Altered factor VII activating protease expression in murine hepatic fibrosis and its influence on hepatic stellate cells

Background: Platelet‐derived growth factor‐BB (PDGF‐BB) is a profibrotic factor in liver fibrosis through its ability to stimulate hepatic stellate cells (HSC). The liver‐derived serine protease factor VII activating protease (FSAP) regulates the activities of PDGF‐BB in a cell‐specific manner. Aims: Our aim was to determine the influence of FSAP on the activation of HSC and to analyse the regulation of FSAP in hepatic fibrogenesis. Methods: The effect of FSAP on PDGF‐stimulated p42/p44 mitogen‐activated protein kinase (MAPK) activation in primary rat HSC was determined by Western blotting. Migration and proliferation of HSC was evaluated in Boyden chamber experiments and ³H‐thymidine incorporation assays respectively. Expression of FSAP was analysed in a CCl₄ mouse model of liver fibrosis by Western blot, quantitative real‐time polymerase chain reaction and immunohistochemistry. Results: FSAP inhibited PDGF‐BB‐stimulated p42/p44 MAPK phosphorylation, proliferation and migration of HSC. FSAP mRNA expression level was increased 3 h after CCl₄ application and decreased after 18 h and, in established fibrosis, after chronic CCl₄ administration. In parallel, there was a decrease in the circulating FSAP protein in chronic fibrosis. Concurrently, the homogenous hepatic expression pattern of FSAP was disturbed. Immunohistochemistry revealed a decrease of FSAP in hepatocytes in inflammatory and fibrotic lesions. Conclusions: Our results demonstrate an inhibitory effect of FSAP on PDGF‐mediated activation of HSC. In addition, FSAP expression is transiently increased in acute‐phase reaction but decreased during chronic fibrogenesis, which in turn may influence PDGF‐BB availability and myofibroblast activity.     

Cell migration: a novel aspect of pancreatic stellate cell biology

BACKGROUND: Pancreatic stellate cells (PSCs), implicated as key mediators of pancreatic fibrogenesis, are found in increased numbers in areas of pancreatic injury. This increase in PSC number may be due to increased local proliferation and/or migration of these cells from adjacent areas. The ability of PSCs to proliferate has been well established but their potential for migration has not been examined. AIMS: Therefore, the aims of this study were to determine whether cultured rat PSCs have the capacity to migrate and, if so, to characterise this migratory capacity with respect to the influence of basement membrane components and the effect of platelet derived growth factor (PDGF, a known stimulant for migration of other cell types). METHODS: Migration of freshly isolated (quiescent) and culture activated (passaged) rat PSCs was assessed across uncoated or Matrigel (a basement membrane-like substance) coated porous membranes (pore size 8 micro m) in the presence or absence of PDGF (10 and 20 ng/ml) in the culture medium. A checkerboard assay was performed to assess whether the effect of PDGF on PSC migration was chemotactic or chemokinetic. RESULTS: Cell migration was observed with both freshly isolated and passaged PSCs. However, compared with passaged (culture activated) cells, migration of freshly isolated cells was delayed, occurring only at or after 48 hours of incubation when the cells displayed an activated phenotype. PSC migration through Matrigel coated membranes was delayed but not prevented by basement membrane components. PSC migration was increased by PDGF and this effect was predominantly chemotactic (that is, in the direction of a positive concentration gradient). CONCLUSIONS: (i) PSCs have the capacity to migrate. (ii) Activation of PSCs appears to be a prerequisite for migration. (iii) PDGF stimulates PSC migration and this effect is predominantly chemotactic. IMPLICATION: Chemotactic factors released during pancreatic injury may stimulate the migration of PSCs through surrounding basement membrane towards affected areas of the gland.     

法舒地尔通过Rho/ROCK信号通路抑制肝星状细胞黏附、迁移和增殖

目的观察Rho／Rho激酶（ROCK）信号转导通路抑制剂——法舒地尔，对肝星状细胞（1ISC）黏附、迁移和增殖的影响。方法将培养的HSC分为以下5组：对照组；法舒地尔12.5μmol／L组；法舒地尔25μmol／L组；法舒地尔50μmol／L组；法舒地尔100μmol／L组。采用甲苯胺蓝染色法测定细胞黏附率，Boyden Chamber小室测定HSC跨膜迁移数量，用四甲基偶氮唑盐法检测细胞增殖，Western blot检测HSC RhoA、p-MLC（Thr18／Ser19）和α-平滑肌肌动蛋白的表达。结果法舒地尔对HSC的黏附、迁移和增殖均具有抑制作用，随着浓度增加，抑制作用加强；法舒地尔抑制HSC的α-平滑肌肌动蛋白表达，并且对Rho／ROCK信号转导通路关键信号分子P-MLC（Thr18／Ser19）的蛋白表达有抑制作用。结论法舒地尔通过抑制Rho／ROCK信号通路细胞骨架的调节作用抑制1ISC黏附、迁移和增殖。     

Inhibition of migration and proliferation of rat vascular smooth muscle cells by a new HMG-CoA reductase inhibitor, pitavastatin.

The migration and proliferation of vascular smooth muscle cells (SMCs) are known to play roles in the pathogenesis of atherosclerosis. Therapy with a reductase inhibitor of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) ("statin") produces significant alterations in various SMC functions. The objectives of the present study were to determine whether pitavastatin, a new chemically synthesized and powerful statin, can affect angiotensin II (Ang II)- and platelet-derived growth factor (PDGF)-induced migration and proliferation of cultured rat vascular SMCs. The effect of pitavastatin on cell viability was also examined in these cells. Migration was evaluated by the Boyden's chamber method using microchemotaxis chambers. As expected, Ang II and PDGF BB potently stimulated cell migration in a concentration-dependent manner. Pitavastatin significantly inhibited Ang II (10(-6) mol/l)-induced migration at the concentrations of 10(-8) and 10(-7) mol/l. Pitavastatin also inhibited PDGF BB (1 ng/ml)-induced migration at concentrations between 10(-9) and 10(-8) mol/l in a relatively concentration-dependent manner. This statin modestly but significantly inhibited Ang II (10(-6) mol/l)- and PDGF BB (1 ng/ml)-induced DNA synthesis at concentrations between 10(-9) and 10(-7) mol/l. In addition, pitavastatin clearly inhibited Ang II (10(-6) mol/l)- and PDGF BB (1 ng/ml)-induced increases of cell number at concentrations between 10(-9) and 10(-7) mol/l. Pitavastatin did not affect lactate dehydrogenase release from these cells at the concentrations used in this experiment. In a trypan blue exclusion test, dead cells stained with trypan blue were not found 24 h after treatment with 10(-9), 10(-8) or 10(-7) mol/l of pitavastatin. These findings suggest that pitavastatin suppresses the migration and proliferation stimulated by Ang II and PDGF BB without affecting cell viability. Pitavastatin may exert an anti-atherogenic effect, in part, through these mechanisms.     

Proanthocyanidin derived from the leaves of Vaccinium virgatum suppresses platelet‐derived growth factor‐induced proliferation of the human hepatic stellate cell line LI90

Aim: Hepatic stellate cell (HSC) proliferation plays a pivotal role in liver fibrogenesis, and agents that suppress HSC activation, including platelet‐derived growth factor (PDGF)‐induced HSC proliferation, are good candidates for antifibrogenic therapies. In this report, we use the LI90 HSC line to elucidate the antifibrogenic effects of proanthocyanidin derived from the leaves of Vaccinium virgatum. Methods: Proanthocyanidin (PAC) was extracted from the leaves of blueberry V. virgatum (BB‐PAC), grape seeds (GS‐PAC) and Croton lechleri (CL‐PAC). These extracts were examined for their effects on PDGF‐BB‐induced LI90 cell proliferation and DNA synthesis. Extracellular signal‐regulated kinase (ERK) and Akt phosphorylation and PDGF receptor‐β (PDGFR‐β) expression were evaluated by western blot analysis. Results: BB‐PAC potently suppressed PDGF‐BB‐induced proliferation and DNA synthesis of LI90 cells. BB‐PAC also suppressed PDGF‐BB‐induced DNA synthesis in primary cultured rat HSC. Moreover, GS‐PAC and CL‐PAC suppressed PDGF‐BB‐induced DNA synthesis in LI90 cells. In contrast, the monomeric PAC catechin and epicatechin and dimeric PAC procyanidin B2 only slightly suppressed PDGF‐BB‐induced DNA synthesis. Western blot analysis showed that BB‐PAC completely or partially inhibited PDGF‐BB‐induced ERK and Akt phosphorylation, respectively. In addition, BB‐PAC partially inhibited the PDGF‐BB‐induced degradation of PDGFR‐β. Conclusion: Our results suggest that BB‐PAC suppresses activated HSC by inhibiting the PDGF signaling pathway. In addition, these results provide novel findings that may facilitate the development of antifibrogenic agents.     

Suppression by eicosapentaenoic acid of oxidized low-density lipoprotein and lysophosphatidylcholine-induced migration in cultured rat vascular smooth muscle cells

The migration of medial smooth muscle cells into the intima is proposed to be an initial process of intimal thickening in atherosclerotic lesions. The present study was designed to determine whether pretreatment with the antiatherogenic agent eicosapentaenoic acid (EPA) inhibits the migration induced by oxidized low-density lipoprotein (LDL) and its major phospholipid component, lysophosphatidylcholine (lyso-PC), in cultured rat vascular smooth muscle cells (VSMCs) using Boyden's chamber method. The effects of EPA pretreatment on angiotensin II (Ang II)- and platelet-derived growth factor BB (PDGF BB)-induced migration were also examined in these cells. Oxidized LDL and lyso-PC induced migration in a concentration-dependent manner. EPA pretreatment clearly suppressed oxidized LDL (200 microg/mL)- and lyso-PC (10(-5) mol/L)-induced migration between 40 and 160 micromol/L. EPA pretreatment also suppressed Ang 11 (10(-7) mol/L)- and PDGF BB (5 ng/mL)-induced migration at a concentration of 80 and 160 micromol/L. However, in a trypan blue exclusion test, dead cells stained with trypan blue were not found 24 hours after treatment with EPA. These results suggest that EPA suppresses VSMC migration induced by oxidized LDL and lyso-PC, as well as Ang II and PDGF BB. These preliminary data concerning the effects of EPA may partly explain the antiatherosclerotic effects of this agent.     

Cellular retinol-binding protein-1 expression in normal and fibrotic/cirrhotic human liver: different patterns of expression in hepatic stellate cells and (myo)fibroblast subpopulations

BACKGROUND/AIMS: Cellular retinol-binding protein-1 (CRBP-1) which is involved in vitamin A metabolism is highly expressed in liver cells, particularly in hepatic stellate cells (HSCs). In this work, the CRBP-1 expression was studied by immunohistochemistry in the different liver cell populations, including HSCs and portal fibroblasts, of normal liver and of fibrotic and cirrhotic liver. METHODS: Normal liver, fibrotic liver in different stages and cirrhotic liver sections were studied. Immunohistochemistry was performed using antibodies against CRBP-1, alpha-smooth muscle actin (SMA), CD 68 and CD 34. RESULTS: In normal liver, quiescent HSCs expressed CRBP-1, while portal fibroblasts did not. In fibrotic or cirrhotic liver, activated HSCs co-expressed CRBP-1 and alpha-SMA; a variable proportion of portal and septal (myo)fibroblasts, more important in cirrhosis, neo-expressed both CRBP-1 and alpha-SMA. Biliary epithelial cells both in normal and pathological situations expressed CRBP-1. Neither Kupffer cells, nor endothelial cells showed CRBP-1 expression. CONCLUSIONS: Our study demonstrates that CRBP-1 is a good marker to identify HSC in normal human liver. Furthermore, in fibrotic or cirrhotic liver, the different patterns of expression for CRBP-1 and alpha-SMA allow the distinction of different subsets of fibroblastic cells involved in fibrogenesis and septa formation.     

抗血小板衍生生长因子受体的核酶对肝星状细胞生物学特性的影响

目的 研究抗血小板衍生生长因子受体β亚单位(PDGFR-β)核酶在肝星状细胞(HSC)内的切割活性及其对HSC生物学特性的影响。 方法 构建抗PDGFR-β核酶的真核表达载体,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆;分别用northern blot、western blot和免疫细胞化学检测PDGFR-β表达,用MTT法检测细胞增殖,免疫细胞化学检测α-F滑肌肌动蛋白(α-sMA)和Ⅰ、Ⅲ型胶原表达,用流式细胞仪、吖啶噔荧光染色和电镜分析细胞凋亡。 结果 转染核酶的HSC的PDGFR-β在mRNA和蛋白水平的表达量均显著降低,仅为对照组的43％～51％(t≥3.95 7,P<0.05);增殖活性显著低于对照组(t≥3.858,P<0.0 5),且对血小板衍生生长因子(PDGF)促增殖效应的敏感性显著减弱;Ⅰ、Ⅲ型胶原和α-SMA的表达显著减少(t≥6.790,P<0.01);凋亡发生率显著高于对照组(x2≥14.157,P<0.01),电镜下可见典型凋亡细胞。 结论 抗PDGFR-β核酶的真核表达载体可在细胞内稳定表达,能有效切割靶RNA,抑制HSC增殖及胶原合成,并诱导其凋亡。为抗肝纤维化治疗提供了新的靶点和手段。     

白细胞介素10对肝星状细胞激活的调节

目的 探讨白细胞介素10(IL-10)通过血小板衍生生长因子(PDGF)和丝裂原活化激酶(MAPK)信号通路蛋白对肝星状细胞(HSC)激活的影响。 方法 将培养的HSC随机分为4组:1组:对照组;2组:加入1 ng/ml IL-10;3组:加入5 ng/ml IL-10;4组:加入25 ng/ml IL-10。培养2d后,逆转录-聚合酶链反应法检测各组细胞中PDGF mRNA的表达;western blot法检测各组细胞PDGF、MAPK信号通路蛋白细胞外信号调节激酶(ERK)和p38以及α-平滑肌肌动蛋白(α-SMA)的表达。 结果 1、5、25 ng/ml的IL-10作用后,与对照组相比HSC的ERK、p38以及α-SMA的表达显著降低(F值分别为240.47、21.39、28.86,P值均<0.01),并呈量效依赖关系;5、25 ng/ml的IL-10可以使PDGF表达显著降低(P值均<0.01),并呈量效依赖关系。 结论 IL-10可通过PDGF/MAPK信号通路抑制肝星状细胞激活。     

Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.     

PDGF-BB对肝星状细胞表达MMP-2及TIMP-1的影响

目的：探讨血小板衍生生长因子－BB（PDGF－BB）对体外培养大鼠肝星状细胞（HSC）表达基质金属蛋白酶－2（MM－2）及金属蛋白酶组织抑制因子－1（TIMP－1）的影响。方法：原位灌流分离HSC，体外培养激活，用PDGF－BB干预，采用半定RT－PCR方法检测各组MMP－2、TIMP－1 mRNA的表达情况。结果：PDGF－BB干预组TIMP－1 mRNA的表达较对照组明显增强（P＜0．01），且随着干预时间的延长其表达逐渐增强（P＜0．01）；而PDGF－BB干预组MMP－2表达与对照组无明显差异（P＞0．05）。结论：PDGF－BB促进肝纤维化与其引起HSC表达TIMP－1增强有关。     

Dose dependent and divergent effects of superoxide anion on cell death, proliferation, and migration of activated human hepatic stellate cells

BACKGROUND AND AIMS: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration. METHODS: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. CONCLUSIONS: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.     

Potential role of chitotriosidase gene in nonalcoholic fatty liver disease evolution

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by steatosis and periportal and lobular inflammation. The molecular mechanisms involved in the anomalous behavior of liver cells have only partially been disclosed. Human Chitotriosidase (Chit) is a member of the chitinase family that it is mainly synthesized by activated macrophages. We investigated chitotriosidase gene expression in Kupffer cells to determine the potential implication of this enzyme in the inflammation and in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS: Seventy-five liver biopsies from 40 subjects with NASH, 20 with simple steatosis, and 15 controls were used to detect CHIT expression, tumor necrosis factor-alpha (TNF-alpha), alpha-smooth muscle actin (alpha-SMA), and lipid peroxidation. RESULTS: CHIT was expressed exclusively by Kupffer cells. The levels of CHIT expression were significantly higher in NASH patients than in simple steatosis patients and in the control group. In addition, we found that CHIT over-expression influenced hepatic stellate cells activation, as demonstrated by the significant correlation between CHIT and alpha-SMA expression in NASH patients. A significant correlation was observed also between CHIT, TNF-alpha and lipid peroxidation in both NASH and simple steatosis. CONCLUSION: These results suggest that CHIT over-produced by Kupffer cells may contribute to the progression of hepatic fibrosis.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

Review: Regulation of liver regeneration by pro-inflammatory cytokines

The liver has tremendous regenerative capacity. This distinguishes it from other vital organs (e.g. the brain, heart and lungs) that cannot replace functional tissue once it has been destroyed. Although hepatocytes rarely proliferate in the healthy adult liver, virtually all surviving hepatocytes replicate at least once after 70% partial hepatectomy. Therefore, partial liver resection has been used to characterize mechanisms that regulate liver regeneration. Residual hepatocytes up-regulate both proliferative and liver-specific gene expression in order to preserve tissue specific function. In addition, hepatocyte proliferation is tightly co-ordinated to complement regenerative responses in hepatic nonparenchymal cells (e.g. endothelia, biliary epithelia, stellate and Kupffer cells), so that the entire organ can be reconstituted within days. Studies with neutralizing antibodies to tumour necrosis factor-α (TNF) clearly demonstrate that, after partial hepatectomy, TNF promotes liver cell proliferation. The present review focuses on the regulation of the hepatocyte proliferative response by pro-inflammatory cytokines.     

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells

AIM: To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21waf1, and p27kip1 expression and phosphorylation of PDGF β-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. RESULTS: EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin Dl and increased p27kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF p-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/ Akt pathways. CONCLUSION: EGCG inhibited PDGFBB-induced proliferation and migration of PSCs through the inhibition of PDGFmediated signaling pathways.