Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.     

Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage

Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1. CONCLUSION: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.     

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.     

Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway

Oxidative stress is involved in hepatic fibrogenesis. Activation of hepatic stellate cells (HSCs), the key effectors in hepatic fibrogenesis, is characterized by overproduction of extracellular matrix. Astragaloside IV, the active component of Radix Astragali, has antioxidant properties and antifibrotic potential in renal fibrosis. Little is known about the role of astragaloside IV in liver and its involvement in hepatic fibrosis. This study aims at evaluating the antifibrotic potential of astragaloside IV and characterizing involved signal transduction pathways in culture-activated HSCs. Our results show that astragaloside IV attenuates oxidative stress in culture-activated HSCs, as demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation, and elevates the level of cellular glutathione by stimulating Nrf2gene expression. Depletion of cellular glutathione by buthionine sulfoximine or abrogation of p38 MAPK by SB-203580 evidently eliminates the inhibitory effects of astragaloside IV on genes relevant to HSC activation. These results demonstrate that astragaloside IV inhibits HSC activation by inhibiting generation of oxidative stress and associated p38 MAPK activation and provide novel insights into the mechanisms of astragaloside IV as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.     

Nicotinamide riboside,an NAD+ precursor,attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

ObjectiveLiver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis.MethodsMale C57BL/6 J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR) for 20 weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro.ResultsNR supplementation significantly reduced body weight of mice only 7 weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of β-oxidation in skeletal muscle and brown adipose tissue.ConclusionNR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.     

Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation

Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down-regulation of two over-expressed miRNAs, miR-27a and 27b allowed culture-activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor α was confirmed to be the target of miR-27a and 27b. These results indicated a new role and mechanism of miR-27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.     

Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation

Chronic liver injury always progresses to fibrosis and eventually to cirrhosis, a massive health care burden worldwide. Delta-like 1 (Dlk1) is well known as an inhibitor of adipocyte differentiation. However, whether it is involved in liver fibrosis remains unclear. Here, we provide the first evidence that Dlk1 is a critical contributor to liver fibrosis through promoting activation of hepatic stellate cells (HSCs) during chronic liver injury. We found that upon liver injury, Dlk1 was dramatically induced and initially expressed in hepatocytes and then into the HSCs by a paracrine manner. It leads to the activation of HSCs, which is considered to be a pivotal event in liver fibrogenesis. Two forms (～50 and ～25 kDa) of the Dlk1 protein were detected by Western blot analysis. In vitro administration of Dlk1 significantly promoted HSC activation, whereas in vivo knockdown of Dlk1 dramatically inhibited HSC activation and the subsequent fibrosis. The large soluble form (～50 kDa) of Dlk1 was shown to contribute to HSC activation. We were encouraged to find the Dlk1-promoted HSC activation and liver fibrosis can be depressed by transplantation of bone marrow-mesenchymal stem cells (BM-MSCs). Furthermore, we demonstrated that FGF2 was up-regulated in BM-MSCs under injury stimulation, and it probably participated in the inhibition of Dlk1 by BM-MSCs. Our findings provide a novel role of Dlk1 in liver fibrosis leading to a better understanding of the molecular basis in fibrosis and cirrhosis and also give insights into the cellular and molecular mechanisms of MSC biology in liver repair.     

Hepatic stellate cells and astrocytes

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.     

Inhibition of EGFR attenuates fibrosis and stellate cell activation in diet-induced model of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. NAFLD begins with steatosis and advances to nonalcoholic steatohepatitis (NASH) and cirrhosis. The molecular mechanisms involved in NAFLD progression are not understood. Based on recent studies showing dysregulation of epidermal growth factor receptor (EGFR) in animal models of liver injury, we sought to determine if inhibition of EGFR mitigates liver fibrosis and HSC activation in NAFLD. We utilized the high fat diet (HFD)-induced murine model of liver injury to study the role of EGFR in NAFLD. The lipid accumulation, oxidative stress, hepatic stellate cell (HSC) activation and matrix deposition were examined in the liver tissues. We also evaluated the EGFR signaling pathway, ROS activation and pro-fibrogenic phenotype in oxidized low density lipoproteins (ox-LDL) challenged cultured HSCs. We demonstrate that EGFR was phosphorylated in liver tissues of HFD murine model of NAFLD. Inhibition of EGFR prevented diet-induced lipid accumulation, oxidative stress, and HSC activation and matrix deposition. In cultured HSCs, we show that ox-LDL caused rapid activation of the EGFR signaling pathway and induce the production of reactive oxygen species. EGFR also mediated HSC activation and promoted a pro-fibrogenic phenotype. In conclusion, our data demonstrate that EGFR plays an important role in NAFLD and is an attractive target for NAFLD therapy.     

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl₄-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.Subject terms: Endocrine system and metabolic diseases, Cell biology     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Retinol uptake and metabolism,and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 M, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

Suppression of hepatic stellate cell activation by microRNA-29b

MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3′UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-β, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.     

Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.Key words: p75 neurotrophin receptor, transforming growth factor-β, neurotrophins, epidermal growth factor, extracellular matrix, collagen, chondroitin sulfate proteoglycans, matrix metalloproteinases, scar, neurons, hepatocytes