Role of fibroblast growth factor type 1 and 2 in carbon tetrachloride-induced hepatic injury and fibrogenesis

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis.     

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.     

Oxidative stress effect on the activation of hepatic stellate cells

Collagen is the most excessive extracellular matrix protein in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (HSCs) have a high level of collagen and a smooth muscle actin (alpha SMA) expression. HSCs play a key role in the pathogenesis of hepatic fibrosis. We analyzed a mechanism leading to HSC activation by evaluating the role of oxidative stress and the expression of NFkB. In vitro study HSCs were proliferated (PCNA:2% vs 68%) and activated (alpha SMA: 5% vs 78%) by ascorbate/FeSO4, and HSCs activated by type I collagen were blocked (PCNA: 97% vs 4%, a SMA: 86% vs 9%) by a-tocopherol. In vivo study means of a SMA positive cells in liver at 400 x HPF were 48.3+/-5.2 and 15.2+/-1.8 and [3H]thymidine uptake of HSC was 529.2+/-284.8 cpm and 223.0+/-86.3 cpm in control and a-tocopherol treated group respectively at 32 hours after CCl4 injection. Nuclear extracts from activated, but not from quiescent, HSCs formed a complex with the NFkB cognate oligonucleotidesand alpha-tocopherol inhibited this bindings. This study indicates that oxidative stress plays an essential role through the induction of NFkB on HSC activation.     

Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells

Collagen degradation by matrix metalloproteinases is the limiting step in reversing liver fibrosis. Although collagen production in cirrhotic livers is increased, the expression and/or activity of matrix metalloproteinases could be normal, increased in early fibrosis, or decreased during advanced liver cirrhosis. Hepatic stellate cells are the main producers of collagens and matrix metalloproteinases in the liver. Therefore, we sought to investigate whether they simultaneously produce alpha1(I) collagen and matrix metalloproteinase-13 mRNAs. In this communication we show that expression of matrix metalloproteinase-13 mRNA is reciprocally modulated by tumor necrosis factor-alpha and transforming growth factor-beta1. When hepatic stellate cells are co-cultured with hepatocytes, matrix metalloproteinase-13 mRNA is up-regulated and alpha1(I) collagen is down-regulated. Injuring hepatocytes with galactosamine further increased matrix metalloproteinase-13 mRNA production. Confocal microscopy and differential centrifugation of co-cultured cells revealed that matrix metalloproteinase-13 is localized mainly within hepatic stellate cells. Studies performed with various hepatic stellate cell lines revealed that they are heterogeneous regarding expression of matrix metalloproteinase-13. Those with myofibroblastic phenotypes produce more type I collagen whereas those resembling freshly isolated hepatic stellate cells express matrix metalloproteinase-13. Overall, these findings strongly support the notion that alpha1(I) collagen and matrix metalloproteinase-13 mRNAs are reciprocally modulated.     

Discoidin domain receptor 1 is a major mediator of inflammation and fibrosis in obstructive nephropathy

The interactions between tubulointerstitial infiltrating cells and the extracellular matrix play an important role in regulating renal fibrosis. Discoidin domain receptor 1 (DDR1) is a nonintegrin tyrosine kinase receptor for collagen implicated in cell adhesion, proliferation, and extracellular matrix remodeling. We have previously demonstrated that transgenic mice lacking DDR1 are protected from hypertension-associated renal fibrosis. The purpose of this study was to determine the role of DDR1 in renal inflammation and fibrosis related to primitive tubulointerstitial injury. After 12 days of unilateral ureteral obstruction (UUO), kidney histopathologic and real-time quantitative PCR analyses were performed in DDR1(-/-) and wild-type mice. DDR1 expression was strongly increased in the obstructed kidney. Wild-type mice developed important perivascular and interstitial inflammation and fibrosis. In comparison, DDR1(-/-) mice displayed reduced accumulation of fibrillar collagen and transforming growth factor β expression. F4/80(+) cell count and proinflammatory cytokines were remarkably blunted in DDR1(-/-) obstructed kidneys. Leukocyte rolling and adhesion evaluated by intravital microscopy were not different between DDR1(-/-) and wild-type mice. Importantly, macrophages isolated from DDR1(-/-) mice presented similar M1/M2 polarization but displayed impaired migration in response to monocyte chemoattractant protein-1. Together, these data suggest that DDR1 plays an important role in the pathogenesis of renal disease via enhanced inflammation. Inhibition of DDR1 expression or activity may represent a novel therapeutic target against the progression of renal diseases.     

Effect of cholinergic denervation on hepatic fibrosis induced by carbon tetrachloride in rats

Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.     

Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis.

In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis. To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by light microscopy. Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs. However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.     

瘦素及I和III型胶原蛋白在肝纤维化大鼠肝组织中的变化

 摘要： 目的 研究瘦素（Leptin）及I、 III型胶原蛋白及基因在肝纤维化模型组织中的动态表达水平。方法 四氯化碳（CCl4）皮下注射法制备肝纤维化模型,分别以Western blot及RT-PCR法检测Leptin及I、 III型胶原蛋白及基因在肝纤维化组织中的动态表达。结果 Leptin以及I、 III型胶原蛋白及基因在正常对照组肝脏中均有微量表达,CCl4注射2周后,三者的表达均开始增强,随着纤维化发展呈梯度增加。其mRNA表达水平在模型组明显高于正常组 (P<0.05);在肝纤维化过程中,Leptin与I型胶原(r=0.595,P=0.017)及Leptin与III型胶原(r=0.478,P=0.011) 的动态改变呈显著正相关。结论 Leptin的表达随着纤维化的程度加重而逐步增强,在肝纤维化过程中,Leptin可能参与了细胞外基质成分（ECM）的合成与降解。     

Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity

Liver cirrhosis is caused by a relative imbalance between synthesis and degradation of collagens. Arg-Gly-Asp (RGD) peptide is a major adhesive domain of several extracellular matrix (ECM) components, such as that involved in the binding of fibronectin to the alpha5beta1 integrin receptor. We previously reported that RGD peptide increased the expression of matrix metalloproteinase in hepatic stellate cells (HSCs) which play a major role in hepatic fibrosis. We evaluated whether RGD-peptides inhibit the progression of liver fibrosis in an animal model of carbon tetrachloride-induced hepatotoxicity. RGD peptide (GRGDS) (1 mg/kg body weight) was injected intraperitoneally (i.p.) 3 times a week for one month. The group treated with control peptide (GRGES) showed pathologically typical hepatic fibrosis, while the RGD-treated group showed minimal fibrotic changes. The liver contents of collagen and hydroxyproline in the RGD-treated group was significantly lower than that of the control group. Collagenase activity measured in liver homogenates was significantly higher in the treated group than in the control group. In an in vitro study using TWNT-4 cells derived from human HSCs, RGD peptide (100 mug/ml) reduced the expression of type I collagen and tissue inhibitor of matrix metalloproteinase-1, and increased that of matrix metalloproteinase-1. These results indicated that RGD peptides inhibited liver fibrosis associated with both decreased collagen production and increased collagenase acitivity, and suggested that RGD peptide might be useful for the therapy of hepatic fibrosis.     

肝再生过程中肝星状细胞及基质金属蛋白酶的动态变化

目的研究大鼠肝大部切除(PH)后再生过程中肝星状细胞(HSCs)的动态变化及肝MMP-2、MMP-9的活性变化,探讨肝星状细胞在肝再生中的作用。方法按Higgins等方法制备肝大部切除动物模型,于术后恢复不同时程取材,免疫组织化学法检测肝HSCs的动态变化,明胶酶谱电泳法检测肝中MMP-2、MMP-9的变化。结果(1)正常肝小叶内HSCs呈网架状分布,PH后Desmin阳性HSCs的数量递减;胶质纤维酸性蛋白(GFAP)阳性HSCs的数量也呈递减趋势。(2)PH后再生过程中,肝脏MMP-2、MMP-9的表达逐渐增多。结论肝再生过程中HSCs的增殖反应较慢且维持的时间短,这不同于肝纤维化等肝病过程中HSCs维持较长时间的激活状态。肝再生过程中基质金属蛋白酶-2,-9的表达发生显著改变,提示基质金属蛋白酶-2,-9参与了肝再生过程。     

Increased iron deposition in rat liver fibrosis induced by a high-dose injection of dimethylnitrosamine

Using a developed rat model of hepatic necrosis and subsequent fibrosis induced by a high-dose intraperitoneal injection of dimethylnitrosamine (DMN), we studied iron deposition and expression of transforming growth factor-beta(1) (TGF-beta(1)) during the development of persistent liver fibrosis. Rats were sacrificed at several timepoints from 6 h to 10 months post-injection and the livers were examined for iron content and distribution, and for expression of alpha-smooth muscle actin, ED-1, TGF-beta(1), and collagen (alpha(2))I. Morphologic evidence of acute submassive hemorrhagic necrosis peaked at 36 h; on day 3 the residual parenchyma contained activated hepatic stellate cells (HSCs) and necrotic areas contained numerous macrophages; and on day 5, necrotic tissues and erythrocytes had been phagocytosed and macrophages contained abundant iron deposits. From days 7 to 10, iron-laden macrophages and activated HSCs (myofibroblasts) populated the fibrous septa in parallel. From week 2 to month 10, closely arranged macrophages and myofibroblasts were found in central-to-central bridging fibrotic tissue. TGF-beta(1) was strongly detected in both macrophages and HSCs during development of liver fibrosis. Our data suggest that increased iron deposition may be involved in the initiation and perpetuation of rat liver fibrosis. Iron-laden macrophages may influence HSCs through the action of TGF-beta(1) in DMN-induced liver fibrosis.     

Liver precursor cells increase hepatic fibrosis induced by chronic carbon tetrachloride intoxication in rats

Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl(4)-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.     

CCl4肝纤维化过程中肝组织金属硫蛋白与肝星状细胞MMP-2表达及活性之间相关性研究

目的： 研究金属硫蛋白（MT）在体内及体外与基质金属蛋白酶-2(MMP-2)表达、活性之间的相关性。方法: 昆明种雄性成年小鼠50只,其中实验鼠 40只,对照鼠10只,用CCl4诱导小鼠肝纤维化模型。向肝星状细胞（HSCs）条件培养基（含有MMP-2）中加不同浓度MT,使之与MMP-2酶学共浴,检测MMP-2活性。制作小鼠肝组织芯片,天狼猩红染色判断肝纤维化程度。免疫组化法测肝组织MMP-2、MT蛋白表达,酶图法测鼠肝组织匀浆和培养基中MMP-2活性。结果: ①模型组小鼠肝组织MMP-2和MT蛋白表达水平均随纤维化进展而呈波浪式交替升高,但二者时相变化相反。 ②模型组小鼠肝组织MMP-2活性随实验时间的增加而逐渐升高,亦有波动。除个别时点外,酶活性与MT表达呈负相关。③肝星状细胞条件培养基与不同浓度的MT直接作用后,MMP-2活性随MT浓度增加活性逐渐降低(P＜0.01)。结论: 在小鼠肝纤维化发生发展过程中,肝组织MT蛋白和MMP-2蛋白表达均有上升趋势,二者间可能存在相互作用;MT蛋白和MMP-2活性呈负相关,前者对后者可能起抑制作用。     

Dual Role of CCR2 in the Constitution and the Resolution of Liver Fibrosis in Mice

Inflammation has been shown to induce the progression of fibrosis in response to liver injury. Among inflammatory cells, macrophages and lymphocytes play major roles in both the constitution and resolution of liver fibrosis. The chemokine receptor CCR2 is involved in the recruitment of monocytes to injury sites, and it is known to be induced during the progression of fibrosis in humans. However, its specific role during this process has not yet been unveiled. We first demonstrated that, compared with wild-type mice, CCR2 knockout animals presented a delay in liver injury after acute CCl₄ injection, accompanied by a reduction in infiltrating macrophage populations. We then induced fibrosis using repeated injections of CCl₄ and observed a significantly lower level of fibrotic scars at the peak of fibrosis in mutant animals compared with control mice. This diminished fibrosis was associated with a reduction in F4/80⁺CD11b⁺ and CD11c⁺ populations at the sites of injury. Subsequent analysis of the kinetics of the resolution of fibrosis showed that fibrosis rapidly regressed in wild-type, but not in CCR2^−/− mice. The persistence of hepatic injury in mutant animals was correlated with sustained tissue inhibitor of metalloproteinase-1 mRNA expression levels and a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-13 expression levels. In conclusion, these findings underline the role of the CCR2 signaling pathway in both the constitution and resolution of liver fibrotic scars.Liver fibrosis and its progression toward cirrhosis is the consequence of chronic liver aggressions provoked by various agents such as viruses, bile acids, or alcohol. It is characterized by an accumulation of extracellular matrix mainly secreted by activated myofibroblasts and hepatic stellate cells (HSCs).^1,2 Although originally considered as an irreversible process, cirrhosis has now been shown to be potentially reversible, provided that the causative agent is removed.³ Current evidence suggests that the process of hepatic fibrosis is driven primarily by the development of an inflammatory reaction in response to parenchymal injury. In agreement with this hypothesis, deletion of specific components of the inflammatory response modifies or attenuates the fibrotic scar in vivo. It has been demonstrated that Kupffer cell inactivation,⁴ B lymphocyte deficiency,⁵ or macrophage depletion⁶ result in lower scarring and reduced activation of HSCs.Molecular mechanisms responsible for reversibility of liver fibrosis are only now starting to be deciphered. Experimental depletion of macrophages at the onset of fibrosis resolution retards extracellular matrix degradation and the loss of activated hepatic stellate cells.⁶ Therefore, macrophages seem to be crucial in both processes: fibrogenesis and fibrosis resolution.Chemokines are known to be critical mediators of inflammatory cell trafficking into sites of injury. Monocyte chemoattractant proteins (MCPs) attract cells through activation of their cognate receptor. MCP-1 is expressed by hepatocytes, endothelial cells, Kupffer cells, and HSCs in response to injury. CC chemokine receptor 2 (CCR2), which is expressed on monocytes/macrophages and on a subpopulation of T lymphocytes, has been found progressively up-regulated during the progression of liver fibrosis in humans.⁷ Moreover, comparison of serial sections of liver biopsies from patients with various degrees of necroinflammatory activity showed that infiltration with monocytes/macrophages is directly correlated with the expression of MCP-1,⁸ for which CCR2 is the main receptor.We hypothesized that signaling through CCR2 is involved in inflammatory cell recruitment during fibrogenesis and/or regression of fibrosis. To test this hypothesis, we compared the establishment and resolution of fibrosis after chronic liver aggression in CCR2-deficient mice versus wild-type mice. Our data show that the absence of CCR2 has opposite consequences during the constitution and the resolution of fibrosis.     

CXCL10 promotes liver fibrosis by prevention of NK cell mediated hepatic stellate cell inactivation

Chemokines, such as CXCL10, promote hepatic inflammation in chronic or acute liver injury through recruitment of leukocytes to the liver parenchyma. The CXCL10 receptor CXCR3, which is expressed on a subset of leukocytes, plays an important part in Th1-dependent inflammatory responses. Here, we investigated the role of CXCL10 in chemically induced liver fibrosis. We used carbon tetrachloride (CCl(4)) to trigger chronic liver damage in wildtype C57BL/6 and CXCL10-deficient mice. Fibrosis severity was assessed by Sirius Red staining and intrahepatic leukocyte subsets were investigated by immunohistochemistry. We have further analyzed hepatic stellate cell (HSC) distribution and activation and investigated the effect of CXCL10 on HSC motility and proliferation. In order to demonstrate a possible therapeutic intervention strategy, we have examined the anti-fibrotic potential of a neutralizing anti-CXCL10 antibody. Upon CCl(4) administration, CXCL10-deficient mice showed massively reduced liver fibrosis, when compared to wildtype mice. CXCL10-deficient mice had less B- and T lymphocyte and dendritic cell infiltrations within the liver and the number and activity of HSCs was reduced. In contrast, natural killer (NK) cells were more abundant in CXCL10-deficient mice and granzyme B expression was increased in areas with high numbers of NK cells. Further detailed analysis revealed that HSCs express CXCR3, respond to CXCL10 and secrete CXCL10 when stimulated with IFNγ. Blockade of CXCL10 with a neutralizing antibody exhibited a significant anti-fibrotic effect. Our data suggest that CXCL10 is a pro-fibrotic factor, which participates in a crosstalk between hepatocytes, HSCs and immune cells. NK cells seem to play an important role in controlling HSC activity and fibrosis. CXCL10 blockade may constitute a possible therapeutic intervention for hepatic fibrosis.     

Plasminogen Deficiency Leads to Impaired Lobular Reorganization and Matrix Accumulation after Chronic Liver Injury

To determine the regulatory role of plasminogen in hepatic repair following a chronic liver injury, we injected carbon tetrachloride (CCl(4)) biweekly into mice lacking plasminogen (Plg(0)) and plasminogen-sufficient littermates (Plg(+)). On gross examination, we found that Plg(0) livers became enlarged and pale with foci of red nodules as early as 4 weeks after CCl(4) injection, while Plg(+) livers appeared minimally affected by 6 weeks. Microscopically, Plg(0) livers had a pronounced pericentral linking, with accumulation of centrilobular eosinophilic material in injured areas, which resulted in a significant increase in liver mass and total protein. Immunohistochemistry revealed that fibrin accumulated progressively in injured regions. However, the combined genetic loss of plasminogen and fibrinogen did not correct the abnormal phenotype. Mason's trichrome staining revealed intense signal in centrilobular regions and electron microscopy showed a marked increase in fibrillary material demonstrating an excessive accumulation of extracellular matrix in Plg(0) mice. The zone-specific increase in matrix components was associated with an increase in the number of activated hepatic stellate cells within injured sites of Plg(0) livers. Taken together, these data suggest that the progressive accumulation of fibrin-unrelated matrix substrates in Plg(0) livers after a chronic injury results from the combined effects of impaired proteolysis and increased matrix production.     

脐带源间充质干细胞移植治疗肝纤维化及肝硬化的相关机制

背景：肝硬化是多种原因引起的肝脏慢性病变,目前尚没有有效的治疗方法,很多研究表明,间充质干细胞对肝纤维化及肝硬化有一定的治疗作用。 目的：研究人脐带源间充质干细胞移植对大鼠肝纤维化及肝硬化的治疗作用及其作用机制。 方法：应用CCl4诱导制备肝纤维化及肝硬化模型,造模后经尾静脉注射人脐带间充质干细胞。细胞移植后采用Beckman Coulter analyzer检测人脐带源间充质干细胞移植对大鼠肝功能的影响;采用天狼猩红染色检测肝组织病理改变;应用免疫组织化学染色、Western blot和real-time Q-PCR方法检测Ⅰ、Ⅲ型胶原、基质金属蛋白酶2、基质金属蛋白酶抑制剂2蛋白与mRNA在大鼠肝组织中的表达。 结果与结论：人脐带源间充质干细胞移植可以改善肝纤维化及肝硬化大鼠的肝功能。人脐带源间充质干细胞移植后,除肝纤维化细胞移植1周组与对应模型组相比差异无显著性意义外,其余各细胞移植组肝脏组织中基质金属蛋白酶2 mRNA及蛋白表达水平明显升高,而Ⅰ、Ⅲ型胶原、基质金属蛋白酶抑制剂2表达水平明显降低。人脐带源间充质干细胞通过上调基质金属蛋白酶2表达,下调基质金属蛋白酶抑制剂2表达,对肝纤维化及肝硬化起到治疗作用;在致病因素持续存在的情况下,人脐带源间充质干细胞移植并不能逆转肝纤维化或者肝硬化,只能延缓肝纤维化或肝硬化的进程。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程