Aberrant basement membrane production by HSCs in MASLD is attenuated by the bile acid analog INT-767

Abstract

Background: The farnesoid X receptor (FXR) is a leading therapeutic target for metabolic dysfunction-associated steatohepatitis (MASH)-related fibrosis. INT-767, a potent FXR agonist, has shown promise in preclinical models. We aimed to define the mechanisms of INT-767 activity in experimental MASH and dissect cellular and molecular targets of FXR agonism in human disease.

Methods: Leptin-deficient ob/ob mice were fed a MASH-inducing diet for 15 weeks before the study started. After baseline liver biopsy and stratification, mice were allocated to INT-767 (10 mg/kg/d) or vehicle treatment for 8 weeks, either alongside an ongoing MASH diet (progression) or following conversion to normal chow (reversal). Effects on extracellular matrix remodeling were analyzed histologically and by RNA-sequencing. Serum fibrosis biomarkers were measured longitudinally. Human liver samples were investigated using bulk and single-cell RNA-sequencing, histology, and cell culture assays.

Results: INT-767 treatment was antifibrotic during MASH progression but not reversal, attenuating the accumulation of type I collagen and basement membrane proteins (type IV collagen and laminin). Circulating levels of PRO-C4, a type IV collagen formation marker, were reduced by INT-767 treatment and correlated with fibrosis. Expression of basement membrane constituents also correlated with fibrosis severity and adverse clinical outcomes in human MASH. Single-cell RNA-sequencing analysis of mouse and human livers, and immunofluorescence staining colocalized FXR and basement membrane expression to myofibroblasts within the fibrotic niche. Treatment of culture-activated primary human HSCs with INT-767 decreased expression of basement membrane components.

Conclusions: These findings highlight the importance of basement membrane remodeling in MASH pathobiology and as a source of circulating biomarkers. Basement membrane deposition by activated HSCs is abrogated by INT-767 treatment and measurement of basement membrane molecules should be included when determining the therapeutic efficacy of FXR agonists.

FIGURE 1

INT-767 treatment inhibits basement membrane expression in AMLN ob/ob-MASH mice. (A) Summary of AMLN ob/ob-MASH mouse model. Lep^ob/ob mice on an AMLN diet for 12 weeks underwent liver biopsy. Mice with fibrosis stage ≥1 and steatosis score ≥2 continued the AMLN diet and were randomized to treatment with either INT-767 (10 mg/kg) or vehicle once daily for a further 8 weeks. (B) RNA-seq analysis of liver tissue demonstrates modules of differentially expressed genes that are downregulated (cluster 1) and upregulated (cluster 2) following INT-767 treatment. (C) Annotation of statistically significantly enriched functional categories of genes in cluster 1 and cluster 2 using the DAVID tool identifies the downregulation of extracellular matrix and basement membrane genes following INT-767 treatment. (D) Comparison of expression levels of stated gene between the vehicle (n = 7) and INT-767 (n = 4) treated mice using RNA-seq data. Levels of significance: *p < 0.05, **p < 0.01, ns p ≥ 0.05 (Mann-Whitney test). Abbreviations: AMLN, Amylin Liver NASH; MASH, metabolic dysfunction–associated steatohepatitis; RPKM, reads per kilobase of transcript per million mapped reads.

FIGURE 2

INT-767 treatment reduces basement membrane deposition in AMLN ob/ob-MASH mice. (A) Representative liver H&E images of AMLN ob/ob-MASH mouse model following treatment with INT-767 or vehicle. Scale bar = 100 μm. (B) Histological quantification of % steatosis in AMLN ob/ob-MASH mice following treatment with INT-767 (n = 14) or vehicle (n = 13). Levels of significance: ****p < 0.0001 (unpaired t test). (C) Histological quantification of NAS in AMLN ob/ob-MASH mice following treatment with INT-767 (n = 14) or vehicle (n = 13). Levels of significance: **p = 0.0024 (Mann-Whitney test). (D) Histological quantification of NAS components in AMLN ob/ob-MASH mice following treatment with INT-767 (n = 14) or vehicle (n = 13). Levels of significance: ****p < 0.0001, ns (inflammation) p = 0.14, ns (ballooning) p = 0.13 (Mann-Whitney test). (E) Representative liver immunohistochemistry images of AMLN ob/ob-MASH mice following treatment with INT-767 or vehicle for stated protein. Scale bar = 100 μm. (F–H) Histological quantification of % type I collagen, type IV collagen, and laminin staining in AMLN ob/ob-MASH mice following treatment with INT-767 (n = 14) or vehicle (n = 13). Levels of significance: ****p < 0.0001 (unpaired t test). Abbreviations: AMLN, Amylin Liver NASH; H&E, hematoxylin and eosin; MASH, metabolic dysfunction–associated steatohepatitis; NAS, nonalcoholic fatty liver disease activity score.

FIGURE 3

Basement membrane deposition in the fibrotic niche of human MASH. (A) RNA-seq analysis of human MASH liver tissue, grouped by histological NASH Clinical Research Network fibrosis stage. Comparison of expression (in counts per million) of stated gene between fibrosis stage 0 (n = 10), stage 1–2 (n = 19), and stage 3–4 (n = 25). Levels of significance: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 (Welch’s ANOVA with Dunnett’s T3 multiple comparison test). (B) Correlation matrix of NR1H4 (FXR) regulon activity profile (NR1H4_RAP) and normalized gene expression counts for stated genes in the complete n = 663 set of cases with RNA-seq from the SteatoSITE data set. Statistically significant correlations (p < 0.05) shown as shaded boxes and nonsignificant correlations marked with an X. Boxes colored by Spearman correlation coefficients. (C, D) Biopsy cases from the SteatoSITE data set (n = 447) with available RNA-seq and no hepatic decompensation–related coding event before the time of the biopsy were used for time-to-event analysis. NR1H4 (FXR) regulon activity profile (C) and normalized gene expression counts for COL4A1 and COL4A2 (D) were used to divide cases into low and high risk of hepatic decompensation (Kaplan-Meier estimator curves with log-rank test p value shown). Abbreviation: MASH, metabolic dysfunction–associated steatohepatitis.

FIGURE 4

Activated HSCs are a candidate in vivo target population for INT-767 in murine MASH. (A) UMAP showing annotation of mouse liver scRNA-seq data (33,168 cells) by cell lineage. (B) UMAP showing whether cells are from healthy (n = 3) or MASH livers (n = 3). (C) Normalized expression level of the stated gene in each mouse liver cell lineage. (D) UMAP showing annotation of 429 mouse liver mesenchymal single cells as portal fibroblasts, quiescent HSCs, activated HSCs, or mesothelia. (E) UMAP showing whether mesenchymal cells are from healthy (n = 3) or MASH livers (n = 3). (F) Normalized expression level of the stated gene in each mouse liver mesenchymal cell type. Abbreviations: MASH, metabolic dysfunction–associated steatohepatitis; MP, mononuclear phagocyte; scRNA-seq, single-cell RNA-sequencing.

FIGURE 5

Human SAMes are a candidate target population for INT-767. (A) UMAP showing annotation of human liver scRNA-seq data (58,358 cells) by cell lineage (B) UMAP showing whether cells are from healthy (n = 5) or cirrhotic livers (n = 5). (C) Dot plot showing normalized expression level (color) and percentage of cells (dot size) expressing the stated gene in each cell lineage. (D) UMAP showing annotation of 2316 human liver mesenchymal single cells as VSMC, HSCs, mesothelia, or SAMes. (E) UMAP showing whether mesenchymal cells are from healthy (n = 5) or cirrhotic livers (n = 5). (F) Normalized expression level of the stated gene in each human liver mesenchymal cell type. Abbreviations: SAMes, scar-associated mesenchymal cells; scRNA-seq, single-cell RNA-sequencing; VSMC, vascular smooth muscle cells.

FIGURE 6

INT-767 regulates basement membrane production by HSCs. (A) Representative immunofluorescence images of human MASH liver tissue stained for NR1H4/FXR and a-SMA. Individual channel images and merged images are shown. White arrows indicating NR1H4⁺ α-SMA⁺ cells; red arrows indicating NR1H4⁺ α-SMA⁻ cells. Scale bar = 50 μm. (B) Culture-activated primary human HSCs treated with INT-767 (n = 8–9) or vehicle control (n = 8–9) from 3 independent experiments. Expression of stated gene quantified by qPCR and expressed relative to mean gene expression for vehicle control. Levels of significance: *p < 0.05, ns (Col1a1) p = 0.22, ns (Col3a1) p = 0.34, ns (Acta2) p = 0.20 (unpaired t test). (C) Deposition of ECM components collagen IV, collagen I + III, and laminin by culture-activated primary human HSCs assessed by immunofluorescent staining in response to stated concentration of INT-767 or vehicle (DMSO) control. Exemplar images from 4 wells at different INT-767 concentrations shown. Data expressed as average total positive pixels per well. Analysis from 4 independent experiments; symbols indicate separate experiments. Levels of significance: ns (collagen I + III) p = 0.58, ns (laminin) p = 0.48 (Friedman test). Abbreviations: ECM, extracellular matrix; MASH, metabolic dysfunction–associated steatohepatitis.