Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes

Abstract

Background:

Cholestatic liver diseases, including primary sclerosing cholangitis, are characterized by biliary fibroinflammation. TGFβ-activated cholangiocytes release signals that recruit immune cells and activate myofibroblasts, promoting inflammation and extracellular matrix (ECM) deposition. TGFβ also regulates stearoyl-CoA desaturase (SCD), an enzyme involved in lipid signaling. Yet, the role of SCD or its inhibitor, Aramchol, in biliary fibroinflammation had not been studied.

Methods and Results:

Mdr2^-/- with established biliary fibrosis and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet-fed mice were treated with Aramchol meglumine (12.5 mg/kg/day). Hepatic fibrosis was assessed by qPCR, Picrosirius red staining, immunofluorescence, and hydroxyproline content. Human H69 or murine large cholangiocyte cell lines stimulated with TGFβ, as well as PSC-derived cholangiocytes (PSC-C), were treated with Aramchol or SCD siRNA. RNA-seq, fibroinflammatory marker expression, peroxisome proliferator-activated receptor (PPAR) activity, and targeted fatty acid profiling were performed. Aramchol treatment significantly reduced hepatic ECM gene expression, inflammatory cytokines (Il6,Tnfa), collagen content, and myofibroblast activation (aSMA staining) in both mouse models. In TGFβ-stimulated H69 cells, Aramchol suppressed hepatic fibrosis pathways and enhanced PPAR signaling. Aramchol also reduced the expression of fibrotic markers, myofibroblast-activating mediators (VEGFA and PDGFB), and IL6, mirroring the effects of SCD knockdown. In PSC-C, Aramchol significantly downregulated SCD, VEGFA and IL6. Conversely, PPARα and -γ activity and fatty acid agonist, linoleic acid levels were increased in cholangiocyte cell lines.

Conclusions:

Aramchol attenuates and prevents biliary fibrosis in mouse models of cholestatic liver disease by inhibiting TGFβ-induced fibroinflammatory mediators and activating PPARa/γ in cholangiocytes. These findings, combined with its favorable clinical safety profile, support the potential of Aramchol as a therapeutic candidate for PSC.

INTRODUCTION

Biliary fibrosis is the predominant pathological process in fibroinflammatory cholangiopathies, notably primary sclerosing cholangitis (PSC) and primary biliary cholangitis. These diseases collectively account for at least 16% of liver transplantation performed in the United States, with an annual cost of $400 million. ¹ Yet, there are no approved treatments for biliary fibrosis, and late-stage cholangiopathies typically require liver transplantation.

A key pathway of fibrosis is TGFβ signaling, a potent activator of hepatic stellate cells (HSCs) and portal fibroblasts into a myofibroblast-like phenotype that secretes extracellular matrix (ECM). ² Immune cells, such as macrophages and cholangiocytes, are the major sources of secreted TGFβ,^3,4 which by autocrine or paracrine mechanisms stimulate cholangiocytes themselves. Cholangiocytes activated by TGFβ recruit transcriptomic regulators, including epigenetic enzymes, which result in the expression of fibroinflammatory signals.^5–7 Activated cholangiocytes secrete TGFβ, inflammatory mediators such as IL6, and other fibrogenic factors such as plasminogen activator inhibitor 1 (PAI-1/SERPINE1), further propagating the fibroinflammatory signaling.^8–10

TGFβ also regulates cellular metabolic and lipid profiles.^11,12 However, how these cellular metabolic changes enable TGFβ-mediated signaling in biliary fibrosis is not completely elucidated. In epithelial cells, TGFβ upregulates the expression of stearoyl-CoA desaturase-1 (SCD),^11–13 an enzyme that catalyzes the rate-limiting step in the formation of mono-unsaturated fatty acids, notably palmitoleate (C16:1n-7) and oleate (C18:1n-9) from palmitic acid (C16:0) and stearic acid (C18:0). SCD has multiple isoforms in mice, SCD1-4, and 2 isoforms in humans (SCD and SCD5). These isoforms share a similar structure and amino acid sequence, but have divergent tissue-specific gene expression. ¹⁴ Mouse Scd1 and Scd2 appear to have important functions in the liver.^15,16 In humans, SCD is the predominant isoform in many organs, including the liver, although SCD5 is also detectable in hepatocytes and pancreas but not cholangiocytes (https://www.proteinatlas.org/ENSG00000145284-SCD5/tissue). ¹⁷ SCD substrates and products have demonstrated both proinflammatory and anti-inflammatory effects.^18–21 In addition, SCD suppresses peroxisome proliferator–activated receptor (PPAR)-γ in various cells.^11,13 These observations suggest cell-specific signaling and require further investigation of SCD in cholangiocytes.

Arachidyl-amido cholanoic acid (Aramchol) was previously shown to both downregulate SCD and upregulate PPARγ expression.^22,23 Aramchol has demonstrated antifibrotic effects in HSCs and hepatocytes, thereby regulating markers of fibrosis in mouse models of metabolic dysfunction–associated steatohepatitis (MASH).^22–24 In clinical trials of MASH, Aramchol demonstrated beneficial effects on steatohepatitis and fibrosis with an excellent safety profile.^25,26 However, the role of Aramchol in cholangiocyte signaling and biliary fibrosis was not previously assessed. In this study, we demonstrate that Aramchol significantly inhibited fibrosis and inflammatory markers in 2 mouse models of biliary fibrosis. Furthermore, we showed that Aramchol inhibited the TGFβ-stimulated fibroinflammatory signals in cholangiocytes while upregulating PPARα/γ activity. Thus, this study provides the rationale for assessing Aramchol in clinical trials of human diseases driven by biliary fibrosis, particularly PSC.

METHODS

Mouse studies

Multidrug resistance 2 knockout (Mdr2^−/−) mice on C57BL/6J background were kindly provided by Dr Daniel Goldenberg (Hadassah-Hebrew University Medical Center, Jerusalem, Israel). For in vivo studies, the salt form of Aramchol, Aramchol meglumine, was used. While Aramchol acid has been used in MASH mouse models, ²⁴ the Aramchol meglumine formulation has not been previously tested in liver disease models. The dose of Aramchol meglumine used in our mouse models is based on unpublished studies of pulmonary fibrosis and colitis mouse models, and preclinical pharmacodynamics of Aramchol meglumine. This dose provides roughly similar moles of Aramchol acid in mice (13.9 µmol/kg) compared with clinical studies in humans (12.25 µmol/kg).^25,26 Male and female 10–16-week-old Mdr2^−/− mice were administered Aramchol meglumine dissolved in water at 12.5 mg/kg or vehicle only by oral gavage daily for 4 weeks. At the end of the experiment, mice were euthanized, and the liver and blood were harvested.

Similarly, wild-type female and male mice were placed on regular chow or 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet for 3 weeks with 1-day breaks of regular chow every week. All mice were orally gavaged with Aramchol meglumine or vehicle as described above.

All mice were housed under 12–12-hour light and dark cycles with water and standard chow ad libitum when not on the DDC diet. All mouse experiments were conducted in strict accordance with protocols approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

Hydroxyproline measurement

The hydroxyproline content of mouse livers was quantified using mass spectrometry by the VCU Lipidomics and Metabolomics core. Briefly, AUCs of hydroxyproline standards were used to generate a standard curve, which was used to measure the concentration of samples. Sample measurements were normalized to their weights (30–50 mg).

Immunofluorescence and imaging

Cells were fixed with formalin for 15 minutes. De-paraffinized liver tissue or cell slides were blocked using Intercept Blocking Buffer (LI-COR #927-70001), followed by overnight incubation with the primary antibody, followed by species-specific fluorophore-conjugated secondary antibodies. Slides were imaged with Axio Observer A1 inverted (Zeiss) and Keyence BZ-X910 microscopes. Mouse liver slides were imaged at 10× and stitched together for further analysis using ImageJ software for area quantification.

RNA-sequencing

RNA sequencing and bioinformatics analysis was conducted with the VCU Genomics and Bioinformatics cores. RNA quality control was performed using Agilent Bioanalyzers. Pathway analyses were performed by Ingenuity Pathway Analysis (Qiagen).

Cell culture

Transformed cholangiocyte cell line, H69 cells, were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, adenine, insulin, epinephrine, T3-T, hydrocortisone, and EGF. PSC patient–derived cholangiocytes (PSC-Cs) were kindly provided by Dr. Nicholas LaRusso (Mayo Clinic) and cultured in the H69 media.^27,28 Mouse large biliary epithelial cells (MLEs) were cultured in complete DMEM media.^29,30 Cells were serum-starved before treatment with 10 ng/mL recombinant TGFβ (R&D Systems #240-B) for 16 hours. For siRNA targeting, cells were transfected with SCD or control siRNAs (Dharmacon, ON-TARGETplus SMARTPool) using the Oligofectamine reagent (Invitrogen #12–252-011). Aramchol acid (Galmed Pharmaceutical) was dissolved in DMSO first, and cells were treated at the indicated doses. For HSC activation, cholangiocytes were stimulated with TGFβ with and without Aramchol overnight, media removed, cells washed 3×, and fresh serum media replaced for another overnight incubation to yield the cholangiocyte conditioned media (CM). Human HSCs (ScienCell) were serum-starved and then incubated overnight with the cholangiocyte CM. For some experiments, nuclear extracts were isolated using a nuclear isolation kit (Cayman Chemical, Cat# 10009277). Nuclear extracts were used to show PPARγ and PPARα binding to PPAR responsive elements (PPREs) using colorimetric kits (Abcam ab133107; Cayman Chemical Cat# 10006855).

Quantitative RT-PCR

Total RNA was extracted from cells using the TRIzol RNA Isolation Reagent (ThermoFisher, 15596026). Reverse transcription was performed with 2 μg RNA using oligo (dT) primer and SuperScript III. Real-time PCR was performed using Sybr Green Master Mix made to a 20 μL final volume per reaction and the QuantStudio 3 Real-Time PCR System (Applied Biosystems) using primer sequences shown in Supplemental Table S1, http://links.lww.com/HC9/C28

ELISA

Mouse and human IL6 ELISA kits with standards were purchased from eBioscience (Human Cat# 88-7066-88, Mouse Cat# 88-7064-88). IL6 was quantified in the cell culture media following the manufacturer's protocols.

Immunoblotting

Cells were lysed, and protein was isolated in RIPA buffer. Protein concentration was determined using the BCA protein assay. Cell-cultured media were briefly spun down before immunoblotting. Protein (10–40 μg) was loaded onto either 4%–20% or 10% Tris-Glycine gels, electrophoresed, and transferred onto nitrocellulose membranes (Scientific Laboratory Supplies) for blotting. The membrane was blocked, incubated with primary antibodies, rinsed with Tris-buffered saline with Tween, and incubated with fluorophore-conjugated secondary antibodies (Supplemental Table S1, http://links.lww.com/HC9/C28). After washing, the membrane was imaged using the iBright 1500 imaging system (Invitrogen).

Extraction and liquid chromatography-tandem mass spectrometry of fatty acids

Cholangiocytes were extracted in 2 mL methanol along with 5 ng of each internal standard, followed by ultrasonication for 30 seconds at room temperature. One milliliter of chloroform was added and incubated at 48 °C overnight. The extract underwent centrifugation, dried using a speed vacuum and reconstituted in 0.3 mL of the starting mobile phase solvent for liquid chromatography-tandem mass spectrometry. A Shimadzu Nexera LC-30 AD binary pump system coupled to a SIL-30AC autoinjector and DGU20A_5R degasser coupled to a Sciex 5500 quadrupole/linear ion trap (QTrap) (SCIEX Framingham) operating in a triple quadrupole mode was used. A 14-minute reversed-phase LC method utilizing a Spelco Acentis Express C18 column (100 × 2.1 mm, 1.7 µm) and a Shimadzu UPLC was used to separate the eicosanoids at a flow rate of 500 µL/min at 50 °C. The column was first equilibrated with 100% Solvent A (acetonitrile: water: formic acid [20:80:0.02]) for 2 minutes and then 10 µL of sample was injected. Solvent A (100%) was used for the first 2 minutes of elution. Solvent B (acetonitrile: isopropanol: formic acid [20:80:0.02]) was increased in a linear gradient to 25% solvent B by 3 minutes, to 30% by 6 minutes, to 55% by 6.1 minutes, to 70% by 10 minutes, and to 100% by 10.1 minutes. Solvent B (100%) was held for 13 minutes, then decreased to 0% by 13.1 minutes and held at 0% for 14 minutes. The eluting eicosanoids were analyzed using a hybrid triple quadrupole linear ion trap mass spectrometer (Sciex 5500 QTRAP) through multiple-reaction monitoring in negative-ion mode. Eicosanoids were monitored using species-specific precursor/product ion MRM pairs. The mass spectrometer parameters were: curtain gas: 30 psi; CAD: High; ion spray voltage: −3500 V; temperature: 300 °C; Gas 1: 40; Gas 2: 60; declustering potential, collision energy, and cell exit potential were optimized per transition.

Statistical analysis

Data are presented as mean with SD. One-way ANOVA with Tukey’s Honestly Significant Difference posttest was used to assess the statistical significance between ≥3 groups with p value<0.05 in 2-tailed analyses. Where data did not have a normal distribution, 1-way ANOVA on ranks (Kruskal-Wallis test) with post hoc Dunn’s test was used. The Student t test was used in 2 group analyses.

RESULTS

Aramchol meglumine downregulates fibrosis and inflammation in mouse models of biliary fibrosis

Aramchol reduced steatosis and fibrosis in a dietary mouse model of MASH. ²⁴ We assessed the effect of Aramchol on fibrosis and inflammation in 2 mouse models of biliary fibrosis. First, in a fibrosis prevention model, age- and sex-matched wild-type mice (10–16 wk old) were placed on the DDC diet with a weekly 1-day break on regular chow, for a total of 3 weeks. Mice received Aramchol meglumine at 12.5 mg/kg/d or vehicle (water) daily by oral gavage. DDC diet resulted in a significant decrease in weight, but there was no significant difference between the Aramchol meglumine and vehicle groups (Supplemental Figure S1A, http://links.lww.com/HC9/C28). In addition, there were no significant changes observed in liver chemistries, including serum hepatic injury markers (ALP, ALT, and AST) or hepatic function tests (total bilirubin and albumin) (Supplemental Figure S1B, http://links.lww.com/HC9/C28). The increase in fibrosis was significantly attenuated with Aramchol meglumine administration as demonstrated by significantly reduced hydroxyproline content (Figure 1A). Consistently, the mRNA expressions of Col1a, αSma/Acta2, and Serpine1 were significantly reduced (Figure 1B). While significant changes in the mRNA expression of Tgfb, Ppara, and Pparg, regulators of profibrosis and antifibrosis pathways, were seen in mice on the DDC diet compared with chow, there were no significant changes in Aramchol meglumine–treated mice compared with vehicle on the DDC diet (Supplemental Figure 2A, http://links.lww.com/HC9/C28). However, canonical target genes of TGFβ signaling, such as TGFβ-induced, Tgfbi, in addition to Serpine1 and markers of fibrosis, were significantly reduced with Aramchol meglumine treatment in the liver of mice on the DDC diet (Supplemental Figure S2A, http://links.lww.com/HC9/C28). These findings suggest that, although TGFβ expression is not affected, Aramchol treatment attenuates downstream TGFβ signaling. Similarly, histological analysis with picrosirius red staining and quantification of positively stained area was significantly reduced with Aramchol meglumine treatment (Figures 1C and D). The DDC diet–induced markers of inflammation, Il6, Tnfa, and Nfkb, and a marker of macrophages, Cd68, were all significantly reduced with Aramchol meglumine treatment (Figure 1E and Supplemental Figure S2B, http://links.lww.com/HC9/C28). Myeloperoxidase (Mpo), a marker of neutrophils, was significantly increased in the liver of mice on the DDC diet, but there was no significant difference with Aramchol meglumine treatment compared with vehicle (Supplemental Figure S2C, http://links.lww.com/HC9/C28). αSma, a marker of activated myofibroblasts, staining of liver sections by immunofluorescence was significantly reduced in Aramchol meglumine–treated mice compared with vehicle only on the DDC diet (Figure 1F). Markers of quiescent HSCs (Reln and Lrat) were upregulated with the DDC diet, with Reln significantly reduced and no change in Lrat expression with Aramchol meglumine treatment (Supplemental Figure S2D, http://links.lww.com/HC9/C28). CK19 immunostaining of liver sections was significantly increased with the DDC diet, which was significantly attenuated in mice treated with Aramchol meglumine (Figure 1F and Supplemental Figure S2E, http://links.lww.com/HC9/C28). Sox9, another cholangiocyte marker, expression was similarly increased with the DDC diet and attenuated with Aramchol meglumine treatment (Supplemental Figure S2F, http://links.lww.com/HC9/C28). These observations indicate that the increase in biliary mass with the DDC diet is significantly attenuated with the treatment of Aramchol meglumine.

FIGURE 1.

(A) The increased liver content of hydroxyproline caused by the DDC diet was significantly reduced with Aramchol meglumine treatment (shortened to Aramchol in the figures) compared with vehicle only. (B) qPCR analysis demonstrated Aramchol meglumine significantly inhibited the DDC diet–induced increase in ECM components, collagen (Col1a) and alpha smooth muscle actin (αSMA), and profibrotic markers Serpine1. (C) The DDC diet–induced increased picrosirius staining of liver sections was significantly attenuated with Aramchol meglumine treatment, with area quantification shown in (D). (E) The DDC diet–induced increased expression of inflammatory markers, Il6, master regulator of inflammation, Nfkb, and macrophage marker, Cd68, was significantly inhibited with Aramchol meglumine treatment. (F) The DDC diet–induced increased IF staining of αSMA (red) surrounding CK19 immunostained bile ducts (green) was significantly reduced with Aramchol meglumine treatment. (N=4 mice per group on regular chow, 10 mice per group on DDC diet. For IF analyses, 3 mice per group on regular chow and 6 mice per group on DDC diet were randomly selected). Abbreviations: DDC, 3,5-diethoxycarboncyl-1,4-dihydrocollidine; ECM, extracellular matrix; IF, immunofluorescence; qPCR, quantitative polymerase chain reaction.

Aramchol has previously been demonstrated to inhibit SCD.^22,23 Therefore, we next analyzed the expression of mouse Scd isoforms in the DDC mouse model of biliary fibrosis. Immunofluorescence assay demonstrated the expression of both SCD1 and SCD2 in the bile ducts (demarcated by cholangiocyte markers, CK19 and CK7) in sister sections of the liver. The expression of SCD1 was not altered in the bile ducts of mice on regular chow or DDC diet determined by selectively quantifying mean immunofluorescence in bile ducts (Figure 2). There were also no differences observed with Aramchol meglumine treatment (Figure 2). In contrast, SCD2 expression was significantly increased in the bile ducts of mice on the DDC diet (Figure 2). However, there were no differences with Aramchol meglumine treatment (Figure 2).

FIGURE 2.

(A) Immunofluorescence using anti-SCD1 and SCD2 antibodies of liver sections of mice treated with vehicle or Aramchol meglumine on regular chow diet. Sister sections were immunostained for biliary markers, CK19 and CK7. (B) Liver sections of mice on DDC diet treated with vehicle or Aramchol meglumine were similarly immunostained for SCD1, SCD2, CK19, and CK7. (C) Mean fluorescence of bile ducts for SCD1 (top) and SCD2 (bottom) showed significantly increased immunofluorescence staining for SCD2 in mice on DDC diet compared with regular chow, without a significant difference between vehicle or Aramchol meglumine–treated groups. (N=4 mice per group on regular chow, 10 mice per group on DDC diet. For IF analyses, 3 mice per group on regular chow and 6 mice per group on DDC diet were randomly selected. *p<0.05 comparing regular chow and DDC groups). Abbreviations: DDC, 3,5-diethoxycarboncyl-1,4-dihydrocollidine; IF, immunofluorescence.

In the second model, Mdr2^−/− mice, which have established fibrosis and inflammation at 10–16 weeks of age, were used as a reversal (treatment) model. Aramchol meglumine at 12.5 mg/kg/d or vehicle only was administered daily for 4 weeks. Overall, the mice did not show any signs of toxicity and tolerated the drug well. There were no differences in weight (Supplemental Figure S3A, http://links.lww.com/HC9/C28) or behavioral changes. Similarly, there were no significant differences in serum ALP, ALT, AST, total bilirubin, or albumin in Aramchol meglumine–treated mice compared with vehicle only (Supplemental Figure S3B, http://links.lww.com/HC9/C28). Aramchol meglumine significantly reduced fibrosis as demonstrated by picrosirius red staining and quantification of the area of positive staining (Figures 3A and B). Consistently, the mRNA expression of extracellular matrix content, Collagen (Col1a1), and α-Smooth muscle actin (αSma/Acta2) was significantly reduced (Figure 3C). Similarly, profibrotic markers, PAI-1/Serpine1 and Timp1, were significantly reduced with Aramchol meglumine treatment (Figure 3C). The canonical targets of TGFβ signaling, Tgfbi, in addition to Serpine1 and markers of fibrosis, were significantly reduced with Aramchol meglumine treatment (Supplemental Figure S4A, http://links.lww.com/HC9/C28). Markers of inflammation, Il6, Tnfa, and Nfkb, were significantly reduced with Aramchol meglumine (Figure 3D). Macrophage marker, Cd68, was also similarly reduced (Figure 3D). Mpo, a marker of neutrophils, trended down with Aramchol meglumine treatment but did not reach statistical significance (p=0.06, Supplemental Figure S4B, http://links.lww.com/HC9/C28). Hydroxyproline content of the liver also trended down with Aramchol meglumine treatment but did not reach statistical significance (p=0.09). Myofibroblast activation was significantly reduced with Aramchol meglumine treatment as demonstrated by immunofluorescence using the αSMA marker (Figure 3F). In contrast, markers of quiescent HSCs (Reln and Lrat) were not significantly altered with Aramchol meglumine treatment (Supplemental Figure S4C, http://links.lww.com/HC9/C28). CK19 immunostaining was significantly reduced with Aramchol meglumine treatment, as was the expression of Sox9, collectively demonstrating a reduction in the biliary mass (Figure 3F and Supplemental Figures S4D and E, http://links.lww.com/HC9/C28).

FIGURE 3.

(A) Representative images of picrosirius red staining of livers from Mdr2^−/− mice showed reduced staining with Aramchol meglumine treatment (shortened to Aramchol in the figures) compared with vehicle, with area quantification using ImageJ shown in (B). (C, D) qPCR analysis of liver lysate demonstrated attenuation of ECM components, Col1a and αSMA/Acta2, profibrotic markers Serpine1 and Timp1, inflammatory markers, Il6, Tnfa, and Nfkb, and macrophage marker, Cd68, with Aramchol meglumine treatment. (E) Hydroxyproline content in the liver trended down with Aramchol meglumine treatment compared with vehicle, but did not reach statistical significance (p=0.09). (F) IF of αSMA (red) showed reduced immunostaining surrounding CK19 immunostained bile ducts (green) with Aramchol meglumine treatment. (N=10 vehicle, 9 Aramchol). Abbreviations: ECM, extracellular matrix; IF, immunofluorescence; qPCR, Quantitative polymerase chain reaction.

Aramchol inhibits the TGFβ-stimulated fibroinflammatory gene expression in cholangiocytes

We have previously demonstrated that TGFβ-activated cholangiocytes express multiple genes associated with fibrosis and inflammation. ³¹ TGFβ may regulate transcriptomics by modulating cellular metabolomics, including upregulation of enzymes involved in synthesizing inflammatory lipids.^11,12 In fact, TGFβ upregulated the expression of SCD in retinal epithelial cells.^11–13 Aramchol was previously shown to both downregulate SCD and inhibit its function.^22,23 Using RNA-seq, we show that Aramchol broadly inhibited the TGFβ transcriptomic modulations (Figure 4A). Comparing TGFβ and Aramchol cotreated H69 cells with TGFβ only, 558 genes were significantly downregulated, while 336 genes were significantly upregulated with Aramchol cotreatment (adjusted p value <0.05). These genes were subjected to Ingenuity Pathway Analysis to identify the most affected signaling pathways (those with the lowest p values). Fibrosis and HSC activation pathways were the most downregulated, while anti-fibroinflammatory PPAR signaling was among the most upregulated pathways (Figure 3B). These observations indicate that Aramchol attenuates the TGFβ-induced gene expression with prominent effects in downregulating fibrotic pathways while upregulating anti-fibroinflammatory signaling. Representative genes of the downregulated pathways, IL6, VEGFA, PDGFB, and plasminogen activator inhibitor-1 (PAI-1, SERPINE1 gene), were analyzed in transformed (H69 and MLE) and human primary PSC cholangiocytes (PSC-Cs). The TGFβ-induced increased expression was significantly inhibited by Aramchol in a dose-dependent manner, both at the transcript and protein level (Figures 4C and D). To determine the effect of cholangiocyte signals on HSC activation, CM from TGFβ-activated cholangiocytes with and without Aramchol cotreatment were used to treat human HSCs. CM from TGFβ-activated cholangiocytes activated HSCs as determined by significantly increased expression of αSma/Acta2, which was significantly reduced in HSCs treated with CM from TGFβ-activated cholangiocytes cotreated with Aramchol (Figure 4E).

FIGURE 4.

(A) RNA-seq analysis of H69 cholangiocytes revealed significant modulation of multiple genes with TGFβ stimulation (columns 1 vs. 2), which were significantly attenuated with Aramchol cotreatment (columns 2 vs. 3). (B) IPA analysis of genes that showed significant changes with Aramchol cotreatment compared with TGFβ only (columns 3 vs. 2 of [A]) identified inhibition of hepatic fibrosis pathways (negative Z-scores) while stimulating PPAR signaling (positive Z-score) by Aramchol cotreatment. (C) qPCR analysis showed that the TGFβ-induced elevated levels of IL6, VEGFA, and PDGFB were significantly and dose-dependently inhibited with Aramchol cotreatment in H69 cholangiocytes. (D) Western blot analysis demonstrated that the TGFβ-induced elevated level of PAI-1 was significantly inhibited with Aramchol cotreatment. (E) CM from H69 cholangiocytes previously stimulated with TGFβ, with and without Aramchol treatment, were used to treat human HSCs. CM from TGFβ-activated cholangiocytes significantly increased the expression of αSma/Acta2, which was significantly reduced in HSCs treated with CM from TGFβ-activated cholangiocytes cotreated with Aramchol. (N=3 at least, *p<0.05 when compared with TGFβ only). Abbreviations: CM, conditioned media; IPA, Ingenuity Pathway Analysis; qPCR, quantitative polymerase chain reaction.

In PSC-Cs, the basal high expression of these genes was markedly downregulated by Aramchol except SERPINE1 (Figure 5A). IL6, which was abundantly released into the cell CM by PSC-C, was significantly reduced by Aramchol determined by a specific ELISA assay (Figure 5B). HSC treatment of CM from PSC-Cs previously treated with Aramchol resulted in significantly reduced expression of αSMA/ACTA2 and COL1A1, indicative of reduced HSC activation (Figure 5C).

FIGURE 5.

(A) qPCR analysis showed that Aramchol treatment of PSC-Cs resulted in significant reductions in the expression of IL6, VEGFA, PDGFB, and SCD but not SERPINE1. (B) ELISA analysis of PSC-C cultured media demonstrated significant reductions in IL6 levels with Aramchol treatment. (C) HSC treatment of CM from PSC-Cs previously treated with Aramchol resulted in significantly reduced expression of αSMA/ACTA2, PDGFRA and COL1A1. (D) qPCR analysis showed that the TGFβ-induced elevated expression of Il6, Vegfa, Pdgfb, and Serpine1 was significantly and dose-dependently inhibited with Aramchol cotreatment in MLEs. (E) ELISA analysis showed that the TGFβ-induced elevated level of IL6 was significantly and dose-dependently inhibited with Aramchol cotreatment in MLEs. (N=3 at least, *p<0.05 when compared with TGFβ only). Abbreviations: CM, conditioned media; MLE, mouse large biliary epithelial cell; PSC-C, PSC patient–derived cholangiocyte; qPCR, quantitative polymerase chain reaction.

Similarly, in MLEs, the TGFβ-induced expressions of Il6, Vegfa, Pdgfb, and Serpine1 were significantly reduced by Aramchol in a dose-dependent manner (Figure 5D), as was the IL6 released into the CM determined by ELISA (Figure 5E). SCD expression was significantly reduced in PSC-Cs but not in the transformed cells (Figures 4C and 5A). These observations indicate that Aramchol markedly inhibits the fibroinflammatory signals emanating from activated cholangiocytes, consistent with previous studies in HSCs. ²³

SCD siRNA knockdown in cholangiocytes shows similar results to Aramchol treatment

Aramchol has been previously shown to downregulate markers of fibrosis in HSCs and hepatocytes by inhibiting SCD.^22,23 Using H69 cholangiocytes, we show that SCD siRNA knockdown has similar effects to Aramchol treatment. SCD was significantly knocked down by ~95%, which resulted in significantly attenuating the TGFβ-induced mRNA expression of IL6 and SERPINE1, and protein levels of IL6 and PAI-1 (Figure 6). These observations indicate that the effects of Aramchol on cholangiocytes, similar to HSCs and hepatocytes, are through inhibition of SCD.

FIGURE 6.

(A) qPCR analysis showed increased expression of SCD with TGFβ treatment of H69 cholangiocytes, which was significantly knocked down (~95%) with specific siRNAs. Furthermore, the TGFβ-induced elevated expression of IL6 and SERPINE1 was significantly inhibited with SCD siRNA knockdown. (B, C) ELISA and western blot analyses showed that the TGFβ-induced elevated expression of IL6 and PAI-1, respectively, in the cell-cultured media was significantly inhibited with SCD siRNA knockdown. (N=3 at least). Abbreviations: qPCR, quantitative polymerase chain reaction; SCD, stearoyl-CoA desaturase.

Aramchol upregulates specific fatty acids and PPARα/γ activity

Previous studies have shown that Scd1 knockdown in T cells modulates lipid profiles, facilitating regulatory T-cell differentiation and reducing autoimmunity in a mouse model of multiple sclerosis. ³² Upregulated lipid species included docosahexaenoic and arachidonic acids (20:4). Docosahexaenoic acid (22:6) activated PPARγ and enhanced regulatory T-cell differentiation. ³² This is consistent with previous studies demonstrating PPAR activating properties of omega-3 and -6 eicosanoids. ³³ TGFβ signaling inhibits PPARα/γ, while activation of PPARα/γ suppresses the fibroinflammatory signals.^34,35 Aramchol has been previously shown to upregulate PPARγ in HSCs.^22–24 Therefore, we assessed the levels of select omega-3 and -6 eicosanoids in cholangiocytes stimulated with TGFβ with and without Aramchol cotreatment using liquid chromatography-tandem mass spectrometry (Figures 7A). The essential fatty acid linoleic acid (18:2) with high cellular concentration was significantly downregulated by TGFβ treatment compared with control, which was partially rescued with Aramchol cotreatment (Figure 7A). Linolenic acid (18:3) at much lower cellular concentration appeared to be increased with Aramchol cotreatment of TGFβ-stimulated cholangiocytes, which was approaching statistical significance (p=0.07, Figure 7A). We tested the effect of these fatty acids on the TGFβ-induced expression of fibroinflammatory signals by cholangiocytes. Linoleic acid (LA) attenuated the TGFβ-stimulated expression of Il6 and Vegfa in a dose-dependent manner (Figure 7B) but paradoxically increased the expression of Serpine1 (Supplemental Figure S5A, http://links.lww.com/HC9/C28). There was no additive suppressive effect when combining linoleic acid with Aramchol cotreatment on Il6 or Vegfa expression (Figure 7C and Supplemental Figure S5B, http://links.lww.com/HC9/C28). These findings indicate that TGFβ suppresses LA levels to allow the expression of some fibroinflammatory signals, while treatment of TGFβ-stimulated cholangiocytes with either LA or Aramchol reverses this effect, at least in part, through the same mechanism.

FIGURE 7.

(A) Free fatty acid quantification of H69 cholangiocyte lysate following stimulation with TGFβ with and without Aramchol cotreatment compared with controls using liquid chromatography-tandem mass spectrometry, showing significant reduction in the levels of LA (18:2) with TGFβ, which is partially rescued with Aramchol cotreatment. Aramchol alone also reduced levels of LA. Aramchol cotreatment of TGFβ-treated cells appeared to increase linolenic acid (18:3) level, which was approaching statistical significance. (B) LA treatment of MLE cholangiocytes resulted in a dose-dependent suppression of TGFβ-stimulated expression of Il6 and Vegfa. (C) Combination of LA (100 µM) and Aramchol (30 µM) did not lead to further suppression of TGFβ-stimulated expression of Il6. (D) In an ELISA system with immobilized DNA containing PPRE sites and PPAR-specific antibodies, Aramchol significantly increased the PPARα and γ binding to PPRE sites under basal conditions, but only significantly increased the PPRE binding of PPARγ with the cotreatment of TGFβ in MLEs. (E) The TGFβ-induced suppression of PPAR-responsive genes, Fabp4 and Aqp7, was also significantly reversed. (F) In H69 cholangiocyte lysates, PPARα binding to PPRE sites was significantly reduced with TGFβ treatment, which was significantly reversed with Aramchol cotreatment. PPARγ binding of PPRE sites was also increased with Aramchol treatment. (G) PPARα/γ responsive genes, PDK4 and AQP3, were significantly reduced with TGFβ, which was partially reversed with Aramchol cotreatment. (N=3 at least, *p<0.05 when compared with TGFβ only). Abbreviations: LA, linoleic acid; MLE, mouse large biliary epithelial cell; PPAR, peroxisome proliferator–activated receptor; PPRE, PPAR responsive element.

Aramchol significantly increased the PPARα and γ binding to PPRE sites under basal conditions, a direct measure of PPAR activity, but significantly increased the activity of only PPARγ when MLEs were costimulated with TGFβ (Figure 7D). Pparg and Ppara expression was significantly downregulated with TGFβ, which was modestly reversed with Aramchol (Supplemental Figure S5C, http://links.lww.com/HC9/C28). Correspondingly, the TGFβ-induced suppression of PPAR-responsive genes was also significantly reversed (Figure 7E). In TGFβ-stimulated H69 cholangiocytes, Aramchol increased the PPARα and γ binding to PPRE sites (Figure 7F). Consistently, PPARα/γ responsive genes were increased (Figure 7G). This was accompanied by a modest increase in the expression of PPARA with Aramchol treatment (Supplemental Figure S5D, http://links.lww.com/HC9/C28).

DISCUSSION

Biliary fibrosis is the predominant process of fibroinflammatory cholestatic liver diseases such as PSC and primary biliary cholangitis, where morbidity and mortality closely correlate with biliary fibrosis. There are currently no treatments for biliary fibrosis, which severely limits therapeutic options for these diseases. Central to the pathogenesis of biliary fibrosis are cholangiocytes that serve as a “hub” of signaling. ³⁶ Injury and inflammatory signals activate cholangiocytes to become highly secretory. Activated cholangiocytes interact with immune cells and myofibroblasts to initiate and propagate biliary fibrosis in the setting of chronic disease. ⁸ However, the molecular mechanisms of activated cholangiocytes that allow for their secretory phenotype and paracrine interactions are not completely elucidated.

In this study, we showed that Aramchol meglumine significantly attenuated biliary fibrosis in 2 mouse models of cholestatic injury and fibrosis, the Mdr2^−/− and the DDC diet model. This effect was demonstrated by reductions in both measures of ECM synthesis (mRNA expression of ECM components in the liver) and the collagen composition of the liver (picrosirius red staining and hydroxyproline content). Interestingly, these reductions in fibrosis were not accompanied by a significant change in liver injury markers such as ALT, AST, and ALP (Supplemental Figures S1 and S2, http://links.lww.com/HC9/C28). This is in contrast to MASH, where Aramchol improves liver enzymes, suggestive of reducing hepatocyte injury by relieving lipotoxicity.^25,26 These observations indicate that Aramchol meglumine does not have a significant protective effect on hepatocytes or cholangiocytes against bile toxicity or cholestasis-induced liver injury. This is unlike bile acid therapies such as ursodeoxycholic acid, with direct reduction of bile and cholestasis toxicity compared with this fatty acid-bile acid conjugate.^37,38 Rather, the effects of Aramchol on fibrosis and inflammation are likely mediated through cellular signaling after the injury, which can be combined with toxicity-reducing modalities.

We hypothesized that the attenuation of biliary fibrosis by Aramchol meglumine resulted from its effects on the cholangiocyte signaling. Accordingly, we tested Aramchol’s effects on the inhibition of pro-fibroinflammatory signals emanating from cholangiocytes. Cholangiocytes activated by TGFβ release a number of inflammatory and fibrogenic stimuli. These signals mediate the interactions and activation of immune cells and myofibroblasts, which collectively propagate inflammation and fibrosis. Inhibiting the signaling pathways that stimulate these signals attenuates biliary fibrosis. ³⁶ Consistently, Aramchol cotreatment of cholangiocytes led to significant attenuation of TGFβ-induced fibroinflammatory signals. This is hypothesized to occur through at least 2 interdependent modalities: inhibition of SCD and activation of PPARα and PPARγ (Figure 8).

FIGURE 8.

TGFβ signaling upregulates SCD, which inhibits PPARα/γ, allowing for the expression of fibroinflammatory signals in cholangiocytes. Aramchol inhibition of SCD allows increased PPARα/γ expression and activity, which inhibits the TGFβ-induced expression of fibroinflammatory signals. Abbreviations: PPAR, peroxisome proliferator–activated receptor; SCD, stearoyl-CoA desaturase.

TGFβ upregulates SCD in epithelial cells, which may be partially responsible for TGFβ-induced downstream effects.^11–13 Aramchol has been previously shown to inhibit SCD in hepatocytes and HSCs.^22–24 As further evidence, we show that SCD siRNA knockdown in cholangiocytes produced similar effects to Aramchol treatment on TGFβ-induced gene expression. These observations support the hypothesis that Aramchol attenuates the fibroinflammatory effects of TGFβ by inhibiting SCD in cholangiocytes. In the mouse model of biliary fibrosis, the mouse Scd2 isoform, but not Scd1, is markedly upregulated in the cholangiocytes compared with control/basal conditions (Figure 2). Inhibition of these enzymes at least partially explains the observed effects of Aramchol in the in vivo models. However, the contribution of each mouse Scd isoform to biliary fibroinflammation remains unclear and requires cholangiocyte-specific genetic knockdown of each isoform in mouse models of biliary fibrosis. We plan to carry out these experiments in the future.

PPAR signaling promotes anti-fibroinflammatory pathways in part by inhibiting TGFβ signaling. On the other end, TGFβ inhibits PPARγ to allow for its fibrogenic signaling.^34,35 Previous studies have shown that SCD activity can inhibit PPARγ.^11–13 In HSCs, Aramchol treatment or SCD siRNA knockdown resulted in the upregulation of PPARγ. ²³ Therefore, the TGFβ inhibition of PPARγ may occur through the upregulation of SCD expression and activity. Increased SCD expression, in turn, may reduce the cellular levels of PPAR agonists such as fatty acids. ³² Several omega-3 and -6 eicosanoid fatty acids have demonstrated PPAR agonism. Therefore, we measured the cellular content of these fatty acids and identified LA as a major target of TGFβ regulation. This was partially reversed with Aramchol cotreatment (Figure 7A). Furthermore, LA cotreatment significantly and dose-dependently suppressed the TGFβ-induced increased expression of some fibroinflammatory readouts, without a significant additional suppressive impact when Aramchol was added to LA (Figure 7). These findings point to Aramchol treatment promoting an activated PPARα/γ state by blocking the reduction of its agonist, LA. Consistently, Aramchol treatment of cholangiocytes upregulated PPARα/γ expression and activity. PPAR activity was determined by both measurement of PPRE binding and assessment of PPAR-responsive genes, which were both modestly upregulated with Aramchol cotreatment. These observations support the interplay of TGFβ, SCD, and PPARγ in cholangiocyte-mediated promotion of biliary fibroinflammation. Nevertheless, additional and alternative mechanisms of Aramchol interference in TGFβ signaling remain a possibility given the modest effects of Aramchol on PPAR activity.

The effects of Aramchol may extend beyond the TGFβ signaling pathway, given the complex interactions of this pathway with several others and the involvement of SCD in other pathways.^16,39 In fact, SCD is implicated in Wnt signaling to promote HSC activation and hepatocyte tumorigenesis. ¹⁵ The canonical Wnt pathway is well-studied in biliary fibrosis and known to overlap with TGFβ signaling. ³⁹

The attenuation of fibrosis in our in vivo models may also be in part due to Aramchol directly inhibiting hepatic myofibroblast ECM synthesis. Indeed, in vitro studies of Aramchol have demonstrated inhibition of HSC production of ECM components. ²³ The beneficial effects of Aramchol may extend to other cell compartments involved in biliary fibrosis, including the cells of the immune system.^36,40,41 Indeed, Scd1 impairs the reparative phenotype of macrophages while stimulating an inflammatory phenotype in models of multiple sclerosis. ⁴² Macrophages have important roles in the pathogenesis of biliary fibrosis, both reparative (resident Kupffer cells) and deleterious (monocyte-derived).^43,44 Scd1 knockdown resulted in increased regulatory T-cell differentiation by activation of PPARγ and reduction of autoimmunity. ³² However, any direct effects of Aramchol or SCD on immune cells and their activation or differentiation have not been shown in detail in models of biliary fibrosis. To distinguish the effects of Aramchol on various cell mediators involved in the pathogenesis of biliary fibrosis would require transgenic mouse models to specifically knock down Aramchol target, Scd isoforms, in these cell compartments.

Aramchol is well-studied in MASH with promising results. ²⁵ It reduces steatohepatitis and fibrosis in mouse models of MASH. ²⁴ In a randomized, double-blind, placebo-controlled phase IIb trial and an open label extension of a phase III study, Aramchol demonstrated significantly increased resolution of steatohepatitis and improvement in fibrosis while decreasing liver injury markers such as serum ALT.^25,26 Importantly, Aramchol was safe, well tolerated, and without an imbalance in adverse events compared with the placebo arm. A similar safety level was observed in a meta-analysis of 3 clinical trials. ⁴⁵

Taken together, Aramchol attenuates biliary fibrosis in 2 mouse models of biliary fibrosis along with antifibrotic effects in cholangiocytes, myofibroblasts, and hepatocytes. These observations, combined with its excellent clinical trial safety data, provide the rationale for further clinical studies of Aramchol in patients with biliary fibrosis, in particular PSC, where treatments are desperately needed.

Supplementary Material

hc9-9-e0748-s001.pdf ^{(791.4KB, pdf)}