Fig. 3. NMVs adhere to HCAEC under flow and enhance monocyte migration.

a, b HCAEC were cultured (72 h) under static, high laminar (HSS) or low oscillatory shear stress (OSS). Fluorescently labelled NMVs (red) were added for 2 h. Cells co-stained for CD31 (green) and TO-PRO-3 (blue). a Representative images (scale bar = 50 µm) and b quantification of NMV adhesion (n = 3). c HCAEC cultured as for a were incubated with fluorescently labelled anti-ICAM-1, changes analysed by flow cytometry and displayed as mean fluorescence intensity (MFI) (n = 5). d, e HCAEC cultured under OSS (72 h) were incubated with anti-ICAM-1 or isotype control antibody (100 µg mL⁻¹) for 1 h before addition of fluorescently labelled NMVs. d Representative images (scale bar = 100 µm) and e quantification of NMV (red) adhesion. Data expressed as percentage of mean of isotype sample (n = 3). f, g After 72 h exposure to OSS, NMVs were perfused over conditioned HCAEC for 2 h. Fluorescently labelled monocytes (red) were added to perfusion media for 2–4 h. f Representative images (scale bar = 100 µm) and g quantification of adherent monocytes (n = 3). Data expressed as percentage of mean number of adherent monocytes per HCAEC in control samples (−NMV). h–j HCAEC cultured on transwells were incubated with (+NMV) or without (−NMV) NMVs for 30 min followed by addition of monocytes. Monocyte transmigration toward CCL2 (5 nmol/L) was measured after 90 min (n = 5). i Monocyte transmigration was repeated in absence of HCAEC (n = 3). j NMVs were treated with anti-CD18 or isotype control for 20 min, washed and the transmigration experiment repeated (n = 4). h–j Data expressed as percentage of mean of control samples (−NMV/isotype) and presented as mean ± SEM. Statistical significance evaluated using a paired t-test (e, i, j) or one-way ANOVA followed by Tukey’s (b, c) or Dunnettʼs (g, h) test. NS not statistically significant, *P < 0.05, **P < 0.01. All n numbers represent independent experiments. Source data are provided in Source Data file.