Summary charts of area at risk and infarct size measurements in male mice hearts.

<p>After hemodynamic measurements, the animals were perfused with triphenyltetrasolium (TPP) and Evans Blue to assess perfused myocardium, area at risk and infracted (necrotic) tissue. Myocardial infarct size (IS) was expressed as a percentage of area at risk (AAR). Values are mean percent± SEM, n = 6 animals/group. A <i>P</i> value <0.05 was considered as statistically significant.</p

Aromatase upregulation by desflurane <i>in vivo</i> and <i>in vitro</i>.

<p>Male mice were treated with 1 MAC desflurane for 15 minutes, then were euthanized 15 minutes later (DES15) or 48 hours later (DES48). The hearts from untreated animals served as native control (Native). Total RNA was extracted from myocardial tissue and aromatase gene (Cyp19a1) expression was analyzed by quantitative real- time PCR (qRT-PCR), n = 5/group (A). Aromatase protein expression <i>in vitro</i> was studied by western blotting. Endothelial cells were treated with oxygen: desflurane (1 MAC) mixture for 15 minutes and collected 15 minutes later (DES0.25), 24 hours later (DES24) or 48 hours later (DES 48). Cells treated with oxygen alone for 15 minutes and collected 15 minutes later, 24 hours later and 48 hours later were assigned as controls (CON0.25, CON 24 and CON 48, respectively). The cells kept under standard conditions served as native control (B). The chart in panel B represents statistical analysis of aromatase signal quantification (n = 4). Aromatase signal in CON group was set to 1 (gray bars) and desflurane influence on aromatase protein expression was compared to CON group (solid bars). GAPDH protein served as internal loading control. Representative aromatase western blot is shown in lower part of panel B. The data are presented as mean± SEM and the <i>P</i> value <0.05 was considered as statistically significant.</p

Levels of parathyroid hormone (A), serum calcium (B) and vitamin D (C) are shown in NC patients and controls at the indicated time points.

<p>Patients with calcification are depicted in red. Data are shown as mean ± standard error of the mean (SEM).</p

Aromatase immunostaining in mouse heart and aorta.

<p>Aromatase protein was abundant in mouse myocardium (B, solid arrows) and in the endothelium (solid arrows) as well as in media (white arrows) in the aorta (D). Aromatase signal was visualized by diaminobenzidine substrate (DAB) combined with hematoxylin/eosin staining. Primary antibody was omitted in negative control staining (A and C).</p

Drug treatment and allograft status of patients and controls at biopsy time points.

<p>Values represent percentages.</p>*<p>p<0.05;</p>+<p>p = 0.056.</p>§<p>ARB’s: angiotensin receptor blockers; ACE-I’s: Angiotensin converting enzyme inhibitors,</p>$<p>any grade of hydronephrosis,</p>&<p>any cGrade>0 according to the BANFF classification.</p

Urinary mineral ions in patients and controls at biopsy time points.

<p>Urinary mineral ions in patients and controls at biopsy time points.</p

Aromatase expression in endothelial and smooth muscle cells.

<p>Aromatase cellular localisation pattern in endothelial (EC) and smooth muscle cells (SMC) was assessed by immunocytochemistry and visualized using confocal imaging technique. Aromatase was immunostained using specific primary antibody and visualized by addition of the secondary antibody conjugated with AlexaFluor594 fluorophore (red). Aromatase was abundant in the cytosolic fraction of endothelial (A) and smooth muscle cells (C). Cell nuclei were detected using lamin A/C antibody and visualized using AlexaFluor488- labelled secondary antibody (green). Merge images B and D show aromatase and lamin A/C co- immunostaining.</p

Urinary concentrations of OPN are shown in NC patients and control transplant patients without signs of calcification at the indicated time points.

<p>Patients with calcification are depicted in red.</p

Serum concentrations of Fetuin A (A) as well as MGP (B) are shown in NC patients (n = 31) in comparison to control transplant patients without signs of calcification (n = 27) at the indicated time points.

<p>Patients with calcification are depicted in red.</p

Aromatase co- localization with endothelial cells and smooth muscle cells in mouse heart studies using confocal microscopy.

<p>Aromatase was detected by immunostaining using specific antibody and visualized by AlexaFluor488- coupled secondary antibody (A and D). Endothelial cell specific marker PECAM1 (CD31) (B) and smooth muscle marker α-smooth muscle actin (SMA) (E) were detected using specific primary antibodies and visualized using AlexFluor594-coupled secondary antibodies (B and E, respectively). Aromatase and PECAM1 co- localization is shown in section C, whereas aromatase and α-smooth muscle actin co- expression in the heart is demonstrated in section F.</p