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

Abstract

Atherosclerotic lesions mainly form in areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Endothelial autophagy is a protective process, stimulated under conditions of high shear stress (HSS, 20 dyn/cm 2 ) when compared to LSS (2 dyn/cm 2 ), and hampers the development these lesions. While previous studies have shown links between autophagy and extracellular vesicle (EV) formation, the precise role of the autophagic machinery in the release and uptake of EVs by endothelial cells remains elusive. Our aim is therefore to decipher the interplay between these processes in cells exposed to either atheroprone or atheroprotective shear stress. Confluent human umbical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours in a culture medium containing EV-free foetal calf serum. Large (>200 nm) and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography; they were then characterized by Western blot analysis of EV markers (CD9, CD63) and tunable resistive pulse sensing. EV uptake was assessed by flow cytometry and confocal microscopy. Levels of large and small EVs are fifty and five times higher in HSS than in LSS conditions, respectively. Surprisingly, inhibiting endothelial autophagy using shRNA targeting ATG5 in order to mimic LSS conditions slightly increases small EV levels. Uptake experiments using fluorescently labelled EVs reveal greater incorporation in LSS conditions compared to HSS. Furthermore, silencing ATG5 appears to increase EV uptake in HSS conditions. Altogether these findings suggest a role of endothelial autophagy during EV release and uptake. Given the therapeutic capacity of EVs and the importance of the autophagic process in vascular health, deciphering the relation between these processes could yield innovative strategies for the treatment of atherosclerosis.