Elsevier

Journal of Hepatology

Volume 71, Issue 3, September 2019, Pages 573-585
Journal of Hepatology

Research Article
Activated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice

https://doi.org/10.1016/j.jhep.2019.04.012Get rights and content

Highlights

  • Hepatic myofibroblasts progressively accumulate in the livers of Mdr2−/− mice.

  • Myofibroblasts mostly originate from hepatic stellate cells or portal fibroblasts.

  • Meanwhile, fibrocytes minimally contribute to myofibroblasts in Mdr2−/− mice.

  • In addition to collagen production, myofibroblasts serve as a source of NADPH oxidase (NOX).

  • Therapeutic blocking of NOX1/4 ameliorates cholestatic fibrosis in Mdr2−/− mice.

Background & Aims

Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2−/−) mice, a genetic model that resembles primary sclerosing cholangitis in patients.

Methods

Mdr2−/− mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2−/− mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2−/− mice aged 12–16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury.

Results

Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2−/− mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2−/− mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2−/− mice. The development of cholestatic liver fibrosis in Mdr2−/− mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2−/− mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis.

Conclusions

Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2−/− mice, and serve as targets for antifibrotic therapy.

Lay summary

Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2−/− mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2−/− mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

Introduction

Chronic liver injury often results in liver fibrosis. The development of liver fibrosis is associated with migration and proliferation of collagen type I-producing myofibroblasts, which are not present in the normal liver. Activated myofibroblasts originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes.[1], [2], [3], [4] Activated HSCs (aHSCs) were implicated in the pathogenesis of experimental toxic liver fibrosis, such as chronic CCl4 administration and alcoholic liver disease,[1], [5] while PFs are predominantly activated in response to cholestatic liver fibrosis, such as bile duct ligation (BDL).2

Several experimental models of cholestatic liver injury have been developed.6 BDL causes rapid activation of PFs, especially at the onset of injury. Although the pathology resulting from BDL resembles that seen in human chronic cholestatic disease, the surgical stress and the severity of cholestatic injury limit the utility of the BDL model. The multidrug resistance gene 2 knockout (Mdr2−/−, also known as Abcb4−/−) mouse is another well-established model of chronic cholestatic liver injury. Deficiency of Mdr2, a canalicular phospholipid flippase, disrupts biliary phospholipid secretion, leading to the increase of potentially toxic bile acid, which induces hepatocyte damage and cholangiopathy,[7], [8], [9] which is characterized by pericholangitis and onion-skin-type periductal fibrosis, resembling the pathological features of primary sclerosing cholangitis (PSC).[10], [11] Despite extensive studies,[12], [13] the contribution of aHSCs and activated PFs (aPFs) to cholestatic fibrosis in Mdr2−/− mice has not been defined.

Under physiological conditions, quiescent HSCs (qHSCs) reside in the space of Disse (which is located between hepatocytes and sinusoidal endothelial cells),1 store vitamin A, and serve as liver pericytes. QHSCs express specific markers, such as glial fibrillary acid protein (GFAP), synaptophysin, nerve growth factor p75 (NGFR1), and lecithin retinol acyltransferase (Lrat).[2], [14], [15] In response to toxic liver injury, HSCs downregulate the expression of vitamin A in lipid droplets, migrate to the pericentral areas, and transdifferentiate into collagen type I and α-smooth muscle actin (αSMA) expressing myofibroblasts.

Portal fibroblasts, which reside around the portal area and maintain the integrity of the biliary tree and portal tract,16 comprise a small population of fibroblasts in the liver under physiological conditions. In response to cholestatic injury, PFs proliferate, get activated, and contribute to collagen type I deposition. APFs can be distinguished from aHSCs by expression of Thy1, Fibulin2, Elastin, Gremlin1, ecto-ATPase nucleoside triphosphate diphosphohydrolase 2, mesothelin (Msln), and mucin 16 (Muc16),[2], [14], [16], [17] and the lack of HSC markers.

Therapeutic approach to cholestatic fibrosis remains challenging; hence, liver transplantation is the only effective therapy for patients with late-stage PSC.18 The activity of NADPH oxidase (NOX), an enzyme system that catalyzes the reduction of molecular oxygen to superoxide, plays an important role in the activation of HSCs and the development of hepatic fibrosis,[19], [20], [21], [22] and treatment with a NOX1/4 dual inhibitor decreases both CCl4-induced hepatotoxic fibrosis and BDL-induced cholestatic fibrosis.23

Here, we characterize the development of cholestatic fibrosis in Mdr2−/− mice. We determined that aPFs and aHSCs (but not fibrocytes) contribute to hepatic myofibroblasts in Mdr2−/− mice during the progression of cholestatic fibrosis. Increased expression of fibrogenic genes was associated with the upregulation of Thy1 and CD34 in aPFs. Although aPFs were in close proximity to proliferating cholangiocytes, cholangiocytes themselves did not express collagen type I. Hepatic expression of NOX was induced in Mdr2−/− mice only upon the development of liver fibrosis. Therapeutic inhibition of NOX1/4 in Mdr2−/− mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

Section snippets

Mice

BALB/c-Mdr2−/− mice (gift of Dr. Frank Lammert)24 were crossed with collagen-α1(I)-GFP (ColGFP) mice.25 LratCre mice (gift of Dr. Robert Schwabe)15 were crossed with Rosa26flox-stop-flox-YFP reporter mice (The Jackson Laboratory, CA, USA). The mice were housed and maintained under specific pathogen-free conditions in a standard environment with a 12-hour light–dark cycle, and fed a diet of normal chow ad libitum, at the animal facilities of University of California San Diego under protocol

Development of fibrosis in Mdr2−/− mice is associated with inflammation, oxidative stress, and ductular reaction

Progression of cholestatic liver fibrosis was analyzed in 4-, 8-, 12-, and 16-week-old Mdr2−/− mice. Extracellular matrix (ECM) deposition was observed in livers of Mdr2−/− mice as early as 4 weeks of age, and reached maximum at 12–16 weeks, as demonstrated by Sirius Red staining (Fig. 1A and B), which was accompanied by expression of fibrogenic genes (Col1a1, αSMA, Desmin, TIMP1, and TGFβR1 mRNA; Fig. 1C) and inflammatory genes (F4/80, IL-1β, and IL-6 mRNA; Fig. 1D). Age-dependent ECM

Discussion

This study demonstrates that both aPFs and aHSCs contribute to the pathogenesis of cholestatic fibrosis in Mdr2−/− mice. APFs were mostly localized in the portal areas, while aHSCs were located in the portal, sinusoidal, and capsular areas. Consistent with a common etiology of cholestatic injury, Thy1+ColGFP+ aPFs in Mdr2−/− mice exhibited a phenotype similar to that observed in aPFs in the BDL model.14 APFs were located in close proximity to proliferating bile ducts, but cholangiocytes

Financial support

This study was supported by the National Institutes of Health R01 DK101737-01A1, U01 AA022614-01A1, R01 DK099205-01A1, P50AA011999, and AI043477 (TK and DAB), and the Herman Lopata Memorial Hepatitis Postdoctoral ALF Fellowship (JX).

Conflict of interest

The authors declare no conflict of interest.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

TN performed the experiments, collected and analyzed the data, and wrote the manuscript. RH, SL, YK, GY, DK, JB, H-YM, JX, XL, and DD performed the experiments. SBR analyzed the data. KI and KT provided support with data collection. TK and DAB provided support, designed the study, and wrote the manuscript.

Acknowledgements

The authors thank Philippe Wiesel and Cedric Szyndralewiez (Genkyotex S.A.) for providing GKT137831, Dennis R. Petersen for providing the antibody against 4-HNE, Ryan McCubbin and Katrin Hochrath (University of California San Diego, La Jolla, CA, USA) for their help with in vivo experiments, and Karin Diggle (University of California San Diego) for her excellent management of the laboratory.

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