Gastroenterology

Gastroenterology

Volume 143, Issue 4, October 2012, Pages 1073-1083.e22
Gastroenterology

Original Research
Basic and Translational—Liver
Deactivation of Hepatic Stellate Cells During Liver Fibrosis Resolution in Mice

https://doi.org/10.1053/j.gastro.2012.06.036Get rights and content

Background & Aims

Activated hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, undergo apoptosis after cessation of liver injury, which contributes to resolution of fibrosis. In this study, we investigated whether HSC deactivation constitutes an additional mechanism of liver fibrosis resolution.

Methods

HSC activation and deactivation were investigated by single-cell PCR and genetic tracking in transgenic mice that expressed a tamoxifen-inducible CreER under control of the endogenous vimentin promoter (Vimentin-CreER).

Results

Single-cell quantitative polymerase chain reaction demonstrated activation of almost the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. Vimentin-CreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter after injection of carbon tetrachloride, in liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from peri-sinusoidal to fibrotic septa. Tracking of mGFP-positive HSCs revealed the persistence of 40%–45% of mGFP expression in livers and isolated HSCs 30–45 days after carbon tetrachloride was no longer administered, despite normalization of fibrogenesis parameters; these findings confirm reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive peri-sinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher levels of responsiveness to fibrogenic stimuli.

Conclusions

In mice, reversal of HSC activation contributes to termination of fibrogenesis during fibrosis resolution, but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation.

Section snippets

Generation of Vimentin-CreER Mice

A CreERFrtNeoFrt cassette was PCR-amplified from a p451 plasmid14 with 60-bp overhangs homologous to the upstream and downstream sequence surrounding ATG site of the mouse vimentin gene. The PCR product was inserted into a BAC containing the mouse vimentin gene by recombineering. After removal of the Neo cassette by arabinose-induced flippase, BAC DNA was microinjected into the pronucleus of fertilized CBA×C57BL/6J oocytes. One of 3 founders showed strong Vimentin-CreER (Vim-CreER) activity

Activation of HSCs in the Injured Liver at a Single-Cell Level

To determine whether activated HSCs can deactivate, we first compared fibrogenic gene expression in CCl4-injured livers and HSCs at different time points after cessation of injury to pre- and peak-injury levels. After 4 injections of CCl4, fibrogenic gene expression in whole liver was elevated 2 days after the last injection, and gradually returned to normal levels with almost normalized levels of fibrogenic gene expression 30 days after the last CCl4 injection (Figure 1A). This regimen also

Discussion

Removal of hepatic myofibroblasts by apoptosis is considered the key mechanism that reduces the number of activated myofibroblasts and allows the liver to return to normal architecture.6 Here we provide evidence that reversal of hepatic myofibroblasts to a quiescent state represents a second mechanism that contributes to the regression of liver fibrosis. Our study used 2 complementary approaches, single-cell qPCR and genetic cell fate tracking, to determine reversal of activated HSCs to a

Conclusion

Our study shows deactivation of roughly half the HSC population during liver fibrosis regression and higher responsiveness of reverted HSCs to recurrent profibrogenic stimulation.

Acknowledgments

Juliane S. Troeger and Ingmar Mederacke contributed equally to this study.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This study was supported by grants R01DK076920 (to RFS), U54CA1261513 (PI of Project 2: RFS), and U54CA163111. Ingmar Mederacke was supported by the German Research Foundation (ME 3723/1-1). Dianne Dapito was supported by 1F31DK091980. Jean-Philippe Pradere was supported by a postdoctoral fellowship from the American Liver Foundation.

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