Abstract

Temporal Influence of Extracellular Vesicle-Depleted Serum on Extracellular Vesicles and Endothelial Cells

Luiz Fernando Cardoso Garcia, First author, ICC-FIOCRUZ/PR

Extracellular vesicles (EVs) are generated by various cell types, including endothelial cells. The presence of EVs in fetal bovine serum (FBS), commonly used in cell culture, has been recognized as a potential confounding factor. To elucidate this, human brain microvascular endothelial cells (HBMEC) were cultured for 2 or 24 hours in the presence of EV-depleted FBS (EVdS). Cell viability, gene and protein expression, and EVs isolated from these cells were assessed. Additionally, EV uptake, ICAM-1 expression, and monocyte adhesion to HBMEC exposed to EVs were also examined. Elevated apoptosis rates in cells cultured with EVdS for 2 and 24 hours was observed. Within 2 hours, there was an upregulation of IL8, followed by the downregulation of IL6 and IL8 after 24 hours. Proteomic analysis revealed that EVs cultured for 2 hours (EV2h) were enriched in proteins associated with ribosomes and carbon metabolism, while those cultured for 24 hours (EV24h) exhibited proteins linked to cell adhesion and platelet activation. Moreover, HBMECs exposed to EV2h displayed increased ICAM-1 expression and monocyte adhesion compared to cells exposed to EV24h. These findings highlight that HBMECs cultured with EVdS produce EVs with distinct physical characteristics and protein content that varies across time.