Gastroenterology

Gastroenterology

Volume 137, Issue 4, October 2009, Pages 1459-1466.e1
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Negligible Contribution of Bone Marrow-Derived Cells to Collagen Production During Hepatic Fibrogenesis in Mice

https://doi.org/10.1053/j.gastro.2009.07.006Get rights and content

Background & Aims

Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients.

Methods

BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of α2(I) collagen gene (COL1A2) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates.

Results

The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli.

Conclusions

By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Section snippets

Mice

All animals used in the present study received humane care, and the experiments were approved by the Animal Experiment Committee of Tokai University. C57BL/6 mice were purchased from CLEA Japan Inc. (Tokyo, Japan). A transgenic mouse strain (COL/LUC) that contains the −17,000 to +54 region of the mouse upstream sequence of α2(I) collagen gene (COL1A2) linked to a firefly luciferase gene was previously described.14 The −17,000 to −15,450 COL1A2 sequence exhibits a strong enhancer activity that

BM-Derived Cells Migrating Into Fibrotic Liver Seldom Differentiate Into α-SMA-Positive Cells in Both Experimental Fibrosis Models

We first examined migration of BM cells into liver tissue in 2 mechanistically distinct fibrosis models introduced into CAG/EGFP-recipient mice. As previously reported,3 bridging fibrosis connecting the neighboring portal areas and central veins was formed, but complete cirrhosis was not established after 30 times of repeated CCl4 injections (Figure 2A). A large number of EGFP-expressing BM-derived cells migrated into the fibrotic liver 2 days after the last CCl4 injection, at peak fibrosis (

Discussion

In the present study, we have revealed an unexpectedly limited role of BM-derived cells in collagen production in 2 mechanistically distinct models of liver fibrosis. Although some of the BM-derived cells exhibited a mesenchymal morphology resembling myofibroblasts, the number of BM-derived α-SMA-positive cells was much smaller than previously reported. More importantly, specific and quantitative analyses of COL1A2 promoter activation by using a combination of EGFP and luciferase reporter genes

Acknowledgments

The authors thank Dr Benoit de Crombrugghe for his generous gift of transgenic collagen reporter mice, Dr Masaru Okabe for transgenic mice that constitutively express enhanced green fluorescent protein, and Dr Kiyoshi Higashi for his continuous support and helpful suggestions throughout the work.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan; a grant from the Scleroderma Research Committee of the Ministry of Health, Labour and Welfare, Japan; and a research grant from Mitsui Life Social Welfare Foundation.

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