Fig. 4: AB-EVs exacerbate vascular calcification.

a Experimental design of the VD3-induced acute vascular calcification mouse models treated with solvent, YB-EVs, or AB-EVs by intravenous injection. b qRT-PCR analysis of Sm22α and αSma expression in abdominal aortas of mice in (a). n = 9 biologically independent animals per group. c–e, Von Kossa and ARS staining images (c) and quantification of the percentages of Von Kossa⁺ (brownish black; d) and ARS⁺ (red; e) areas. Scale bar: 200 μm. n = 10 biologically independent animals per group. f Vascular calcium content measurement. n = 10 biologically independent animals per group. g–h, RUNX2 immunofluorescence staining images (g) and quantification of the percentage of RUNX2⁺ (red) areas (h). Scale bar: 200 μm. n = 10 biologically independent animals per group. i qRT-PCR analysis of Alpl expression. n = 9 per group. j Experimental design of the adenine-induced chronic vascular calcification mouse models treated with solvent, YB-EVs, or AB-EVs by intravenous injection. k qRT-PCR analysis of Sm22α and αSma expression in abdominal aortas of mice in (j). n = 9 biologically independent animals per group. l–n Von Kossa staining images (l) and quantification of the percentages of Von Kossa⁺ (brownish black; m) and ARS⁺ (red; n) areas. Scale bar: 200 μm. n = 10 biologically independent animals per group. o Vascular calcium content measurement. n = 10 biologically independent animals per group. p-q, RUNX2 immunofluorescence staining images (p) and quantification of the percentage of RUNX2⁺ (red) areas (q). Scale bar: 200 μm. n = 10 biologically independent animals per group. r qRT-PCR for Alpl expression. n = 9 biologically independent animals per group. s Fluorescence microscopy analysis of femur and abdominal aorta sections from mice treated with solvent or DiO (green)-labeled AB-EVs by intramedullary injection. CB: cortical bone; BM: bone marrow. Scale bar: 200 μm (for bone) or 50 μm (for vessel). n = 3 biologically independent animals per group. t–v, Von Kossa staining images (t), quantification of the percentage of Von Kossa⁺ areas (brownish black; u), and vascular calcium content measurement (v) in abdominal aortas from the VD3-induced acute vascular calcification mouse models receiving solvent or AB-EVs treatment by intramedullary injection. Scale bar: 200 μm. n = 5 biologically independent animals per group. Experiment in s was repeated independently three times with similar results. The illustrated results represented one of the three independent experiments. The other experiments were performed with at least five biological replicates per group without independent repetition. Data were presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test (a–t) or unpaired, two-tailed Student’s t-test (u–v). Source data are provided as a Source Data file.