Single-cell transcriptomics of hepatic stellate cells uncover crucial pathways and key regulators involved in non-alcoholic steatohepatitis

Background Fibrosis is an important pathological process in the development of non-alcoholic steatohepatitis (NASH), and the activation of hepatic stellate cell (HSC) is a central event in liver fibrosis. However, the transcriptomic change of activated HSCs (aHSCs) and resting HSCs (rHSCs) in NASH patients has not been assessed. This study aimed to identify transcriptomic signature of HSCs during the development of NASH and the underlying key functional pathways. Methods NASH-associated transcriptomic change of HSCs was defined by single-cell RNA-sequencing (scRNA-seq) analysis, and those top upregulated genes were identified as NASH-associated transcriptomic signatures. Those functional pathways involved in the NASH-associated transcriptomic change of aHSCs were explored by weighted gene co-expression network analysis (WGCNA) and functional enrichment analyses. Key regulators were explored by upstream regulator analysis and transcription factor enrichment analysis. Results scRNA-seq analysis identified numerous differentially expressed genes in both rHSCs and aHSCs between NASH patients and healthy controls. Both scRNA-seq analysis and in-vivo experiments showed the existence of rHSCs (mainly expressing a-SMA) in the normal liver and the increased aHSCs (mainly expressing collagen 1) in the fibrosis liver tissues. NASH-associated transcriptomic signature of rHSC (NASHrHSCsignature) and NASH-associated transcriptomic signature of aHSC (NASHaHSCsignature) were identified. WGCNA revealed the main pathways correlated with the transcriptomic change of aHSCs. Several key upstream regulators and transcription factors for determining the functional change of aHSCs in NASH were identified. Conclusion This study developed a useful transcriptomic signature with the potential in assessing fibrosis severity in the development of NASH. This study also identified the main pathways in the activation of HSCs during the development of NASH.

[1]  Shou-Li Yuan,et al.  Differences of core genes in liver fibrosis and hepatocellular carcinoma: Evidence from integrated bioinformatics and immunohistochemical analysis , 2022, World journal of gastrointestinal oncology.

[2]  Xiaofan Lai,et al.  Asporin Promotes TGF-β-induced Lung Myofibroblast Differentiation by Facilitating Rab11-dependent Recycling of TβRI. , 2021, American journal of respiratory cell and molecular biology.

[3]  Dongmei Wang,et al.  Identification of transcriptomic signatures and crucial pathways involved in non-alcoholic steatohepatitis , 2021, Endocrine.

[4]  A. Federico,et al.  Immunity as Cornerstone of Non-Alcoholic Fatty Liver Disease: The Contribution of Oxidative Stress in the Disease Progression , 2021, International journal of molecular sciences.

[5]  P. Gual,et al.  Chronic Inflammation in Non-Alcoholic Steatohepatitis: Molecular Mechanisms and Therapeutic Strategies , 2020, Frontiers in Endocrinology.

[6]  N. Garg,et al.  Trypanosoma cruzi Induces the PARP1/AP-1 Pathway for Upregulation of Metalloproteinases and Transforming Growth Factor β in Macrophages: Role in Cardiac Fibroblast Differentiation and Fibrosis in Chagas Disease , 2020, mBio.

[7]  R. Schwabe,et al.  Mechanisms of Fibrosis Development in Nonalcoholic Steatohepatitis , 2020 .

[8]  Platelet-Derived Growth Factor Receptor Beta , 2020, Definitions.

[9]  Platelet-Derived Growth Factor Receptor Alpha , 2020, Definitions.

[10]  Zhehu Jin,et al.  Syndecan-1 regulates extracellular matrix expression in keloid fibroblasts via TGF-β1/Smad and MAPK signaling pathways. , 2020, Life sciences.

[11]  Felix Alonso-Valenteen,et al.  Syndecan-1 promotes lung fibrosis by regulating epithelial reprogramming through extracellular vesicles. , 2019, JCI insight.

[12]  Hongliang Li,et al.  The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis , 2019, Hepatology.

[13]  P. Gual,et al.  Natural Killer Cells and Type 1 Innate Lymphoid Cells Are New Actors in Non-alcoholic Fatty Liver Disease , 2019, Front. Immunol..

[14]  Z. Younossi.  Non-alcoholic fatty liver disease - A global public health perspective. , 2019, Journal of hepatology.

[15]  S. Mandrup,et al.  Transcriptional regulation of Hepatic Stellate Cell activation in NASH , 2019, Scientific Reports.

[16]  Z. Younossi,et al.  Burden of Illness and Economic Model for Patients With Nonalcoholic Steatohepatitis in the United States , 2019, Hepatology.

[17]  Christoph Hafemeister,et al.  Comprehensive integration of single cell data , 2018, bioRxiv.

[18]  D. Schuppan,et al.  The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis , 2018, Nature Reviews Gastroenterology & Hepatology.

[19]  Antonio Felix Conde-Martin,et al.  Fibrosis Severity as a Determinant of Cause-Specific Mortality in Patients With Advanced Nonalcoholic Fatty Liver Disease: A Multi-National Cohort Study. , 2018, Gastroenterology.

[20]  B. Neuschwander‐Tetri,et al.  Mechanisms of NAFLD development and therapeutic strategies , 2018, Nature Medicine.

[21]  Shelly C. Lu,et al.  NASH Leading Cause of Liver Transplant in Women: Updated Analysis of Indications For Liver Transplant and Ethnic and Gender Variances , 2018, The American Journal of Gastroenterology.

[22]  D. S̆timac,et al.  Nonalcoholic fatty liver disease and liver transplantation - Where do we stand? , 2018, World journal of gastroenterology.

[23]  R. Hultcrantz,et al.  Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD. , 2017, Journal of hepatology.

[24]  S. Friedman,et al.  Hepatic stellate cells as key target in liver fibrosis. , 2017, Advanced drug delivery reviews.

[25]  E. Hatzimichael,et al.  Expression patterns of the activator protein-1 (AP-1) family members in lymphoid neoplasms , 2017, Clinical and Experimental Medicine.

[26]  S. Friedman,et al.  Mechanisms of hepatic stellate cell activation , 2017, Nature Reviews Gastroenterology &Hepatology.

[27]  V. Wong,et al.  Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta‐analysis , 2017, Hepatology.

[28]  Alex Gutteridge,et al.  Interpreting transcriptional changes using causal graphs: new methods and their practical utility on public networks , 2016, BMC Bioinformatics.

[29]  Q. Anstee,et al.  Nonalcoholic Fatty Liver Disease: Pathogenesis and Disease Spectrum. , 2016, Annual review of pathology.

[30]  E. Bjornsson,et al.  Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. , 2015, Gastroenterology.

[31]  M. Karsdal,et al.  Novel insights into the function and dynamics of extracellular matrix in liver fibrosis. , 2015, American journal of physiology. Gastrointestinal and liver physiology.

[32]  T. Kisseleva,et al.  Bone marrow-derived fibrocytes contribute to liver fibrosis , 2015, Experimental biology and medicine.

[33]  A. Diehl,et al.  Fibrosis in Nonalcoholic Fatty Liver Disease: Mechanisms and Clinical Implications , 2015, Seminars in Liver Disease.

[34]  R. Schwabe,et al.  Origin and Function of Myofibroblasts in the Liver , 2015, Seminars in Liver Disease.

[35]  D. Abraham,et al.  Failed Degradation of JunB Contributes to Overproduction of Type I Collagen and Development of Dermal Fibrosis in Patients With Systemic Sclerosis , 2014, Arthritis & rheumatology.

[36]  L. Bach.  IGFBP6 (insulin-like growth factor binding protein 6) , 2014 .

[37]  M. Nomizu,et al.  Identification of fibronectin binding sites in dermatopontin and their biological function. , 2014, Journal of dermatological science.

[38]  M. Nomizu,et al.  Functional peptide of dermatopontin produces fibrinogen fibrils and modifies its biological activity. , 2014, Journal of dermatological science.

[39]  E. Brunt,et al.  Role of liver biopsy in nonalcoholic fatty liver disease. , 2014, World journal of gastroenterology.

[40]  Stein Aerts,et al.  iRegulon: From a Gene List to a Gene Regulatory Network Using Large Motif and Track Collections , 2014, PLoS Comput. Biol..

[41]  R. Schwabe,et al.  Fate-tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its etiology , 2013, Nature Communications.

[42]  S. Friedman,et al.  Hepatic stellate cells and liver fibrosis. , 2013, Comprehensive Physiology.

[43]  Justin Guinney,et al.  GSVA: gene set variation analysis for microarray and RNA-Seq data , 2013, BMC Bioinformatics.

[44]  G. Gores,et al.  Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis , 2012, Laboratory Investigation.

[45]  Song Liu,et al.  Jcb: Article , 2022 .

[46]  W. Cheung,et al.  FHL2 (four and a half LIM domains 2) , 2011 .

[47]  T. Shimada,et al.  Dermatopontin Interacts with Fibronectin, Promotes Fibronectin Fibril Formation, and Enhances Cell Adhesion* , 2011, The Journal of Biological Chemistry.

[48]  M. Leshno,et al.  Syndecan 1 (CD138) serum levels: a novel biomarker in predicting liver fibrosis stage in patients with hepatitis C , 2009, Liver international : official journal of the International Association for the Study of the Liver.

[49]  Charlyn J. Suarez,et al.  Integrated Weighted Gene Co-expression Network Analysis with an Application to Chronic Fatigue Syndrome , 2008, BMC Systems Biology.

[50]  R. Farràs,et al.  Regulation and function of JunB in cell proliferation. , 2008, Biochemical Society transactions.

[51]  P. Roughley,et al.  Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues , 2008, Arthritis research & therapy.

[52]  L. Ventéo,et al.  Expression of lumican, a small leucine‐rich proteoglycan with antitumour activity, in human malignant melanoma , 2007, Clinical and experimental dermatology.

[53]  G. Farrell,et al.  Nonalcoholic fatty liver disease: From steatosis to cirrhosis , 2006, Hepatology.

[54]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[55]  G. Jacquemin.  [Nonalcoholic steatohepatitis: a review]. , 2003, Revue medicale de Liege.

[56]  P. Roughley,et al.  Lumican is a major proteoglycan component of the bone matrix. , 2002, Matrix biology : journal of the International Society for Matrix Biology.

[57]  J. Hassell,et al.  Regulation of corneal collagen fibrillogenesis in vitro by corneal proteoglycan (lumican and decorin) core proteins. , 1993, Experimental eye research.

[58]  A. Charchanti,et al.  Expression of Syndecan-1 in Chronic Liver Diseases: Correlation With Hepatic Fibrosis , 2021, In Vivo.

[59]  L. Henry,et al.  Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention , 2018, Nature Reviews Gastroenterology & Hepatology.

[60]  H. Lee,et al.  Association between non-alcoholic fatty liver disease and cancer incidence rate. , 2017, Journal of hepatology.

[61]  S. Friedman.  Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. , 2008, Physiological reviews.