Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-β stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-β differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I. J. Cell. Physiol. 174:240–250, 1998. © 1998 Wiley-Liss, Inc.     

CD133+ hepatic stellate cells are progenitor cells

Hepatic stellate cells (HSC) play an important role in the development of liver fibrosis. Here, we report that HSC express the stem/progenitor cell marker CD133 and exhibit properties of progenitor cells. CD133+ HSC of rats were selected by specific antibodies and magnetic cell sorting. Selected cells displayed typical markers of HSC, endothelial progenitor cells (EPC), and monocytes. In cell culture, CD133+ HSC transformed into alpha-smooth muscle actin positive myofibroblast-like cells, whereas application of cytokines known to facilitate EPC differentiation into endothelial cells led to the formation of branched tube-like structures and induced expression of the endothelial cell markers endothelial nitric oxide synthase and vascular-endothelial cadherin. Moreover, cytokines that guide stem cells to develop hepatocytes led to the appearance of rotund cells and expression of the hepatocyte markers alpha-fetoprotein and albumin. It is concluded that CD133+ HSC are a not yet recognized progenitor cell compartment with characteristics of early EPC. Their potential to differentiate into endothelial or hepatocyte lineages suggests important functions of CD133+ HSC during liver regeneration.     

Anti-fibrotic effect of thymoquinone on hepatic stellate cells

Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl₄-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect.     

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.     

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.     

核受体FXR在肝星状细胞及肝纤维化中的作用

法尼醇X受体(farnesoid X receptor,FXR)是核受体超家族的重要成员,以作为胆汁酸受体被大家所熟知.除了调节胆汁酸的合成和转运,FXR在调控葡萄糖代谢、甘油三酯代谢、炎症、凝血等方面也发挥重要作用.肝纤维化是慢性肝脏疾病发展成肝硬化的共同途径,终末期肝纤维化将导致肝硬化、肝衰竭,甚至危及生命.肝星状...     

Antifibrotic constituents of Alnus firma on hepatic stellate cells

Suppression of hepatic stellate cell (HSC) growth and activation have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. In the course of screening antifibrotic activity of natural products, the methanolic extract of Alnus firma barks (Betulaceae) showed inhibitory activity of cell proliferation on HSC-T6 cells. A new triterpenoid characterized as lup-20(29) en-2,28-diol-3-yl caffeate (13) was isolated with 12 known diarylheptanoids (1-12) from the barks of A. firma using bioactivity-guided fractionation. Among these compounds, 2 and 13 significantly inhibited the proliferation of HSCs in dose- and time-dependent manners at concentrations from 10 to 100 μM. Taken together, antifibrotic activities of A. firma and its active constituents might suggest the therapeutic potentials against liver fibrosis.     

Impaired proteolysis of collagen I inhibits proliferation of hepatic stellate cells: implications for regulation of liver fibrosis

Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.     

Molecular basis for calcium signaling in hepatic stellate cells

Progressive liver fibrosis (with the resultant cirrhosis) is the primary cause of chronic liver failure. Hepatic stellate cells (HSCs) are critically important mediators of liver fibrosis. In the healthy liver, HSCs are quiescent lipid-storing cells limited to the perisinusoidal endothelium. However, in the injured liver, HSCs undergo myofibroblastic transdifferentiation (activation), which is a critical step in the development of organ fibrosis. HSCs express P2Y receptors linking extracellular ATP to inositol (1,4,5)-trisphosphate-mediated cytosolic Ca(2+) signals. Here, we report that HSCs express only the type I inositol (1,4,5)-trisphosphate receptor and that the receptor shifts into the nucleus and cell extensions upon activation. These cell extensions, furthermore, express sufficient machinery to enable local application of ATP to evoke highly localized Ca(2+) signals that induce localized contractions. These autonomous units of subcellular signaling and response reveal a new level of subcellular organization, which, in turn, establishes a novel paradigm for the local control of fibrogenesis in the liver.     

FRNK质粒转染抑制肝星状细胞增殖机制研究

目的：探讨黏着斑激酶相关非激酶（FRNK）质粒瞬时转染肝星状细胞（HSC）,对纤维连接蛋白（FN）刺激的HSC增殖的影响及其机制。方法：应用体外细胞培养技术,脂质体介导法进行FRNK质粒转染;Westernblot技术检测FRNK、黏着斑激酶（FAK）、p-raX（Tyr^397）蛋白的表达情况,鉴定转染效果;改良MTF法测定HSC增殖;流式细胞术（FCM）检测细胞周期时相。结果：FRNK质粒转染HSC48h时,FRNK蛋白表达最强（P〈0．01）;FAK蛋白转染前后无明显差异（P〉0．01）;FRNK质粒组p-FAK（Tyr^397）蛋白含量（0．40±0．14）较空质粒组（1．89±0．25）显著下降（P〈0．01）;FRNK在HSC大量表达后,于转染12h、24h、48h的HSC增殖抑制率分别为20．07％、26．16％和29．77％（P〈0．01）,呈时间依赖性关系;FRNK质粒组G0／G1期细胞数（71．4±2．81）较空质粒组（48．9±1．66）明显增加（P〈0．01）。结论：脂质体介导下FRNK质粒转染,可使外源性FRNK在HSC内大量表达,抑制FAK磷酸化,阻滞HSC周期时相于G0／G1期,抑制HSC增殖。     

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.     

On the presence of hepatic stellate cells in portal spaces

Previous studies in mice with hypervitaminosis A have demonstrated that fat-storing cells (hepatic stellate cells-HSCs) participate in schistosomal granuloma fibrogenesis. The origin of such cells in portal areas, away from the Disse spaces, was herein investigated. HSCs were identified in frozen sections of the liver by means of Sudan III staining. They appeared as red-stained cells disposed along the sinusoids of normal mice, but were never found within portal spaces. However, in the chronically inflamed portal spaces of Capillaria hepatica-infected mice, Sudan III-positive cells were frequently present among leukocytes and fibroblast-like cells. Thus, there are no resident HSCs in portal spaces, but their presence there in chronic inflammatory processes indicates that they are able to migrate from peri-sinusoidal areas in order to reach the portal areas.     

Isolation and culture of hepatic stellate cells from mouse liver

Hepatic stellate cells （HSCs） are the primary extracellular matrix-producing cells within the river and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in river disease. The volume of the mouse river is much smaller than that of the rat river, which makes it much more difficult to isolate mouse HSCs （mHSCs） than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with riposomeencapsulated dichloromethylene diphosphate （CL2MDP） to selectively eliminate Kupffer cells from the river. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution （PBS）-liposome group, the yield of mHSCs was （1.37 ±0.23） × 10^6/g river, the cell purity was （90.18 ± 1.61）%, and the cell survival rate was （94.51 ±1.61）%. While in the CL2MDP-liposome group, the yield of mHSCs was （1.62 ±0.34）× 10^6/g liver, the cell purity was （94.44 ± 1.89）%, and the cell survival rate was （94.41 ±1.50）%. Based on the yield and purity of mHSCs, the CL2MDP-riposome treatment was superior to the PBS-liposome treatment （P 〈 0.05, P 〈 0.01）. This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.     

Maintenance of hepatocyte functions in coculture with hepatic stellate cells

Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine.     

肝星型细胞分离方法和功能研究进展

肝星型细胞(Hepatic stellate cells,HSCs),又叫储脂细胞(Fat-storing cells,FSCs)或脂肪细胞(lipocytes),是肝脏固有的非实质细胞类型之一,存在于狄氏腔内,以脂滴的形式储存人体维生素A总量的50%–80%。原代HSCs分离方法,目前主要集中于密度梯度离心法结合离心淘洗、HSCs高侧向角的流式分选法、紫外激发的自发荧光或特异性抗体标记的流式细胞术等,将为HSCs生理和病理研究提供坚实的基础。近年来,HSCs的研究蓬勃发展,合作领域不断拓宽。生理状态下,HSCs处于静息状态,合成细胞外基质(Extracellular matrix,ECM)并维持其稳态,同时广泛摄取和储存维生素A,并具有调节肝细胞再生的功能;而病理状态下,HSCs在肝损伤和持续性刺激条件下被激活,增殖活性明显增强,脂滴减少或消失,ECM合成也明显增加,具有收缩性,同时分泌多种促炎因子和粘附分子,并向肌成纤维细胞转变,表明HSCs的活化是肝纤维化发生发展过程中的关键环节之一。有关HSCs的分离和功能研究一直是肝脏细胞学和肝脏病理学研究的热点之一。文中我们将系统总结和探讨HSCs的分离方法和改进策略,及其功能研究进展和具有潜在价值的研究方向。     

Fibronectin dependent upregulation of matrix metalloproteinases in hepatic stellate cells

Activation and transition of hepatic stellate cells (HSCs) to myofibroblast (MFB)-like cells is influenced by growth factors, cytokines and matrix proteins like fibronectin (FN). To examine whether the FN-dependent transition of HSCs is mediated through FN receptor, a marker function, such as matrix metallo-proteinase (MMP) production by HSCs in primary culture was studied. An upregulation of MMP production by HSCs maintained on FN was observed. FN-dependent upregulation of MMPs was significantly reduced when cells were pre-treated with antibodies to alpha5beta1 integrin. Treatment of cells with genistein, a protein kinase C inhibitor completely blocked the gelatinase production by HSCs, indicating that the FN-dependent upregulation of MMPs is mediated through integrins and it involves tyrosine phosphorylation dependent signaling pathways.