Plasma Extracellular Vesicles Contain Protein Biomarkers for Capturing Stages of Metabolic Dysfunction-Associated Steatotic Liver Disease: A Preliminary Exploratory Study

Metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing in both prevalence and severity, highlighting the need for non-invasive biomarkers to assess disease activity. Extracellular vesicles (EVs), which carry molecular cargo from their cells of origin, hold promise as accessible biomarkers. We performed proteomic profiling of plasma-derived EVs from 70 patients with MASLD to identify protein signatures associated with key histological features (steatosis, metabolic dysfunction-associated steatohepatitis (MASH), and fibrosis). These proteins were subsequently correlated with magnetic resonance (MR)-based liver imaging. Plasma EV protein profile differed between mild (S1) and advanced steatosis (S3). H4C1, OIT3, and ANPEP were elevated in S3, while CCDC25 and KLHL41 were decreased (|log₂ fold change| > 1, <i>p</i> < 0.05). KLHL41 had a weak-to-moderate correlation with proton density fat fraction (PDFF) (R = -0.34, <i>p</i> = 0.016). GP1BA was upregulated in MASH (log₂ fold change = 1.13, <i>p</i> = 0.03) but showed weak correlation with cT1, an imaging parameter for steatohepatitis (R = 0.22, <i>p</i> = 0.173). In fibrosis, complement component 7 (C7) was elevated in advanced (≥F3) vs. mild fibrosis (<F2) (log₂ fold change = 0.95, adjusted <i>p</i> = 0.002) and correlated with MR elastography-derived liver stiffness (R = 0.38, <i>p</i> = 0.004). The AUC of C7 for differentiating <F2 vs. ≥F2 and <F3 vs. ≥F3 was 0.80 (95% CI: 0.69-0.91) and 0.83 (95% CI: 0.72-0.93), respectively. In conclusion, plasma EVs contain distinct protein signatures associated with steatosis, steatohepatitis, and fibrosis in MASLD. These preliminary findings support the potential utility of plasma EVs as non-invasive biomarkers and provide insights into disease pathophysiology. However, further validation in larger, independent cohorts is necessary to confirm these associations and establish their clinical relevance.