Journal: bioRxiv

Article Title: Atheroprone shear stress stimulates noxious endothelial extracellular vesicle uptake by MCAM and PECAM-1 cell adhesion molecules

doi: 10.1101/2022.12.31.522373

Figure Lengend Snippet: (A) Fluorescently-labeled SVEC4-10 LSS-lEVs (red) were injected into the mouse bloodstream. Animals were sacrificed after 30 min and aortas were harvested. Endothelial cells (green; Cadherin-5 staining) were imaged by confocal microscopy. (B) Fluorescently-labeled HUVEC LSS-lEVs (red) were incubated for 90 min with HUVECs exposed to HSS or LSS conditions for 24 h. Cells were fixed, stained for Cadherin-5 (green) and imaged by confocal microscopy. (C) Fluorescently-labeled HUVEC-derived LSS-lEVs were incubated for varying periods min with HUVECs exposed to HSS (blue) or LSS (red) conditions for 24 h. Representative quantifications of EV signal per cell from confocal images captured at different time points (15 fields per condition, *P<0.05, ****P<0.0001, One-way Anova). (D) Fluorescently-labeled HUVEC-derived HSS-lEVs (blue) or LSS-lEVs (red) were incubated for 90 min with HUVECs exposed to HSS or LSS conditions for 24 h. Representative quantifications of EV signal per cell from confocal images (15-17 fields per condition, ***P<0.001, ****P<0.0001, One-way Anova). (E) Fluorescently-labeled HUVEC-derived HSS-(blue) or LSS-(red) lEVs (left) or sEVs (right) were incubated for 90 min with HUVECs exposed to HSS or LSS conditions for 24 h. % of cells positive for EV signal was analyzed by flow cytometry. Data represent means ± SEM of 7 independent experiments, *P<0.05, **P<0.01, ***P<0.001, One-way Anova. (F) Fluorescently-labeled HUVEC-derived LSS lEVs or sEVs were incubated with HUVECs exposed to LSS conditions for 24 h. Cells were and visualized by image flow cytometry. (G) Fluorescently-labeled HUVEC-derived LSS lEVs (left) or sEVs (right) were incubated for 90 min, in the presence of endocytosis inhibitors, with HUVECs exposed to LSS conditions for 24 h. % of cells positive for EV signal, relative to Cont, was analyzed by flow cytometry. Data represent means ± SEM, relative to Cont, of 6 independent experiments, *P<0.05, **P<0.01, *** P<0.001, Friedman test. (H) Fluorescently-labeled lEVs derived from HUVECs exposed to LSS (Endo-LSS) or HSS (Endo-LSS), peripheral blood mononuclear cells (PMBC), neutrophils, platelets, red blood cells (RBC) or platelet free plasma (PFP) were incubated for 90 min with HUVECs exposed to LSS conditions for 24 h. % of cells positive for EV signal was analyzed by flow cytometry. Data represent means ± SEM of 4 independent experiments, ***P<0.001, ****P<0.0001, One-way Anova.

Article Snippet: Confluent Human Umbilical Vein Endothelial Cells (HUVEC; passage 2-4; 20 different primary cultures; PromoCell) were cultured on 0,2 % gelatin-coated slides, in Endothelial Cell Basal Medium (ECBM, PromoCell), supplemented with 2 % Fetal Calf Serum (PromoCell), growth factors (0.4% ECGS, 0.1 ng/mL EGF, 1 ng/mL ß-FGF), heparin (90 μg/mL), hydrocortisone (1 μg/mL), Amphotericin B (10 μg/L, Gibco), Streptomycin (100 IU/mL, Gibco) and Penicillin (100 IU/mL, Gibco).

Techniques: Labeling, Injection, Staining, Confocal Microscopy, Incubation, Derivative Assay, Flow Cytometry, Clinical Proteomics