Endocytosis of lysosomal enzymes by non-parenchymal rat liver cells. Comparative study of lysosomal-enzyme uptake by hepatocytes and non-parenchymal liver cells.
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ABSTRACT:
SUBMITTER: Ullrich K
PROVIDER: S-EPMC1161311 | biostudies-other | 1979 Aug
REPOSITORIES: biostudies-other
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Project description:Chronic liver disease is characterized by progressive hepatic fibrosis leading to the formation of cirrhosis irrespective of the etiology with no effective treatment currently available. Liver stiffness (LS) is currently the best clinical predictor of this fibrosis progression irrespective of the etiology. LS and hepatocytes-nonparenchymal cells (NPC) interactions are two variables known to be important in regulating hepatic function during liver fibrosis, but little is known about the interplay of these cues. Here, we use polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to study how cell-cell interaction and stiffness regulates hepatocytes function. Specifically, primary rat hepatocytes were cocultured with NIH-3T3 fibroblasts on soft (2 kPa) and stiff substrates that recreates physiologic (2 kPa) and cirrhotic liver stiffness (55 kPa). Urea synthesis by primary hepatocytes depended on the presence of fibroblast and was independent of the substrate stiffness. However, albumin synthesis and Cytochrome P450 enzyme activity increased in hepatocytes on soft substrates and when in coculture with a fibroblast. Western blot analysis of hepatic markers, E-cadherin, confirmed that hepatocytes on soft substrates in coculture promoted better maintenance of the hepatic phenotype. These findings indicate the role of stiffness in regulating the hepatocytes interactions with NPCs necessary for maintenance of hepatocytes function.
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Project description:Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.
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Project description:BACKGROUND & AIMS:Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS:Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS:Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION:Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY:The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).
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Project description:Background & aimsLiver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.MethodsHepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.ResultsCell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 ?m latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of ?-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.ConclusionsOur isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.
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Project description:Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.
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Project description:During liver development, hepatoblasts and liver non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) constitute the liver bud where they proliferate and differentiate. Accordingly, we reasoned that liver NPCs would support the maturation of hepatocytes derived from human induced pluripotent stem cells (hiPSCs), which usually exhibit limited functions. We found that the transforming growth factor β and Rho signaling pathways, respectively, regulated the proliferation and maturation of LSEC and HSC progenitors isolated from mouse fetal livers. Based on these results, we have established culture systems to generate LSECs and HSCs from hiPSCs. These hiPSC-derived NPCs exhibited distinctive phenotypes and promoted self-renewal of hiPSC-derived liver progenitor cells (LPCs) over the long term in the two-dimensional culture system without exogenous cytokines and hepatic maturation of hiPSC-derived LPCs. Thus, a functional human liver model can be constructed in vitro from the LPCs, LSECs, and HSCs derived from hiPSCs.
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Project description:1. Hepatic uptake of low-density lipoprotein (LDL) in parenchymal cells and non-parenchymal cells was studied in control-fed and cholesterol-fed rabbits after intravenous injection of radioiodinated native LDL (125I-TC-LDL) and methylated LDL (131I-TC-MetLDL). 2. LDL was taken up by rabbit liver parenchymal cells, as well as by endothelial and Kupffer cells. Parenchymal cells, however, were responsible for 92% of the hepatic LDL uptake. 3. Of LDL in the hepatocytes, 89% was taken up via the B,E receptor, whereas 16% and 32% of the uptake of LDL in liver endothelial cells and Kupffer cells, respectively, was B,E receptor-dependent. 4. Cholesterol feeding markedly reduced B,E receptor-mediated uptake of LDL in parenchymal liver cells and in Kupffer cells, to 19% and 29% of controls, respectively. Total uptake of LDL in liver endothelial cells was increased about 2-fold. This increased uptake is probably mediated via the scavenger receptor. The B,E receptor-independent association of LDL with parenchymal cells was not affected by the cholesterol feeding. 5. It is concluded that the B,E receptor is located in parenchymal as well as in the non-parenchymal rabbit liver cells, and that this receptor is down-regulated by cholesterol feeding. Parenchymal cells are the main site of hepatic uptake of LDL, both under normal conditions and when the number of B,E receptors is down-regulated by cholesterol feeding. In addition, LDL is taken up by B,E receptor-independent mechanism(s) in rabbit liver parenchymal, endothelial and Kupffer cells. The non-parenchymal liver cells may play a quantitatively important role when the concentration of circulating LDL is maintained at a high level in plasma, being responsible for 26% of hepatic uptake of LDL in cholesterol-fed rabbits as compared with 8% in control-fed rabbits. The proportion of hepatic LDL uptake in endothelial cells was greater than 5-fold higher in the diet-induced hypercholesterolaemic rabbits than in controls.
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Project description:The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.
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Project description:Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.
| S-EPMC4393274 | biostudies-literature
Project description:In order to assess the relative importance of the receptor for low-density lipoprotein (LDL) (apo-B,E receptor) in the various liver cell types for the catabolism of lipoproteins in vivo, human LDL was labelled with [14C]sucrose. Up to 4.5h after intravenous injection, [14C]sucrose becomes associated with liver almost linearly with time. During this time the liver is responsible for 70-80% of the removal of LDL from blood. A comparison of the uptake of [14C]sucrose-labelled LDL and reductive-methylated [14C]sucrose-labelled LDL ([14C]sucrose-labelled Me-LDL) by the liver shows that methylation leads to a 65% decrease of the LDL uptake. This indicated that 65% of the LDL uptake by liver is mediated by a specific apo-B,E receptor. Parenchymal and non-parenchymal liver cells were isolated at various times after intravenous injection of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL. Non-parenchymal liver cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells when expressed per mg of cell protein. This factor is independent of the time after injection of LDL. Taking into account the relative protein contribution of the various liver cell types to the total liver, it can be calculated that non-parenchymal cells are responsible for 71% of the total liver uptake of [14C]sucrose-labelled LDL. A comparison of the cellular uptake of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL after 4.5h circulation indicates that 79% of the uptake of LDL by non-parenchymal cells is receptor-dependent. With parenchymal cells no significant difference in uptake between [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL was found. A further separation of the nonparenchymal cells into Kupffer and endothelial cells by centrifugal elutriation shows that within the non-parenchymal-cell preparation solely the Kupffer cells are responsible for the receptor-dependent uptake of LDL. It is concluded that in rats the Kupffer cell is the main cell type responsible for the receptor-dependent catabolism of lipoproteins containing only apolipoprotein B.
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