Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells

Sphingolipids play a relevant role in cell-cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat-storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [(14)C]galactose, [(14)C]serine, or [(14)C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a-pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long-chain fatty acids in retinol-induced lipocytes as compared to the insulin/indomethacin induced ones. Time-course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long-chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX-cells.     

Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha

Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 +/- 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.     

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl₄. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1⁺ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1⁺ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl₄-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

Background  

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.     

Astragaloside IV synergizes with ferulic acid to suppress hepatic stellate cells activation in vitro

Because hepatic fibrosis usually involves more than one pathological process, combination therapy with modalities that target aberrant signaling cascade in activated hepatic stellate cells (HSCs) represents an alternative strategy. This study evaluates the hypothesis that astragaloside IV (AS-IV) and ferulic acid (FA) synergize to inhibit HSCs activation via simultaneous activating nuclear factor erythroid-2-related factor-2 (Nrf2) and blocking transforming growth factor-β (TGF-β) pathways. The combination of FA and AS-IV, hereafter referred to as the AS-IV/FA, at suboptimal concentrations synergistically inhibited HSCs activation, as measured by expressions of α-smooth muscle actin (α-SMA), collagen α type I (Col I) and fibronectin. Nrf2 nuclear accumulation, glutathione (GSH) increase, and reactive oxygen species (ROS) reduction by AS-IV were not potentiated by co-treatment with FA. Similarly, inhibition of TGF-β1 secretion and Smad activity by FA also was not enhanced by combined treatment with AS-IV. AS-IV/FA synergistically suppresses the p38 mitogen-activated protein kinase (MAPK) activity. Inhibition of HSCs activation by AS-IV/FA could be completely blocked by TGF-βs-neutralizing antibody plus shRNA-mediated knockdown of Nrf2. Dual blockade of the TGF-β1/Smad pathway by FA and activation of Nrf2/ARE pathway by AS-IV contributed to the synergistic effects of this combination treatment. These results suggest that combinatorial treatments that target different pathway may afford a more effective strategy to inhibit HSC activation.     

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in co-culture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1–2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells

Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability.     

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM β-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 μM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-¹⁴C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain’s composition.     

Involvement of C/EBP-alpha gene in in vitro activation of rat hepatic stellate cells

Hepatic stellate cells (HSCs) play key roles in hepatic fibrosis. One of the most striking alterations in activated HSCs is loss of cytoplasmic lipid droplets. However, the association of lipid storage with the activation of HSCs remains unclear. CCAAT/enhancer-binding proteins family (C/EBPs), especially C/EBP-alpha, controls differentiation of adipocytes. We suggested that C/EBP-alpha gene may be involved in HSCs activation. The present results showed that the expression levels of C/EBP-alpha and C/EBP-beta genes declined in activated HSCs. Over-expression of C/EBP-alpha gene in activated HSCs: (1) inhibited HSCs proliferation, extracellular matrix-producing, alpha-smooth muscle actin gene expression, and induced rebound of cytoplasmic lipid droplets; (2) reduced retinoic acid receptor-beta, C/EBP-delta and -beta gene expressions, but increased the active form C/EBP-beta PSer(105), and induced retinoid X receptor-alpha gene expression; and (3) did not affect the protein level of p16INK4a, p21Cip1/WAF1 or p27Kip1. In conclusions, C/EBP-alpha gene is involved in in vitro activation of rat HSCs.     

Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM), namely, fibrillar collagens in the hepatic stellate cells (HSCs). Earlier, we developed an antigene approach, using a type alpha1(I) collagen gene promoter specific triplex-forming oligonucleotide (TFO) to inhibit collagen gene expression. In this paper, to enhance overall delivery of TFOs to the liver and more specifically to HSCs, we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) by phosphorylating p-nitrophenyl-alpha-d-mannopyranoside, reducing its nitro group, and reacting it with thiophosgene to produce p-isothiocyanatophenyl-6-phospho-alpha-d-mannopyranoside (itcM6P) for conjugation with BSA. (33)P-TFO was conjugated with M6P-BSA via a disulfide bond, and the stability of the (M6P)(20)-BSA-TFO conjugate was determined. Following tail vein injection into rats, (M6P)(20)-BSA-(33)P-TFO rapidly cleared from the circulation and accumulated mainly in the liver. Almost 66% of the injected (M6P)(20)-BSA-(33)P-TFO accumulated in the liver at 30 min postinjection, which was significantly higher than that deposited after injection of (33)P-TFO. A large proportion of the injected (M6P)(20)-BSA-(33)P-TFO was taken up by the HSCs as evidenced by determination of radioactivity in the digested liver cells upon liver perfusion and separation on a Nycodenz gradient. Therefore, this TFO conjugate may be used for the treatment of liver fibrosis.     

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARγ) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARγ ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1α(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARγ through an adenovirus-expressing PPARγ (Ad-PPARγ), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARγ is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARγ in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARγ in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARγ. It is likely that PPARγ reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.     

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar–parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation. Accepted: 16 September 1999