine (CDE diet). In a first study using the CDE-model, we designed a time line set up whereby mice were sacrificed at certain time points in order to determine the chronology of the events taking place during LPC-mediated liver regeneration. Hereby, we were able to demonstrate that HSC/MF are rapidly activated (within three days) after the initiation of the diet. These mesenchymal cells were found to proliferate and to deposit ECM around the portal area before the appearance of LPC. Several days later, LPC started to proliferate and to form branching structures while elongating from the pre-existing canals of Hering, the connective structures between hepatocytic canaliculi and bile ductules, and invading the parenchyma. During this parenchymal invasion, we documented that the HSC/MF were wrapped around the LPC. Moreover, the HSC/MF and their ECM deposition were preceding the LPC at the edges of migration. In addition, the quantity of HSC/MF/ECM was shown to increase in parallel with the accumulation of LPC. Taken together, the mesenchymal contribution (both HSC/MF and ECM) seems to be a requisite for the appearance, the proliferation and the migration of the LPC during this attempt of regeneration. In a following study, we focused on the role of resident liver macrophages or Kupffer cells (KC) in the LPC-mediated liver regeneration as inflammation is also accompanying the progenitor cell response. In normal liver, KC were uniformly scattered in the parenchyma. At day 3 of the CDE model, KC were preferentially located around the portal area. Later, KC were migrating from portal to central area (day 7) to be found mainly in the region around the central veins (day 10). This showed that activated KC are also preceding migrating LPC, seemingly even in advance of the HSC/MF. We postulated that KC could potentially influence the LPC response and investigated this by depleting the macrophage population in the CDE model. Removal of the KC did not influence the proliferative capacity of the LPC. Instead, this had an effect on the migratory phenotype of the LPC, which were found to be rounder, strongly interconnected and closer to the portal tract in comparison to non-depleted controls. In addition, some LPC of KC-depleted animals appeared organised in pseudo-ducts leading to the suggestion that these cells were in a more differentiated stage and leaning towards a biliary phenotype. This was in contrast to KC-containing CDE progenitor cells which were arranged in filaments without notable differentiation. KC are therefore documented to play a role not in the activation or proliferation of the LPC but in their migration and differentiation.(SBIM 3) -- UCL, 201

Insulin resistance in hepatocytes and sinusoidal liver cells: mechanisms and consequences.

Hepatic insulin resistance is an important underlying cause of the metabolic syndrome that manifests itself in diseases such as diabetes type II, atherosclerosis or non-alcoholic fatty liver disease (NAFLD). In this paper, we summarize comprehensively the current state of knowledge pertaining to the molecular mechanisms that lead to insulin resistance in hepatocytes and sinusoidal liver cells. In hepatocytes, the insulin resistant state is brought about by at least one, but more likely by a combination, of the following pathological alterations: hyperglycaemia and hyperinsulinaemia, formation of advanced glycation end-products, increased free fatty acids and their metabolites, oxidative stress and altered profiles of adipocytokines. Insulin resistance in hepatocytes distorts directly glucose metabolism, especially the control over glucose output into the circulation and interferes with cell survival and proliferation, while hepatic fatty acid synthesis remains stimulated by compensatory hyperinsulinaemia, resulting in steatosis. Very few studies have addressed insulin resistance in sinusoidal liver cells. These cells are not simply bystanders and passive witnesses of the changes affecting the hepatocytes. They are target cells that will respond to the pathological alterations occurring in the insulin resistant state. They are also effector cells that may exacerbate insulin resistance in hepatocytes by increasing oxidative stress and by secreting cytokines such as TNF and IL-6. Moreover, activation of sinusoidal endothelial cells, Kupffer cells and stellate cells will lead to chemo-attraction of inflammatory cells. Finally, activation of stellate cells will set in motion a fibrogenic response that paves the way to cirrhosis

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model.

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ∼100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67(+)), while more numerous (∼25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes

Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.

In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area, migrate inside the lobule, and undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. We analyzed LPC expansion and matrix accumulation in a choline-deficient, ethionine-supplemented (CDE) model of LPC proliferation. After day 3, CDE induced collagen deposits in the periportal area. Expansion of LPCs as assessed by increased number of cytokeratin 19 (CK19)-positive cells was first observed at day 7, while ECM accumulated 10 times more than in controls. Thereafter, LPCs and ECM increased in parallel. Furthermore, ECM not only accumulates prior to the increase in number of LPCs, but is also found in front of LPCs along the porto-venous gradient of lobular invasion. Double immunostaining revealed that LPCs are embedded in ECM at all times. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by alpha-smooth muscle actin (alpha-SMA)-positive cells. Gene expression analyses confirmed these observations. The expression of CK19, alpha-fetoprotein, E-cadherin, and CD49f messenger RNA (mRNA), largely overexpressed by LPCs, significantly increased between day 7 and day 10. By contrast, at day 3 there was a rapid burst in the expression of components of the ECM, collagen I and laminin, as well as in alpha-SMA and connective tissue growth factor expression. Conclusion: Our data demonstrate that, in a CDE model, ECM deposition and activation of matrix-producing cells occurred as an initial phase, prior to LPC expansion, and in front of LPCs along the porto-veinous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation and differentiation of liver progenitor cells. (HEPATOLOGY 2009.)

Hepatic stellate cells improve engraftment of human primary hepatocytes: A pre-clinical transplantation study in animal model.

Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether co-transplantation of hepatocytes with hepatic stellate cells (HSC) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSC (quiescent or after culture-activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin positive (huAlb(+)) hepatocytes were found scattered. When co-transplanted in a mixture with HSC or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2 to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSC increased the number of hepatocytes entrapped in the host liver at an early time point post-transplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks post-transplantation, we found no accumulation of αSMA(+) activated HSC or collagen deposition. To follow the fate of transplanted HSC, HSC derived from GFP(+) mice were injected into GFP(-) littermate: 17 hours post-transplant, GFP(+) HSC were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Co-culture with HSC improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSC. In vivo, co-transplantation of hepatocytes with HSC into a healthy liver recipient does not generate fibrosis but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing