Exploring the crosstalk between endothelial autophagy and extracellular vesicle biology: Potential role in atherosclerosis

Abstract

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express an inflammatory phenotype. Our team demonstrated that endothelial autophagy is protective, stimulated by high shear stress (HSS) conditions when compared to LSS, hampering the development of these lesions. Endothelial EVs have been shown to regulate inflammation and senescence, thus playing a crucial role in vascular homeostasis. While previous studies found links between autophagy and EV formation, the exact role of autophagy in the release and uptake of EVs remains elusive. Thus, our aim is to decipher the interplay between these processes in endothelial cells. Confluent HUVECs were exposed to LSS (2 dyn/cm 2 ) or HSS (20 dyn/cm 2 ) for 24 hours. Large (> 200 nm) and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by Western blot, TRPS, flow cytometry and proteomics. Uptake experiments were performed using fluorescently labelled EVs. Large and small EV levels were 50 and 5 times higher in HSS than in LSS conditions, respectively. In vivo and in vitro assays revealed more EV uptake by cells exposed to LSS compared to HSS. Interestingly, endothelial LSS-EVs had greater affinity for HUVECs than HSS-EVs or EVs derived from other cell types. Proteomics showed that LSS-EVs were enriched in adhesion proteins PECAM1 and MCAM. Blocking these proteins on EVs reduced their uptake by HUVECs. Finally, inhibiting autophagy in parent cells raised adhesion protein levels in HSS-EVs and caused them to be more readily taken up. These findings suggest that autophagy modulation, by shear stress, plays a crucial role during EV formation. Given the importance of EVs and autophagy in vascular health, deciphering the relation between these processes may yield new strategies for early detection and treatment of atherosclerosis.