Leptin enhances alpha1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.     

DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells

Dilinoleoylphosphatidylcholine (DLPC), the active component of polyenylphosphatidylcholine extracted from soybeans, decreases collagen accumulation induced by TGF-beta1 in cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant effects and TGF-beta1 generates oxidative stress, we evaluated whether the antifibrogenic effect of DLPC is linked to its antioxidant action. In passage 1 culture of rat HSCs, TGF-beta1 induced a concentration-dependent increase in procollagen-alpha(1)(I) mRNA levels and enhanced intracellular H(2)O(2) and superoxide anion formation and lipid peroxidation but decreased GSH levels. These changes were prevented by DLPC. Upregulation of collagen mRNA by TGF-beta1 was likewise inhibited by catalase and p38 MAPK inhibitor SB-203580, suggesting involvement of H(2)O(2) and p38 MAPK signaling in this process. TGF-beta1 or addition of H(2)O(2) to HSCs activated p38 MAPK with a rise in procollagen mRNA level; these changes were blocked by catalase and SB-203580 and likewise by DLPC. alpha-Smooth muscle actin abundance in HSCs was not altered by TGF-beta1 treatment (with or without DLPC), indicating that downregulation of procollagen mRNA by DLPC was not due to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-beta1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated H(2)O(2)-dependent p38 MAPK activation, which explains its antifibrogenic effect. DLPC, an innocuous phospholipid, may be considered for prevention and treatment of liver fibrosis.     

Activation of the human alpha1(I) collagen promoter by leptin is not mediated by transforming growth factor beta responsive elements

Leptin increases human alpha1 (I) collagen mRNA and type I collagen production and enhances hepatic fibrosis in animal models of hepatic fibrosis. These effects of leptin on fibrogenesis may be mediated by TGFbeta1, since leptin increases the TGFbeta type II receptor and augments the effect of TGFbeta1 on collagen production by stellate cells. In this study, leptin increased the activity of the human alpha1 (I) collagen promoter in transfected stellate cells. Leptin did not further enhance the activation of the promoter induced by TGFbeta1. Leptin had no effects on the transfected TGFbeta-responsive p3TP-LUX plasmid, which contains 3 CAGA elements that are essential and sufficient for the induction by TGFbeta. Leptin did not increase significantly the binding of proteins to two TGFbeta1 responsive elements in the human alpha1 (I) collagen promoter. In conclusion, this study shows that leptin activates the alpha1 (I) collagen gene and that this effect is not mediated by TGFbeta responsive elements.     

A novel synthetic oleanolic acid derivative (CPU-II<Subscript>2</Subscript>) attenuates liver fibrosis in mice through regulating the function of hepatic stellate cells

Regulation on the function of the hepatic stellate cells (HSCs) is one of the proposed therapeutic approaches to liver fibrosis. In the present study, we examined the in vitro and in vivo effects of CPU-II₂, a novel synthetic oleanolic acid (OLA) derivative with nitrate, on hepatic fibrosis. This compound alleviated CCl₄-induced hepatic fibrosis in mice with a decrease in hepatic hydroxyproline (Hyp) content and histological changes. CPU-II₂ also attenuated the mRNA expression of α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) induced by CCl₄ in mice and reduced both mRNA and protein levels of α-SMA in HSC-T6 cells. Interestingly, CPU-II₂ did not affect the survival of HSC-T6 cells but decreased the expression of procollagen-α1 (I) in HSC-T6 cells through down-regulating the phosphorylation of p38 MAPK. Conclusion: CPU-II₂ attenuates the development of liver fibrosis rather by regulating the function of HSCs through p38 MAPK pathway than by damaging the stellate cells.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways

Leptin is recognized as a profibrogenic hormone in the liver, but the mechanisms involved have not been clarified. The tissue inhibitor of metalloproteinase (TIMP)-1, which acts through inhibition of collagen degradation, is synthesized by activated hepatic stellate cells (HSC) in response to fibrogenic substances. The capacity of leptin to induce TIMP-1 and its signaling molecules were investigated in a human HSC cell line, LX-2. Leptin stimulated TIMP-1 protein, mRNA, and promoter activity. JAK1 and -2, as well as STAT3 and -5, were activated. After leptin, there was increased expression of tyrosine 1141-phosphorylated leptin receptor, which may contribute to STAT3 activation. AG 490, a JAK inhibitor, blocked JAK phosphorylation with concomitant inhibition of STAT activation, TIMP-1 mRNA expression, and promoter activity. Leptin also induced an oxidative stress, which was inhibited by AG 490, indicating a JAK mediation process. ERK1/2 MAPK and p38 were activated, which was prevented by catalase, indicating an H2O2-dependent mechanism. Catalase treatment resulted in total suppression of TIMP-1 mRNA expression and promoter activity. SB203580, a p38 inhibitor, prevented p38 activation and reduced TIMP-1 message half-life with down-regulation of TIMP-1 mRNA. These changes were reproduced by overexpression of the dominant negative p38alpha and p38beta mutants. PD098059, an ERK1/2 inhibitor, opposed ERK1/2 activation and TIMP-1 promoter activity, leading to TIMP-1 mRNA down-regulation. Thus, leptin has a direct action on liver fibrogenesis by stimulating TIMP-1 production in activated HSC. This process appears to be mediated by the JAK/STAT pathway via the leptin receptor long form and the H2O2-dependent p38 and ERK1/2 pathways via activated JAK.     

Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-beta and MAPK

Accumulation of extracellular matrix (ECM) is a hallmark feature of vascular disease. We have previously shown that hyperglycemia induces the expression of B(2)-kinin receptors in vascular smooth muscle cells (VSMC) and that bradykinin (BK) and hyperglycemia synergize to stimulate ECM production. The present study examined the cellular mechanisms through which BK contributes to VSMC fibrosis. VSMC treated with BK (10(-8) M) for 24 h significantly increased alpha(2)(I) collagen mRNA levels. In addition, BK produced a two- to threefold increase in alpha(2)(I) collagen promoter activity in VSMC transfected with a plasmid containing the alpha(2)(I) collagen promoter. Furthermore, treatment of VSMC with BK for 24 h produced a two- to threefold increase in the secretion rate of tissue inhibitor of metalloproteinase 1 (TIMP-1). The increase in alpha(2)(I) collagen mRNA levels and alpha(2)(I) collagen promoter activity, as well as TIMP-1 secretion, in response to BK were blocked by anti-transforming growth factor-beta (anti-TGF-beta) neutralizing antibodies. BK (10(-8) M) increased the endogenous production of TGF-beta1 mRNA and protein levels. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 inhibited the increase of alpha(2)(I) collagen promoter activity, TIMP-1 production, and TGF-beta1 protein levels observed in response to BK. These findings provide the first evidence that BK induces collagen type I and TIMP-1 production via autocrine activation of TGF-beta1 and implicate MAPK pathway as a key player in VSMC fibrosis in response of BK.     

Hypoxia induces the activation of human hepatic stellate cells LX-2 through TGF-beta signaling pathway

Hypoxia is a common environmental stress factor and is also associated with various physiological and pathological conditions such as fibrogenesis. The activation of hepatic stellate cells (HSCs) is the key event in the liver fibrogenesis. In this study, the behavior of human HSCs LX-2 in low oxygen tension (1% O2) was analyzed. Upon hypoxia, the expression of HIF-1alpha and VEGF gene was induced. The result of Western blotting showed that the expression of alpha-SMA was increased by hypoxic stimulation. Furthermore, the expression of MMP-2 and TIMP-1 genes was increased. Hypoxia also elevated the protein expression of the collagen type I in LX-2 cells. The analysis of TGF-beta/Smad signaling pathway showed that hypoxia potentiated the expression of TGF-beta1 and the phosphorylation status of Smad2. Gene expression profiles of LX-2 cells induced by hypoxia were obtained by using cDNA microarray technique.     

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

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

MFAP2 promotes HSCs activation through FBN1/TGF-β/Smad3 pathway

Liver fibrosis is a chronic inflammatory process characterized by the accumulation of extracellular matrix (ECM), which contributes to cirrhosis and hepatocellular carcinoma. Increasing evidence suggests that the activation of hepatic stellate cells (HSCs) under an inflammatory state leads to the secretion of collagens, which can cause cirrhosis. In this study, we analysed data from the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs) between quiescent and fibrotic HSCs. We found that Microfibril Associated Protein 2 (MFAP2) was elevated in carbon tetrachloride (CCl4)-induced liver fibrosis and Transforming Growth Factor-Beta 1 (TGF-β1)-activated HSCs. Knockdown of MFAP2 inhibited HSC proliferation and partially attenuated TGF-β-stimulated fibrogenesis markers. Bioinformatics analysis revealed that Fibrillin-1 (FBN1) was correlated with MFAP2, and the expression of FBN1 was significantly upregulated after MFAP2 overexpression. Silencing MFAP2 partially attenuated the activation of HSCs by inhibiting HSC proliferation and decreasing collagen deposits. In vitro results showed that the inhibition of MFAP2 alleviated hepatic fibrosis by inhibiting the activation and inducing the apoptosis of active HSCs in a CCl4-induced mouse model. In conclusion, our results suggest that MFAP2 is a potential target for the clinical treatment of liver fibrosis.     

剪切应力环境下的内皮祖细胞对肝星状细胞增殖和生物功能的影响

目的：研究流体剪切应力条件下的内皮祖细胞（EPCs）对肝星状细胞（HSCs）增殖、粘附、迁移、凋亡等生物学功能以及成纤维化因子α-平滑肌肌动蛋白（α-SMA）、胶原I （Col-I）、胶原III （Col-III）表达的影响。方法：将HSCs与EPCs分别接种于共培养小室的上层和下层,共培养24 h后,给EPCs细胞施加12 dyne/cm²剪切应力,持续24 h。消化细胞,采用CCK-8法检测HSCs的增殖;流式细胞术检测HSCs的凋亡率;细胞贴壁法检测HSCs的粘附功能;Boyden小室检测HSCs的迁移;荧光定量PCR法及Western blot分别检测HSCs的α-SMA、Col-I、Col-III mRNA和蛋白质的表达情况。结果：在剪切应力条件下,EPCs生态小境能明显抑制HSCs的增殖、粘附和迁移能力,促进HSCs凋亡,下调HSCs中Col-I、Col-III mRNA和蛋白质的表达。结论：在剪切应力条件下,EPCs生态小境对HSCs纤维化的发展具有一定抑制作用。     

猪血清攻击后肝纤维化大鼠α-SMA、TIMP-1在肝组织内持续表达

目的猪血清法肝纤维化模型中,致纤维化因子停止作用于肝脏后α-SMA、TIMP-1阳性HSCs表型与组织中I型胶原表达的相关性。方法猪血清10周法制作大鼠肝纤维化模型,于造模6周、10周、14周和20周,免疫组织化学观察α-SMA、I型胶原,肝组织原位杂交法检测TIMP-1阳性表型HSCs在肝组织中的分布,计算机图像分析系统测定阳性比率,SPSS11.0分析各参数在肝纤维化进程中相关性。结果α-SMA于造模第6周、10周即有表达,主要分布于汇管区血管或中央静脉内膜下。于造模第14周、20周时持续表达。TIMP-1于造成模第6周时,表达也限于汇管区血管和中央静脉内膜下。造模第10周、14周时阳性表达率明显增加,向肝组织内伸展,并持续维持高阳性表达至造模第20周。α-SMA和TIMP-1在造模过程中随肝纤维化的进程呈显著正相关（r=0.989,P=0.000）,二者分别与Ⅰ型胶原呈显著正相关（r=0.893,P=0.000;r=0.923,P=0.000）。结论猪血清攻击法肝纤维化大鼠模型中,致纤维化因子启动肝纤维化进程,但不随致纤维化因子的终止而终止。所以,抗肝纤维化治疗成为治疗本病的关键。