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

Abstract

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 (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) 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 α-smooth muscle actin (αSMA). In AAF/CCl(4)-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 CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-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-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ, contributed to the accumulation of αSMA-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.

Figure 1

Liver injury and histopathology. A) Serum ALAT activity was increased to a level which was similar in CCl₄ and AAF/CCl₄-treated rats and significantly higher than in AAF-treated rats. Elevation of fluorometric caspase-3 activity was similar in rat liver treated either by CCl₄ or AAF/CCl₄ and was significantly higher in AAF-treated animals. B) The liver weight to body weight ratio was significantly increased in AAF/CCl₄-treated rats after 6 weeks of treatment as compared to the other groups. # (P<0.05) denotes significant difference from control diet and ** (P<0.001) between AAF/CCl₄ and CCl₄-treated animals. C) Representative H&E staining of liver sections after 4 (W4) and 6 weeks (W6) of treatment (original magnifications ×40 and ×400 as indicated) show steatosis and ballooned hepatocytes (arrows) in CCl₄-treated animals (n=9) with formation of centro-central bridges dividing the hepatic parenchyma into lobules centered by portal tracts. In group AAF/CCl₄ (n=9), centrilobular injury was associated with expansion of basophilic cells extending from portal areas and leading to nodules. Proliferation of these cells with small ovoid nuclei in periportal areas was also observed in AAF-treated rats (n=4). PT denotes portal tracts.

Figure 2

LPC compartment activation. A) Representative immunofluorescent detection of CK19-positive cells in liver sections revealed an accumulation of LPC in rats treated with AAF (n=4) and AAF/CCl₄ (n=9), arising from portal areas at 4 weeks and expanding inside the lobule after 6 weeks of treatment (original magnification ×200). Insets show higher magnification of CK19 labeling. Such an accumulation of LPC was not detected in CCl₄ group (n=9) where CK19 labeling was restricted to bile duct cells. B) Real-time PCR revealed an increase of CK19 mRNA expression only in AAF and AAF/CCl₄ groups. C) AFP mRNA detected by in situ hybridization (AFP AS probe) was restricted to cells of the ductular reaction in AAF and AAF/CCl₄-treated animals. No specific signal was observed using AFP sense probe (original magnification ×400). * denotes portal tract. Images are representative of three independent experiments from three different rat liver sections.

Figure 3

Liver fibrosis. A) Picrosirius red staining revealed progressive liver fibrosis induced by CCl₄ at week 4, with formation of centro-central fibrous septa at week 6. In AAF/CCl₄-treated rats, marked fibrosis with porto-central bridging was observed at week 4, leading to cirrhosis at week 6. In portal areas of animals from AAF group, a faint picrosirius red staining was observed around LPC expansion. Original magnification ×100. Images are representative of 4 (AAF) to 9 (CCl₄ or AAF/CCl₄) liver sections. B) After 6 weeks, immunofluorescent collagen 1 detection revealed strong matrix deposition in perinodular areas from cirrhotic rats liver treated by AAF/CCl₄. A lower collagen accumulation was detected in fibrous septa after CCl₄ administration, and a weak labeling was observed in the AAF-induced ductular reaction. Representative fluorescence photomicrographs of two experiments on liver sections from 3 rats in each group at magnification ×200. C) Quantitative RT-PCR analysis showed a gradual induction of interstitial collagen type 1 and 3 mRNA enhanced in AAF/CCl₄-treated rats as compared to CCl₄ treatment alone. Induction of collagen 4 mRNA was shown only in AAF and AAF/CCl₄ groups associated with LPC expansion. D) Total liver collagen content determined by hydroxyproline (OH-Pro) assay was higher in AAF/CCl₄-treated rats.

Figure 4

Macrophage recruitment and inflammation. A) CD68 immunofluorescent detection (original magnification ×200) revealed positive cells all over liver parenchyma in AAF group, in fibrous septa after CCl₄ treatment and in the cirrhotic liver of AAF/CCl₄-treated animals. Images are representative of 4 (AAF) to 6 (CCl₄ or AAF/CCl₄) liver sections. * denotes portal tracts. B) Quantification of CD68-positive cells in liver from animals did not reveal a significant difference in the number of hepatic macrophages in CCl₄ and AAF/CCl₄ groups after 4 or 6 weeks of treatment. Data are means± SEM of separate analysis on two fields for each liver section from 4 (AAF) to 6 (CCl₄ or AAF/CCl₄) separate rats. Quantitative RT-PCR did not demonstrate quantitative differences in C) TNFα or D) CCR2 mRNA expression, revealing that inflammatory response was similar in the 3 groups.

Figure 5

HSC or portal fibroblast activation. A) Immunohistochemical detection of αSMA-positive cells revealed few activated HSC delineating portal vessels after 6 weeks of CCl₄ administration alone. In contrast, numerous positive cells were detected in AAF/CCl₄-treated rats around portal areas at week 2, in fibrous septa at week 4 and in perinodular areas at week 6. No αSMA-positive cell was observed in the ductular reaction in AAF group. Images are representative of 4 (AAF) to 9 (CCl₄ or AAF/CCl₄) liver sections at original magnification ×100. B) After 6 weeks of CCl₄ treatment, higher magnification (×400) clearly showed αSMA-positive cells in centro-central fibrous septa containing marked collagen deposition revealed by picrosirius red staining. By contrast, in AAF/CCl₄-treated rats, activation of HSC or portal fibroblasts was early detected at week 2, around the LPC arising in periportal areas faintly stained by picrosirius red. At week 6, αSMA labeling was noted in bridging fibrous septa with large amount of collagen deposition.

Figure 6

Detection of fibrogenic liver cells. A) Immunofluorescent detection of αSMA and desmin after 6 weeks of treatment confirms activation of HSC into αSMA-expressing myofibroblasts in cirrhotic liver of AAF/CCl₄-treated rats. Very few αSMA-positive HSC were noted in fibrous septa after CCl₄ administration alone. Lack of activated HSC was observed around the portal areas in the AAF-induced ductular reaction. Representative fluorescence photomicrographs of two experiments on liver sections from 2 rats in each group at original magnification ×200. B) Quantitative RT-PCR analysis revealed an enhanced induction of TGFβ mRNA expression in AAF and AAF/CCl₄-treated animals and C) TGFβ mRNA detection by in situ hybridization showed a strong positive signal in LPC in duct-like structures (inset contains sense probe labeling) in both groups (original magnification ×400). D) Double-immunofluorescent detection of TGFβ (green) and CK19 (red) proteins in liver sections from AAF/CCl₄-treated rats, revealed TGFβ-expressing LPC in merged imaged by confocal analysis. Images are representative of two independent experiments from three different rats in each group.

Figure 7

Immunolocalization of mesenchymal proteins on CK19-positive LPC from AAF/CCl₄-treated animals after 6 weeks of treatment. A) Representative images of double immunofluorescence for CK19 (green) and myofibroblastic markers (red) αSMA, desmin, FSP1 and collagen 1 of two experiments from two rats (original magnification ×200). No merging signals between any mesenchymal marker and CK19 could be obtained in LPC. B) Confocal analysis confirmed the absence of mesenchymal markers in CK19-positive LPC that are surrounded by desmin- and αSMA-positive cells (×630).