Characterization and Proteome of Circulating Extracellular Vesicles as Potential Biomarkers for NASH

Abstract

Nonalcoholic fatty liver disease (NAFLD) is currently one of most common forms of chronic liver disease globally. NAFLD represents a wide spectrum of liver involvement from nonprogressive isolated steatosis to nonalcoholic steatohepatitis (NASH), characterized by liver necroinflammation and fibrosis and currently one of the top causes of end‐stage liver disease and hepatocellular carcinoma. At present, there is a lack of effective treatments, and a central barrier to the development of therapies is the requirement for an invasive liver biopsy for diagnosis of NASH. Discovery of reliable, noninvasive biomarkers are urgently needed. In this study, we tested whether circulating extracellular vesicles (EVs), cell‐derived small membrane‐surrounded structures with a rich cargo of bioactive molecules, may serve as reliable noninvasive “liquid biopsies” for NASH diagnosis and assessment of disease severity. Total circulating EVs and hepatocyte‐derived EVs were isolated by differential centrifugation and size‐exclusion chromatography from serum samples of healthy individuals, patients with precirrhotic NASH, and patients with cirrhotic NASH. EVs were further characterized by flow cytometry, electron microscopy, western blotting, and dynamic light scattering assays before performing a proteomics analysis. Our findings suggest that levels of total and hepatocyte‐derived EVs correlate with NASH clinical characteristics and disease severity. Additionally, using proteomics data, we developed understandable, powerful, and unique EV‐based proteomic signatures for potential diagnosis of advanced NASH. Conclusion: Our study shows that the quantity and protein constituents of circulating EVs provide strong evidence for EV protein–based liquid biopsies for NAFLD/NASH diagnosis.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses

“…Markers were selected based on the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines for confirming the presence of EV-enriched proteins and include those derived from the plasma membrane, endosomal pathway, and cytosol [21]. The panel was comprised of tetraspanins CD9, CD63 and CD81, and the cytosolic protein tumour susceptibility gene 101 (TSG101), as well as the hepatocyte-specific surface protein asialoglycoprotein receptor 1 (ASGR1), which is known to be expressed on EVs derived from this cell type [4,22]. The expression of protein markers was quantified by LCMS and assessed for variability between subjects in AM and PM EVs ( Figure 3D-H).…”

Controversies and Opportunities in the Use of Inflammatory Markers for Diagnosis or Risk Prediction in Fatty Liver Disease

“…Besides such changing cargo, also changes in the absolute numbers of circulating EVs may be observed during disease. For example, an elevated number of circulating EVs is observed in NASH patients, with the EV-numbers strongly correlating to NASH clinical characteristics and disease severity ( 139 ). These findings reflect the elevated number of circulating EVs identified in mice with diet-induced NASH ( 140 ).…”

Novel high-throughput applications for NAFLD diagnostics and biomarker discovery

“…The diagnostic accuracy presented AUROCs between 0.93 and 1. Moreover, Povero et al [24] recently described the application of SOMAScan® to the proteomic characterization of circulating extracellular vesicles (EVs). The data demonstrated that the quantity and protein constituents of circulating EVs provided strong evidence for the utility of EV proteinbased liquid biopsies for NAFLD/NASH diagnosis.…”