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Stellate Cells (stellate + cell)
Kinds of Stellate Cells Terms modified by Stellate Cells Selected AbstractsAdipogenic Phenotype of Hepatic Stellate CellsALCOHOLISM, Issue 2005Hide Tsukamoto Abstract: Transdifferentiation of hepatic stellate cells (HSC) constitutes a major cellular event in the genesis of alcoholic liver fibrosis and cirrhosis and molecular mechanisms underlying this process is incompletely understood. Our laboratory proposed several years ago that HSC quiescence requires the transcriptional program known to be integral to preadipocyte to adipocyte differentiation. In support of the hypothesis, our research demonstrates the expression of adipogenic transcription factors (C/EBPs, PPAR,, SREBP-1c, LXR,) and adipocyte-specific genes (adipsin, resistin) are high in quiescent HSC and depleted in activated HSC. Three gain-of-function approaches have been taken to test this notion: the treatment of activated HSC with the adipocyte differentiation cocktail; ectopic expression of PPAR, or SREBP-1c. All three treatments coordinately upregulate a panel of putative adipogenic transcrition factors and cause morphologic and biochemical reversal of activated HSC to quiescent cells. These findings establish a new conceptual framework for the treatment of liver fibrosis and propose an intriguing notion concerning the plasticity of HSC. [source] Distinct Mechanism of Small-for-Size Fatty Liver Graft Injury,Wnt4 Signaling Activates Hepatic Stellate CellsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2010Q. Cheng In this study, we aimed to investigate the significance of hepatic stellate cells (HSCs) activation in small-for-size fatty liver graft injury and to explore the underlying molecular mechanism in a rat liver transplantation model. A rat orthotopic liver transplantation model using fatty grafts (40% of fatty changes) and cirrhotic recipients was applied. Intragraft gene expression profiles, ultrastructure features and HSCs activation were compared among the rats received different types of grafts (whole vs. small-for-size, normal vs. fatty). The distinct molecular signature of small-for-size fatty graft injury was identified by cDNA microarray screening and confirmed by RT-PCR detection. In vitro functional studies were further conducted to investigate the direct effect of specific molecular signature on HSCs activation. HSCs activation was predominantly present in small-for-size fatty grafts during the first 2 weeks after transplantation, and was strongly correlated with progressive hepatic sinusoidal damage and significant upregulation of intragraft Wnt4 signaling pathway. In vitro suppression of Wnt4 expression could inhibit HSC activation directly. In conclusion, upregulation of Wnt4 signaling led to direct HSC activation and subsequently induced small-for-size fatty liver grafts injury. Discovery of this distinct mechanism may lay the foundation for prophylactic treatment for marginal graft injury in living donor liver transplantation. [source] In vivo and in vitro Interactions between Human Colon Carcinoma Cells and Hepatic Stellate CellsCANCER SCIENCE, Issue 12 2000Sadatoshi Shimizu Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM-H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed ,-smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM-H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture-induced activation contained platelet-derived growth factor (PDGF)-AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF),, and could augment LM-H3-cell proliferation and migration. Neutralizing antibodies against PDGF-AA and PDGF-BB and those against PDGF-BB and HGF inhibited proliferation and migration, respectively, of LM-H3 cells, whereas antibody against TGF-, had no effect. LM-H3 cells expressed PDGF receptors-, and -, and c-met. Conditioned medium of LM-H3 cells contained PDGF-AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti-PDGF-AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis. [source] Melatonin reduces dimethylnitrosamine-induced liver fibrosis in ratsJOURNAL OF PINEAL RESEARCH, Issue 2 2004Veysel Tahan Abstract:, Increased deposition of the extracellular matrix components, particularly collagen, is a central phenomenon in liver fibrosis. Stellate cells, the central mediators in the pathogenesis of fibrosis are activated by free radicals, and synthesize collagen. Melatonin is a potent physiological scavenger of hydroxyl radicals. Melatonin has also been shown to be involved in the inhibitory regulation of collagen content in tissues. At present, no effective treatment of liver fibrosis is available for clinical use. We aimed to test the effects of melatonin on dimethylnitrosamine (DMN)-induced liver damage in rats. Wistar albino rats were injected with DMN intraperitoneally. Following a single dose of 40 mg/kg DMN, either saline (DMN) or 100 mg/kg daily melatonin was administered for 14 days. In other rats, physiologic saline or melatonin were injected for 14 days, following a single injection of saline as control. Hepatic fibrotic changes were evaluated biochemically by measuring tissue hydroxyproline levels and histopathogical examination. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione (GSH) and superoxide dismutase (SOD) levels were evaluated in blood and tissue homogenates. DMN caused hepatic fibrotic changes, whereas melatonin suppressed these changes in five of 14 rats (P < 0.05). DMN administration resulted in increased hydroxyproline and MDA levels, and decreased GSH and SOD levels, whereas melatonin reversed these effects. When melatonin was administered alone, no significant changes in biochemical parameters were noted. In conclusion, the present study suggests that melatonin functions as a potent fibrosuppressant and antioxidant, and may be a therapeutic choice. [source] Morphological mechanisms for regulating blood flow through hepatic sinusoidsLIVER INTERNATIONAL, Issue 1 2000Robert S. McCuskey Abstract: This review summarizes what is known about the various morphological sites that regulate the distribution of blood flow to and from the sinusoids in the hepatic microvascular system. These sites potentially include the various segments of the afferent portal venules and hepatic arterioles, the sinusoids themselves, and central and hepatic venules. Given the paucity of smooth muscle in the walls of these vessels, various sinusoidal lining cells have been suggested to play a role in regulating the diameters of sinusoids and influencing the distribution and velocity of blood flow in these vessels. While sinusoidal endothelial cells have been demonstrated to be contractile and to exhibit sphincter function, attention has recently focused on the perisinusoidal stellate cell as the cell responsible for controlling the sinusoidal diameter. A very recent study, however, suggested that the principal site of vasoconstriction elicited by ET-1 was the pre-terminal portal venule. This raised the question of whether or not the diameters of sinusoids might decrease due to passive recoil when inflow is reduced or eliminated and intra-sinusoidal pressure falls. In more recent in vivo microscopic studies, clamping of the portal vein dramatically reduced sinusoidal blood flow as well as the diameters of sinusoids. The sinusoidal lumens rapidly returned to their initial diameters upon restoration of portal blood flow suggesting that sinusoidal blood pressure normally distends the sinusoidal wall which can recoil when the pressure drops. Stellate cells may be responsible for this reaction given the nature of their attachment to parenchymal cells by obliquely oriented microprojections from the lateral edges of their subendothelial processes. This suggests that care must be exercised when interpreting the mechanism for the reduction of sinusoidal diameters following drug administration without knowledge of changes occurring to the portal venous and hepatic inflow. [source] Vitamin A distribution and content in tissues of the lamprey, Lampetra japonicaTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2004Heidi L. Wold Abstract Vitamin A (retinol and retinyl ester) distribution and content in tissues of a lamprey (Lampetra japonica) were analyzed by morphological methods, namely, gold chloride staining, fluorescence microscopy to detect specific vitamin A autofluorescence, and electron microscopy, as well as high-performance liquid chromatography (HPLC). Hepatic stellate cells showed an abundance of vitamin A stored in lipid droplets in their cytoplasm. Similar cells storing vitamin A were present in the intestine, kidney, gill, and heart in both female and male lampreys. Morphological data obtained by gold chloride staining method, fluorescence microscopy, transmission electron microscopy, and HPLC quantification of retinol were consistent. The highest level of total retinol measured by HPLC was found in the intestine. The second and third highest concentrations of vitamin A were found in the liver and the kidney, respectively. These vitamin A-storing cells were not epithelial cells, but mesoderm-derived cells. We propose as a hypothesis that these cells belong to the stellate cell system (family) that stores vitamin A and regulates homeostasis of the vitamin in the whole body in the lamprey. Fibroblastic cells in the skin and somatic muscle stored little vitamin A. These results indicate that there is difference in the vitamin A-storing capacity between the splanchnic and intermediate mesoderm-derived cells (stellate cells) and somatic and dorsal mesoderm-derived cells (fibroblasts) in the lamprey. Stellate cells derived from the splanchnic and intermediate mesoderm have high capacity and fibroblasts derived from the somatic and dorsal mesoderm have low capacity for the storage of vitamin A in the lamprey. Anat Rec Part A 276A:134,142, 2004. © 2004 Wiley-Liss, Inc. [source] Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis,HEPATOLOGY, Issue 1 2009Karlin Raja Karlmark In addition to liver-resident Kupffer cells, infiltrating immune cells have recently been linked to the development of liver fibrosis. Blood monocytes are circulating precursors of tissue macrophages and can be divided into two functionally distinct subpopulations in mice: Gr1hi (Ly6Chi) and Gr1lo (Ly6Clo) monocytes. The role of these monocyte subsets in hepatic fibrosis and the mechanisms of their differential recruitment into the injured liver are unknown. We therefore characterized subpopulations of infiltrating monocytes in acute and chronic carbon tetrachloride (CCl4)-induced liver injury in mice using flow cytometry and immunohistochemistry. Inflammatory Gr1hi but not Gr1lo monocytes are massively recruited into the liver upon toxic injury constituting an up to 10-fold increase in CD11b+F4/80+ intrahepatic macrophages. Comparing wild-type with C-C chemokine receptor (CCR2)-deficient and CCR2/CCR6,deficient mice revealed that CCR2 critically controls intrahepatic Gr1hi monocyte accumulation by mediating their egress from bone marrow. During chronic liver damage, intrahepatic CD11b+F4/80+Gr1+ monocyte-derived cells differentiate preferentially into inducible nitric oxide synthase,producing macrophages exerting proinflammatory and profibrogenic actions, such as promoting hepatic stellate cell (HSC) activation, T helper 1,T cell differentiation and transforming growth factor , (TGF-,) release. Impaired monocyte subset recruitment in Ccr2,/, and Ccr2,/,Ccr6,/, mice results in reduced HSC activation and diminished liver fibrosis. Moreover, adoptively transferred Gr1hi monocytes traffic into the injured liver and promote fibrosis progression in wild-type and Ccr2,/,Ccr6,/, mice, which are otherwise protected from hepatic fibrosis. Intrahepatic CD11b+F4/80+Gr1+ monocyte-derived macrophages purified from CCl4 -treated animals, but not naïve bone marrow monocytes or control lymphocytes, directly activate HSCs in a TGF-,,dependent manner in vitro. Conclusion: Inflammatory Gr1+ monocytes, recruited into the injured liver via CCR2-dependent bone marrow egress, promote the progression of liver fibrosis. Thus, they may represent an interesting novel target for antifibrotic strategies. (HEPATOLOGY 2009;50:261,274.) [source] Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouseHEPATOLOGY, Issue 4 2002Hitoshi Yoshiji It has been suggested that the tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in spontaneous resolution of liver fibrosis. The aim of this study was to investigate whether TIMP-1 altered spontaneous resolution of liver fibrosis in conjunction with matrix metalloproteinases (MMP) inhibition and hepatic stellate cell (HSC) activation. The livers of liver-targeted TIMP-1 transgenic (TIMP-Tg) and control hybrid (Cont) mice were harvested at 0, 3, 7, and 28 days following spontaneous recovery from CCl4 -induced liver fibrosis. The extent of fibrosis resolution, MMP expression, ,-smooth-muscle actin (,-SMA) positive cells, and procollagen-(I) messenger RNA (mRNA) in the liver were assessed at the respective periods in both groups. We also examined the effect of TIMP-1 on HSC apoptosis. The TIMP-Tg mice showed significantly attenuated resolution of spontaneous liver fibrosis compared with the Cont mice. The hydroxyproline content, number of ,-SMA positive cells, and procollagen-(I) mRNA rapidly decreased with time in the Cont mice, whereas these markers were little changed in TIMP-Tg mice. The level of the active form of metalloproteinases-2 (MMP-2) in the TIMP-Tg mice was less than that in the Cont mice. TIMP-1 markedly decreased the nonparenchyma apoptotic cells in the liver fibrosis resolution model, and it also inhibited HSC apoptosis associated with suppression of caspase-3 activity in vitro. In conclusion, TIMP-1 significantly attenuated spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC. [source] Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse modelHEPATOLOGY, Issue 6 2000Hitoshi Yoshiji Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/enhancer. A model of CCl4 -induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(,1)-collagen-I, (,2)-collagen-IV, and ,-smooth muscle actin (,-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl4, however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl4 -induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl4 -treated TIMP-Tg-mice with a pattern similar to that of ,-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development. [source] Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitisJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 12 2008Haruko Sugino Abstract Background and Aim:, The effect of polaprezinc, a zinc-carnosine chelate compound, on the development of non-alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. Methods:, Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. Results:, MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor-,, transforming growth factor-,1 and procollagen ,1(I). The mRNA expression levels of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro-inflammatory cytokines. Polaprezinc affected the MCD diet-enhanced expression of TIMP-1 even when administered relatively late. Conclusion:, These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH. [source] Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehydeJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2001Silvia C Quiroz Abstract Background and Aim: The role of endotoxin in alcohol-induced liver damage is well recognized. How pre-exposure to endotoxin might affect alcohol injury is not known. We herein studied the effect of endotoxin pretreatment on hepatic stellate cell (HSC) response to ethanol and acetaldehyde. Methods: Rat HSC (CFSC-2G) were exposed to media supplemented with 1 ,g/mL lipopolysaccharide (LPS). This was followed by a 24 h exposure to media containing LPS plus 50 mmol/L ethanol or 175 ,mol/L acetaldehyde. Lipid peroxidation, collagen, and tumor necrosis factor (TNF)-,, interleukin (IL)-1,, IL-6 and transforming growth factor (TGF)-,1 secretion were determined at the end of both periods of exposure. Results: Lipopolysaccharide pretreatment did not modify lipid peroxidation induced by ethanol or acetaldehyde alone. Glutathione (GSH) content decreased to 4.2 ± 0.5 and 16.3 ± 0.8 nmol protein after exposure to ethanol or acetaldehyde alone, and decreased further with LPS pretreatment (2.4 ± 0.2 and 2.7 ± 0.3 nmol/mg protein, respectively). Oxidized GSH (GSSG) content increased in ethanol and acetaldehyde LPS-pretreated cells only. Collagen secretion increased to 988 ± 82 and 1169 ± 91 ,g/106 cells after exposure to acetaldehyde or LPS alone. Lipopolysaccharide pretreatment enhanced collagen secretion significantly in both ethanol- and acetaldehyde-treated cells (969 ± 56 and 1360 ± 72 ,g/106 cells, respectively). Interleukin-6 production increased to 288 ± 48, 1195 ± 86 and 247 ± 35 pg/mL per 106 cells after ethanol, acetaldehyde and LPS exposure, and increased further with LPS pretreatment in ethanol-exposed cells (680 ± 23 pg/mL 106 cells). Conclusion: Lipopolysaccharide pretreatment of HSC adds to the damage produced by ethanol and acetaldehyde by diminishing GSH content and increasing GSSG content, collagen and IL-6 secretion. [source] Systematic review: hepatic fibrosis , regression with therapyALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 10 2008C. D. ZOIS Summary Background, Hepatic fibrosis occurs in response to chronic liver injury, regardless of the cause. An impressive amount of knowledge concerning the pathogenesis and treatment of liver fibrosis has emerged over the past few years. The hallmark of this event is the activation of the hepatic stellate cell. The latter event causes accumulation of extracellular matrix and formation of scar, leading to deterioration in hepatic function. Aim, To assess chronic liver injury, many invasive and non-invasive methods have been suggested. Methods, Although transient elastography, image analysis of fractal geometry and fibrotest with actitest have been used in clinical practice, liver biopsy remains the recommended choice, especially when histological staging of fibrosis or response to treatment is needed. Conclusions, The recent advances in anti-viral therapy have resulted in many reports on fibrosis and even on cirrhosis regression, especially early and in young people. A number of new agents have been suggested for the treatment of fibrosis, with promising results in animals; however, their efficacy in humans remains to be elucidated. The investigation of heterogeneity and plasticity of hepatic stellate cells is a topic of scientific interest and may result in improvements in patient management. [source] Morphological mechanisms for regulating blood flow through hepatic sinusoidsLIVER INTERNATIONAL, Issue 1 2000Robert S. McCuskey Abstract: This review summarizes what is known about the various morphological sites that regulate the distribution of blood flow to and from the sinusoids in the hepatic microvascular system. These sites potentially include the various segments of the afferent portal venules and hepatic arterioles, the sinusoids themselves, and central and hepatic venules. Given the paucity of smooth muscle in the walls of these vessels, various sinusoidal lining cells have been suggested to play a role in regulating the diameters of sinusoids and influencing the distribution and velocity of blood flow in these vessels. While sinusoidal endothelial cells have been demonstrated to be contractile and to exhibit sphincter function, attention has recently focused on the perisinusoidal stellate cell as the cell responsible for controlling the sinusoidal diameter. A very recent study, however, suggested that the principal site of vasoconstriction elicited by ET-1 was the pre-terminal portal venule. This raised the question of whether or not the diameters of sinusoids might decrease due to passive recoil when inflow is reduced or eliminated and intra-sinusoidal pressure falls. In more recent in vivo microscopic studies, clamping of the portal vein dramatically reduced sinusoidal blood flow as well as the diameters of sinusoids. The sinusoidal lumens rapidly returned to their initial diameters upon restoration of portal blood flow suggesting that sinusoidal blood pressure normally distends the sinusoidal wall which can recoil when the pressure drops. Stellate cells may be responsible for this reaction given the nature of their attachment to parenchymal cells by obliquely oriented microprojections from the lateral edges of their subendothelial processes. This suggests that care must be exercised when interpreting the mechanism for the reduction of sinusoidal diameters following drug administration without knowledge of changes occurring to the portal venous and hepatic inflow. [source] Inhibitory effects of antisense oligonucleotides on the expression of procollagen type III gene in mouse hepatic stellate cells transformed by simian virus 40PATHOLOGY INTERNATIONAL, Issue 12 2000Satoshi Horie The effects of phosphorothioate antisense oligonucleotides (ASO), complementary to the AUG start region, the junctional region of the intron and exon, and to exon of the procollagen type III gene, were investigated in a mouse hepatic stellate cell (HSC) line transformed by the simian virus 40 gene, SV68c-IS cells. ASO were transfected by lipofection. Immunohistochemistry, western and northern blotting showed inhibitory effects on procollagen type III gene expression by ASO that were complementary to the AUG start region and the junctional region of the intron and exon 2. However, ASO complementary to the exon 2 and 3, junctional region of the intron and exon 3, and sense oligonucleotides complementary to each ASO did not show any inhibitory effects. The effects of ASO complementary to the AUG start region were greater than those of ASO complementary to the junctional region. The effects of ASO were transient and a large amount of ASO was required to induce inhibitory effects without lipofection. ASO were effective in inhibiting the expression of the procollagen type III gene in the HSC which is well known to play a critical role in liver fibrosis. [source] Origin and Endpoint of the Olfactory Nerve Fibers: As Described by Santiago Ramón y Cajal,THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 7 2008Catherine Levine Illustration by author Catherine Levine inspired by the original drawing by Santiago Ramón y Cajal, featured in the article Origin and endpoint of the olfactory nerve fibers: As described by Santiago Ramón y Cajal. Depicted are large tufted cells and granule cells, a large stellate cell, a row of mitral cells and the arborization on the olfactory glomeruli, with olfactory nerve fibers streaming through the cartilage formation of the cribriform plate. See Levine et al., Anatomical Record 291:741,750. [source] Mechanical stretch induces TGF-, synthesis in hepatic stellate cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2004R. Sakata Abstract Background, It is known that mechanical stress induces extracellular matrix via transforming growth factor-, (TGF-,) synthesis in vascular smooth muscle cells. Activated hepatic stellate cells (HSCs) are an important source of TGF-, in the liver. However, it remains unclear whether mechanical stress induces TGF-, in HSCs. The Rho small GTP-binding protein (Rho) has recently emerged as an important regulator of actin and cytoskeleton. We examined whether TGF-, is expressed in stretched HSCs and whether Rho is involved in stretch-induced TGF-, synthesis. Materials and methods, A cultured human HSC cell line, LI90, was used for this study. Hepatic stellate cells were cyclically stretched using the Flexercell® strain unit. Concentration of TGF-, in the conditioned medium was estimated by a bioassay using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Transforming growth factor-, mRNA expression of HSCs was estimated by a reverse-transcription polymerase chain reaction. Replication-defective adenoviral vectors expressing a dominant negative type of Rho was utilized to suppress its effect on HSCs. Results, Transforming growth factor-, concentration of the conditioned media of stretched HSCs showed time-dependent increases as compared to nonstretched HSCs from 2 h to 24 h. Transforming growth factor-, mRNA expression in stretched HSCs was increased compared with that in nonstretched HSCs. Transfection of dominant negative Rho inhibited the stretch-induced TGF-, synthesis. Conclusions, Mechanical stretch enhanced TGF-, expression on mRNA and protein level in HSCs. Rho was closely related to stretch-induced TGF-, synthesis in HSCs. [source] Dendritic cells: Understanding immunogenicityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue S1 2007Ralph Abstract The impetus for the discovery of dendritic cells in 1972 was to understand immunogenicity, the capacity of an antigenic substance to provoke immunity. During experiments to characterize "accessory" cells that enhanced immunity, we spotted unusual stellate cells in mouse spleen. They had a distinct capacity to form and retract processes or dendrites and were named dendritic cells (DC). DC proved to be different from other cell types and to be peculiarly immunogenic when loaded with antigens. When Langerhans cells were studied, immunogenicity was found to involve two steps: antigen presentation by immature DC and maturation to elicit immunity. Antigen-bearing DC were also immunogenic in vivo and were therefore termed "nature's adjuvants". Several labs then learned to generate large numbers of DC from progenitors, which accelerated DC research. Tolerogenicity via DC, including the control of foxp3+ suppressor T cells, was recently discovered. Two areas of current research that I find intriguing are to identify mechanisms for antigen uptake and processing, and for the control of different types of immunity and tolerance. These subjects should be studied in vivo with clinically relevant antigens, so that the activities of DC can be better integrated into the prevention and treatment of disease in patients. [source] Growth of ameloblast-lineage cells in a three-dimensional Matrigel environmentEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Wu Li Enamel organ epithelial cells grow in culture as two distinct cell populations , either stellate-shaped or polygonal-shaped cells. The polygonal cells have an ameloblast cell phenotype and are difficult to grow in culture beyond two passages. This study was designed to determine the effects of a Matrigel three-dimensional (3D) environment on polygonal cells, as compared with stellate cells, derived from porcine tooth enamel organ. Enamel organs were dissected free from the unerupted molars of 30-kg pigs and then grown in LCH-8e media, either with or without serum. Cells grown in serum-free media were primarily polygonal shaped, whereas cells grown in media containing serum were stellate shaped. Both types of cells were grown in a 3D Matrigel matrix. In addition, polygonal-shaped cells were mixed with hydroxyapatite powder and transplanted subcutaneously into nude mice. Polygonal-shaped epithelial cells formed cell groups, similar to epithelial pearls, both in vitro and in vivo. The stellate-shaped cells, in contrast, did not form similar structures, but remained suspended in the Matrigel and gradually disappeared from the culture. These results suggest that a Matrigel environment, rich in basement membrane and matrix proteins, selects for polygonal-shaped ameloblast-lineage cells and induces the formation of epithelial pearls. [source] CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice,HEPATOLOGY, Issue 4 2010Tomonori Aoyama Chronic liver disease is associated with hepatocyte injury, inflammation, and fibrosis. Chemokines and chemokine receptors are key factors for the migration of inflammatory cells such as macrophages and noninflammatory cells such as hepatic stellate cells (HSCs). The expression of CX3CR1 and its ligand, CX3CL1, is up-regulated in chronic liver diseases such as chronic hepatitis C. However, the precise role of CX3CR1 in the liver is still unclear. Here we investigated the role of the CX3CL1-CX3CR1 interaction in a carbon tetrachloride (CCl4),induced liver inflammation and fibrosis model. CX3CR1 was dominantly expressed in Kupffer cells in the liver. In contrast, the main source of CX3CL1 was HSCs. Mice deficient in CX3CR1 showed significant increases in inflammatory cell recruitment and cytokine production [including tumor necrosis factor , (TNF-,); monocyte chemoattractant protein 1; macrophage inflammatory protein 1,; and regulated upon activation, normal T cell expressed, and secreted (RANTES)] after CCl4 treatment versus wild-type (WT) mice. This suggested that CX3CR1 signaling prevented liver inflammation. Kupffer cells in CX3CR1-deficient mice after CCl4 treatment showed increased expression of TNF-, and transforming growth factor , and reduced expression of the anti-inflammatory markers interleukin-10 (IL-10) and arginase-1. Coculture experiments showed that HSCs experienced significantly greater activation by Kupffer cells from CCl4 -treated CX3CR1-deficient mice versus WT mice. Indeed, augmented fibrosis was observed in CX3CR1-deficient mice versus WT mice after CCl4 treatment. Finally, CX3CL1 treatment induced the expression of IL-10 and arginase-1 in WT cultured Kupffer cells through CX3CR1, which in turn suppressed HSC activation. Conclusion: The CX3CL1-CX3CR1 interaction inhibits inflammatory properties in Kupffer cells/macrophages and results in decreased liver inflammation and fibrosis. (Hepatology 2010) [source] Tumor necrosis factor,like weak inducer of apoptosis is a mitogen for liver progenitor cells,,HEPATOLOGY, Issue 1 2010Janina E. E. Tirnitz-Parker Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor,like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin,positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NF,B) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NF,B subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NF,B-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration. (HEPATOLOGY 2010) [source] CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis,HEPATOLOGY, Issue 4 2010Mirko Moreno Zaldivar Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4,/, and wild-type mice were subjected to two models of chronic liver injury (CCl4 and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus,induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl4 and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf -, [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4,/, mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8+ T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. Conclusion: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies. (HEPATOLOGY 2010.) [source] CCR2 promotes hepatic fibrosis in mice,HEPATOLOGY, Issue 1 2009Ekihiro Seki Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)-1, MCP-2, and MCP-3, in hepatic fibrosis. Hepatic CCR2, MCP-1, MCP-2, and MCP-3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL- and CCl4 -induced fibrosis was markedly reduced in CCR2,/, mice as assessed through collagen deposition, ,-smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild-type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2,/, BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2,/, or its downstream mediator p47phox,/, did not display extracellular signal-regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP-1, MCP-2, and MCP-3. Conclusion: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis. (HEPATOLOGY 2009.) [source] The interleukin-17 pathway is involved in human alcoholic liver disease,,HEPATOLOGY, Issue 2 2009Arnaud Lemmers Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin-17 (IL-17) is known to enhance neutrophil recruitment. We studied the IL-17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL-17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme-linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL-17 staining and co-staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL-17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL-17 by chemotaxis assays. IL-17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL-17, and their CD4+ T lymphocytes disclosed an IL-17,secreting phenotype. In the liver, IL-17,secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL-17+ cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL-17+ cells infiltrates correlated to model for end-stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL-17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL-17 stimulation in a dose-dependent manner through IL-8 and growth related oncogen , (GRO-,) secretion in vitro. Conclusion: Human alcoholic liver disease is characterized by the activation of the IL-17 pathway. In alcoholic hepatitis, liver infiltration with IL-17,secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597). (HEPATOLOGY 2009;49:646,657.) [source] Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis,HEPATOLOGY, Issue 3 2008Nidal Muhanna Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. Conclusion: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis. (HEPATOLOGY 2008.) [source] Multidrug resistance,associated proteins are crucial for the viability of activated rat hepatic stellate cells,,HEPATOLOGY, Issue 2 2008Rebekka A. Hannivoort Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance,associated protein (Mrp)-type and multidrug resistance protein (Mdr),type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl4),induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl4 -treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. Conclusion: Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases. (HEPATOLOGY 2008.) [source] Angiotensin II type 1 receptor blocker inhibits fibrosis in rat nonalcoholic steatohepatitis,HEPATOLOGY, Issue 6 2007Akira Hirose Nonalcoholic steatohepatitis (NASH) is now the most frequent cause of chronic liver impairment in developed countries and is a suggested causative factor in the development of cryptogenic cirrhosis and hepatocellular carcinoma. At present there is no effective and accepted therapy for NASH. The renin-angiotensin system is involved in hepatic fibrosis through activation of hepatic stellate cells, major fibrogenic cells in the liver. Hepatic stellate cells are activated by liver injury to express excessive matrix proteins and profibrogenic cytokines such as transforming growth factor,beta 1. Medicines that inhibit this pathway may be of therapeutic potential in NASH. Using a methionine-choline,deficient rat model of NASH, we studied the potential utility of an angiotensin II type 1 receptor blocker (ARB), olmesartan, on biochemical, histologic, and antioxidant measures of disease activity. ARB significantly attenuated increases in aspartate aminotransferase, activation of hepatic stellate cells, oxidative stress, expression of transforming growth factor,beta 1, expression of collagen genes, and liver fibrosis. Conclusion: Our observations strongly suggest a potential preventive role for ARB in the progression of nonalcoholic steatohepatitis. (HEPATOLOGY 2007.) [source] Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver,HEPATOLOGY, Issue 6 2005Yosuke Osawa Tumor necrosis factor (TNF) receptor, and Fas-mediated apoptosis are major death processes of hepatocytes in liver disease. Although antiapoptotic effects in the injured liver promote chronic hepatitis and carcinogenesis, scant information is known about these mechanisms. To explore this issue, we compared acute liver injury after TNF-, or anti-Fas antibody (Jo2) between livers from sham-operated mice and chronic injured liver via bile duct ligation (BDL). BDL inhibited hepatocyte apoptosis induced by TNF-, but not by Jo2. On the other hand, BDL inhibited the massive hemorrhage seen in livers treated with either TNF-, or Jo2. Inactivation of AKT blocked the antiapoptotic effect of BDL. Sphingosine kinase knockout mice also lost the antihemorrhagic effect of BDL and attenuated the antiapoptotic effects of BDL. In bile duct,ligated livers, hepatic stellate cells (HSCs) were activated and produced tissue inhibitor of metalloproteinase 1 in a sphingosine kinase (SphK)-1,dependent mechanism. In conclusion, BDL exerts antiapoptotic effects that appear to require activation of AKT in hepatocytes and SphK in HSCs.(HEPATOLOGY 2005;42:1320,1328.) [source] Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line,HEPATOLOGY, Issue 4 2005Xin Maggie Wang Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces,stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-,1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:935,945.) [source] Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct,ligated rats,HEPATOLOGY, Issue 5 2005Ramón Bataller Recent evidence indicates that the renin,angiotensin system (RAS) plays a major role in liver fibrosis. Here, we investigate whether the circulatory RAS, which is frequently activated in patients with chronic liver disease, contributes to fibrosis progression. To test this hypothesis, we increased circulatory angiotensin II (Ang II) levels in rats undergoing biliary fibrosis. Saline or Ang II (25 ng/kg/h) were infused into bile duct,ligated rats for 2 weeks through a subcutaneous pump. Ang II infusion increased serum levels of Ang II and augmented bile duct ligation,induced liver injury, as assessed by elevated liver serum enzymes. Moreover, it increased the hepatic concentration of inflammatory proteins (tumor necrosis factor , and interleukin 1,) and the infiltration of CD43-positive inflammatory cells. Ang II infusion also favored the development of vascular thrombosis and increased the procoagulant activity of tissue factor in the liver. Livers from bile duct,ligated rats infused with Ang II showed increased transforming growth factor ,1 content, collagen deposition, accumulation of smooth muscle ,-actin,positive cells, and lipid peroxidation products. Moreover, Ang II infusion stimulated phosphorylation of c-Jun and p42/44 mitogen-activated protein kinase and increased proliferation of bile duct cells. In cultured rat hepatic stellate cells (HSCs), Ang II (10,8 mol/L) increased intracellular calcium and stimulated reactive oxygen species formation, cellular proliferation and secretion of proinflammatory cytokines. Moreover, Ang II stimulated the procoagulant activity of HSCs, a newly described biological function for these cells. In conclusion, increased systemic Ang II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. (HEPATOLOGY 2005;41:1046,1055.) [source] A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populationsHEPATOLOGY, Issue 5 2004Scott T. Magness Activation of hepatic stellate cells (HSCs) and other resident mesenchymal cells into myofibroblasts expressing alpha smooth muscle actin (,SMA) and collagen I is a key event in liver fibrogenesis. However, the temporal expression profiles of ,SMA and collagen I genes in these cells is unknown. To address this question, we studied ,SMA and collagen ,1(I) transcriptional patterns in primary cultures of HSCs, and additionally, in an in vivo model of secondary biliary fibrosis using transgenic mice that express the Discomsoma sp. red fluorescent protein (RFP) and the enhanced green fluorescent protein (EGFP) reporter genes under direction of the mouse ,SMA and collagen ,1(I) promoter/enhancers, respectively. The ,SMA-RFP mice were crossed with collagen-EGFP mice to generate double transgenic mice. Reporter gene expression in cultured HSCs demonstrated that both transgenes were induced at day 3 with continued expression through day 14. Interestingly, ,SMA and collagen ,1(I) transgenes were not coexpressed in all cells. Flow cytometry analysis showed three different patterns of gene expression: ,SMA-RFP positive cells, collagen-EGFP positive cells, and cells expressing both transgenes. ,SMA-only and ,SMA/collagen expressing cells showed higher expression levels of synaptophysin, reelin, MMP13, TIMP1, and ICAM-1 compared to collagen-only expressing cells, as assessed by real-time PCR. Following bile duct ligation, ,SMA and collagen ,1(I) transgenes were differentially expressed by peribiliary, parenchymal and vascular fibrogenic cells. Peribiliary cells preferentially expressed collagen ,1(I), while parenchymal myofibroblasts expressed both ,SMA and collagen ,1(I). In conclusion, these data demonstrate heterogeneity of gene expression in myofibroblastic cells during active fibrogenesis. These reporter mice provide a useful tool to further characterize fibrogenic cell types and to evaluate antifibrotic drugs. (HEPATOLOGY 2004.) [source] | |||||||||||||||||||||||||||||||||||||