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Multi-Modal Comparison of human Hepatic Stellate Cells identifies novel therapeutic targets for Metabolic Dysfunction-Associated Steatotic Liver Disease

ABSTRACT: Background and aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) progresses from steatosis (Metabolic dysfunction-associated steatotic liver, MASL) to Metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Activation of Hepatic Stellate Cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. Here we compared gene expression and chromatin accessibility profiles of human HSCs in NORMAL, MASL, and MASH livers at single cell resolution. Methods: 18 human livers were profiled using single-nucleus (sn) RNA-seq and snATAC-seq. High priority targets were identified and then tested in 2D human HSCs, 3D human liver spheroids, and HSC-specific gene knockout mice. Results: This study identified novel gene regulatory mechanisms underlying MASL- and MASH-associated HSC heterogeneity and outlined potential strategies for anti-fibrotic therapy. Specifically, MASH-enriched HSC-subcluster hA1, represented by highly fibrogenic population of myofibroblasts, served as a critical source of extracellular matrix protein (ECM) in MASH, and its activation was regulated via a cross-talk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors (TFs). Additionally, we identified a set of core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive ECM production in hA1 HSCs. The pathological relevance of the selected hA1 targets, such as SERPINE1 and others, was demonstrated using siRNA-based HSC-specific gene knockdown or pharmacological inhibitor of SERPINE1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice with MASH. Conclusion: We identified potential targets for anti-fibrotic therapy of MASH in patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE244832 | GEO | 2024/11/13

REPOSITORIES: GEO

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Project description:BACKGROUND AND AIM: We previously established that endoplasmic reticulum stress leads to unfolded protein response (UPR) that promotes liver fibrosis by activating hepatic stellate cell (HSC). We aimed to determine the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways, in the fibrogenic HSC activation. METHODS: XBP1 expression was measured by qPCR in tunicamycin-treated human HSC lines (TWNT4 and LX2) and culture-activated human primary HSCs. XBP1 was overexpressed in primary human HSCs and the HSC lines, and fibrogenic genes (COL1A1, ACTA2, PDGFRB, MMP2, TIMP1) and encoded proteins were assessed by qPCR and Western blotting, respectively. Autophagy was inhibited by knockdown of ATG7. Genome-wide transcriptome profiling was performed to determine modulated molecular pathway by XBP1. In vivo XBP1 activation was assessed in transcriptome datasets of fibrotic mouse models and immunohistochemistry of human liver tissues. RESULTS: XBP1 overexpression accompanied both tunicamycin- and culture-based HSC activation. Ectopic overexpression of XBP1 induced fibrogenic gene expression in the HSC lines, which was inhibited by knockdown of ATG7. UPR pathway together with PDGFRB pathway, a hallmark of HSC activation, were induced in transcriptome profiles of XBP1-transduced HSC lines. Some known fibrogenic pathways such as transforming growth factor (TGF)-beta pathway were not induced, suggesting partial contribution of XBP1 to the entire fibrogenic HSC activation machinery. XBP1 target gene signature defined in our XBP1-overexpressing HSC lines was significantly induced in liver tissue transcriptome profiles of carbon tetrachloride-treated or bile duct-ligated mouse models, and XBP1 and alpha-smooth muscle actin (encoded by ACTA2, another hallmark of HSC activation) were co-localized in human fibrotic liver tissues, collectively supporting involvement of XBP1 in HSC activation and fibrogesis in vivo. CONCLUSIONS: XBP1-mediated UPR is at least partially responsible for fibrogenic HSC activation via autophagy.

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Multi-Modal Comparison of human Hepatic Stellate Cells identifies novel therapeutic targets for Metabolic Dysfunction-Associated Steatotic Liver Disease

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