Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction

Background

In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.

Methodology

HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.

Principal Findings

The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.

Conclusions

Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.     

MicroRNA-29a protects against acute liver injury in a mouse model of obstructive jaundice via inhibition of the extrinsic apoptosis pathway

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis, as demonstrated in human liver cirrhosis, and that its downregulation influences the activation of hepatic stellate cells. In addition, both cleaved caspase-3 production and apoptosis play a role in cholestatic liver injury. However, it is unknown if miR-29 is effective in modulating the extent of injury. We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type (WT) littermates to clarify the role of miR-29 in hepatic injury and fibrogenesis, using the bile duct-ligation (BDL) mouse model. After BDL, all three members of the miR-29 family were significantly downregulated in the livers of WT mice, and miR-29b and miR-29c were significantly downregulated in the livers of the miR-29aTg mice. Liver function, as measured by alanine transaminase and aspartate transaminase activity, was significantly improved in the miR-29aTg mice than in the WT littermates, following 1 week of obstructive jaundice. In addition, overexpression of miR-29a was associated with a significant downregulation of the expression of collagen-1α1, collagen-4α1, phospho-FADD, cleaved caspase-8, cleaved caspase-3, Bax, Bcl-2, PARP, and nuclear factor-κB, as well as an upregulation of phospho-AKT expression. In addition, there were significantly fewer TUNEL-positive liver cells in the miR-29aTg group than in the WT littermates after BDL. Our results indicate that miR-29a decreases cholestatic liver injury and fibrosis after BDL, at least partially, by modulating the extrinsic rather than intrinsic pathway of apoptosis.     

MiR-21 Simultaneously Regulates ERK1 Signaling in HSC Activation and Hepatocyte EMT in Hepatic Fibrosis

Background

MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.

Methods

Quantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α.

Results

The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.

Conclusions

MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.     

X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4

Background & Aims

Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers.

Methods

Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.

Results

X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.

Conclusions

X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.     

Inhibition of Tyrosine Kinase Receptor Tie2 Reverts HCV-Induced Hepatic Stellate Cell Activation

Background

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease (CLD) and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC) is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD.

Methods

Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons). The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined.

Results

Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody) or signaling (by selective AKT and MAPK inhibitors) significantly reduced alpha-smooth muscle actin (α-SMA) expression and the invasive potential of HCV-conditioned HSC.

Conclusions

These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.     

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

Background & Aim

MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis.

Methods

Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed.

Results

MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice.

Conclusions

Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis.     

Rapamycin Ameliorates Inflammation and Fibrosis in the Early Phase of Cirrhotic Portal Hypertension in Rats through Inhibition of mTORC1 but Not mTORC2

Objective

Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension.

Methods

Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1β) were assessed by western blot.

Results

The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin.

Conclusions

The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.     

Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-β

Aim

Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-β, the precursor of the most fibrogenic cytokine TGF-β.

Methods

After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-β activation using primary cultured HSCs and liver sinusoidal ECs (LSECs).

Results

In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-β via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-β present in LSEC conditioned medium and subsequent HSC activation.

Conclusion

Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-β that activated on the surface of HSCs by PLK, thereby resultant active TGF-β stimulates the activation of HSCs.     

Deficiency in augmenter of liver regeneration accelerates liver fibrosis by promoting migration of hepatic stellate cell

Background

Augmenter of liver regeneration (ALR) protects liver from various injuries, however, the association of ALR with liver fibrosis, particularly its effect on hepatic stellate cells (HSC), remains unclear. In this study, we investigated the impact of ALR on the activation of HSC, a pivotal event in occurrence of liver fibrosis.

Spironolactone lowers portal hypertension by inhibiting liver fibrosis, ROCK-2 activity and activating NO/PKG pathway in the bile-duct-ligated rat

Objective

Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension.

Methods

Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson''s trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo.

Results

Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector- protein kinase G (PKG) in the liver.

Conclusion

Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.     

Activation of protein serine/threonine phosphatase PP2Cα efficiently prevents liver fibrosis

Background

Over-activation of TGFβ signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2Cα was reported to negatively regulate TGFβ signaling pathway and cell cycle. Our study aimed to investigate the role of PP2Cα in liver fibrogenesis.

Methodology/Principal Findings

The effects of PP2Cα activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl₄)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2Cα by overexpression or the new discovered small molecular activator NPLC0393 terminated TGFβ-Smad3 and TGFβ-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased α-smooth muscle actin (α-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl₄- and BDL-induced mice.

Conclusions/Significance

Our findings suggested that PP2Cα activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease.     

The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

Background

The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life''s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.

Aim

In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.

Methods

LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.

Results

Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

Conclusions

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.     

Activated Effects of Parathyroid Hormone-Related Protein on Human Hepatic Stellate Cells

Background & Aims

After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines.

Methods

TGF-β1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1–36) or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media.

Results

In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs.

Conclusions

PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.     

Schistosoma japonicum Soluble Egg Antigens Facilitate Hepatic Stellate Cell Apoptosis by Downregulating Akt Expression and Upregulating p53 and DR5 Expression

Background

The induction of hepatic stellate cell (HSC) apoptosis has potential as a potent strategy to diminish the progression of liver fibrosis. Previous studies have demonstrated the ability of soluble egg antigens (SEA) from schistosomes to inhibit HSC activation and to induce apoptosis in vitro. In this study, we aimed to explore the mechanism of SEA-induced apoptosis in HSCs.

Methodology/Principal Findings

In this study, we found that SEA could upregulate p53 and DR5 and downregulate the p-Akt. The apoptosis of HSCs induced by SEA could be reduced in HSCs that were treated with p53-specific siRNA and in HSCs that were treated with DR5-specific shRNA. In addition, {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516, which enhances the expression of Akt, could also decrease the SEA-induced HSC apoptosis. We also found that the increased expression of p53 and DR5 induced by SEA through Mdm2 were reduced by {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516.

Conclusions/Significance

Our data suggest that SEA can induce HSC apoptosis by downregulating Akt expression and upregulating p53-dependent DR5 expression.     

A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

Background

In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis.

Methodology/Principal Findings

Chronic treatment with the hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ^+/+ mice with CCl₄-induced severe liver fibrosis, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis.

Conclusions/Significance

These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.     

In Vivo Tracking and Comparison of the Therapeutic Effects of MSCs and HSCs for Liver Injury

Background

Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been studied for damaged liver repair; however, the conclusions drawn regarding their homing capacity to the injured liver are conflicting. Besides, the relative utility and synergistic effects of these two cell types on the injured liver remain unclear.

Methodology/Principal Findings

MSCs, HSCs and the combination of both cells were obtained from the bone marrow of male mice expressing enhanced green fluorescent protein(EGFP)and injected into the female mice with or without liver fibrosis. The distribution of the stem cells, survival rates, liver function, hepatocyte regeneration, growth factors and cytokines of the recipient mice were analyzed. We found that the liver content of the EGFP-donor cells was significantly higher in the MSCs group than in the HSCs or MSCs+HSCs group. The survival rate for the MSCs group was significantly higher than that of the HSCs or MSCs+HSCs group; all surpassed the control group. After MSC-transplantation, the injured livers were maximally restored, with less collagen than the controls. The fibrotic areas had decreased to a lesser extent in the mice transplanted with HSCs or MSCs+HSCs. Compared with mice in the HSCs group, the mice that received MSCs had better improved liver function. MSCs exhibited more remarkable paracrine effects and immunomodulatory properties on hepatic stellate cells and native hepatocytes in the treatment of the liver pathology. Synergistic actions of MSCs and HSCs were most likely not observed because the stem cells in liver were detected mostly as single cells, and single MSCs are insufficient to provide a beneficial niche for HSCs.

Conclusions/Significance

MSCs exhibited a greater homing capability for the injured liver and modulated fibrosis and inflammation more effectively than did HSCs. Synergistic effects of MSCs and HSCs were not observed in liver injury.     

Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

Background

Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl₄)-induced liver injury rats.

Methods

Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated.

Results

Oral administration of MGP significantly alleviated DMN- or CCl₄-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression.

Conclusions

The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.