Fig. 2

A-EC/sEVs inhibited the osteogenic differentiation of HA-VSMCs in vitro. A Representative fluorescence micrograph of PKH-26-labelled sEVs (red) internalised by primary HA-VSMCs, while blue represents the nucleus. The labelled sEVs were co-incubated with HA-VSMCs for 12 h. B Effect of A-EC/sEVs on the viability of HA-VSMCs (sEVs-1 and sEVs-2 indicate 50 and 100 μg/mL of sEVs, respectively). CCK-8 values were measured at 48 h post co-incubation. C Representative western blot image showing the effect of A-EC/sEVs on the protein levels of Runx2 and BMP2 in β-GP-induced HA-VSMCs after 48 h co-incubation. D, E The mRNA levels of Runx2 and BMP2 in HA-VSMCs as measured by qPCR. ^##P < 0.01; data are presented as the mean ± SD of three replicates. F Representative images showing the transwell co-incubation assay of HA-VSMCs and HUVECs pre-treated with or without AGEs or GW4869, respectively, for 48 h. The protein levels of Runx2 and BMP2 in HA-VSMCs. G, H The mRNA levels of Runx2 and BMP2 in HA-VSMCs. ^##P < 0.01, ^#P < 0.05; data are presented as the mean ± SD of three replicates. Alkaline phosphatase (ALP) staining (I) and ALP activity (J) of calcified HA-VSMCs treated with A-EC/sEVs, EC/sEVs or sEVs from HUVECs pre-treated with GW4869 before AGEs treatment (GW4869/A-EC/sEVs) for 7 days. Alizarin Red staining (ARS) (K) and calcium content (L) of calcified HA-VSMCs treated with A-EC/sEVs, EC/sEVs or GW4869/A-EC/sEVs for 21 days. Representative micrographs are shown, scale bar represents 50 µm. ^##P < 0.01, ^#P < 0.05