In silico Discovery and Translational Characterization of Biomarkers for Fibrosis in Metabolic Dysfunction-Associated Steatotic Liver Disease

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease, affecting over 30% of adults globally, with projections rising to 55% by 2040 alongside obesity and metabolic syndromes. Hepatic fibrosis is the best predictor of MASLD-related morbidity and mortality, but liver biopsy, the gold standard for staging, is invasive and costly. Thus, there is a pressing need for non-invasive biomarkers for fibrosis diagnosis. This study employed integrative in silico, in vivo, and in vitro approaches to identify protein-coding genes as potential biomarkers, while elucidating molecular mechanisms of fibrosis in MASLD. Transcriptomic datasets were re-analysed, comparing early (F0-F1) versus moderate/advanced fibrosis (F2-F4) in MASLD liver biopsies. Plasma-secreted DEGs were identify using a new in silico funnel strategy, with top-ranked genes analysed by qPCR in morbidly obese liver samples stratified by fibrosis and by ELISA in plasma from a bariatric cohort. Of 106 DEGs, 22 encoded potentially circulating proteins. Eight candidates, including EFEMP1, LTBP2, LUM, DPT, CHI3L1, SPON1, FBLN5 and CCL20, underwent validation. EFEMP1 showed consistent differences in liver mRNA and protein expression between early and advanced fibrosis and increased plasma levels with fibrosis severity. Plasma Fibulin-3 (EFEMP1) outperformed common fibrosis scores in detecting significant fibrosis, with accuracy further enhanced when combined with standard clinical parameters such as platelets, gamma-glutamyl transferase and hepatic steatosis index To gain mechanistic insight, integrative network analysis was performed using transcriptomic data from fibrotic liver tissues obtained via Genevestigator and Network Analyst. Mechanistically, network analysis of fibrotic liver transcriptomes revealed EFEMP1’s integration with key regulators of ECM remodeling and fibrogenic pathways, underscoring its relevance in fibrogenesis. Furthermore, GADD45G gene exhibited the highest degree of connectivity within the PPI interaction network, indicating its potential role as a regulatory hub protein coding gene in fibrosis progression, and it was part of the 39 DEGs across all the fibrosis stages. In the HuH7-LX2 co-culture model, free fatty acid and TGF-β stimulation upregulated EFEMP1, indicating responsiveness to metabolic and profibrotic signals. EFEMP1 silencing modestly altered α-SMA while increasing collagen and disrupting expression of ECM regulators MMP2 and TIMP2, supporting EFEMP1’s role as a dynamic regulator of fibrosis and its potential as a circulating fibrosis biomarker in MASLD. Liver transplantation remains the definitive treatment option for patients with end-stage chronic liver disease which includes MASLD, offering improved survival and quality of life when medical therapy is no longer effective. We also explored epigenetic and transcriptomic changes during liver transplantation from brain-dead donors to investigate ischemia-reperfusion injury (IRI). Serial biopsies during procurement and post-reperfusion underwent genome-wide DNA methylation and transcriptomic profiling, revealing significant methylation changes, particularly post-reperfusion, impacting promoters involved in RNA processing and immune regulation. Transcriptome analysis showed activation of TNF-α, NF-κB, and interleukin pathways with enrichment of innate immune cells, indicating coordinated epigenetic and transcriptional reprogramming during IRI. Together, these findings advance the potential clinical utility of EFEMP1 as a non-invasive fibrosis biomarker in MASLD and underscore the critical role of epigenetic mechanisms in post-transplant liver inflammation, paving the way for biomarker-guided management and therapeutic interventions in liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease, affecting over 30% of adults globally, with projections rising to 55% by 2040 alongside obesity and metabolic syndromes. Hepatic fibrosis is the best predictor of MASLD-related morbidity and mortality, but liver biopsy, the gold standard for staging, is invasive and costly. Thus, there is a pressing need for non-invasive biomarkers for fibrosis diagnosis. This study employed integrative in silico, in vivo, and in vitro approaches to identify protein-coding genes as potential biomarkers, while elucidating molecular mechanisms of fibrosis in MASLD. Transcriptomic datasets were re-analysed, comparing early (F0-F1) versus moderate/advanced fibrosis (F2-F4) in MASLD liver biopsies. Plasma-secreted DEGs were identify using a new in silico funnel strategy, with top-ranked genes analysed by qPCR in morbidly obese liver samples stratified by fibrosis and by ELISA in plasma from a bariatric cohort. Of 106 DEGs, 22 encoded potentially circulating proteins. Eight candidates, including EFEMP1, LTBP2, LUM, DPT, CHI3L1, SPON1, FBLN5 and CCL20, underwent validation. EFEMP1 showed consistent differences in liver mRNA and protein expression between early and advanced fibrosis and increased plasma levels with fibrosis severity. Plasma Fibulin-3 (EFEMP1) outperformed common fibrosis scores in detecting significant fibrosis, with accuracy further enhanced when combined with standard clinical parameters such as platelets, gamma-glutamyl transferase and hepatic steatosis index To gain mechanistic insight, integrative network analysis was performed using transcriptomic data from fibrotic liver tissues obtained via Genevestigator and Network Analyst. Mechanistically, network analysis of fibrotic liver transcriptomes revealed EFEMP1’s integration with key regulators of ECM remodeling and fibrogenic pathways, underscoring its relevance in fibrogenesis. Furthermore, GADD45G gene exhibited the highest degree of connectivity within the PPI interaction network, indicating its potential role as a regulatory hub protein coding gene in fibrosis progression, and it was part of the 39 DEGs across all the fibrosis stages. In the HuH7-LX2 co-culture model, free fatty acid and TGF-β stimulation upregulated EFEMP1, indicating responsiveness to metabolic and profibrotic signals. EFEMP1 silencing modestly altered α-SMA while increasing collagen and disrupting expression of ECM regulators MMP2 and TIMP2, supporting EFEMP1’s role as a dynamic regulator of fibrosis and its potential as a circulating fibrosis biomarker in MASLD. Liver transplantation remains the definitive treatment option for patients with end-stage chronic liver disease which includes MASLD, offering improved survival and quality of life when medical therapy is no longer effective. We also explored epigenetic and transcriptomic changes during liver transplantation from brain-dead donors to investigate ischemia-reperfusion injury (IRI). Serial biopsies during procurement and post-reperfusion underwent genome-wide DNA methylation and transcriptomic profiling, revealing significant methylation changes, particularly post-reperfusion, impacting promoters involved in RNA processing and immune regulation. Transcriptome analysis showed activation of TNF-α, NF-κB, and interleukin pathways with enrichment of innate immune cells, indicating coordinated epigenetic and transcriptional reprogramming during IRI. Together, these findings advance the potential clinical utility of EFEMP1 as a non-invasive fibrosis biomarker in MASLD and underscore the critical role of epigenetic mechanisms in post-transplant liver inflammation, paving the way for biomarker-guided management and therapeutic interventions in liver disease.