Alix Mediated Selective Packaging of β-Catenin into Extracellular Vesicles Enhances Their Pro-Angiogenic 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 co-localization 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 (KD) and Alix-overexpressing (OE) MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-OE-MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-KD-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 pro-angiogenic potency. Modified.