^{Division of Gastroenterology and Hepatology, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea;}

^{Department of Hepatology, Qilu Hospital Shandong University, Shandong 250012, China;}

^{Innovation Center for Immunoregulation and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan;}

^{MedImmune Ltd., Cambridge CB21 6GH, United Kingdom; and}

^{Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan}

^{To whom correspondence should be addressed. Email:}ude.dscu@avelessikt.

Edited^* by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved June 23, 2014 (received for review January 6, 2014)

Author contributions: K.I., C.J., M.Z., D.A.B., and T.K. designed research; K.I., C.J., M.Z., M.C., T.J.M.-M., T.J.P., X.L., J.X., P.W., Y.-H.P., F.M., L.A.M., and T.K. performed research; M.A., A.F.H., T.I., C.K.G., and D.A.B. contributed new reagents/analytic tools; K.I., C.J., M.Z., M.C., T.J.M.-M., T.J.P., X.L., J.X., Y.-H.P., F.M., M.A., and C.K.G. analyzed data; and D.A.B. and T.K. wrote the paper.

^{K.I., C.J., and M.Z. contributed equally to this work.}

Edited^* by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved June 23, 2014 (received for review January 6, 2014)

Freely available online through the PNAS open access option.

Significance

Liver resident activated hepatic stellate cells (aHSCs), and activated portal fibroblasts (aPFs) are the major source of the fibrous scar in the liver. aPFs have been implicated in liver fibrosis caused by cholestatic liver injury, whereas fibrosis in hepatotoxic liver injury is attributed to aHSCs. However, the contribution of aPFs to cholestatic fibrosis is not well characterized because of difficulties in cell purification and the lack of identified aPF-specific markers. We have developed a novel flow cytometry-based method of aPFs purification from the nonparenchymal cell fraction of collagen-α1(I)-GFP mice and have identified potential aPF-specific markers. The goal of this study is to determine whether aPFs contribute to cholestatic liver fibrosis and identify the mechanism(s) of their activation.

Keywords: ECM deposition, markers of fibrogenic myofibroblasts

Significance

Abstract

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl₄) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP^{cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl}₄ liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Abstract

Chronic liver injury of many etiologies results in liver fibrosis. There are two general types of chronic liver diseases, hepatocellular (injury to hepatocytes, such as chronic viral hepatitis and nonalcoholic steatohepatitis) and cholestatic (obstruction to bile flow, such as primary biliary cirrhosis and primary sclerosing cholangitis) (1). Experimental rodent models of liver fibrosis mimic these two types of chronic liver injuries: Repeated carbon tetrachloride (CCl₄) administration produces hepatocelluar injury, and common bile duct ligation (BDL) produces cholestatic injury (2). In all chronic liver diseases, myofibroblasts are embedded in the fibrous scar and are the source of this excessive extracellular matrix (ECM). Myofibroblasts, which are not present in normal liver, are characterized by distinct morphology, contractility with intracellular stress fibers [α-smooth muscle actin (α-SMA), nonmuscle myosin, and vimentin], and secretion of extracellular matrix (fibronectin and fibrillar collagens) (1, 2).

The cells of origin of hepatic myofibroblasts are unresolved, and perhaps the fibrosis induced by different types of liver injury results from different fibrogenic cells. Hepatic myofibroblasts may originate from bone marrow (BM)-derived mesenchymal cells and fibrocytes, but only a small contribution of BM-derived cells to the myofibroblast population has been detected in experimental liver fibrosis (3 –5). Another potential source of myofibroblast is epithelial-to-mesenchymal transition (EMT), in which epithelial cells acquire a mesenchymal phenotype and may give rise to fully differentiated myofibroblasts. However, recent cell fate mapping studies have failed to detect any hepatic myofibroblasts originating from hepatocytes, cholangiocytes, or epithelial progenitor cells (3, 6 –10). Thus, the major sources of myofibroblasts in liver fibrosis are the endogenous liver mesenchymal cells, which consist of portal fibroblasts and hepatic stellate cells.

Quiescent hepatic stellate cells (qHSCs) are located in the space of Disse, store retinoids in lipid droplets, and express neural markers, such as glial fibrillary acidic protein (GFAP), synaptophisin, and nerve growth factor receptor p75 (1). In response to injury, qHSCs down-regulate vitamin A-containing lipid droplets and neural markers, and differentiate into α-SMA–expressing myofibroblasts (1, 2). Portal fibroblasts normally comprise a small population of the fibroblastic cells that surround the portal vein to maintain integrity of portal tract. They were first described as “mesenchymal cells not related to sinusoids,” and since then have been called “periductular fibroblasts” or portal/periportal mesenchymal cells” (11) and implicated by association in the pathogenesis of cholestatic liver injury. In response to chronic injury, portal fibroblasts may proliferate, differentiate into α-SMA–expressing myofibroblasts, and synthesize extracellular matrix (11 –14).

The contribution of portal fibroblasts (PFs) to liver fibrosis of different etiologies is not well understood, mainly because of difficulties in isolating PFs and myofibroblasts. The most widely used method of PF isolation from rats is based on liver perfusion with enzymatic digestion followed by size selection (15). Cell outgrowth from dissected bile segments is still used to isolate mouse PFs, and after 10–14 d in culture, PFs undergo progressive myofibroblastic activation (16). The disadvantage of this technique is that it requires multiple passaging and prolong culturing (11). A more physiological method of PF culturing in a precision-cut liver slice is designed to maintain cell–cell and cell–matrix interactions and mimic natural microenvironment of PFs, but it does not enable the study of purified PFs (17). Therefore, only a few markers of PFs are available to identify PFs in the myofibroblast population, including gremlin, Thy1, fibulin 2, interleukin 6 (IL-6), elastin, the ecto-AT-Pase nucleoside triphosphate diphosphohydrolase-2 (NTPD2), and coffilin 1. In addition, the lack of desmin, cytoglobin, α2-macroglobulin, neural proteins (GFAP, p75, synaptophysin), and lipid droplets distinguishes PFs from HSCs (1, 17 –21).

Our study uses transgenic reporter mice and new flow cytometry protocols to identify the origin of myofibroblasts and quantify their numbers in two murine models of chronic liver injury (BDL and CCl₄). Our study demonstrates that the origin of the myofibroblasts is determined by the type of liver injury. As previously reported using other methodologies, HSCs are the major source of myofibroblasts in CCl₄ liver injury. In contrast, most of the myofibroblasts at the onset of BDL-induced liver injury originate from activated PFs (aPFs).

Using the whole mouse genome microarray, expression of signature genes was determined for BDL-aPFs. Expression of genes previously identified as PF-specific (underlined) was confirmed. Fold induction (compared with the highest value observed in BDL- or CCl₄-aHSCs) is shown for each gene. Full list of genes is shown in Fig. S7.

Click here to view.

Acknowledgments

We thank Dr. Sato and Dr. Uehiro for initial technical assistance with laser capture microdissection and Tom Kisby and Rebecca Dunmore for technical assistance with the in vitro studies using human HSCs. This work was supported by National Institutes of Health Grants DK088837, GM41804, AA15055, DK72237, AI0777802, and P50 AA011999; the Japanese Ministry of Health, Labour, and Welfare; and the American Liver Foundation.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400062111/-/DCSupplemental.

Footnotes

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