Endoplasmic reticulum stress does not contribute to steatohepatitis in obese and insulin resistant high-fat diet fed foz/foz mice

Non-alcoholic fatty liver (steatosis) and steatohepatitis [non-alcoholic steatohepatitis (NASH)] are hepatic complications of the metabolic syndrome. Endoplasmic reticulum (ER) stress is proposed as a crucial disease mechanism in obese and insulin-resistant animals (such as ob/ob mice) with simple steatosis, but its role in NASH remains controversial. We therefore evaluated the role of ER stress as a disease mechanism in foz/foz mice, which develop both the metabolic and histological features that mimic human NASH. We explored ER stress markers in the liver of foz/foz mice in response to a high-fat diet (HFD) at several time points. We then evaluated the effect of treatment with an ER stress inducer tunicamycin, or conversely with the ER protectant tauroursodeoxycholic acid (TUDCA), on the metabolic and hepatic features. foz/foz mice are obese, glucose intolerant and develop NASH characterized by steatosis, inflammation, ballooned hepatocytes and apoptosis from 6 weeks of HFD feeding. This was not associated with activation of the upstream unfolded protein response [phospho-eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme 1α (IRE1α) activity and spliced X-box-binding protein 1 (Xbp1)]. Activation of c-Jun N-terminal kinase (JNK) and up-regulation of activating transcription factor-4 (Atf4) and CCAAT/enhancer-binding protein-homologous protein (Chop) transcripts were however compatible with a ‘pathological’ response to ER stress. We tested this by using intervention experiments. Induction of chronic ER stress failed to worsen obesity, glucose intolerance and NASH pathology in HFD-fed foz/foz mice. In addition, the ER protectant TUDCA, although reducing steatosis, failed to improve glucose intolerance, hepatic inflammation and apoptosis in HFD-fed foz/foz mice. These results show that signals driving hepatic inflammation, apoptosis and insulin resistance are independent of ER stress in obese diabetic mice with steatohepatitis

Paracrine cellular senescence exacerbates biliary injury and impairs regeneration

Senescence has been suggested as causing biliary cholangiopathies but how this is regulated is unclear. Here, the authors generate a mouse model of biliary senescence by deleting Mdm2 in bile ducts and show that inhibiting TGFβ limits senescence-dependent aggravation of cholangiopathies

Cholangiocytes act as Facultative Liver Stem Cells during Impaired Hepatocyte Regeneration

After liver injury, regeneration occurs through self-replication of hepatocytes. In severe liver injury, hepatocyte proliferation is impaired - a feature of human chronic liver disease. It is unclear whether other liver cell types can regenerate hepatocytes. Here we use two independent systems to impair hepatocyte proliferation during liver injury to evaluate the contribution of non-hepatocytes to parenchymal regeneration. First, loss of β1-integrin in hepatocytes with liver injury triggered a ductular reaction of cholangiocyte origin, with approximately 25% of hepatocytes being derived from a non-hepatocyte origin. Second, cholangiocytes were lineage traced with concurrent inhibition of hepatocyte proliferation by β1-integrin knockdown or p21 overexpression, resulting in the significant emergence of cholangiocyte-derived hepatocytes. We describe a model of combined liver injury and inhibition of hepatocyte proliferation that causes physiologically significant levels of regeneration of functional hepatocytes from biliary cells

Liver cell therapy: is this the end of the beginning?

The prevalence of liver diseases is increasing globally. Orthotopic liver transplantation is widely used to treat liver disease upon organ failure. The complexity of this procedure and finite numbers of healthy organ donors have prompted research into alternative therapeutic options to treat liver disease. This includes the transplantation of liver cells to promote regeneration. While successful, the routine supply of good quality human liver cells is limited. Therefore, renewable and scalable sources of these cells are sought. Liver progenitor and pluripotent stem cells offer potential cell sources that could be used clinically. This review discusses recent approaches in liver cell transplantation and requirements to improve the process, with the ultimate goal being efficient organ regeneration. We also discuss the potential off-target effects of cell-based therapies, and the advantages and drawbacks of current pre-clinical animal models used to study organ senescence, repopulation and regeneration

Origin and fate of liver progenitor cells

The liver has the extraordinary capacity to self-regenerate following acute and chronic injury. Nevertheless, in terminal-stages of liver disease, this capacity is abrogated and liver transplantation is the only possible rescue. These days, the long-standing shortage of donor livers has encouraged researchers to progress in the development of new therapeutic options for diseases with liver failure. Hepatocyte turnover is low in the adult healthy liver, but in case of injury, hepatocytes vigorously replicate and proliferate to recover the liver mass. When hepatocyte function is impaired, then a reservoir of liver progenitor cells (LPC) activates and expands. The origin of those LPC remains unclear. However, LPC are believed to have the bipotential capacity to generate hepatocytes or biliary cells depending on the nature of the cellular damage in order to restore the hepatic cell loss and function. Although such bipotentiality has been greatly demonstrated in in vitro and ex vitro studies, there is a lack of evidence regarding to whether that differentiation process occurs in vivo. Using in vivo genetic lineage tracing experiments, here we demonstrate that LPC originate from the embryonic ductal plate cells, and that they are able to generate functional hepatocytes in a mouse model of chronic liver injury. We show for the first time that in specific conditions of liver injury, the liver progenitor cell compartment proliferates and gives rise to new mature hepatocytes. In addition, we confirm that hepatocytes are responsible for the maintenance of normal liver homeostasis and for the recovery of the liver mass after acute liver injury induced by physical insult or chemical toxicity. Likewise, hepatocytes mediate hepatic regeneration in drugs (paracetamol), chemicals and toxins-induced chronic liver injury. Furthermore, we demonstrate that the LPC’s plasticity towards the hepatocytic or biliary lineage is modulated by and dependent on the microenvironment, in particular, extracellular matrix, laminin of the basement membrane and Kupffer cells (the resident liver macrophages). In conclusion, our findings indicate that in certain circumstances LPC participate to liver regeneration and wound healing by generating functional hepatocytes, and that stimulation of the differentiation process is feasible by controlling their niche. Therefore, this opens new ways to improve functional liver recovery and the outcome of patients suffering from liver failure.(SBIM 3) -- UCL, 201

Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice

BACKGROUND & AIMS: Self-renewal of mature hepatocytes promotes homeostasis and regeneration of adult liver. However, recent studies have indicated that liver progenitor cells (LPC) could give rise to hepatic epithelial cells during normal turnover of the liver and after acute injury. We investigated the capacity of LPC to differentiate into hepatocytes in vivo and contribute to liver regeneration. METHODS: We performed lineage tracing experiments, using mice that express tamoxifen-inducible Cre recombinase under control of osteopontin regulatory region crossed with yelow fluorescent protein reporter mice, to follow the fate of LPC and biliary cells. Adult mice received partial (two-thirds) hepatectomy, acute or chronic administration of carbon tetrachloride (CCl(4)), choline-deficient diet supplemented with ethionine, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. RESULTS: LPC and/or biliary cells generated 0.78% and 2.45% of hepatocytes during and upon recovery of mice from liver injury, respectively. Repopulation efficiency by LPC and/or biliary cells increased when extracellular matrix and laminin deposition were reduced. The newly formed hepatocytes integrated into hepatic cords, formed biliary canaliculi, expressed hepato-specific enzymes, accumulated glycogen, and proliferated in response to partial hepatectomy, as neighboring native hepatocytes. By contrast, LPC did not contribute to hepatocyte regeneration during normal liver homeostasis, in response to surgical or toxic loss of liver mass, during chronic liver injury (CCl(4)-induced), or during ductular reactions. CONCLUSIONS: LPC or biliary cells terminally differentiate into functional hepatocytes in mice with liver injury

Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha.

UnlabelledMatrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of liver-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150 Pa and increased to 1-6 kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α), whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase. In addition, blockade of the Rho/Rho-associated protein kinase pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix.ConclusionFibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/Rho-associated protein kinase pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. (Hepatology 2016;64:261-275)

In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis

Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis