Aiting Yang

Antifibrotic Effects of a Recombinant Adeno-Associated Virus Carrying Small Interfering RNA Targeting TIMP-1 in Rat Liver Fibrosis

Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA-TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA-TIMP-1. In the carbon tetrachloride model, rAAV/siRNA-TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA-TIMP-1 treatment significantly alleviated bile duct ligation-induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA-TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA-TIMP-1 may be an effective antifibrotic gene therapy agent.

Primary isolated hepatic oval cells maintain progenitor cell phenotypes after two-year prolonged cultivation

BACKGROUND & AIMS Although expandable hepatic progenitors provide renewable cell sources for treatment of hepatic disorders, long-term cultivation of hepatic progenitors may affect proliferation and differentiation abilities, and even initiate the formation of malignant cancer stem cells. This study aims to determine characteristics of primary cultured hepatic oval cells after prolonged cultivation in vitro. METHODS Hepatic oval cells isolated from rats fed with a choline-deficient, ethionine-supplemented diet were continuously propagated every 5-7 days, to 100 passages over two years. Hepatocytic differentiation was induced by sodium butyrate and characterized using western blot, periodic acid Schiff assays, albumin secretion and urea production. Proliferation capacity was evaluated using growth-curve and cell-cycle analysis; anchorage-independent growth and tumorigenicity were determined using soft agar and xenograft assay. RESULTS After 2 years of serial passages, hepatic oval cells with typical epithelial morphology continuously expressed OV-6, BD-1, BD-2, and Dlk as markers for hepatic progenitors, cytokeratin 19 as a cholangiocyte marker, and alpha-fetoprotein and albumin as hepatocyte markers. Furthermore, sodium butyrate could induce these cells to become glycogen-storage cells with the functions of albumin secretion and ureagenesis from ammonia clearance, indicating hepatocytic differentiation. Although proliferation slightly accelerated after the 50th passage, hepatic oval cells stayed diploid cells with features of chromosomal stability, which did not acquire anchorage-independent growth capacity and caused no tumor in immunodeficient mice, suggesting no spontaneous malignant transformation. CONCLUSIONS Hepatic oval cells retain the progenitor cell features without spontaneous malignant transformation after prolonged cultivation, and thus may serve as an expandable cell source for future exploitation of stem cell technology.

EGF Suppresses the Initiation and Drives the Reversion of TGF‐β1‐induced Transition in Hepatic Oval Cells Showing the Plasticity of Progenitor Cells

Transforming growth factor‐β1 (TGF‐β1) induces hepatic progenitors to tumor initiating cells through epithelial‐mesenchymal transition (EMT), thus raising an important drawback for stem cell–based therapy. How to block and reverse TGF‐β1‐induced transition is crucial for progenitors’ clinical application and carcinogenic prevention. Rat adult hepatic progenitors, hepatic oval cells, experienced E‐cadherin to N‐cadherin switch and changed to α‐smooth muscle actin (α‐SMA) positive cells after TGF‐β1 incubation, indicating EMT. When TGF‐β1 plus EGF were co‐administrated to these cells, EGF dose‐dependently suppressed the cadherin switch and α‐SMA expression. Interestingly, if EGF was applied to TGF‐β1‐pretreated cells, the cells that have experienced EMT could return to their epithelial phenotype. Abruption of EGF receptor revealed that EGF exerted its blockage and reversal effects through phosphorylation of ERK1/2 and Akt. These findings suggest an important attribute of EGF on opposing and reversing TGF‐β1 effects, indicating the plasticity of hepatic progenitors. J. Cell. Physiol. 230: 2362–2370, 2015.

Increasing Newly Diagnosed Rate and Changing Risk Factors of HCV in Yanbian Prefecture, a High Endemic Area in China

Background The newly diagnosed rate of HCV infection is increasing in China. However, the risk factors have not been fully identified. Here, a survey was performed in Yanbian Prefecture, a high-endemic area in China. Methods We identified newly diagnosed HCV infection in 2007–2011, using the local National Disease Supervision Information Management System from the Chinese Center for Disease Control and Prevention. We determined the risk factors using a case-control survey by questionnaire. Results Yanbian Prefecture had a rapid increase in the yearly newly diagnosed rate of HCV infection from 32.6 to 72.1/100.000 from the year 2007 to 2011. People aged 50–64 years had a high HCV infection of 43.4%, but only 0.3% of cases were reported in those aged less than 20 years. Cosmetic treatment, family history, blood transfusion, and dental treatment were independent risk factors for HCV infection. Unexpectedly, cosmetic treatments [odd ratio (OR) = 5.15, 95% confidence interval (CI) = 2.31–11.48, P = 0.00] and family history (OR = 4.68, 95% CI = 2.67–8.75, P = 0.00) showed a higher risk than the conventional risk factors of blood transfusion (OR = 4.49, 95% CI = 1.95–10.37, P = 0.001) and dental treatment (OR = 2.98, 95% CI = 1.42–6.25, P = 0.00). To further analyze the intrafamilial transmission, we found that spouses of HCV patients had an increased risk for acquiring HCV (OR = 5.75, 95% CI: 1.94–17.07), without significant association between either HCV RNA viral load (P = 0.29) or genotype (P = 0.43). Conclusions HCV infection was increased in Yanbian Prefecture. Cosmetic treatment was a higher risk factor than medical procedure. HCV infection had a clear family clustering phenomenon, especially between spouses.

Connective tissue growth factor induces hepatic progenitor cells to differentiate into hepatocytes

Connective tissue growth factor (CTGF) plays an important role in the proliferation of hepatic progenitors, however, little is known concerning the mechanism(s) through which it influences their differentiation. The differentiation of hepatic progenitors (WB-F344), either stimulated with recombinant CTGF or stably transfected with a CTGF overexpression plasmid, was investigated. Expression of the differentiation markers α-fetoprotein (AFP), albumin (ALB) and cytokeratin-19 (CK-19) was assessed. To confirm the effects of CTGF on progenitor differentiation, cells were treated with an inhibitor (WP631) of CTGF. Treatment of WB-F344 cells with recombinant CTGF for 24 h did not change the survival rate significantly, but the progenitors were enlarged with a decreased nuclear/cytoplasmic ratio. CTGF downregulated the expression of the fetal hepatocyte marker, AFP, while it upregulated the mature hepatocyte cell marker, ALB. The effect of CTGF overexpression plasmid on WB-F344 cell differentiation was consistent with a pattern of direct CTGF stimulation, including decreased AFP and increased ALB expression. Furthermore, the suppression of CTGF induction by an inhibitor was associated with significant inhibition of hepatic progenitor cell differentiation into hepatocytes. Importantly, we showed that differentiated WB-F344 cells by CTGF had in vitro functions characteristic of hepatocytes, including ALB production, glycogen storage and cytochrome P450 activity. Both recombinant CTGF and the CTGF overexpression plasmid induced hepatic progenitor differentiation into hepatocytes. This was suppressed by the CTGF inhibitor.

TGF-β1 Induces the Dual Regulation of Hepatic Progenitor Cells with Both Anti- and Proliver Fibrosis

Transforming growth factor-beta 1 (TGF-β1) plays a central role in hepatic progenitor cells- (HPCs-) mediated liver repair and fibrosis. However, different effects of TGF-β1 on progenitor cells have not been described. In this study, both in vitro (HPCs cocultured with hepatic stellate cells (HSCs) in transwells) and in vivo (CCl4-injured liver fibrosis rat) systems were used to evaluate the impacts. We found that HPCs pretreated with TGF-β1 for 12 hours inhibited the activation of HSCs, while sensitization for 48 hours increased the activation of HSCs. Consistent with these in vitro results, the in vivo fibrosis rat model showed the same time-dependent dual effect of TGF-β1. Regression of liver fibrosis as well as normalization of serum aminotransferase and albumin levels was detected in the rats transplanted with HPCs pretreated with TGF-β1 for 12 hours. In contrast, severe liver fibrosis and elevated collagen-1 levels were detected in the rats transplanted with HPCs pretreated with TGF-β1 for 48 hours. Furthermore, the TGF-β1-pretreated HPCs were shown to deactivate HSCs via enhancing SERPINE1 expression. Inhibition of SERPINE1 reversed the deactivation response in a dose-dependent manner.

The Characteristics Variation of Hepatic Progenitors after TGF-β1-Induced Transition and EGF-Induced Reversion

Profibrogenesis cytokine, transforming growth factor- (TGF-) β1, induces hepatic progenitors experiencing epithelial to mesenchymal transition (EMT) to matrix synthesis cells, even tumor initiating cells. Our previous data found that epidermal growth factor (EGF) blocks and reverses TGF-β1-induced transition. The aim of this study is to determine the characteristic changes of hepatic progenitors after TGF-β1-induced transition and EGF-induced reversion. Hepatic oval cells, rat hepatic progenitors, were isolated from rats fed a choline-deficient diet supplemented with ethionine. TGF-β1-containing medium was used for inducing EMT, while EGF-containing medium was used for reversing EMT. During TGF-β1-induced transition and EGF-induced reversion, hepatic oval cells sustained their progenitor cell marker expression, including α-fetoprotein, albumin, and cytokeratin-19. The proliferation ability and differentiation potential of these cells were suppressed by TGF-β1, while EGF resumed these capacities to the level similar to the control cells. RNA microarray analysis showed that most of the genes with significant changes after TGF-β1 incubation were recovered by EGF. Signal pathway analysis revealed that TGF-β1 impaired the pathways of cell cycle and cytochrome P450 detoxification, and EGF reverted TGF-β1 effects through activating MAPK and PI3K-Akt pathway. EGF reverses the characteristics impaired by TGF-β1 in hepatic oval cells, serving as a protective cytokine to hepatic progenitors.

Baseline Hepatitis B Virus DNA Level is a Promising Factor for Predicting the 3 (rd) Month Virological Response to Entecavir Therapy: A Study of Strict Defined Hepatitis B virus Induced Cirrhosis.

Background: Cirrhosis is a common complication of chronic hepatitis B. It remains unclear if viral and biochemical parameters at baseline affect virological response to entecavir and therefore warrant investigation. In the present study, we aimed to evaluate the efficacy of entecavir therapy by monitoring virological response at the end of the 3rd month of treatment and try to figure out whether baseline factors could help predict it in a cohort of hepatitis B virus (HBV) compensated cirrhosis patients and to determine the cut-off value of a predicting parameter. Methods: A total of 91 nucleos(t)ide-naïve patients with HBV induced cirrhosis (compensatory stage) were enrolled in a prospective cohort. HBV DNA and alanine aminotransferase (ALT) were tested at baseline and monitored every 3–6 months after starting therapy. Results: Of all 91 patients, the median follow-up time was 12 (9–24) months. Overall, 64 patients (70.3%) achieved virological response in the 3rd month. Univariate analysis showed that the 3rd month virological response can be predicted by baseline HBV DNA levels (P < 0.001, odds ratio [OR]: 2.13, 95% confidence interval [CI]: 1.44–3.15), ALT value (P = 0.023, OR: 1.01, 95% CI: 1.00–1.01) and hepatitis B e antigen (HBeAg) negativity (P = 0.016, OR: 0.30, 95% CI: 0.11–0.80). Multiple regression analysis showed baseline HBV DNA level was the only parameter related to full virological response. Higher baseline HBV DNA strata indicated a higher probability that HBV DNA remains detectable at the 3rd month (P = 0.001). Area under receiver operating characteristic curve for determining the 3rd month virological response by baseline HBV DNA was 77.6% (95% CI: 66.7–85.2%), with a best cut-off value of 5.8 log10. Conclusions: Baseline HBV DNA, HBeAg negativity, and ALT were independent factors contributing to virological response at the 3rd month. Further, multiple regression showed that HBV DNA level was the only parameter predicting full virological response as early as the 3rd month, in this cirrhosis cohort.

Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking

Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1) levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing arrested expression of α-SMA, elastin and collagen Ι. Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51% decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.

Inhibitory effects of HNF4α on migration/maltransformation of hepatic progenitors: HNF4α-overexpressing hepatic progenitors for liver repopulation

BackgroundAlthough they are expandable in vitro, hepatic progenitors are immature cells and share many immunomarkers with hepatocellular carcinoma, raising potential concerns regarding maltransformation after transplantation. This study investigated the effects of hepatic nuclear factor (HNF) 4α on the proliferation, migration, and maltransformation of hepatic progenitors and determined the feasibility of using these manipulated cells for transplantation.MethodsThe effects of HNF4α on rat hepatic progenitors (i.e. hepatic oval cells) were analyzed by HNF4α overexpression and HNF4α shRNA. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice injured by carbon chloride (CCl4) were then transplanted with control, HNF4α-overexpressing or HNF4α-suppressing hepatic oval cells. Finally, the engraftment of these cells in the recipient liver was analyzed.ResultsRat hepatic progenitors (i.e. hepatic oval cells) expressed HNF4α, although less than that in hepatocytes. When HNF4α was overexpressed in these cells, the proliferation and migration of hepatic oval cells were reduced; but when HNF4α was suppressed by shRNA, the proliferation and migration, and even anchorage-independent growth, of these cells were accelerated. RNA microarray and gene functional analysis revealed that suppressing HNF4α not only impaired many biosynthesis and metabolism pathways of hepatocytes but also increased pathways for cancer. When transplanted into CCl4-injured NOD/SCID mice, few HNF4α-suppressing hepatic oval cells localized into the liver, while control cells and HNF4α-overexpressing cells engrafted into the liver and differentiated into albumin-positive hepatocytes. Interestingly, the hepatocytes derived from HNF4α-overexpressing cells were less migrative and expressed less c-Myc than the cells derived from control cells.ConclusionHNF4α constrains proliferation, migration, and maltransformation of hepatic progenitors, and HNF4α-overexpressing hepatic progenitors serve as an optimal candidate for cell transplantation.