Secreted phospholipase activity alters the post-secretory stability of extracellular vesicles

Background Extracellular vesicle (EV)-mediated signaling and EV-based delivery require that vesicles remain stable after release. In the post-secretory phase, released EVs encounter extracellular fluids before reaching recipient cells, yet the enzymatic mechanisms that modify EV stability in these environments remain poorly defined. Results Using bronchoalveolar lavage fluid (BALF) as a physiologically relevant extracellular fluid model, we examined whether released EVs are enzymatically altered during the post-secretory extracellular phase. Exposure of EV preparations to BALF redistributed particle-size profiles toward smaller size fractions, increasing the 0–200 nm fraction from 26.1% to 46.1% and reducing the 200–400 nm fraction from 65.3% to 44.5%. This shift was reproduced by recombinant group V secreted phospholipase A2 (sPLA2) and attenuated by pharmacological sPLA2 inhibition, linking BALF-induced EV remodeling to sPLA2-sensitive phospholipid hydrolysis. Consistently, sPLA2 exposure generated an arachidonic acid (AA)-associated lipid signal, supporting EV membrane phospholipid hydrolysis. In vivo, adeno-associated virus-mediated expression of catalytically active sPLA2g5 shifted endogenous BALF EV profiles and reduced exogenous EV accumulation in BALF macrophages, whereas the catalytically inactive H67A mutant did not reproduce this effect. In lipopolysaccharide-induced pulmonary inflammation, Pla2g5 expression was increased and exogenous EV accumulation in BALF macrophages was reduced, and this reduction was attenuated by sPLA2 inhibition. Conclusions These findings identify sPLA2-sensitive phospholipid hydrolysis as an extracellular mechanism that regulates post-secretory EV stability and indicate that phospholipase-active extracellular fluid environments can influence EV persistence and recipient-cell accumulation.