Introduction: Metabolic associated fatty liver disease (MAFLD) is the most common chronic liver disease globally, yet minimally-invasive diagnostic tools and approved drug treatments are lacking. Plasma EVs have emerged as a promising source of biomarkers to monitor disease. We aimed to evaluate the capacity for liver-derived EVs to convey alterations in circulating miRNA profile compared to non-EV and non-liver-specific sources. Since MAFLD can impair hepatic drug clearance, we also characterised expression of drug metabolising enzyme CYP3A4 in liver-derived EVs.
Methods: EVs were isolated from plasma of patients across MAFLD stages (simple steatosis SS, steatohepatitis NASH, n=15) and controls (n=13) by size exclusion chromatography and anti-ASGR1 immunoprecipitation (IP). Targeted proteomics assessed presence of EV markers and contaminants, and abundance of markers from hepatic and non-hepatic origin. Changes in ASGR1+ EV CYP3A4 protein expression was assessed in control, SS and NASH subjects. Total circulating and vesicular RNA were isolated by phenol-chloroform extraction and expression of miR -122, -192 and -128-3p quantified by RT-qPCR.
Results: Anti-ASGR1 IP successfully captured plasma EVs of hepatic origin. Patterns of biomarker expression varied in each source of RNA (total/global EV/ASGR1+ EV). Selective analysis of ASGR1+ EVs produced a clear directional trend with increasing miRNA expression in disease (c-statistics 0.78-0.84). A significant decreasing trend in CYP3A4 expression was observed in ASGR1+ EVs with increasing MAFLD severity (p=0.049).
Conclusions: ASGR1+ EVs represent a valuable source of circulating liver-specific miRNA biomarkers compared to total plasma or global EV. ASGR1+ EV convey the impact of MAFLD on hepatic drug clearance.