CCl4-induced toxicity and its mechanisms have been extensively investigated after oral administration to rodents. A large number of rat models have been reported to develop both liver cirrhosis and HCC using CCl4 or diethylnitrosamine. In the current study, we generated well-differentiated HCCs in the livers of all CCl4-treated strain A/J mice. Fibrosis but not cirrhosis was identified in these livers, even in those with HCCs. The cessation of oral gavage of CCl4 after 17 weeks may explain why cirrhosis did not develop although HCC clearly developed before any signs of cirrhosis. In previous reports of a rat model, liver cirrhosis induced by toxic agents preceded the development of HCC, like in humans [17, 18]. In this study, HCCs were obviously generated in fibrotic, not cirrhotic, livers.
Many investigators have previously reported their findings in mice treated with CCl4, each with different mouse strains and CCl4 administration schedules. We therefore reviewed eight published reports of mouse models of liver "cirrhosis" induced only by CCl4 to assess fibrosis, regenerative nodules, and cirrhosis [19–26]. One report of a transgenic mouse model was also reviewed . Of the nine models previously reported in the literature, only the model by Xue et al. shows incomplete cirrhosis (Ishak score = 5) with portal fibrosis, bridging fibrosis, and regenerative nodules [23, 28]. Two other models show changes similar to ours [19, 24], all the others showed only limited portal fibrosis and bridging fibrosis with no regenerative nodules. Thus, it may be more difficult to induce cirrhosis in mice.
The mechanism mobilizing the normally quiescent hepatocyte into mitogenesis in injured liver is poorly understood, although accumulating data suggest that EGF and TGF-α, specific ligands of EGFR, play a central role in initiating and/or sustaining the early growth response program [15, 28, 29]. We obtained evidence that both mRNA and protein expression of EGF significantly decreased during liver injury, but increased during repair after the withdrawal of CCl4. The transcription of EGF was shown to be extremely low in control livers but was highly elevated in cirrhotic livers of human patients, and EGF expression was increased significantly during the course of cirrhosis development in a rat model [12, 30]. Therefore, histopathological changes and EGF expression patterns observed in this mouse model were different from those previously reported in rats and humans. Consistent with previously reported rat models of liver cirrhosis, EGFR expression was shown to decrease in this mouse study, whereas it has been reported to increase in human cirrhosis patients [31, 32]. However, these results are difficult to interpret as decreased levels of total EGFR observed in animal models might be due to ligand-mediated receptor endocytosis in response to elevated signaling.
Combined with our histopathological analysis of previously published reports, our results indicate that there are species-specific differences in liver regeneration in response to cytokines and growth factors. Previous investigators have provided evidence of the differences in the response to hepatotoxicity in the livers of rodents. High levels of inhalation exposure to CCl4 resulted in liver cirrhosis in rats but in neither fibrosis nor cirrhosis in mice . Furthermore, inflammation of the liver was almost absent in rats after common bile duct ligation but was more pronounced in mice . It was also reported that the lack of circulating EGF in sialoadenectomized mice did not decrease the proportion of hepatocytes that replicate during liver regeneration after partial hepatectomy , whereas removal of the salivary glands in rats led to complete blockage of liver regeneration . It would appear that other EGFR ligands might be critically involved in liver regeneration in mice instead of EGF [28, 37]. For example, both TGF-α and AREG increased in injured livers in this study. TGF-α has previously been shown to increase in previous reports of rats and humans and AREG has previously been shown to participate in the development of mouse liver fibrosis . Importantly, AREG has also been shown to contribute to the neoplastic phenotype of human HCC cells (Castillo 2006 Cancer Research) and therefore the CCL4 model could be used to further study the role of AREG in hepatocarcinogenesis.
We observed that the expression of both desmin and GFAP, known to be markers of HSCs, were significantly elevated in injured liver samples. This finding implicates increased numbers of HSCs in the livers of CCl4-treated mice. In a recent study, Kordes et al. demonstrated that 20-40% of HSCs expressed CD133 and exhibited properties of progenitor cells in rats . Motivated by this report, we also examined CD133 expression in the injured livers of mice. The mRNA expression of CD133 was observed to be up-regulated in more injured liver as was desmin and GFAP. Further, immunohistochemical staining revealed that CD133, desmin and α-SMA localized together in CCl4-treated mice livers. CD133 has been recognized as a cell surface marker of stem/progenitor cells. However, the mRNA expression of VEGFR2, reported to be one of the markers of both early and mature EPCs was not associated with the degree of the liver injury. Our results indicated that CD133 positive cells probably correspond to HSCs, not EPCs, and that CD133+ HSCs increased through chronic liver injury in CCl4-treated mice. Finally, CD133 expression returned to control levels in the surrounding non-cancerous tissues but remained elevated in HCCs.
CD133+ cells have been identified in the ductular reactions of chronically damaged human livers . CD133 has been implicated as a marker for cancer stem cells of epithelial origin , and consistent with this, CD133+ cells isolated from human HCC cell lines have been reported to have cancer stem cell properties [41, 42]. Further, CD133 expression has been reported to be associated with poor disease-free survival in HCC patients but was negatively associated with HBsAg, implicating a non-viral origin of CD133 expression in HCC. Here, we have shown that a chemical toxicity induces CD133 expression in the liver. Overall, our results suggest an association between CD133 upregulation and tumor generation although details must be elucidated by further investigation.
Our results suggest that the use of CCl4-induced chronic liver injury in mice has important differences compared to human cirrhosis. In humans, HCCs typically develop within the setting of cirrhosis; however, in this study, no evidence of cirrhosis was seen, and HCCs developed within a setting of fibrosis. Furthermore, EGF expression decreased significantly during chronic liver injury, whereas it has been shown to increase during development of human cirrhosis. These results suggest that species-specific differences exist with respect to the histopathological and molecular pathogenesis of chronic liver disease. The dramatic up-regulation of CD133 is a notable finding, and the contribution of CD133+ HSCs to liver regeneration and tumor progression await further studies in rodents and humans.