Alix-mediated selective packaging of β-catenin into extracellular vesicles enhances their proangiogenic function.

The function of extracellular vesicles (EVs) is determined by the molecular cargo they carry from their parent cells. Although apoptosis-linked gene 2-interacting protein X (Alix) is known to regulate EV cargo loading and functional properties, the specific mechanisms underlying its role in mediating β-catenin sorting and function remain unclear. In this study, we first observed the colocalization of Alix and β-catenin through immunofluorescence staining. To assess whether the interaction between Alix and β-catenin affects the function of mesenchymal stem cell (MSC)-derived EVs, we generated Alix-knockdown and Alix-overexpressing MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-overexpressing MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-knockdown MSCs suppressed angiogenesis. Mechanistically, we confirmed that the Alix-β-catenin interaction selectively enhances β-catenin enrichment within EVs. In conclusion, our findings demonstrate that Alix plays a critical role in selectively packaging β-catenin into EVs, thereby enhancing their proangiogenic potency.