# Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

## TL;DR

This paper reveals a new pathway where a specific protein signal leads to the release of large cell vesicles containing a growth factor that promotes blood vessel formation.

## Contribution

The study identifies a novel PI3K–Rab18‐GDP signaling pathway that drives unconventional secretion of SHH-containing XLEVs with pro-angiogenic activity.

## Key findings

- PI3K–Rab18-GDP signaling promotes secretion of SHH-rich XLEVs from mesenchymal stem cells and fibroblasts.
- Rab18-GDP recruits Hsp90α and nSMase2 to facilitate polarized release of SHH-XLEVs.
- SHH-containing XLEVs stimulate endothelial network formation, linking PI3K signaling to angiogenesis.

## Abstract

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.

Human mesenchymal stem cells conditionally secrete sonic hedgehog (SHH)‐containing extra‐large extracellular vesicles (XLEVs) upon PI3K activation, which enriches the GDP‐bound form of Rab18. Rab18‐GDP drives perinuclear maturation and polarized vertical release of SHH‐XLEVs via Hsp90α‐ and nSMase2‐dependent mechanisms. These angiogenic XLEVs robustly stimulate endothelial network formation, revealing an unconventional secretory pathway linking PI3K signaling to regenerative angiogenesis.