Telomere dysfunction promotes cholangiocyte senescence and biliary fibrosis in primary sclerosing cholangitis.

Cellular senescence and biliary fibrosis are prototypical features of obliterative cholangiopathies, such as primary sclerosing cholangitis (PSC). Telomere dysfunction can lead to senescence either through telomere erosion or damaged telomeres. Our goal was to investigate a mechanistic relationship between telomere damage and biliary fibrosis in PSC. Telomere attrition was observed in the bile ducts of patients with PSC along with a reduction in telomerase reverse transcriptase (TERT) expression, compared with that in normal livers. Similarly, liver tissue from mouse models of biliary fibrosis showed telomere attrition with increased damage at telomeres measured as telomere-associated foci (TAF). Cellular models of senescence induction increased the TAF in cholangiocytes. This coincided with decreased TERT expression and increased senescence, which was rescued by modulating TERT levels. Epigenetic analysis revealed increased acquisition of repressive histone methylation at the TERT promoter, which correlated with decreased TERT transcription. Cholangiocyte-selective deletion of TERT in mice exacerbated fibrosis, whereas androgen therapy toward telomerase rescued liver fibrosis and liver function in a genetic mouse model of PSC. Our results demonstrate a mechanistic role for telomere dysfunction in cellular senescence and fibrosis that characterize PSC. This suggests that PSC may be, in part, a telomere biology disorder, and identifies TERT as a potential therapeutic target.

Biodegradable biliary stents coated with mesenchymal stromal cells in a porcine choledochojejunostomy model.

BACKGROUND AIMS: Roux en y anastomosis is a preferred method of biliary reconstruction in liver transplantation that involves living donors or pediatric patients. However, biliary stricture is a frequent and serious complication, accounting for up to 40% of biliary complications in these patients. Previously, we demonstrated that extraluminal delivery of adipose-derived (AD) mesenchymal stromal cells (MSCs) decreased peri-biliary fibrosis and increased neo-angiogenesis in a porcine model of duct-to-duct biliary anastomosis. In this study, we used a porcine model of Roux en y anastomosis to evaluate the beneficial impact of a novel intraluminal MSC delivery system. METHODS: Nine animals were divided into three groups: no stent (group 1), bare stent (group 2) and stent coated with AD-MSCs (group 3). All animals underwent cholecystectomy with roux en y choledochojejunostomy. Two animals per group were followed for 4 weeks and one animal per group was followed for 8 weeks. Cholangiograms and blood were sampled at baseline and the end of study. Biliary tissue was collected and examined by Masson trichrome staining and immunohistochemical staining for MSC markers (CD34 and CD44) and for neo-angiogenesis (CD31). RESULTS: Two of three animals in group 1 developed an anastomotic site stricture. No strictures were observed in the animals of group 2 or group 3. CD34 and CD44 staining showed that AD-MSCs engrafted successfully at the anastomotic site by intraluminal delivery (group 3). Furthermore, biliary tissue from group 3 showed significantly less fibrosis and increased angiogenesis compared with the other groups. CONCLUSIONS: Intraluminal delivery of AD-MSCs resulted in successful biliary engraftment of AD-MSCs as well as reduced peri-biliary fibrosis and increased neo-angiogenesis.

Organoids and regenerative hepatology.

The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions.

Traf2 and NCK Interacting Kinase Is a Critical Regulator of Procollagen I Trafficking and Hepatic Fibrogenesis in Mice.

Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress-induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence-based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase-interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB-0846 or RNA interference-mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB-0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB-0846 displayed reduced CCl4 -induced fibrogenesis compared to CCl4 alone, although α-smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo.

Induced Pluripotent Stem Cells From Subjects With Primary Sclerosing Cholangitis Develop a Senescence Phenotype Following Biliary Differentiation.

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient-specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three-dimensional matrigel-based culture to induce formation of cholangioids. RNA sequencing (RNA-seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA-seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three-dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC-derived cholangioids. Congruent with this, PSC-derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin-6, and chemokine (C-C motif) ligand 2. Conditioned media (CM) from PSC-derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease-specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids.

Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex.

BACKGROUND & AIMS: Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. METHODS: We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. RESULTS: BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. CONCLUSION: Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. LAY SUMMARY: We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-ß Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.

Hepatic stellate cell activation promotes alcohol-induced steatohepatitis through Igfbp3 and SerpinA12.

BACKGROUND & AIMS: Steatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice. METHODS: Mice with HSC-selective deletion of NRP (ColCre/Nrp1loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1loxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated. RESULTS: ColCre/Nrp1loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/Nrp1loxP mice compared to supernatant from Nrp1loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis. CONCLUSION: Selective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling. LAY SUMMARY: Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

Autologous Adipose Tissue-Derived Mesenchymal Stem Cells Introduced by Biliary Stents or Local Immersion in Porcine Bile Duct Anastomoses.

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose-derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC-seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin-eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose-derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

Author Correction: Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Epigenetic Mechanisms of Pancreatobiliary Fibrosis.

PURPOSE OF REVIEW: The goal of this manuscript is to review the current literature related to fibrogenesis in the pancreatobiliary system and how this process contributes to pancreatic and biliary diseases. In particular, we seek to define the current state of knowledge regarding the epigenetic mechanisms that govern and regulate tissue fibrosis in these organs. A better understanding of these underlying molecular events will set the stage for future epigenetic therapeutics. RECENT FINDINGS: We highlight the significant advances that have been made in defining the pathogenesis of pancreatobiliary fibrosis as it relates to chronic pancreatitis, pancreatic cancer, and the fibro-obliterative cholangiopathies. We also review the cell types involved as well as concepts related to epithelial-mesenchymal crosstalk. Furthermore, we outline important signaling pathways (e.g., TGFß) and diverse epigenetic processes (i.e., DNA methylation, non-coding RNAs, histone modifications, and 3D chromatin remodeling) that regulate fibrogenic gene networks in these conditions. We review a growing body of scientific evidence linking epigenetic regulatory events to fibrotic disease states in the pancreas and biliary system. Advances in this understudied area will be critical toward developing epigenetic pharmacological approaches that may lead to more effective treatments for these devastating and difficult to treat disorders.

Proteasomal Degradation of Enhancer of Zeste Homologue 2 in Cholangiocytes Promotes Biliary Fibrosis.

During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor ß (TGF-ß). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-ß signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-ß. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-ß response. EZH2 overexpression suppressed TGF-ß-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-ß. TGF-ß also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-ß. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-ß-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2-/- mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-ß regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.

Cholangiocyte pathobiology.

Cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts, are highly specialized cells residing in a complex anatomic niche where they participate in bile production and homeostasis. Cholangiocytes are damaged in a variety of human diseases termed cholangiopathies, often causing advanced liver failure. The regulation of cholangiocyte transport properties is increasingly understood, as is their anatomical and functional heterogeneity along the biliary tract. Furthermore, cholangiocytes are pivotal in liver regeneration, especially when hepatocyte regeneration is compromised. The role of cholangiocytes in innate and adaptive immune responses, a critical subject relevant to immune-mediated cholangiopathies, is also emerging. Finally, reactive ductular cells are present in many cholestatic and other liver diseases. In chronic disease states, this repair response contributes to liver inflammation, fibrosis and carcinogenesis and is a subject of intense investigation. This Review highlights advances in cholangiocyte research, especially their role in development and liver regeneration, their functional and biochemical heterogeneity, their activation and involvement in inflammation and fibrosis and their engagement with the immune system. We aim to focus further attention on cholangiocyte pathobiology and the search for new disease-modifying therapies targeting the cholangiopathies.

3D Printing for Bio-Synthetic Biliary Stents.

Three-dimensional (3D) printing is an additive manufacturing method that holds great potential in a variety of future patient-specific medical technologies. This project validated a novel crosslinked polyvinyl alcohol (XL-PVA) 3D printed stent infused with collagen, human placental mesenchymal stem cells (PMSCs), and cholangiocytes. The biofabrication method in the present study examined 3D printing and collagen injection molding for rapid prototyping of customized living biliary stents with clinical applications in the setting of malignant and benign bile duct obstructions. XL-PVA stents showed hydrophilic swelling and addition of radiocontrast to the stent matrix improved radiographic opacity. Collagen loaded with PMSCs contracted tightly around hydrophilic stents and dense choloangiocyte coatings were verified through histology and fluorescence microscopy. It is anticipated that design elements used in these stents may enable appropriate stent placement, provide protection of the stent-stem cell matrix against bile constituents, and potentially limit biofilm development. Overall, this approach may allow physicians to create personalized bio-integrating stents for use in biliary procedures and lays a foundation for new patient-specific stent fabrication techniques.

Enhancer of Zeste Homologue 2 Inhibition Attenuates TGF-ß Dependent Hepatic Stellate Cell Activation and Liver Fibrosis.

BACKGROUND & AIMS: Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor ß (TGF-ß) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-ß and PDGF signaling pathways. METHODS: We performed a transcriptomic comparison of HSCs treated with TGF-ß or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators. RESULTS: Enhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-ß but not with PDGF-BB. Indeed, EZH2 inhibition using either a pharmacologic (GSK-503) or a genetic (small interfering RNA) approach caused a significant attenuation of TGF-ß-induced fibronectin, collagen 1α1, and α-smooth muscle actin, both at messenger RNA and protein levels. Conversely, adenoviral overexpression of EZH2 in HSCs resulted in a significant stimulation of fibronectin protein and messenger RNA levels in TGF-ß-treated cells. Finally, we conducted in vivo experiments with mice chronically treated with carbon tetrachloride or bile duct ligation. Administration of GSK-503 to mice receiving either carbon tetrachloride or bile duct ligation led to attenuated fibrosis as assessed by Trichrome and Sirius red stains, hydroxyproline, and α-smooth muscle actin/collagen protein assays. CONCLUSIONS: TGF-ß and PDGF share redundant and distinct transcriptomic targets, with the former predominating in HSC activation. The EZH2 histone methyltransferase is preferentially involved in the TGF-ß as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-ß-treated HSCs and reduces liver fibrosis in vivo. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE119606 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119606).