Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.     

Hepatic differentiation of adult and fetal liver stromal cells in vitro

The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. For example, in patients with alcoholic cirrhosis repair mechanisms are not activated and only organ transplantation or advanced methods of regenerative medicine can help such patients. Clinical trials including patients with various forms of liver disease have shown promising results of transplantation of autologous bone marrow stem cells. However, improvement of the effectiveness of such treatment requires optimization of sources of progenitor cells. In this study we have isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, performed their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. Stromal cells isolated from the fetal liver were used for comparative evaluation. During hepatic differentiation both types of cells expressed hepatic markers and secreted albumin. These results can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies proliferate for a long time in a culture and this provides opportunity to produce them in large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

自噬在肝再生中的作用

自噬是存在于真核细胞内的一种溶酶体依赖性的降解途径,在肝脏生理和病理过程中发挥着重要作用。肝脏具有强大的再生能力,在受到急、慢性损伤时,残肝细胞将会被激活进入细胞周期进行细胞增殖,以补偿丢失的肝组织和恢复肝功能。文章阐述了各种类型损伤之后的肝再生与自噬的关系。在物理性、酒精、食源性等因素引起的肝损伤中,肝脏通过启动自噬来促进肝再生;在化学性损伤的肝再生模型中,自噬在其中的作用仍然有争议;在病毒感染之后的肝再生中,一些嗜肝病毒（如丙肝病毒和乙肝病毒等）反而利用自噬来促进病毒颗粒复制,抑制肝再生。对自噬和肝再生机制的研究,将有助于进一步阐明再生过程,为治疗肝脏疾病提供新方法。     

Distribution of hepatic phosphofructokinase isozymes in parenchymal and sinusoidal cells.

The distribution profile of the isozymes of phosphofructokinase (PFK) in different cell types of rat liver is established using the techniques of electrophoresis and immunodiffusion. Agarose gel electrophoresis of the extracts of parenchymal cells, Kupffer or sinusoidal cells, and whole liver indicated that two PFK isozymes are present in whole liver and that the faster moving hepatic PFK isozyme is present only in parenchymal cells; whereas, the slower moving hepatic PFK isozyme is only in sinusoidal cells. Immunodiffusion studies using antiserum specific for the major hepatic PFK isozyme (PFK-L₂) revealed that PFK-L₂ is present only in whole liver or parenchymal cell extracts and is absent from sinusoidal cells. It is apparent that the other hepatic PFK isozyme (PFK-L₁) is normally found only in sinusoidal cells.     

Liver ultrastructural changes following portal circulation disturbances

Liver cell changes produced in rats by the ligature of the portal vein and of the spleen pedicle were studied by electron microscopy. There were differences in the liver response to the various types of circulatory disturbances. The earliest and most marked lesions of hepatic cells were noticed in the case of portal vein ligature, and occurred at the level of rough endoplasmic reticulum, mitochondria and lysosomes. No significant changes in Kupffer cells. When the spleen pedicle was ligated, the hepatic cell changes were less obvious, but the Kupffer cells changes were more prominent, testifying and increased hetero- and autophagy.     

Generation,characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013. © 2012 Wiley Periodicals, Inc.     

Uptake of lactosylated low-density lipoprotein by galactose-specific receptors in rat liver.

The liver contains two types of galactose receptors, specific for Kupffer and parenchymal cells respectively. These receptors are only expressed in the liver, and therefore are attractive targets for the specific delivery of drugs. We provided low-density lipoprotein (LDL), a particle with a diameter of 23 nm in which a variety of drugs can be incorporated, with terminal galactose residues by lactosylation. Radioiodinated LDL, lactosylated to various extents (60-400 mol of lactose/ mol of LDL), was injected into rats. The plasma clearance and hepatic uptake of radioactivity were correlated with the extent of lactosylation. Highly lactosylated LDL (greater than 300 lactose/LDL) is completely cleared from the blood by liver within 10 min. Pre-injection with N-acetylgalactosamine blocks liver uptake, which indicates that the hepatic recognition sites are galactose-specific. The hepatic uptake occurs mainly by parenchymal and Kupffer cells. At a low degree of lactosylation, approx. 60 lactose/LDL, the specific uptake (ng/mg of cell protein) is 28 times higher in Kupffer cells than in parenchymal cells. However, because of their much larger mass, parenchymal cells are the main site of uptake. At high degrees of lactosylation (greater than 300 lactose/LDL), the specific uptake in Kupffer cells is 70-95 times that in parenchymal cells. Under these conditions, Kupffer cells are, despite their much smaller mass, the main site of uptake. Thus not only the size but also the surface density of galactose on lactosylated LDL is important for the balance of uptake between Kupffer and parenchymal cells. This knowledge should allow us to design particulate galactose-bearing carriers for the rapid transport of various drugs to either parenchymal cells or Kupffer cells.     

Liver cell heterogeneity: functions of non-parenchymal cells.

The normal hepatic sinusoid is formed or lined by four different cell types, each with its specific phenotypic characteristics, functions and topography. Endothelial cells constitute the closed lining or wall of the capillary. They contain small fenestrations to allow the free diffusion of substances, but not of particles like chylomicrons, between the blood and the hepatocyte surface. This filtering effect regulates the fat uptake by the liver. Sinusoidal endothelial cells also have a pronounced endocytotic capacity which makes them an important part of the reticuloendothelial system. They are also active in the secretion of bioactive factors and extracellular matrix components of the liver. Recently, a zonal heterogeneity of the endothelial lining has been reported with regard to its filtering capacity (fenestration) and binding capacity for lectins and cells. Kupffer cells are intrasinusoidally located tissue macrophages with a pronounced endocytotic capacity. They are potent mediators of the inflammatory response by the secretion of a variety of bioactive factors and play an important part in the immune defense. A zonal heterogeneity has been established with regard to the endocytotic capacity and cytotoxic function. Pit cells are now known to represent a liver-associated population of large granular lymphocytes. They have the capacity to kill tumor cells and probably also play a role in the antiviral defense of the liver. In addition, pit cells may have a growth-regulatory function of the liver. They are known to be numerically more prominent in the periportal region, as is also the case for Kupffer cells. Fat-storing or Ito cells are present in the perisinusoidal space of Disse and are thought to represent the main hepatic source of extracellular matrix components. They are also the main site of vitamin-A storage. Fat-storing cells are more numerous in the periportal region than in the central region of the hepatic acinus. The periportal cells also store higher amounts of vitamin A. Sinusoidal cells may be considered to represent a functional unit at the border line between the hepatocytes or parenchymal cells and the blood. They participate in various liver functions and liver pathologies and our knowledge about this is growing. The heterogeneity of these cell types and possible cooperations between them and the hepatocytes may add to our understanding of liver functions.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

Roles of heterogenous hepatic macrophages in the progression of liver diseases

Hepatic macrophages are key immune cells associated with the broad ranges of liver diseases including steatosis, inflammation and fibrosis. Hepatic macrophages interact with other immune cells and orchestrate hepatic immune circumstances. Recently, the heterogenous populations of hepatic macrophages have been discovered termed residential Kupffer cells and monocyte-derived macrophages, and identified their distinct population dynamics during the progression of various liver diseases. Liver injury lead to Kupffer cells activation with induction of inflammatory cytokines and chemokines, which triggers recruitment of inflammatory monocyte-derived macrophages. To understand liver pathology, the functions of different subtypes of liver macrophages should be regarded with different perspectives. In this review, we summarize recent advances in the roles of hepatic macrophages under liver damages and suggest hepatic macrophages as promising therapeutic targets for treating liver diseases.     

Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved

Fibrosis, defined as the excessive deposition of extracellular matrix in an organ, is the main complication of chronic liver damage. Its endpoint is cirrhosis, which is responsible for significant morbidity and mortality. The accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of fibroblasts, which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver. They are produced by the activation of precursor cells, such as hepatic stellate cells and portal fibroblasts. These fibrogenic cells are distributed differently in the hepatic lobule: the hepatic stellate cells resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes, whereas the portal fibroblasts are embedded in the portal tract connective tissue around portal structures (vessels and biliary structures). Differences have been reported between these two fibrogenic cell populations, in the mechanisms leading to myofibroblastic differentiation, activation and "deactivation", but confirmation is required. Second-layer cells surrounding centrolobular veins, fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved in fibrogenesis. It is now widely accepted that the various types of lesion (e.g., lesions caused by alcohol abuse and viral hepatitis) leading to liver fibrosis involve specific fibrogenic cell subpopulations. The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of fibrosis/cirrhosis.     

The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

Bcl-3 promotes TNF-induced hepatocyte apoptosis by regulating the deubiquitination of RIP1

Tumor necrosis factor-α (TNF) is described as a main regulator of cell survival and apoptosis in multiple types of cells, including hepatocytes. Dysregulation in TNF-induced apoptosis is associated with many autoimmune diseases and various liver diseases. Here, we demonstrated a crucial role of Bcl-3, an IκB family member, in regulating TNF-induced hepatic cell death. Specifically, we found that the presence of Bcl-3 promoted TNF-induced cell death in the liver, while Bcl-3 deficiency protected mice against TNF/D-GalN induced hepatoxicity and lethality. Consistently, Bcl-3-depleted hepatic cells exhibited decreased sensitivity to TNF-induced apoptosis when stimulated with TNF/CHX. Mechanistically, the in vitro results showed that Bcl-3 interacted with the deubiquitinase CYLD to synergistically switch the ubiquitination status of RIP1 and facilitate the formation of death-inducing Complex II. This complex further resulted in activation of the caspase cascade to induce apoptosis. By revealing this novel role of Bcl-3 in regulating TNF-induced hepatic cell death, this study provides a potential therapeutic target for liver diseases caused by TNF-related apoptosis.Subject terms: Protein folding, Genetics research     

Hepatic regeneration from hematopoietic stem cells

In recent years, numerous investigators have reported novel cellular fates of multipotent stem or progenitor cells. In this review, we discuss the unexpected observations that hematopoietic stem cells can contribute to the hepatocyte lineage in humans and in rodent models of liver disease and regeneration. A key unresolved issue regarding hepatic regeneration from hematopoietic stem cells is whether the mechanism occurs through transdetermination, cell fusion, or other processes. A better understanding of the various stem or progenitor cells of the hepatic lineage may facilitate cellular transplantation approaches for the correction of hepatic function in patients with end-stage liver disease.     

Role of Oncostatin M in hematopoiesis and liver development

Definitive hematopoietic stem cells (HSCs) first appear in the aorta/gonad/mesonephros (AGM) region and migrate to the fetal liver where they massively produce hematopoietic cells before establishing hematopoiesis in the bone marrow at a perinatal stage. In the AGM region, Oncostatin M (OSM) enhances the development of both hematopoietic and endothelial cells by possibly stimulating their common precursors, so-called hemangioblasts. During development of HSCs in the AGM region, the liver primodium is formed at the foregut and accepts HSCs. While fetal hepatic cells function as hematopoietic microenvironment for expansion of hematopoietic cells during mid to late gestation, they do not possess most of the metabolic functions of adult liver. Along with the expansion of hematopoietic cells in fetal liver, OSM is produced by hematopoietic cells and induces differentiation of fetal hepatic cells, conferring various metabolic activities of adult liver. Matured hepatic cells then lose the ability to support hematopoiesis. Thus, OSM appears to coordinate the development of liver and hematopoiesis in the fetus.     

Colonization of the rat liver by syngeneic tumor cells. An experimental approach by in vivo and in situ studies

Syngeneic colon carcinoma cells and glioma cells were injected into the portal vein of BD IX rats. After various time periods the animals were sacrificed and the livers and lungs were fixed and prepared for histology. Atypical cells were observed in the liver 4 and 7 days after the injection of tumor cells, whereas distinct colonies of both colon carcinoma and glioma cells were demonstrated after 14 days. Lung metastases of both tumor cell types were seen after 14 and 30 days. Furthermore, injection of glioma and carcinoma cells into the tail vein gave detectable lung metastases after 7 and 4 days respectively. Intraperitoneal injection of tumor cells resulted in the accumulation of large tumor masses, particularly in the mesentery. By in situ perfusions of the liver with tumor cells included in the perfusion medium it was possible to establish that all the tumor cells were arrested in the course of 4 min. In contrast, normal rat leukocytes were not trapped in the liver, whereas trypsin-treated leukocytes were, suggesting the importance of trypsin-sensitive structures for binding to hepatic tissue. The binding of both glioma and carcinoma cells to the liver and the ensuing growth of tumor nodules in this organ indicate a lack of specificity on part of the malignant cell types for metastasis to the liver in the rat. Both tumor cell types colonized the first organ encountered after injection.     

Study of hepatocyte differentiation using embryonic stem cells

The liver has many crucial functions including metabolizing dietary molecules, detoxifying compounds, and storing glycogen. The hepatocytes, comprising most of the liver organ, progressively modify their gene expression profile during the fetal development according to their roles in the different phases of development. Embryonic stem (ES) cells serve as a major tool in understanding liver development. These cells may also serve as a source of hepatic cells for cellular therapy. In this review, we aim to summarize the research that has been performed in the field of hepatocyte differentiation from mouse and human ES cells. We discuss the various methodologies for the differentiation of ES cells towards hepatic cells using either spontaneous or directed differentiation protocols. Although many protocols for differentiating ES cells to hepatic cells have been developed, the analysis of their status is not trivial and can lead to various conclusions. Hence, we discuss the issues of analyzing hepatocytes by means of the specificity of the markers for hepatocytes and the status of the cells as fetal or adult hepatocytes.