Society: AASLD

Background: Chronic cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), often lead to end-stage liver disease requiring liver transplantation. PSC also significantly increases the risk of cholangiocarcinoma (CCA). There is currently no effective therapeutic agent for PSC due to a limited understanding of its pathogenesis. Exosomes carry a variety of bioactive molecules, including miRNAs, lipids and proteins. The dysregulation of miRNAs is closely related to various pathological aspects of PSC. In this study, we aimed to investigate the expression profiles and function of exosomal miRNAs under cholestatic conditions.
Methods: One-year-old Mdr2^-/- mice (FVB background) and age-/gender- matched wild-type (WT) mice were used in this study. The serum exosomes were isolated using qEV isolation system (from IZON). Total RNA was isolated from the serum exosomes and liver tissues using Trizol. Total RNA transcriptome was determined by RNAseq. The miRNA profiles in the exosomes and livers were analyzed using the NanoString nCounter® miRNA Expression panel. TargetScan and mirnet2.0 were used to identify the potential target genes. Protein-protein interaction (PPI) was constructed based on the STRING database. Hub genes under cholestatic conditions were visualized by Cytoscape software. The human CCA patient data were downloaded from the Cancer Genome Atlas (TCGA) database.
Results: Bioinformatic analysis identified 74 differentially expressed miRNAs (DE-miRs) and 137 DE-miRs in the male and female Mdr2^-/- mice compared to corresponding WT controls, respectively. The identified DE-miRs were used to predict the target genes. GO and KEGG analysis further showed that these DE-miRs were linked to multiple signaling pathways related to cholestatic liver injury. We also identified 431 up-, and 1106 down- DE genes (DEGs) in the Mdr2^-/- mice compared to WT mice. From the PPI network analysis of the DEGs, the top 100 hug genes were determined. We identified 11 upregulated DE-miRs in Mdr2^-/- mice, which overlapped with the predicted miRs based on the the top 100 hug genes. Interestingly, among the overlapping DE-miRs, two miRs, miR-16 and let-7e, were also upregulated in the serum exosomes of Mdr2^-/- mice. Among the 100 hug genes, we identified nine downregulated genes that were potential targets of miR-16 and let-7e. Further analysis of TCGA CCA data showed upregulation of miR-16 and let-7e, and 7 out of the 9 identified targeted genes were also significantly downregulated in CCA compared to healthy controls.
Conclusion: This study identified potential DE-miRs in the liver and serum exosomes which may contribute to cholestatic liver injury. This study supports the possible use of exosomal miRs as diagnostic and prognostic markers and therapeutic targets for cholestatic liver diseases.

<b>Fig 1. Liver DE-miRs and DEGs identification in Mdr2<sup>-/-</sup> mice compared to corresponding WT controls, target prediction and analysis of these DE-miRs and DEGs, and the overlapping miRNAs in cholestatic liver injury.</b><br /> (A)<b> </b>Liver DE-miRs expression profiles between male Mdr2<sup>-/-</sup> and WT controls. (B) Liver DE-miRs expression profiles between female Mdr2<sup>-/- </sup>and WT controls. (C)<b> </b>Liver DEGs expression profiles between Mdr2<sup>-/-</sup> and WT controls. (D) The top 50 hug genes of the PPI network of upregulated DEGs and the top 50 hug genes of the PPI network of downregulated DEGs. (E) Predicted network of miRNAs – 100 identified hug genes of DEGs between Mdr2<sup>-/- </sup>and WT controls. (F) Identification of 11 overlapping upregulated miRNAs in cholestatic liver injury. FC, fold change; miR, micro RNA. DE, differentially expressed, Mdr2<sup>-/-</sup> , multidrug resistance 2 gene knockout; WT, wild type.

Fig 1. Liver DE-miRs and DEGs identification in Mdr2^-/- mice compared to corresponding WT controls, target prediction and analysis of these DE-miRs and DEGs, and the overlapping miRNAs in cholestatic liver injury.
(A) Liver DE-miRs expression profiles between male Mdr2^-/- and WT controls. (B) Liver DE-miRs expression profiles between female Mdr2^-/-and WT controls. (C) Liver DEGs expression profiles between Mdr2^-/- and WT controls. (D) The top 50 hug genes of the PPI network of upregulated DEGs and the top 50 hug genes of the PPI network of downregulated DEGs. (E) Predicted network of miRNAs – 100 identified hug genes of DEGs between Mdr2^-/-and WT controls. (F) Identification of 11 overlapping upregulated miRNAs in cholestatic liver injury. FC, fold change; miR, micro RNA. DE, differentially expressed, Mdr2^-/- , multidrug resistance 2 gene knockout; WT, wild type.

<b>Fig 2. Exosome DE-miRs identification in Mdr2<sup>-/- </sup>mice compared to corresponding WT controls, the overlapping DE-miRs in the liver and serum exosomes, and comparison of the expression levels of these DE-miRs and target genes between normal and CHOL samples in TCGA CCA database.</b><br /> (A) Exosome DE-miRs expression profiles between female Mdr2<sup>-/-</sup> and WT controls. (B) Identification of 2 overlapping upregulated DE-miRs in the serum exosomes which may contribute to cholestatic liver injury. (C) The expression levels of these 2 overlapping upregulated DE-miRs between normal and CHOL samples. (D) Heat map, the expression levels of these target hug genes of 2 overlapping upregulated DE-miRs between normal and CHOL samples.

Fig 2. Exosome DE-miRs identification in Mdr2^-/-mice compared to corresponding WT controls, the overlapping DE-miRs in the liver and serum exosomes, and comparison of the expression levels of these DE-miRs and target genes between normal and CHOL samples in TCGA CCA database.
(A) Exosome DE-miRs expression profiles between female Mdr2^-/- and WT controls. (B) Identification of 2 overlapping upregulated DE-miRs in the serum exosomes which may contribute to cholestatic liver injury. (C) The expression levels of these 2 overlapping upregulated DE-miRs between normal and CHOL samples. (D) Heat map, the expression levels of these target hug genes of 2 overlapping upregulated DE-miRs between normal and CHOL samples.

Background: Following biliary injury/senescence in rodent models of cholestatic liver injury and in patients with PSC, mast cells (MCs) infiltrate the liver and migrate toward cholangiocytes. Proteases such as chymase and tryptase are indicative of MC origination and function, while activated MCs release histamine (HA) that promotes liver inflammation. In human PSC, chymase gene expression is significantly higher than tryptase in these patients. MC chymase induces detrimental effects in cardiac dysfunction, asthma, and lung fibrosis. In vivo, inhibition of chymase reverses acute liver failure and CCl₄-induced damage. Similarly, in a PSC mouse model, Mdr2^-/-, has increased chymase- and tryptase-positive MC presence at baseline, but after treatment with cromolyn sodium (to block MC activation), these mice display reduced biliary damage/senescence, hepatic fibrosis, and inflammation. Aim: To determine the effects of INVA8001, a specific chymase inhibitor, on PSC phenotypes in Mdr2^-/- mice. Methods: 10 wk male Mdr2^-/- mice were treated with INVA8001 (20 mg/kg BW/day) for 14 days by daily IP injection. Serum, liver, cholangiocytes, and cholangiocyte supernatants were collected. FVB/NJ (wild-type, WT) and untreated Mdr2^-/- mice were utilized for comparison. Liver damage was assessed by H&E staining in liver and total bile acids (TBAs) were measured in serum by EIA. Changes in intrahepatic bile duct mass (i.e., ductular reaction, DR) were determined by semiquantitative CK-19 immunohistochemistry (IHC) and biliary senescence by immunofluorescence (IF) for p16 (costained with CK-19) and qPCR for p16, p21, and p18 in total liver mRNA. Hepatic fibrosis and collagen deposition were evaluated by quantitative Fast Green/Sirius Red staining and qPCR for collagen type 1a and TIMP1 in total liver samples. MC presence was determined by staining for mouse MC protease-1 (marks chymase-positive MCs) and tryptase beta 2 (marks tryptase-positive MCs) and activation by HA serum content and qPCR for mMCP-1 and FCeR1 in total liver. Changes in senescence-associated secretory phenotypes (SASPs) were determined by serum TGF-β1 content using EIA and total liver expression by qPCR. Results: Inhibition with INVA8001 ameliorated DR, hepatic damage, senescence/SASP, and fibrosis along with chymase-positive MC infiltration and activation in Mdr2^-/- mice compared to WT and untreated Mdr2^-/- mice. Conclusion: Specific inhibition of chymase targets PSC phenotypes ameliorating pathogenesis by decreased biliary senescence and SASP factors. Our studies demonstrate the damaging effects of chymase-positive MCs in PSC and the therapeutic benefit of INVA8001 treatment with potential applicability to other liver diseases wherein MCs promote pathogenesis.

Krishnan Nandabalan

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