Unconventional Secretion of Angiogenic Sonic Hedgehog-Containing Extra-Large Extracellular Vesicles is Driven by...

Extra-large extracellular vesicles (XLEVs), with diameters > 600 nm, are increasingly recognised as mediators of specialized modes of intercellular communication; however, the molecular mechanisms governing their biogenesis and functional regulation remain poorly understood. Here, we show that PI3K-Rab18-GDP signalling promotes the secretion of XLEVs from human mesenchymal stem cells (hMSCs) and fibroblasts. These vesicles are highly enriched in sonic hedgehog (SHH) and display potent pro-angiogenic activity. We further demonstrate that Rab18 functions as a key regulator of this pathway specifically in its GDP-bound form, which can be enriched by the Rab inhibitor CID1067700 or by pharmacological activation of PI3K using SF1670. Rab18-GDP preferentially accumulates in the perinuclear region, where it promotes the formation of SHH-XLEV precursors from endosomal compartments. Mechanistically, PI3K-Rab18-GDP signalling recruits heat shock protein 90α (Hsp90α) and neutral sphingomyelinase 2 (nSMase2), facilitating polarized release of SHH-XLEVs from the perinuclear-plasma membrane interface, accompanied by an Hsp90α-enriched extracellular assembly. Together, these findings identify a PI3K-Rab18-GDP-dependent secretory pathway for SHH-XLEVs and provide a framework for understanding how XLEV biogenesis is coupled to SHH-associated angiogenic signalling in developmental and regenerative contexts.