DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl₄‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.     

大鼠纤维化肝组织中SHP1表达与肝星状细胞活化及增殖的关系*

目的: 探讨大鼠肝纤维化病理过程中肝组织及在体肝星状细胞 (HSC)的含SH2结构域的蛋白酪氨酸磷酸酶1 (SHP1)表达变化与在体HSC活化及增殖的关系。方法: 随机将50只健康雄性SD大鼠分为对照组(10只)、模型组(40只),采用腹腔注射四氯化碳法建立大鼠肝纤维化模型,Masson三色染色及HE染色检测大鼠肝脏组织的病理组织学变化,SHP1与α-平滑肌肌动蛋白 (α-SMA)免疫荧光双标记检测大鼠肝组织中活化HSC的SHP1表达,免疫组织化学染色检测大鼠肝组织的α-SMA及SHP1表达,并分别对大鼠肝组织的SHP1表达及大鼠肝组织中活化HSC的SHP1表达与大鼠肝组织的α-SMA表达进行Pearson’s相关性分析。结果: 大鼠肝纤维化模型成功构建,随着造模时间延长,大鼠肝纤维化逐渐加重。与对照组大鼠肝组织的SHP1阳性表达平均光密度值 (MOD) (0.08±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的SHP1阳性表达MOD (0.11±0.01、0.14±0.01、0.16±0.01、0.19±0.01)显著增加(P＜0.05),并逐渐升高(P＜0.05)。与对照组大鼠肝组织的α-SMA阳性表达MOD (0.04±0.01)比较,造模不同时间(2周、4周、6周、8周)大鼠纤维化肝组织的α-SMA阳性表达MOD (0.06±0.01、 0.09±0.01、0.12±0.01、0.16±0.02)明显增加(P＜0.05),并逐渐升高(P＜0.05),即在体HSC的活化及增殖逐渐加快(α-SMA是HSC的活化标志)。SHP1与α-SMA免疫荧光双标记检测显示,造模2周、4周、6周、8周大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比(26.49%±3.44%、37.14%±4.57%、44.90%±2.94%、58.09%±5.33%)逐渐升高(P＜0.05)。上述大鼠纤维化肝组织的SHP1表达及大鼠纤维化肝组织中表达SHP1的活化HSC占总的活化HSC的百分比均与大鼠纤维化肝组织的α-SMA表达呈显著正相关(r值为0.926, 0.984,P＜0.05)。结论: 在大鼠肝纤维病理过程中,肝组织及在体HSC 的SHP1表达与在体HSC的活化及增殖呈显著正相关。     

CX3CL1/fractalkine shedding by human hepatic stellate cells: contribution to chronic inflammation in the liver

Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro . We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.     

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。     

细胞珠蛋白对肝星状细胞氧化损伤的保护作用

为揭示细胞珠蛋白对肝星状细胞氧化损伤的保护作用及相关机制,通过siRNA干扰内源性细胞珠蛋白基因,利用重组细胞珠蛋白作用于完全活化的人肝星状细胞系LX-2及大鼠原代肝星状细胞,并在LX-2细胞内过表达细胞珠蛋白,考察在过氧化氢及铁过载两种不同作用机制的氧化反应模型中细胞的增殖性及细胞内超氧化物水平。结果表明内源性细胞珠蛋白对于两种氧化反应导致的肝星状细胞损伤都具有显著性的保护作用,证明其在活化肝星状细胞内的表达上调是其应对氧化应激的保护性措施;重组细胞珠蛋白不仅能保护完全活化的LX-2细胞免受氧化应激损伤,并且能抑制未完全活化的原代肝星状细胞过度增殖以及保护其被过度损伤;重组细胞珠蛋白对细胞内的活性氧清除效果不理想,可能与其进出细胞缺乏相应的主动运输机制有关。进一步在LX-2细胞内过表达细胞珠蛋白对无论是铁过载或是过氧化氢引起的氧化反应均能发挥较好的保护性作用。为加速肝纤维化药物新靶点开发提供了理论依据。     

Potential natural mTOR inhibitors screened by in silico approach and suppress hepatic stellate cells activation

The mammalian target of rapamycin (mTOR), an atypical serine/threonine kinase, plays a central role in the regulation of cell proliferation, growth, differentiation, migration, and survival. In this study, the 3-D structure of the mTOR (PDB ID: 2FAP) was used for the docking of 47 natural compounds and compared with pharmacophore model of 14 known mTOR inhibitors to identify the novel and specific natural inhibitor. The top four compounds, rutin, curcumin, antroquinonol, and benzyl cinnamate, have been selected based on their PLP score and further validated with hepatic stellate cells NHSC and THSC. Curcumin and antroquinonol significantly inhibited NHSC and THSC cells proliferation in a dose-dependent manner, whereas rutin and benzyl cinnamate showed less alteration of cell viability. Rutin inhibited the phosphorylation of mTOR (p-mTOR) and p-p70 S6 K in NHSC and THSC cells by Western blotting. Additionally, p-p70 S6 K protein was significantly decreased by incubation with benzyl cinnamate and curcumin in THSC cells. Taken together, this result suggests that rutin is a potential mTOR inhibitor in screen hits of molecular docking to hamper the activation of HSC and further applications in the treatment of liver fibrosis.     

Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism

Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G₁ phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor β signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.     

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.     

Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O₂⁻) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O₂⁻ production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O₂⁻ production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.     

Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin

Activation of hepatic stellate cells (HSCs) is an integral component of the wound‐healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen‐rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito‐respiration, mito‐membrane potential (Δψm) and cellular ‘bioenergetic signature’ distinguish fibrogenic HSCs from normal, less‐active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered ‘bioenergetic signature’ of fibrogenic HSCs. Importantly, the distinctive elevation in mito‐Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less‐active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito‐Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.     

The immunoreactivity of glial fibrillary acidic protein in mesangial cells and podocytes of the glomeruli of rat kidney in vivo and in culture

In this study the presence of glial fibrillary acidic protein (GFAP) in kidney is for the first time demonstrated in cryostat sections and cultures of isolated glomerular explants derived from rat kidneys. In double immunolabelling analysis of adult rat kidney sections using antiserum against GFAP and monoclonal antibody (mAb) against vimentin or desmin, the presence of immunoreactivity for GFAP could be observed in the glomerulus of the kidney and vascular cells situated in the peritubular space which expressed vimentin and desmin. Labelling of the sections with absorbed antiserum against GFAP completely abolished the staining in all these cells. The mAb against GFAP, clone GF12.24 which is known to label GFAP both in neural and non-neural cells, recognised its antigen only in the cells located in glomeruli. The investigations performed on early 2- or 3-day-old cultures from glomerular explants revealed different patterns of staining for GFAP in mesangial cells and podocytes: weak filamentous in mesangial cells and a strong non-filamentous perinuclear pattern in podocytes. Due to prominent perinuclear expression in podocytes GFAP may be considered as a marker of these cells. A different pattern of distribution of immunoreactivity for GFAP in podocytes and mesangial cells might be due to function-related posttranslational modifications of GFAP resulting in assembly or disassembly of GFAP filaments. The different pattern of staining for GFAP in the podocytes and mesangial cells, cells which exert a different influence on the capillaries of the glomeruli, suggests a role for GFAP in regulation of the tension and permeability of vascular walls. Previous investigations and present studies hint at GFAP as being a general marker of perivascular cells.     

Biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality

Fibrosis is a frequent, life-threatening complication of most chronic liver diseases. Despite major achievements in the understanding of its pathogenesis, the translation of this knowledge into clinical practice is still limited. In particular, non-invasive and reliable (serum-) biomarkers indicating the activity of fibrogenesis are scarce. Class I biomarkers are defined as serum components having a direct relation to the mechanism of fibrogenesis, either as secreted matrix-related components of activated hepatic stellate cells and fibroblasts or as mediators of extracellular matrix (ECM) synthesis or turnover. They reflect primarily the activity of the fibrogenic process. Many of them, however, proved to be disappointing with regard to sensitivity and specificity. Up to now hyaluronan turned out to be the relative best type I serum marker. Class II biomarkers comprise in general rather simple standard laboratory tests, which are grouped into panels. They fulfil most criteria for detection and staging of fibrosis and to a lesser extent grading of fibrogenic activity. More than 20 scores are currently available, among which Fibrotest is the most popular one. However, the diagnostic use of many of these scores is still limited and standardization of the assays is only partially realized. Combining of panel markers in sequential algorithms might increase their diagnostic validity. The translation of genetic pre-disposition biomarkers into clinical practice has not yet started, but some polymorphisms indicate a link to progression and outcome of fibrogenesis. Parallel to serum markers non-invasive physical techniques, for example, transient elastography, are developed, which can be combined with serum tests and profiling of serum proteins and glycans.     

Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α₅β₁, heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α₅, β₁, αv or β₃ integrin subunits were measured in days 1–7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α₅β₁, HSPG or FN were investigated using an in vitro cell adhesion assay. Co‐incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 (‘CCN2_1–4’) or residues 247–349 containing module 4 alone (‘CCN2₄’) supported day 3 cell adhesion in an integrin α₅β₁‐ and HSPG‐dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α₅β₁‐dependent manner by CCN2_1–4 or CCN2₄, whereas FN promoted HSPG‐dependent HSC adhesion to CCN2_1–4 or CCN2₄. These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α₅β₁, a novel CCN2 receptor in primary culture HSC which interacts co‐operatively with HSPG or FN.