Fig. 4

miR-126-5p silencing reversed A-EC/sEVs-mediated anti-calcification effects on VSMCs by restoring the activation of smad1/5/9 signalling. We transfected miR-126-5p inhibitor to silence miR-126-5p expression in HUVEC with or without AGEs treatment, including 4 groups: negative control (NC), miR-126-5p inhibitor, A-NC, A-miR-126-5p inhibitor. A The transfection efficiency examined by qPCR in HUVECs. B CCK-8 assay showing the viability of HUVECs after 48 h co-incubation. ^##P < 0.01, Data were presented as mean ± SD of 3 replicates. We isolated sEVs from the A-NC or A-miR-126-5p inhibitor group, termed A-EC^NC/sEVs or A-EC^126inhibitor/sEVs, respectively. C Analysis by qPCR showing the level of miR-126-5p in four sEVs (EC/sEVs, A-EC/sEVs, A-EC^NC/sEVs or A-EC^126inhibitor/sEVs). ^##P < 0.01; data were presented as the mean ± SD of three replicates. D Analysis by qPCR showing the level of miR-126-5p in HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. E–G The mRNA and protein level of Runx2 and BMP2 in calcified HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. H ALP staining and ARS staining of calcified HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. Representative micrographs are shown, scale bar represents 50 µm. I, J ALP activity and calcium content of calcified HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. ^##P < 0.01. K The protein level of BMPR1B in calcified HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. L Representative fluorescence micrographs showing the nuclear translocation of p-smad1/5/9 (green) in calcified HA-VSMCs treated with A-EC/sEVs or A-EC^126inhibitor/sEVs. Nuclei were counterstained with DAPI (blue). Scale bar represents 50 µm