Heparan Sulphate Glycosaminoglycan Chains Contribute to the Tethering of Coronal Factors and Are Important for Extracellular Vesicle‐Mediated Fibroblast Activation

ABSTRACT Extracellular vesicles (EVs) play a critical role in intercellular communication, yet the contribution of the EV corona and associated surface structures, such as heparan sulphate glycosaminoglycan (HS GAG) chains, to EV function remains poorly understood. In this study, we highlight a hitherto unknown requirement of HS GAG chains for the simultaneous delivery of a myriad assortment of growth factors by EVs. We demonstrate an attenuated function following enzymatic removal of HS GAG chains from the surface of prostate cancer (PCa)‐derived EVs, using heparinase III (HepIII). Our results confirm that digestion of HS GAG chains is specific and does not compromise EV integrity regarding size or tetraspanin expression. Enzymatic removal of HS GAG chains did, however, substantially altered the vesicular protein profile, reducing the expression of factors such as midkine, CYR61 and TFPI implicating HS GAG chains as a mode of tethering these factors to the EV surface. Importantly, EV‐associated HS GAG chains are required for functional delivery of such factors, resulting in successful activation of cellular signalling pathways for SCF, IGF‐1, midkine and VEGF in recipient fibroblasts. Furthermore, HS GAG chain removal attenuated EV‐induced fibroblast production of pro‐angiogenic factors VEGF and angiogenin as well as inflammatory factors VCAM‐1 and IL‐1α alpha/IL‐1F1, underscoring the role of vesicular HS GAG chains in mediating functional outcomes. These findings highlight the importance of EV surface HS GAG chains in growth factor delivery and signalling, providing new insights into the EV corona and its relevance in pathological processes relating to modulation of the tissue microenvironment.