Research ArticleActivated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice
Graphical abstract
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.
References (35)
- et al.
Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis
J Hepatol
(2006) - et al.
Animal models of biliary injury and altered bile acid metabolism
Biochim Biophys Acta
(2018) - et al.
Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice
Gastroenterology
(2004) - et al.
Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis
J Hepatol
(2016) - et al.
The cholangiopathies: disorders of biliary epithelia
Gastroenterology
(2004) - et al.
Characterization of animal models for primary sclerosing cholangitis (PSC)
J Hepatol
(2014) - et al.
Ursodeoxycholate inhibits mast cell activation and reverses biliary injury and fibrosis in Mdr2
Lab Invest
(2018) The portal fibroblast: not just a poor man’s stellate cell
Gastroenterology
(2014)- et al.
Portal fibroblasts regulate the proliferation of bile duct epithelia via expression of NTPDase2
J Biol Chem
(2005) - et al.
DNase I-hypersensitive sites enhance alpha1(I) collagen gene expression in hepatic stellate cells
Hepatology
(2003)
Characterization of hepatic stellate cells, portal fibroblasts, and mesothelial cells in normal and fibrotic livers
J Hepatol
Fibrillin-1 expression in normal and fibrotic rat liver and in cultured hepatic fibroblastic cells: modulation by mechanical stress and role in cell adhesion
Lab Invest
Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency
J Biol Chem
A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis
Gastroenterology
Liver fibrosis
J Clin Invest
Origin of myofibroblasts in the fibrotic liver in mice
Proc Natl Acad Sci U S A
Origin and function of myofibroblasts in the liver
Semin Liver Dis
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