Characterization of Cellular Sources and Circulating Levels of Extracellular Vesicles in a Dietary Murine Model of Nonalcoholic Steatohepatitis.

Circulating extracellular vesicles (EVs) are a novel and emerging biomarker for nonalcoholic steatohepatitis (NASH). It has been demonstrated that total circulating EVs and hepatocyte-derived EVs are elevated in male mice with diet-induced NASH. How hepatocyte-derived EVs change over time and other cellular sources of EVs in NASH have not been determined. Our objective was to define the quantitative evolution of hepatocyte-derived, macrophage-derived, neutrophil-derived, and platelet-derived EVs in male and female mice with dietary NASH. Fluorescently labeled antibodies and a nanoscale flow cytometer were used to detect plasma levels of EVs. Asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) are markers of hepatocyte-derived EVs; galectin 3 is a marker of macrophage-derived EVs; common epitope on lymphocyte antigen 6 complex, locus G/C1 (Ly-6G and Ly-6C) is a marker of neutrophil-derived EVs; and clusters of differentiation 61 (CD61) is a marker of platelet-derived EVs. Nonalcoholic fatty liver disease activity score (NAS) was calculated using hematoxylin and eosin-stained liver sections, and magnetic resonance imaging (MRI) was used for measurement of the fat fraction and elastography. Hepatocyte-derived EVs increased in both male and female mice at 12 and 10 weeks of feeding, respectively, and remained elevated at 24 weeks in both male and female mice and at 48 weeks in male mice and 36 weeks in female mice. Macrophage- and neutrophil-derived EVs were significantly elevated at 24 weeks of dietary feeding concomitant with the histologic presence of inflammatory foci in the liver. In fat-, fructose-, and cholesterol- (FFC) fed male mice, platelet-derived EVs were elevated at 12, 24, and 48 weeks, whereas in female mice, platelet derived EVs were significantly elevated at 24 weeks. Hepatocyte-, macrophage- and neutrophil-derived EVs correlated well with the histologic NAS. Circulating cell-type-specific EVs may be a novel biomarker for NASH diagnosis and longitudinal follow up.