Unconventional secretion of angiogenic sonic-hedgehog-containing large extracellular vesicles is stimulated via the PI3K–Rab18-GDP pathway

ABSTRACT Large extracellular vesicles (lEVs), with diameters >600 nm, play essential roles in special types of intercellular communications. However, information on the molecular mechanism underlying their roles and on their relevance remains scarce. Here we show that PI3K–Rab18-GDP signaling facilitates the secretion of lEVs from human mesenchymal stem cells (hMSCs) and fibroblasts. These EVs contain a large amount of sonic hedgehog (SHH) protein and promote angiogenesis. Rab18 serves as the master facilitator of this secretion only in its GDP-bound form, which can be enriched by the Rab inhibitor CID1067700 or by the PI3K agonist SF1670. Rab18-GDP accumulates in the cell center and develops SHH-lEV precursors from perinuclear endosomes. Rab18-GDP recruits heat shock protein 90α (Hsp90α) and neutral sphingomyelinase 2 (nSMase2) and facilitates vertical secretion of SHH-lEVs with an Hsp90α corona from the cell center. This secretory machinery may play an important role in developmental and regenerative PI3K-mediated angiogenesis. Competing Interest Statement A Japanese patent (No. 2022-078750 (PCT EP4524239A1)) has been filed.