Elsevier

Journal of Hepatology

Volume 64, Issue 5, May 2016, Pages 1137-1146
Journal of Hepatology

Research Article
Characterization of hepatic stellate cells, portal fibroblasts, and mesothelial cells in normal and fibrotic livers

https://doi.org/10.1016/j.jhep.2016.01.010Get rights and content

Background & Aims

Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs) to myofibroblasts is not fully understood due to insufficient availability of markers and isolation methods. The present study aimed to isolate these cells, characterize their phenotypes, and examine their contribution to myofibroblasts in liver fibrosis.

Methods

Liver fibrosis was induced in Collagen1a1-green fluorescent protein (Col1a1GFP) mice by bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, or CCl4 injections. Combining vitamin A (VitA) lipid autofluorescence and expression of GFP and glycoprotein M6a (GPM6A), we separated HSCs, PFs, and MCs from normal and fibrotic livers by fluorescence-activated cell sorting (FACS).

Results

Normal Col1a1GFP livers broadly expressed GFP in HSCs, PFs, and MCs. Isolated VitA+ HSCs expressed reelin, whereas VitA−GFP+GPM6A− PFs expressed ectonucleoside triphosphate diphosphohydrolase-2 and elastin. VitA−GFP+GPM6A+ MCs expressed keratin 19, mesothelin, and uroplakin 1b. Transforming growth factor (TGF)-β1 treatment induced the transformation of HSCs, PFs, and MCs into myofibroblasts in culture. TGF-β1 suppressed cyclin D1 mRNA expression in PFs but not in HSCs and MCs. In biliary fibrosis, PFs adjacent to the bile duct expressed α-smooth muscle actin. FACS analysis revealed that HSCs are the major source of GFP+ myofibroblasts in the injured Col1a1GFP mice after DDC or CCl4 treatment. Although PFs partly contributed to GFP+ myofibroblasts in the BDL model, HSCs were still dominant source of myofibroblasts.

Conclusion

HSCs, PFs, and MCs have distinct phenotypes, and PFs partly contribute to myofibroblasts in the portal triad in biliary fibrosis.

Introduction

Hepatic stellate cells (HSCs) reside in the space of Disse in the liver and store vitamin A (VitA) lipids as retinyl ester [1], [2]. Upon liver injury, HSCs transform into myofibroblasts that express α-smooth muscle actin (ACTA2) [1], [2]. Myofibroblasts form fibrous scars, actively synthesize extracellular matrices and proinflammatory cytokines, and participate in the progression from an injured liver to fibrosis and cirrhosis. Cell lineage tracing indicated that HSCs are mesodermal in origin and are the major source of myofibroblasts [3], [4], [5]. Clinical cases and animal studies suggest that fibrosis is reversible [6], [7], [8], [9]. During fibrosis regression, activated HSCs undergo apoptosis or revert to quiescent HSCs [6], [8], [9]. Thus, the suppression of HSC activation has been considered to be a therapeutic target for treating liver fibrosis.

In addition to HSCs, different types of liver mesenchymal cells also differentiate into myofibroblasts in fibrosis. Portal fibroblasts (PFs) around the bile duct in the portal tract express COL15A1, elastin (ELN), ectonucleoside triphosphate diphosphohydrolase-2 (ENTPD2/NTPDase2/CD39L1), and THY1 and do not store VitA lipids in the rat liver [10], [11], [12], [13], [14]. In biliary fibrosis, PFs are believed to be the source of myofibroblasts in the portal area. In addition to PFs, second-layer cells (SLCs) in the central vein and capsular fibroblasts (CFs) beneath the mesothelium were characterized based on their morphology and location in the liver [15]. However, little is known about how these cells contribute to fibrosis because the availability of markers and isolation methods for each cell type is limited.

Mesothelial cells (MCs) form a single epithelial cell sheet and cover the liver surface [16]. MCs express glycoprotein M6A (GPM6A), mesothelin (MSLN), and podoplanin (PDPN) [16], [17], [18]. During liver development, mesodermal MCs migrate inward from the liver surface and give rise to both HSCs and PFs [19]. Upon liver injury, MCs give rise to HSCs or myofibroblasts near the liver surface, depending on the etiology [16], [18]. Similar to HSC activation, MCs change their phenotype to myofibroblasts in response to transforming growth factor-β (TGF-β) [16].

In the present study, we separated HSCs, PFs, and MCs by fluorescence-activated cell sorting (FACS) from collagen1a1 promoter-green fluorescent protein (Col1a1GFP) transgenic mouse livers and quantified contribution of these cells to myofibroblasts in liver fibrosis.

Section snippets

Mouse models

Col1a1GFP mice were obtained from Dr. David Brenner [20]. Uroplakin 1b-red fluorescent protein (Upk1bRFP) knock-in mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Fibrosis was induced by bile duct ligation (BDL) for 3 weeks, 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 1 month, or CCl4 injection 12 times as previously described [4], [16]. All animal experiments were performed in accordance with the NIH guidelines under the protocol approved by the IACUC at the

Broad expression of GFP in the Col1a1GFP mouse liver

Immunohistochemistry showed that the normal Col1a1GFP liver expresses GFP in some desmin (DES)+ HSCs in the sinusoid, as well as in the PFs adjacent to the bile duct and smooth muscle cells (SMCs) in the hepatic artery and portal vein (Fig. 1A). GFP expression was also observed in possible DES+ SLCs around the central vein as well as in DES+ CFs beneath MCs (Fig. 1B, C). GFP expression was observed in MCs expressing GPM6A which is an MC marker (Fig. 1D). No GPM6A expression was observed in the

Discussion

During liver fibrosis, myofibroblasts actively synthesize collagen and participate in liver fibrosis. Depending on the etiology, genesis of ACTA2+ myofibroblasts varies, e.g., myofibroblasts appear around the central vein in the CCl4 model, whereas around the bile duct in biliary fibrosis induced by BDL and DDC models. Although HSCs seem to be the major source of myofibroblasts in liver fibrosis [5], PFs are suggested to be another source in biliary fibrosis [10], [11], [12], [13], [14]. In

Financial support

This work was supported by NIH grant R01AA020753 (to K.A.), pilot project funding (to K.A.) from P50AA011999, pilot project funding (to K.A.) from P30DK048522, training program (to I.L.) from T32HD060549, U01DK084538 (to K.W.), and the Robert E. and May R. Wright foundation award (to K.A).

Conflict of interest

The authors who have taken part in this study declared that they do not have any conflict of interest with respect to this manuscript.

Authors’ contributions

I. Lua study concept and design; acquisition of data; analysis and interpretation of data; critical revision of the manuscript for important intellectual content; obtained funding. Y. Li acquisition of data; analysis and interpretation of data. J. Zagory acquisition of data; material support. K. Wang acquisition of data; material support. J. Sévigny material support. S. French acquisition of data; analysis and interpretation of data; material support. K. Asahina study concept and design;

Acknowledgements

We thank Drs. David Brenner, Ekihiro Seki, and Yuval Rinkevich for providing the mice and Meng Li for microarray analysis.

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