Figure 4: Presence of Extracellular Vesicles in HAVICs

(A) HAVICs exhibited calcium-containing cellular vesicles. 1: Phase-contrast image; 2: membrane lipophilic Vybrant DiO dye stains (Thermo Fisher Scientific, Carlsbad, California) of vesicles of various sizes (green); 3: high-calcium-containing vesicles (arrows) stained with Calcium Orange AM (Thermo Fisher Scientific) (red); 4: superimposed image of 1, 3, and nuclear (blue) stained with NucBlue reagent (Thermo Fisher Scientific). (B) Cluster of extracellular vesicles on HAVICs observed using scanning electron microscopy (arrows). (C) HAVICs were treated with 50 μg/ml purified Lp(a) for 7 days in serum-free DMEM. The medium was freshly changed at days 3 and 5. The harvested media (days 3 and 7) were subjected to extracellular vesicle profiling using NanoSight NS500 (Malvern Instruments, Malvern, United Kingdom). Lp(a)-treated cells show higher particle concentration than nontreated, with *p = 0.04 at day 3 in DMEM, and **p = 0.0001 at day 7 in OSM; n = 3 for each group. (D) Extracellular vesicle size profiling shows different particle concentration vs. particle size profiling of Lp(a)-treated HAVICs at 7 days. Each condition was sampled 5 times for size profiling using NanoSight NS500. Data were combined and presented as average particle concentration vs. particle size (mean ± SEM). Abbreviations as in Figures 1 and 2.