Fig 2: HP-stimulated HUVEC-Exos promote VSMC calcification. (A) Expression levels of OPN and Runx2 in VSMCs were measured by western blot analysis. (B) Reverse transcription-quantitative PCR analysis of OPN and Runx2 expression in VSMCs. (C) ALP activity assays were performed. (D) Ca content in VSMCs was measured. (E) VSMCs were stained for mineralization using Alizarin red S. Scale bar, 100 µm. **P<0.01, ***P<0.001, ****P<0.0001 compared with the Ctrl group. ALP, alkaline phosphatase; Ca, calcium; Ctrl, control; HP, high phosphorus; HUVEC-Exos, exosomes from HUVECs; NP, no phosphorus; OPN, osteopontin; Runx2, runt-related transcription factor 2; VSMC, vascular smooth muscle cell.

Fig 3: Wnt/ß-catenin signaling pathway is involved in STAT1-mediated VSMC calcification. STAT1-overexpressing VSMCs were treated with 3 mM phosphorus for 7 days, with or without 100 ng/ml Dkk-1 added for the last 3 days of this period. si-STAT1 VSMCs were treated in the presence of 3 mM phosphorus for 7 days, with or without 5 mmol/l LiCl for the last 3 days of this period. (A) Expression levels of STAT1 in VSMCs were measured by western blot analysis. (B) Expression levels of OPN, Runx2, Wnt-3a and ß-catenin were measured by western blot analysis. (C) Reverse transcription-quantitative PCR analysis of OPN, Runx2, Wnt-3a and ß-catenin in VSMCs. (D) Mineralization was examined using Alizarin Red S staining. Scale bar, 100 µm. (E) Ca content in VSMCs was measured. (F) ALP activity was tested. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ALP, alkaline phosphatase; Ca, calcium; Ctrl, control; NC, negative control; o/e, overexpression; OPN, osteopontin; Runx2, runt-related transcription factor 2; si, small interfering RNA; VSMC, vascular smooth muscle cell; Dkk-1, Dickkopf-1; LiCl, lithium chloride.

Fig 4: STAT1 inhibition alleviates HP-induced VSMC calcification via the Wnt/ß-catenin signaling pathway. VSMCs were treated with si-STAT1 and si-NC and then incubated with HP (3 mM) to induce calcification or fresh conditioned medium (Ctrl) for 7 days. (A) Effect of si-STAT1 on protein levels of STAT1. Based on the knockdown effect, si-STAT1-01 was selected for further experiments. (B) Expression levels of OPN and Runx2 were measured by western blot analysis in VSMCs. (C) RT-qPCR analysis of OPN and Runx2 mRNA expression in VSMCs. (D) Alizarin Red S staining indicated the mineralization. Scale bar, 100 µm. (E) Ca content in VSMCs was measured. (F) ALP activity was tested. (G) Expression levels of Wnt-3a and ß-catenin were measured by western blot analysis. (H) RT-qPCR analysis of Wnt-3a and ß-catenin expression in VSMCs. **P<0.01, ***P<0.001, ****P<0.0001 compared with the Ctrl group. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 compared with the HP group. ALP, alkaline phosphatase; Ca, calcium; Ctrl, control; HP, high phosphorus; NC, negative control; OPN, osteopontin; RT-qPCR, reverse transcription-quantitative PCR; Runx2, runt-related transcription factor 2; si, small interfering RNA; VSMC, vascular smooth muscle cell.

Fig 5: miR-1303 mediates osteogenic differentiation of HBMSCs by sponging COCH. A The predicted binding site between the miR-1303 and COCH, and the luciferase activity of the WT COCH and Mut COCH in 293T cells treated with mimics miR-1303 or mimics NC. B Transfection efficacy after miR-1303 interference is determined by qRT-PCR. C–G Western blotting (C) and quantitative analysis of COCH (D), RUNX2 (E), Osterix (F), and COL1A1 (G) in HBMSCs treated with si-miR-1303 and si-miR-1303 plus si-COCH. H, I Staining of calcium deposition by Alizarin Red (H) and quantitative analysis of staining areas (I) in HBMSCs treated with si-miR-1303 and si-miR-1303 plus si-COCH. Scale bars, 200 µm. J, K Staining of ALP (J) and quantitative analysis of staining areas (K) in HBMSCs treated with si-miR-1303 and si-miR-1303 plus si-COCH. Scale bars, 200 µm. L, M Immunofluorescence staining of RUNX2 (L) and quantitative analysis of RUNX2-positive nuclei (M) in HBMSCs treated with si-miR-1303 and si-miR-1303 plus si-COCH. Scale bars, 100 µm. N, O Immunofluorescence staining of Osterix (N) and quantitative analysis of RUNX2-positive nuclei (O) in HBMSCs treated with si-miR-1303 and si-miR-1303 plus si-COCH. Scale bars, 100 µm. A Values are shown as mean ± SD. ****P < 0.0001, two-way ANOVA. B, D–G, I, K, M, O Values are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA