A red wine polyphenolic extract reduces the activation phenotype of cultured human liver myofibroblasts

AIM： To test the effect of a standardized red wine polyphenolic extract （RWPE） on the phenotype of human liver myofibroblasts in culture. METHODS： Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-（4,5-dimethylthiazol-2-yl）-2,5 diphenyltetrazolium bromide （MTT） assay. Signaling events were analyzed by western blot with phosphospecific antibodies. Matrix-metalloproteinase activity was measured with gel zymography. RESULTS： We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor BB （PDGF-BB）. Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix- metalloproteinases-1.CONCLUSION： Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis.     

Hepatitis C virus proteins do not directly trigger fibrogenic events in cultured human liver myofibroblasts

Summary. Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts, the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core, a structural protein, and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.     

Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts

Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.     

Expression of leukemia inhibitory factor (LIF) and its receptor gp190 in human liver and in cultured human liver myofibroblasts. Cloning of new isoforms of LIF mRNA

BACKGROUND: The cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver. RESULTS: LIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4. CONCLUSIONS: We show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation.     

Experimental study of bioartificial liver with cultured human liver cells

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螺内酯抑制心肌成纤维细胞增殖的实验研究

目的:研究螺内酯对培养的心肌成纤维细胞增殖的作用,揭示螺内酯抑制心肌纤维化的可能机制。方法:进行新西兰白兔心脏心肌成纤维细胞的培养;测定细胞计数,细胞活性(WST-1分解)和DNA合成(BrdU掺入),并以流式细胞仪测定细胞周期等以表示细胞增殖。结果:不同浓度的螺内酯(2.5×10-7~5×10-5mol/L)分别作用1、3、7 d后,心肌成纤维细胞的数量明显减少,并呈剂量-时间-效应(P<0.01)。细胞经不同浓度螺内酯(2.5×10-7~5×10-5mol/L)作用1 d后,DNA合成、活细胞代谢活性和细胞周期的DNA合成期(S) DNA合成后期(G2) 分裂期(M)的百分比明显减少(P<0.01)。结论:螺内酯可能通过抑制心肌成纤维细胞的增殖而抑制心肌纤维化。     

A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis

BACKGROUND & AIMS: Myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. We have analyzed the origin of myofibroblasts within fibrotic liver in 2 scenarios: (1) 7 male patients (hepatitis B; hepatitis B and D; Wilson's disease; hepatitis B, D, and C; and 3 with hepatitis C) who received liver transplants from female donors and subsequently developed liver fibrosis and (2) a female patient who received a bone marrow transplant from a male donor and subsequently developed hepatitis C-induced cirrhosis. METHODS: Through the use of in situ hybridization for the Y chromosome, we have tracked male cells of extrahepatic origin. The phenotype of these male cells was examined by immunohistochemistry using a panel of antibodies against alpha-smooth muscle actin (alpha SMA), vimentin, fibulin-2, and leukocyte common antigen (CD45). Confocal microscopy was performed to confirm the location of the Y chromosome probe within the myofibroblast nuclei. RESULTS: Significant numbers of Y chromosome-positive cells in fibrotic areas were found to be positive for alpha-SMA, vimentin, and fibulin-2 and negative for CD45, thus having a myofibroblast phenotype. In the liver transplant cases, 6.8%-22.2% of alpha-SMA-positive myofibroblasts contained the Y chromosome. In the female recipient of a bone marrow transplant from a male donor, 12.4% of the myofibroblasts were Y chromosome positive, indicating a bone marrow origin. CONCLUSIONS: There is a significant contribution to liver cirrhosis in humans from extrahepatically derived myofibroblasts in liver disease of diverse etiology.     

Targeting liver myofibroblasts: a novel approach in anti-fibrogenic therapy

Chronic liver disease results in a liver-scarring response termed fibrosis. Excessive scarring leads to cirrhosis, which is associated with high morbidity and mortality. The only treatment for liver cirrhosis is liver transplantation; therefore, much attention has been directed toward therapies that will slow or reverse fibrosis. Although anti-fibrogenic therapies have been shown to be effective in experimental animal models, licensed therapies have yet to emerge. A potential problem for any anti-fibrogenic therapy in the liver is the existence of the body’s major drug metabolising cell (the hepatocyte) adjacent to the primary fibrosis-causing cell, the myofibroblast. This article reviews the development of a human recombinant single-chain antibody (scAb) that binds to the surface of myofibroblasts. This antibody binds specifically to myofibroblasts in fibrotic mouse livers. When conjugated with a compound that stimulates myofibroblast apoptosis, the antibody directs the specific apoptosis of myofibroblasts with greater specificity and efficacy than the free compound. The antibody also reduces the adverse effect of liver macrophage apoptosis and—in contrast to the free compound—reversed fibrosis in the sustained injury model used. These data suggest that specifically stimulating the apoptosis of liver myofibroblasts using a targeting antibody has potential in the treatment of liver fibrosis.     

Transforming growth factor-β-induced collagen synthesis by human liver myofibroblasts is inhibited by α2-macroglobulin

Background: Transforming growth factor-β (TGFβ) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFβ in different systems has been shown to be influenced by α₂-macroglubulin (α₂M), a serum protein with strong protease-scavenging and cytokine-binding properties.Aims: In the present study, α₂M derived from normal human plasma has been tested for its ability to modulate the TGFβ-induced collagen production by human liver fat-storing cells (FSC), which had transformed into α-smooth muscle actin-expressing myofibroblasts in culture.Methods: α₂M has been tested after activation with methylamine (α₂M-Me), an in vitro equivalent of protease activated α₂M. The binding of ¹²⁵I-TGFβ1 to activated forms of α₂M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of ³H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of α₂M was studied by means of immunofluorescence.Results: TGFβ (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFβ. α₂M-Me reduced this TGFβ-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 μg/ml α₂M-Me onward. Upon addition of 100 μg/ml α₂M-Me the effect of TGFβ was reduced by 60% to 128±31% (mean±SD) of control values in the absence of TGFβ. Human liver myofibroblasts endocytosed α₂M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFβ-activity by α₂M-Me may be explained by receptor-mediated clearance of α₂M-TGFβ complexes by the cells.Conclusions: TGFβ-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated α₂M. From these results, we hypothesize that α₂M may have an antifibrogenic effect in vivo by interference with TGFβ-induced matrix synthesis during liver fibrosis.     

Origin of myofibroblasts in the fibrotic liver in mice

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl₄) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP⁺ cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl₄ liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.Chronic liver injury of many etiologies results in liver fibrosis. There are two general types of chronic liver diseases, hepatocellular (injury to hepatocytes, such as chronic viral hepatitis and nonalcoholic steatohepatitis) and cholestatic (obstruction to bile flow, such as primary biliary cirrhosis and primary sclerosing cholangitis) (1). Experimental rodent models of liver fibrosis mimic these two types of chronic liver injuries: Repeated carbon tetrachloride (CCl₄) administration produces hepatocelluar injury, and common bile duct ligation (BDL) produces cholestatic injury (2). In all chronic liver diseases, myofibroblasts are embedded in the fibrous scar and are the source of this excessive extracellular matrix (ECM). Myofibroblasts, which are not present in normal liver, are characterized by distinct morphology, contractility with intracellular stress fibers [α-smooth muscle actin (α-SMA), nonmuscle myosin, and vimentin], and secretion of extracellular matrix (fibronectin and fibrillar collagens) (1, 2).The cells of origin of hepatic myofibroblasts are unresolved, and perhaps the fibrosis induced by different types of liver injury results from different fibrogenic cells. Hepatic myofibroblasts may originate from bone marrow (BM)-derived mesenchymal cells and fibrocytes, but only a small contribution of BM-derived cells to the myofibroblast population has been detected in experimental liver fibrosis (3–5). Another potential source of myofibroblast is epithelial-to-mesenchymal transition (EMT), in which epithelial cells acquire a mesenchymal phenotype and may give rise to fully differentiated myofibroblasts. However, recent cell fate mapping studies have failed to detect any hepatic myofibroblasts originating from hepatocytes, cholangiocytes, or epithelial progenitor cells (3, 6–10). Thus, the major sources of myofibroblasts in liver fibrosis are the endogenous liver mesenchymal cells, which consist of portal fibroblasts and hepatic stellate cells.Quiescent hepatic stellate cells (qHSCs) are located in the space of Disse, store retinoids in lipid droplets, and express neural markers, such as glial fibrillary acidic protein (GFAP), synaptophisin, and nerve growth factor receptor p75 (1). In response to injury, qHSCs down-regulate vitamin A-containing lipid droplets and neural markers, and differentiate into α-SMA–expressing myofibroblasts (1, 2). Portal fibroblasts normally comprise a small population of the fibroblastic cells that surround the portal vein to maintain integrity of portal tract. They were first described as “mesenchymal cells not related to sinusoids,” and since then have been called “periductular fibroblasts” or portal/periportal mesenchymal cells” (11) and implicated by association in the pathogenesis of cholestatic liver injury. In response to chronic injury, portal fibroblasts may proliferate, differentiate into α-SMA–expressing myofibroblasts, and synthesize extracellular matrix (11–14).The contribution of portal fibroblasts (PFs) to liver fibrosis of different etiologies is not well understood, mainly because of difficulties in isolating PFs and myofibroblasts. The most widely used method of PF isolation from rats is based on liver perfusion with enzymatic digestion followed by size selection (15). Cell outgrowth from dissected bile segments is still used to isolate mouse PFs, and after 10–14 d in culture, PFs undergo progressive myofibroblastic activation (16). The disadvantage of this technique is that it requires multiple passaging and prolong culturing (11). A more physiological method of PF culturing in a precision-cut liver slice is designed to maintain cell–cell and cell–matrix interactions and mimic natural microenvironment of PFs, but it does not enable the study of purified PFs (17). Therefore, only a few markers of PFs are available to identify PFs in the myofibroblast population, including gremlin, Thy1, fibulin 2, interleukin 6 (IL-6), elastin, the ecto-AT-Pase nucleoside triphosphate diphosphohydrolase-2 (NTPD2), and coffilin 1. In addition, the lack of desmin, cytoglobin, α2-macroglobulin, neural proteins (GFAP, p75, synaptophysin), and lipid droplets distinguishes PFs from HSCs (1, 17–21).Our study uses transgenic reporter mice and new flow cytometry protocols to identify the origin of myofibroblasts and quantify their numbers in two murine models of chronic liver injury (BDL and CCl₄). Our study demonstrates that the origin of the myofibroblasts is determined by the type of liver injury. As previously reported using other methodologies, HSCs are the major source of myofibroblasts in CCl₄ liver injury. In contrast, most of the myofibroblasts at the onset of BDL-induced liver injury originate from activated PFs (aPFs).     

The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver

BACKGROUND/AIMS: The development of the intrahepatic bile ducts most likely requires interactions between epithelial and mesenchymal cells. In view of the epithelial-mesenchymal interactions between portal myofibroblasts (pMFs) and biliary epithelial cells in adult diseases of the bile ducts, we investigated the presence and function of pMFs during the development of intrahepatic bile ducts, as well as the development of intrahepatic branches of the hepatic artery. METHODS: We performed haematoxylin-eosin-stainings and immunohistochemistry for alpha-smooth-muscle actin, cytokeratin 19 and vimentin on serial sections of 45 fetal and postnatal liver biopsies. RESULTS: The mesenchyme of portal tracts in the ductal plate stage devoid of a hepatic artery branch, contained numerous and diffusely scattered pMFs. Portal tracts with a hepatic artery branch were always larger than those without and showed a decreasing number of pMFs. In the remodeling stage, all portal tracts contained a hepatic artery branch, and pMFs were restricted to the periductal mesenchyme. These periductal pMFs disappeared after full incorporation of the bile duct. CONCLUSION: Our findings strongly suggest interactions between pMFs and epithelial cells of the developing bile ducts. The development of the intrahepatic arterial branches always precedes the incorporation of the tubular segments of the ductal plate.     

Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat

To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.     

肝内肌成纤维细胞和星状细胞在肝纤维化中的作用

肝纤维化是不同病因长期作用于肝脏所致损伤后修复反应,发病机制主要是肝内纤维生成,细胞活化、增殖,合成大量细胞外基质(extracellur matrix.ECM),并伴有ECM降解不足,最终导致其在肝内大量积聚.近来有研究提示除肝星状细胞(hepatic satellite cell,HSC)外,肌成纤维细胞(myofibroblast,MF)可能是另一类参与肝纤维化进程并发挥重要作用的细胞,进一步确证上述研究结果将助于针对不同类型肝纤维化寻找和采取不同的干预方法,以更有效地阻断肝纤维化的进展.     

Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta

The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor beta (TGF-beta)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-beta₁. MFB were obtained by subculture of FSC. The TGF-beta concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-beta was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-beta₁. The exclusive role of active TGF-beta in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-beta receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-beta. (Hepatology 1996 Mar;23(3):571-81)     

Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts

BACKGROUND/AIMS: Myofibroblasts are the primary cells responsible for increased matrix deposition in hepatic fibrosis. Activation of hepatic stellate cells and portal fibroblasts to myofibroblasts during cholestatic liver injury is accompanied by increased expression of the activation marker, alpha-smooth muscle actin (SMA), and collagen genes. In contrast to our understanding of injury, the cellular mechanisms of liver repair are not well defined. This study was designed to examine the morphological relationship between bile duct hyperplasia, matrix deposition and myofibroblast phenotype in a model of chronic cholestatic liver injury and repair. METHODS: Reversible extrahepatic obstruction was accomplished in rats using a soft vessel loop suspended from the anterior abdominal wall: duct manipulation alone was performed in sham-operated controls. After 7 days, rats were either sacrificed or decompressed by release of the loop and subsequently sacrificed 2-10 days after reversal. Liver sections were obtained for in situ hybridization for procollagen alpha1(I) mRNA, immunohistochemical staining for SMA and cytokeratin 19, and histochemical staining for reticulin. RESULTS: Cholestatic livers demonstrated bile duct hyperplasia, which reversed to normal within 10 days after decompression. Fibrosis was also substantially reduced during this period. SMA-positive myofibroblasts were abundant and localized to regions adjacent to proliferating ducts and excess matrix in the obstructed animals. Decompressed livers showed a dramatic time-dependent reduction in the number of SMA-positive cells and in the expression of procollagen I mRNA. CONCLUSIONS: Our results show that the disappearance of bile duct hyperplasia after biliary decompression is accompanied by a similarly rapid loss of SMA-positive myofibroblasts. Both cellular events may abrogate enhanced matrix synthesis and allow repair to occur.