10 research outputs found

    Harmful Effects of the Azathioprine Metabolite 6-Mercaptopurine in Vascular Cells: Induction of Mineralization

    No full text
    <div><p>Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells <i>in vitro</i>. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in <i>in vitro</i> and <i>ex vivo</i> models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.</p></div

    Involvement of ROS.

    No full text
    <p>(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 H<sub>2</sub>DCFDA-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.</p

    mRNA expression of osteogenic proteins.

    No full text
    <p>(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.</p

    6-MP-induced calcification <i>in vitro</i> and <i>ex vivo</i>.

    No full text
    <p>(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. <sup>#</sup>p<0.05 vs. CM. ALP: alkaline phosphatase, CM: calcifying medium, 6-MP: 6-mercaptopurine.</p

    Effects of 6-MP metabolites.

    No full text
    <p>(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 <sup>#</sup>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.</p

    Molecular masses of Up<sub>4</sub>U fragments obtained by MALDI-TOF-TOF mass spectrometry (<b>Figure 1</b><b>.B)</b>.

    No full text
    <p>The first column shows the fragment masses measured by MALDI-TOF-TOF mass spectrometry; second column shows the fragments mass of Up<sub>4</sub>U isolated from the endothelial secretome; the third column the fragments mass of Up<sub>4</sub>U isolated from plasma; the fourth column shows the fragment masses calculated from their respective structures; the fifth column shows the fragments masses of synthesised Up<sub>4</sub>U. M<sup>+</sup> = protonated parent ion; Ú =  uracil; U = uridine; p = phosphate group, e.g. Up<sub>3</sub> =  UTP; w/o = without.</p

    Figure 1

    No full text
    <p>(<b>A</b>) Angiogenic effects of endothelial secretome in the rat embryo chorioallantoic membrane. The primitive placenta and yolk sac of rat embryos cultured during organogenesis under negative control conditions (HBSS and bovine serum) (I). The corresponding vascular system is underdeveloped and could be improved by angiogenic factors like VEGF as positive control (II). More complex and structured blood vessels and red staining caused by red blood cells in the blood vessels (marked by arrows). Morphologic evaluation of angiogenic effect of the endothelial secretome (III), of the endothelial secretome after incubation with alkaline phosphate (IV), and of the endothelial secretome after incubation with alkaline phosphate in the presence of suramin (V). (<b>B</b>) MALDI-TOF-TOF mass spectrum of the fraction from the analytical reversed-phase chromatography. (<b>C</b>) Enhanced vascularisation of rat embryonic yolk sac membranes induced by increasing Up<sub>4</sub>U concentrations after 48 h of culture. Typical result out of 5 similar experiments. (D) Effect of increasing Up<sub>4</sub>U concentration on proliferation rate of human endothelial cells (n = 7). (<b>E</b>) Reversed phase chromatography of the fraction of human plasma containing the remaining nucleotides after exclusion of mononucleotides. (<b>F</b>) Up<sub>4</sub>U release of cultivated endothelial cells after stimulation by a cone-and-plate viscometer with shear stress of 3 N m<sup>-2</sup> (n = 11).</p

    Figure 4

    No full text
    <p>(<b>A</b>)Effect of Up<sub>4</sub>U on phosphorylation of MAPK. Phosphorylation of MEK1, ERK1/2, Akt, and p38 measured by Luminex™ technique before (open bar) and after stimulation with Up<sub>4</sub>U for 10 min (filled bar). Ratio of phospho/total were normalized to protein content of the lysates and demonstrated as percent stimulation relative to control (*p<0.05; n = 5). (<b>B</b>)Effect of PD98059, U0126, SB2021902, and GSK on Up<sub>4</sub>U induced proliferation rate in vascular smooth muscle cells (<b>C</b>)Up<sub>4</sub>U amount of secretome from in-vivo/ex-vivo stimulated aortic rings. (*p<0.05; n = 4).</p

    Figure 2

    No full text
    <p>(<b>A</b>)Effect of increasing Up<sub>4</sub>U concentrations on proliferation rate of vascular smooth muscle cells in the absence of PDGF (n = 6). (<b>B</b>)Effect of increasing Up<sub>4</sub>U concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>−6</sup> mol L<sup>-1</sup> PDGF each; n = 3). (<b>C</b>)Effect of increasing UTP concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>−6</sup> mol L<sup>-1</sup> PDGF each; n = 3). (<b>D</b>)Effect of increasing UDP concentrations on proliferation rate of vascular smooth muscle cells in the presence of PDGF (10<sup>−6</sup> mol L<sup>-1</sup> PDGF each; n = 3). (<b>E</b>)Effect of Up<sub>4</sub>U (10<sup>−7</sup> mol L<sup>-1</sup>) or ATPγS (10<sup>−7</sup> mol L<sup>-1</sup>) in the presence of PDGF (10<sup>−6</sup> mol L<sup>-1</sup> PDGF each; n = 3) and suramin (10<sup>−4</sup> mol L<sup>-1</sup>), PPADS (10<sup>−5</sup> mol L<sup>-1</sup>), MRS2179 (10<sup>−5</sup> mol L<sup>-1</sup>) or RBII (10<sup>−5</sup> mol L<sup>-1</sup>) on proliferation rate of vascular smooth muscle cells.</p

    Figure 3

    No full text
    <p>(<b>A</b>)Effect of increasing Up<sub>4</sub>U concentrations on migration rate of endothelial cells in the absence (open bar) and presence (filled bar) of suramin (n = 6). (<b>B</b>)Effect of increasing UTP concentrations on migration rate of endothelial cells (n = 3). (<b>C</b>)Effect of increasing ATP concentrations on migration rate of endothelial cells (n = 3). (<b>D</b>) Effect of increasing Up<sub>4</sub>U concentrations on tube-formation rate of endothelial cells (5 10<sup>−5</sup> mol L<sup>-1</sup> Up<sub>4</sub>U and 10<sup>−7</sup> mol L<sup>-1</sup> PDGF as indicated in the figure; n = 12). (<b>E</b>)Effect of Up<sub>4</sub>U on phenotype of endothelial cells. Representative microscopic images of endothelial cell were exposed to (a) control conditions, (b) PDGF, (c) Up<sub>4</sub>U, and (d) PDGF and Up<sub>4</sub>U for 6 h incubation time (5 10<sup>−5</sup> mol L<sup>-1</sup> Up<sub>4</sub>U and 10<sup>−7</sup> mol L<sup>-1</sup> PDGF (10ng ml<sup>-1</sup>)). (<b>F</b>)Quantification of 3-dimensional <i>in vitro</i> angiogenesis with collagen gel-embedded spheroids of EC. Spheroids were embedded into collagen gels with Up<sub>4</sub>U and with or without VEGF. The cumulative length of all the sprouts originating from an individual spheroid was quantified after 24 h by semiautomatic image analysis.</p
    corecore