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

Laboratory Investigation (2012) 92, 135–150 & 2012 USCAP, Inc All rights reserved 0023-6837/12 $32.00 Liver precursor cells increase hepatic fibrosis induced by chronic carbon tetrachloride intoxication in rats Marie-Noële Chobert1,2,*, Dominique Couchie2,3,*, Agnès Fourcot2, Elie-Serge Zafrani1,2,4, Yannick Laperche1,2, Philippe Mavier2 and Arthur Brouillet1,2 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 (CCl4) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl4 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 a-smooth muscle actin (aSMA). In AAF/CCl4-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 CCl4-treated animals. Enhanced fibrosis in AAF/CCl4 group was accompanied by ductule forming LPC expanding from portal areas, aSMA-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-b (TGFb) without any expression of aSMA, desmin or fibroblastspecific protein-1, demonstrating that LPC did not undergo an epithelial–mesenchymal transition. In this new experimental model, LPC, by expressing TGFb, contributed to the accumulation of aSMA-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. Laboratory Investigation (2012) 92, 135–150; doi:10.1038/labinvest.2011.143; published online 26 September 2011 KEYWORDS: epithelial–mesenchymal transition; fibrosis; liver progenitor cells; TGFb Hepatic fibrosis is the major complication of virtually all types of chronic liver damage (eg. viral, alcoholic liver disease or non-alcoholic fatty liver disease). In humans, fibrosis that ultimately leads to cirrhosis initiates predominantly in the portal areas even though the primary targets of injury are hepatocytes within the lobule.1 Impairment of the replicative capacity of most remnant hepatocytes induces an alternate regenerative process from liver progenitor cells (LPC). These cells (also called oval cells in rodent) are located in the most peripheral branches of the biliary tree (canal of Hering).2 Once activated, LPC proliferate in the portal region and migrate into the hepatic lobule where they undergo further differentiation into hepatocytes or bile duct cells to repopulate the hepatic parenchyma.3,4 This proliferative response characterized by the appearance of bile-duct-like structures in humans is referred to atypical ductular reaction.5 LPC have been of increasing interest in latest years since they could have a role not only in regeneration but also in fibrogenesis and carcinogenesis.6 In experimental models and various human liver diseases, LPC expand in close proximity to a-smooth muscle actin (aSMA)-positive cells deriving from either sinusoidal hepatic stellate cells (HSC) or portal fibroblasts depending upon the location of the injury in the lobule.2,7 There are increasing data demonstrating an intimate cross-talk between LPC and aSMA-positive myofibroblasts3,8–11 and several studies in rodent models and human diseases have pointed out a strong relationship between severity of fibrosis and intensity of the ductular 1 INSERM, U955, Equipe 17, Institut Mondor de Recherche Biomédicale, Créteil, France; 2Université Paris-Est, Créteil, France; 3INSERM, U955, Equipe 21, Créteil, France and 4Département de Pathologie, AP-HP, Groupe Hospitalier Henri Mondor, Créteil, France Correspondence: Dr A Brouillet, PhD, INSERM, U955, Equipe 17, Institut Mondor de Recherche Biomédicale, Créteil F-94010, France. E-mail: *These authors contributed equally to this work. Received 17 March 2011; revised 3 August 2011; accepted 4 August 2011 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 January 2012 135 LPC aggravate liver CCl4-induced fibrosis M-N Chobert et al reaction.12–15 Interactions between LPC and sinusoidal HSC or portal fibroblasts are poorly understood but their activation could occur independently, successively or in tandem through similar stimuli.16 First, it can be considered that secretion of profibrogenic cytokines and growth factors by LPC could participate to portal fibrosis by inducing HSC or portal fibroblast activation,17,18 but this implication of LPC in fibrosis has not been clearly shown in vivo. On the opposite, activation of HSC or portal fibroblasts into myofibroblasts producing extracellular matrix (ECM) and growth factors precedes LPC expansion and differentiation in periportal regions along a porto-venous axis.6,11 Recently, it has been shown that inhibition of HSC activation greatly diminished LPC expansion.19 Therefore, HSC or portal fibroblast activation seems a prerequisite for LPC expansion, as already demonstrated in other stem cell niches.20 Additionally, myofibroblast-LPC cross-talk could indirectly enhance fibrogenesis by recruiting cells of innate immune response, such as leukocytes required for wound healing.21 Among the large variety of factors secreted by myofibroblasts, transforming growth factor-b (TGFb), the most potent fibrogenic cytokine, is also a well-established mediator of epithelial–mesenchymal transition (EMT), which contributes to fibrosis following injury in several organs including the liver.22–26 To investigate the fibrogenic potential of LPC, we established a new model in the rat in which persistent LPC expansion induced by a chronic 2-acetylaminofluorene (AAF) treatment was combined to the well-established model of liver fibrosis using chronic carbon tetrachloride (CCl4) injection.1 Metabolism of CCl4 into highly reactive CCl3 radicals by cytochrome P450 is responsible for centrilobular hepatocellular necrosis, which triggers matrix deposition starting around the central veins, with gradual formation of septa bridging neighboring central veins, without any ductular reaction.25 In our model, administration of AAF before and during CCl4 treatment blocks the proliferative hepatocyte response caused by CCl4-induced necrosis27 and leads to the emer (...truncated)