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. 2024 Aug 28;42:52–67. doi: 10.1016/j.bioactmat.2024.08.021

Fig. 4.

Fig. 4

MT-upregulated exosomal miR-302d-5p antagonized VSMC calcification and senescence. (A–D) Dicer, an essential enzyme for miRNAs maturation was silenced in ECs before MT treatment, then Veh-ECs-Exos and MT-ECs-Exos were harvested for VSMCs incubation. (A) The expression levels of Runx2, BMP2, P21 of VSMCs treated by Veh-ECs-Exos or MT-ECs-Exos with or without miRNAs knock-down were detected by Western blot. (B) The calcium nodules in VSMCs treated by Veh-ECs-Exos or MT-ECs-Exos with or without miRNAs knock-down was measured by Alizarin red S staning (upper panel, scale bar = 200 μm), the senescence VSMCs were measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). (C) The ALP activity in VSMCs incubated with Veh-ECs-Exos or MT-ECs-Exos with or without miRNAs knock-down was measured by an ALP kit. (D) The calcium content in VSMCs treated with Veh-ECs-Exos or MT-ECs-Exos with or without miRNAs knock-down was measured by the o-cresolphthalein method. (E) The differentially expressed miRNAs (a cut-off of absolute fold change ≥2.0 and p < 0.05) between Veh-ECs-Exos and MT-ECs-Exos according to microarray analysis. (F) qRT-PCR quantitative results of miRNAs expression levels of Veh-ECs-Exos and MT-ECs-Exos. (G) Expression levels of miR-302d-5p in VSMCs treated with Veh-ECs-Exos and MT-ECs-Exos. (H) Expression levels of miR-302d-5p in exosomes derived from miR-302d-5p over-expressed or down-expressed ECs. (I) Microscopy analysis was used to verify the florescent signal colocalization of FAM-miR-302d-5p in green, PKH26 in red and DAPI in blue (scale bar = 50 μm). (J) The expression level of Runx2, BMP2, P21 of VSMCs incubated with miR-302d-5p knockdown MT-ECs-Exos was determined by Western blot. (K) The ALP activity in VSMCs incubated with miR-302d-5p knockdown MT-ECs-Exos was measured by an ALP kit. (L) The calcium content in VSMCs treated with miR-302d-5p knockdown MT-ECs-Exos was measured by the o-cresolphthalein method. (M) The calcium nodules in VSMCs treated with miR-302d-5p knockdown MT-ECs-Exos was measured by Alizarin red S staning (upper panel, scale bar = 200 μm), the senescence VSMCs was measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). (N) The expression level of Runx2, BMP2, P21 of VSMCs incubated with miR-302d-5p knock-in Exos was determined by Western blot. (O) The ALP activity in VSMCs incubated with miR-302d-5p knock-in Exos was measured by an ALP kit. (P) The calcium content in VSMCs treated with miR-302d-5p knock-in Exos was measured by the o-cresolphthalein method. (Q) The calcium nodules in VSMCs treated with miR-302d-5p knock-in Exos was measured by Alizarin red S staining (upper panel, scale bar = 200 μm), the senescence VSMCs was measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.0001.