Stellate cell expression of SPARC-related modular calcium-binding protein 2 is associated with human non-alcoholic fatty liver disease severity
Frederik T. Larsen,
Daniel Hansen,
Mike K. Terkelsen,
Sofie M. Bendixen,
Fabio Avolio,
Charlotte W. Wernberg,
Mette M. Lauridsen,
Lea L. Grønkjaer,
Birgitte G. Jacobsen,
Ellen G. Klinggaard,
Susanne Mandrup,
Tina Di Caterino,
Majken S. Siersbæk,
Vineesh Indira Chandran,
Jonas H. Graversen,
Aleksander Krag,
Lars Grøntved,
Kim Ravnskjaer
Affiliations
Frederik T. Larsen
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Daniel Hansen
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Mike K. Terkelsen
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Sofie M. Bendixen
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Fabio Avolio
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Charlotte W. Wernberg
Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Department of Gastroenterology and Hepatology, University Hospital of Southern Denmark, Esbjerg, Denmark; Center for Liver Research (FLASH), Department of Gastroenterology and Hepatology, Odense University Hospital, Odense, Denmark
Mette M. Lauridsen
Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Department of Gastroenterology and Hepatology, University Hospital of Southern Denmark, Esbjerg, Denmark
Lea L. Grønkjaer
Department of Gastroenterology and Hepatology, University Hospital of Southern Denmark, Esbjerg, Denmark
Birgitte G. Jacobsen
Department of Gastroenterology and Hepatology, University Hospital of Southern Denmark, Esbjerg, Denmark
Ellen G. Klinggaard
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Susanne Mandrup
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Tina Di Caterino
Department of Pathology, Odense University Hospital, Odense, Denmark
Majken S. Siersbæk
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Vineesh Indira Chandran
Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark
Jonas H. Graversen
Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark
Aleksander Krag
Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Center for Liver Research (FLASH), Department of Gastroenterology and Hepatology, Odense University Hospital, Odense, Denmark
Lars Grøntved
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark
Kim Ravnskjaer
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Center for Functional Genomics and Tissue Plasticity (ATLAS), University of Southern Denmark, Odense, Denmark; Corresponding author. Address: Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, Denmark. Tel.: +45 65508906/+45 93979317.
Background & Aims: Histological assessment of liver biopsies is the gold standard for diagnosis of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), despite its well-established limitations. Therefore, non-invasive biomarkers that can offer an integrated view of the liver are needed to improve diagnosis and reduce sampling bias. Hepatic stellate cells (HSCs) are central in the development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods: We performed RNA-sequencing on liver biopsies from a histologically characterised cohort of obese patients (n = 30, BMI >35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings using single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results: Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared to no-NAFLD (p.adj <0.001). Single-cell RNA-sequencing data indicated that SMOC2 was primarily expressed by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p <0.001), with a predictive accuracy of AUROC 0.88. Conclusions: Increased SMOC2 in plasma appears to reflect HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the most common forms of chronic liver diseases. Currently, liver biopsies are the gold standard for diagnosing NAFLD. Blood-based biomarkers to complement liver biopsies for diagnosis of NAFLD are required. We found that activated hepatic stellate cells, a cell type central to NAFLD pathogenesis, upregulate expression of the secreted protein SPARC-related modular calcium-binding protein 2 (SMOC2). SMOC2 was elevated in blood samples from patients with NASH and may hold promise as a blood-based biomarker for the diagnosis of NAFLD.
