Multi-modal analysis of human hepatic stellate cells identifies novel therapeutic targets for metabolic dysfunction-associated steatotic liver disease

Hyun Young Kim; Sara Brin Rosenthal; Xiao Liu; Charlene Miciano; Xiaomeng Hou; Michael Miller; Justin Buchanan; Olivier B Poirion; Daisy Chilin-Fuentes; Cuijuan Han; Mojgan Housseini; Raquel Carvalho-Gontijo Weber; Sadatsugu Sakane; Wonseok Lee; Huayi Zhao; Karin Diggle; Sebastian Preissl; Christopher K Glass; Bing Ren; Allen Wang; David A Brenner; Tatiana Kisseleva

doi:10.1016/j.jhep.2024.10.044

Multi-modal analysis of human hepatic stellate cells identifies novel therapeutic targets for metabolic dysfunction-associated steatotic liver disease

J Hepatol. 2024 Nov 8:S0168-8278(24)02667-9. doi: 10.1016/j.jhep.2024.10.044. Online ahead of print.

Authors

Hyun Young Kim¹, Sara Brin Rosenthal², Xiao Liu¹, Charlene Miciano³, Xiaomeng Hou³, Michael Miller³, Justin Buchanan³, Olivier B Poirion³, Daisy Chilin-Fuentes⁴, Cuijuan Han⁴, Mojgan Housseini⁵, Raquel Carvalho-Gontijo Weber¹, Sadatsugu Sakane¹, Wonseok Lee¹, Huayi Zhao¹, Karin Diggle⁴, Sebastian Preissl⁶, Christopher K Glass⁷, Bing Ren³, Allen Wang⁸, David A Brenner⁹, Tatiana Kisseleva¹⁰

Affiliations

¹ Department of Medicine, University of California San Diego, La Jolla, California, USA; Department of Surgery, University of California San Diego, La Jolla, California, USA.
² Center for Computational Biology and Bioinformatics, University of California San Diego, La Jolla, California, USA.
³ Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA; Center for Epigenomics, University of California San Diego, La Jolla, California, USA.
⁴ Department of Medicine, University of California San Diego, La Jolla, California, USA.
⁵ Department of Pathology, University of California San Diego, La Jolla, California, USA.
⁶ Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA; Center for Epigenomics, University of California San Diego, La Jolla, California, USA; Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁷ Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
⁸ Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA; Center for Epigenomics, University of California San Diego, La Jolla, California, USA. Electronic address: a5wang@health.ucsd.edu.
⁹ Department of Medicine, University of California San Diego, La Jolla, California, USA; Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA. Electronic address: dbrenner@sbpdiscovery.org.
¹⁰ Department of Surgery, University of California San Diego, La Jolla, California, USA. Electronic address: tkisseleva@health.ucsd.edu.

PMID: 39522884
DOI: 10.1016/j.jhep.2024.10.044

Abstract

Background & aims: Metabolic dysfunction-associated steatotic liver disease ranges from metabolic dysfunction-associated steatotic liver (MASL) to metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. We compared transcriptome and chromatin accessibility of human HSCs from NORMAL, MASL, and MASH livers at single-cell resolution. We aimed to identify genes that are upregulated in activated HSCs and to determine which of these genes are key in the pathogenesis of MASH fibrosis.

Methods: Eighteen human livers were profiled using single-nucleus (sn)RNA-seq and snATAC-seq. High priority targets were identified, then tested in 2D human HSC cultures, 3D human liver spheroids, and HSC-specific gene knockout mice.

Results: MASH-enriched activated HSC subclusters are the major source of extracellular matrix proteins. We identified a set of concurrently upregulated and more accessible core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive activation of HSCs. Expression of these genes was regulated via crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors. The pathological relevance of the selected targets, such as SERPINE1 (PAI-1), was demonstrated using dsiRNA-based HSC-specific gene knockdown or pharmacological inhibition of PAI-1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice.

Conclusion: This study identified novel gene targets and regulatory mechanisms underlying activation of MASH fibrogenic HSCs and demonstrated that genetic or pharmacological inhibition of select genes suppressed liver fibrosis.

Impact and implications: Herein, we present the results of a multi-modal sequencing analysis of human hepatic stellate cells (HSCs) from NORMAL, MASL (metabolic dysfunction-associated steatotic liver), and metabolic dysfunction-associated steatohepatitis (MASH) livers. We identified additional subclusters that were not detected by previous studies and characterized the mechanism by which HSCs are activated in MASH livers, including the transcriptional machinery that induces the transdifferentiation of HSCs into myofibroblasts. For the first time, we described the pathogenic role of activated HSC-derived PAI-1 (a product of the SERPINE1 gene) in the development of MASH liver fibrosis. Targeting the RUNX1/2-SERPINE1 axis could be a novel strategy for the treatment of liver fibrosis in patients.

Keywords: MASH; MASLD; activation of human HSCs; liver fibrosis; snRNA-seq and snATAC-seq.