Figure 1.
6-MP-induced calcification in vitro and ex vivo.
(A–D) VSMCs were cultured in control medium or CM±6-MP (100 µmol/L) for 21 days. (A) Mineral deposits were visualized via Alizarin red staining. One representative experiment is shown (n = 5). (B) VSMCs were treated with 6-MP (1 µmol/L–1 mmol/L) for up to 21 days and viability/proliferation was measured. (C) Calcium content (n>6) or (D) ALP enzyme activity (n>6) was quantified and normalized to protein content. (E,F) Rat aortic rings were incubated in control medium or CM ± 6-MP (100 µmol/L) for 14 days. (E) One aortic ring treated with each type of stimulation was used for histochemical analysis. Slices were stained with Alizarin Red to visualize calcium deposition. (F) Calcium content was quantified and normalized to the dry weight of aortic rings (n>6). Data represent means±SEM, *p<0.05 vs. control. #p<0.05 vs. CM. ALP: alkaline phosphatase, CM: calcifying medium, 6-MP: 6-mercaptopurine.
Figure 2.
mRNA expression of osteogenic proteins.
(A–C) VSMCs were stimulated with 6-MP as indicated and mRNA expression was detected after 48 h. Data represent means±SEM, n≥6,*p<0.05 vs. control. (D) VSMCs were stimulated with 6-MP for 48 h. Nuclear proteins were extracted. Cbfa1, Cbfa1-phospho and TATA-bp were detected via Western blot. Representative images and relative band intensities of 3 independent blots of Cbfa1-phospho are shown. (E) MEK1 and ERK1/2 activation was detected via Bio-Plex (n≥6). Values are given as % of control and are normalized to total kinase. (F) mRNA expression of cbfa1 after 48 h treatment with 6-MP (100 µmol/L) ± U0126 (1 µmol/L) (n>6). Data represent means±SEM, *p<0.05 vs. control. ALP: alkaline phosphatase, cbfa1: core binding factor alpha-1, 6-MP: 6-mercaptopurine, OCN: osteocalcin.
Figure 3.
(A) Metabolism scheme of the prodrug AZA. (B) Expression of Xdh (351 bp), HPRT1 (168 bp), IMPDH1 (505 bp), TPMT (104 bp), and β-actin (76 bp) in unstimulated VSMCs. (C,D) Effect of 6-MP (100 µmol/L), allopurinol (5 µmol/L) and tiron (100 µmol/L) stimulation on VSMC (C) ALP mRNA expression and (D) ALP enzyme activity. Enzyme activity was normalized to the protein content of the cells. Data represents means±SEM, n≥6, *p<0.05 vs. control #p<0.05 vs. 6-MP. (E,F) VSMCs were stimulated with 6-MP (100 µmol/L), 6-TGNs (each 10 µmol/L), 6-MTIMP (10 µmol/L) or 6-TU (10 µmol/L) for 48 h and (E) cbfa1, and (F) ALP mRNA expression levels were analyzed. Data represents means±SEM, n≥6, *p<0.05 vs. control. (G,H) VSMCs were incubated as indicated (100 µmol/L 6-MP, 6-TGNs [each 10 µmol/L], 6-MTIMP or 6-TU [each 10 µmol/L] for 21 days) and (G) calcium content and (H) ALP enzyme activity were measured. Data represents means±SEM, n≥6,*p<0.05 vs. control. AZA: azathioprine, cbfa1: core binding factor alpha-1, GST: glutathione S-transferase, HPRT: hypoxanthine guanine phosphoribosyltransferase, IMPDH1: inosine monophosphate dehydrogenase 1, 6-MP: 6-mercaptopurine, 6-MTIMP: 6-methylthioinosine monophosphate, 6-TGDP: 6-thioguanosine diphosphate, 6-TGMP: 6-thioguanosine monophosphate, 6-TGNs: 6-thioguanine nucleotides, 6-TGTP: 6-thioguanosine triphosphate, 6-TIMP: 6-thioinosine monophosphate, TPMT: thiopurinemethyltransferase, 6-TU: 6-thiouric acid, Xdh: Xanthine dehydrogenase, XO: xanthine oxidase.
Figure 4.
(A,B,E,F) VSMCs were stimulated as indicated for 30 min before labeling cells with DHE. Superoxide production was (A) visualized via fluorescence microscopy (representative images from 3 independent experiments) or (B,E,F) quantified in a fluorescence plate reader (n≥6). (C,D) Hydrogen peroxide production is measured in H2DCFDA-labeled cells via flow cytometry. (C) Representative histograms of flow data (grey: control, white: 6-MP). (D) Quantification of fluorescence intensity by % of labeled control (n≥6). (E) Stimulation with 6-MP (100 µmol/L) or its metabolites (each 10 µmol/L). (F) Stimulation with 6-MP (100 µmol/L) alone or costimulation with inhibitors (tiron [1 mmol/L] and allopurinol [1 µmol/L]). DHE: dihydroethidium, 6-MP: 6-mercaptopurine, 6-MTIMP: 6-methylthioinosine monophosphate, 6-TGDP: 6-thioguanosine diphosphate, 6-TGMP: 6-thioguanosine monophosphate, 6-TGN: 6-thioguanine nucleotide, 6-TGTP: 6-thioguanosine triphosphate, 6-TU: 6-thiouric acid.