Actin Positive Hepatic Stellate Cells Research Articles

Ecto‐5′‐nucleotidase (CD73) ‐mediated extracellular adenosine production plays a critical role in hepatic fibrosis

Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A(2A) receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl(4), ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl(4) or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl(4), ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl(4) or TAA. There were far fewer alpha-smooth muscle actin positive hepatic stellate cells in CCl(4)-treated KO mice than that in WT mice. After CCl(4) treatment, the mRNA level of A(1), A(2A), A(2B), and A(3) adenosine receptors, tumor necrosis factor-alpha, interleukin (IL) -1beta, IL-13r alpha1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1 alpha1, Col3 alpha1, and transforming growth factor-beta1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13r alpha2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.

Hepatic fibrosis in patients with chronic hepatitis B virus infection can be partially reversed by IFN-γ

Background. Hepatic fibrosis due to chronic hepatitis B virus (HBV) infection has an enormous socio-economic impact. Besides strategies aiming at virus elimination, prevention or reversal of liver fibrosis is amenible. Given the antifibrotic activity of IFN-γ, a randomized open-labeled multicenter trial was initiated to test IFN-γ in HBV infection. Methods. HBs-antigen positive patients with biopsy proven hepatic fibrosis (n=99, stages 2–4 according to Scheuer criterion) were treated with diammone glycyrrhizinate and potassium magnesium aspartate.Treatment with 50g IFN-γ i.m. on a daily basis for three months and on alternate days the subsequent six months was performed in 66 randomly assigned patients. Efficacy was evaluated by liver biopsy and serologic markers. Results. 54 patients in the IFN-γ group and 29 patients in the control group completed the study protocol. The hepatic fibrosis score was significantly reduced in 63 percent of IFN-γ treated patients compared to 24.1 percent in the control group as assessed by a semiquantitative scoring system evaluating both liver architecture and fibrotic deposits. Mean values for total fibrosis score decreased from 13.8±5.8 to 10.1±5.1 in the IFN-γ group, whereas they were unchanged in controls (13.2±6.8 versus 12.6±4.8 after 9 months). The Scheuer fibrosis scoring system revealed 12 out of 54 patients improved 1 stage(s) in the IFN-γ compared to 1/29 in the control group. Antifibrotic activity may be attributed to decreased TGF-β signaling via phospho-Smad2 and reduced number of activated, α smooth muscle actin positive hepatic stellate cells. Conclusions. Prolonged IFN-γ treatment over 9 months significantly improves the fibrosis score in patients with chronic HBV infection most likely by antagonizing profibrogenic TGF-β effects.

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and α-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and α-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic α-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and α-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when α-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and α-SM actin expression. In both cell populations, transforming growth factor-β1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.