Fig 1.
Characterizations of vascular calcification and identification of aortic calcification associated miRNAs of OPG deficient mice.
A, The phenotype of OPG-/- mice were determined by using Dual Source CT; OPG-/- mice showed pronounced calcium loss in spine and obvious calcification in aorta, heart and internal organs at 12 weeks of age (n = 3). B,H&E(Hematoxylin and Eosin) staining and Alizarin Red staining showed that OPG-/- mice exhibited obvious aortic calcification at 12 weeks after birth; arrows represent the calcified nodules (4×, scale length 200 μm) and corresponding magnified ones (20×, scale length 50 μm) in aortic artery (n = 3). The artery staining of correspondingwild type mice (WT)showed no obvious calcification (upper, 4×; lower, 10×. scale length 200 μm). C, The expression of ALP and Runx2 in aortic artery were determined by immunohistochemistry analysis in wild type and OPG-/- mice at the age of 12 weeks (20×) (n = 3), scale length was 50 μm. D, The level of plasma ALP and Runx2 in mice was determined by enzyme-linked immunosorbent assay, and the level of plasma miR-32 was determined by real-time PCR. ALP, Runx2 and miR-32 showed an increased level in plasma of OPG-/- mice at the ages of 12 weeks, compared to those in WT mice (n = 3). E, The cluster maps for the differentially expressed miRNAs; comparisons were made between OPG-/- mice and WT mice at 12 weeks of age (n = 3). F, The differential expression miRNAs were confirmed with OPG-/- mice and WT mice at 12 weeks of age by quantitative reverse transcription polymerase chain reaction (Q-PCR) (n = 3). G, The expression and distribution of miRNA-32 in aortic arteries of OPG-/- and OPG+/+ mice were determined by in situ hybridization (20×), scale length was 50 μm. *P < 0.05, **P < 0.01 compared with WT mice group, data are shown as means±SEM and Mann-whiteney U testis used for comparison.
Fig 2.
The miRNAs expression during osteogenic differentiation of MVSMCs.
A, MVSMCs were cultured in DMEM containing CM (calcification medium) for 14 and 21 days, and cells were stained with Alizarin red for the detection of calcification nodules (20×), scale length was 50 μm. B, The mRNA level of miR-32expression in CM-treated MVMSCs were detected by Q-PCR, and data are shown as mean±SEM of experiments performed in triplicate (n = 3). *P< 0.05, **P < 0.01 versus control(Con).
Fig 3.
MiR-32 induces the expression of vascular calcification markers.
A&B, The miR-32 mimics(pre-miR32),negative control of miR-32 mimics (pre-con), miR-32 inhibitors (anti-miR32), or negative control of miR-32 inhibitors (anti-con) were transfected into MVMSCs for 4 days and miR-32 expression level was determined by Q-PCR (A) and Northern Blot (B) (n = 3). C, The effect of miR-32 on the BMP2, Runx2, OPN and MGP mRNA level in MVSMCs were detected by Q-PCR at the time point of 4 days after the transfection of miR-32 mimics and miR-32 inhibitorsin MVMSCs (n = 3). D&E, The effect of miR-32 on ALP activity and calcium content of MVSMCs were also determined at the time point of 4 days after miR-32 mimics transfection(n = 3). Data are shown as mean±SEM of a representative experiment performed in triplicate, *P< 0.05, ** P< 0.01.
Fig 4.
MiR-32 activates PI3K-Akt pathway by targeting toPTENand enhances Runx2 expression and phosphorylation.
A, The miR-32 mimics (pre-miR32),negative control of miR-32 mimics (pre-con), miR-32 inhibitors (anti-miR32), or negative control of miR-32 inhibitors (anti-con)were transfected into MVSMCs and the cells were lysed and analyzed by immunobloting with the indicated antibody. *P< 0.05, **P< 0.01, ***P< 0.001, compared to control groups. B, Effect of miR-32 on PTEN expression by luciferase reporter assay. MVSMCs were cotransfected with pre-miR32 (or anti-miR32) and PTEN 3’UTR wild-type vectors (PTEN 3’UTR-WT) or PTEN 3’UTR mutant vectors (PTEN 3’UTR-MUT). Luciferase activity was normalized by the ratio of firefly and renilla luciferase signals. C, PI3K signaling is involved in miR-32 induced Runx2 expression and phosphorylation. MVSMCs were transfected with indicated concentration of miR-32 mimics or pre-con, with or without LY294002 and total protein extracts were analyzed by Western blot.Data of panel Aare analyzed with ANOVA with Bonferroni correction, and student t test is used for data anaysis of panel B&C. *P< 0.05,**P< 0.01.
Fig 5.
MiR-32 shows increased level in plasma of patients with coronary artery calcification.
A,The representative images of aortic stenosis of CADpatients confirmed by coronary arteriongraphy (n = 66). a&c (two pictures upper) represent the coronary artery before coronary arteriongraphy and b&d (two pictures lower) represent the coronary artery after coronary arteriongraphy; a&b, CAD patients without coronary calcification; c&d, CAD with calcification.B,Representatives of transverse (a&c, two left pictures) and maximum intensity projection (b&d, two right pictures) images of coronary arteries by CT scanning; a&b, examples of no coronary calcium; c&d, examples of coronary calcification; arrows indicate the calcification of coronary artery.C,Plasma from 66 patients with coronary heart disease of which 33 patients showed calcification in coronary artery confirmed by CT scanning were collected for miRNA-32 detection by Q-PCR. Data are shown as mean±SEM of experiments. D, The plasmaALP activity of patients was detected as described in methods, and data are shown as mean±SEM of experiments (n = 66). E,the scattering spots image showed the positive correlation between miRNA-32 and ALP activity in plasma of patients with coronary artery calcification (n = 33), which was performed by Spearman's Rank Correlation test.