Additional file 1: of Design and rationale of the ATHENA study – A 12-month, multicentre, prospective study evaluating the outcomes of a de novo everolimus-based regimen in combination with reduced cyclosporine or tacrolimus versus a standard regimen in kidney transplant patients: study protocol for a randomised controlled trial

List of ethics committees. (PDF 184 kb

Cell functions of mesenchymal stromal cells upon treatment with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Chemotactic response towards a 5 ng/ml gradient of each growth factor. Bars represent the mean+SEM of 3 independent experiments, 3 replicates each. Control was set to 1. <b>B,</b> Proliferation measured by BrdU incorporation after stimulation with 10 ng/ml of each growth factor for 24 h. Bars represent the mean+SEM of 3 independent experiments, 5 replicates each. Control was set to 1. <b>C,</b> Total collagen content in cell culture supernatants after 6 days of treatment with 10 ng/ml of each growth factor. Bars represent the mean+SEM of 6 independent experiments, 2 replicates each. *P<0.05, **P<0.01, ***P<0.001. <b>D,</b> Illustration of the induction of specific cell types of vascular smooth muscle cell (VSMC) and fibroblast lineages by systemic sclerosis microenvironment defining growth factors.</p

mTOR complex protein expressions and rictor phosphorylation at Ser1235.

<p>Cardiomyocytes were pretreated for 30 min with 20 nM rapamycin and then stimulated with 10 nM IGF-1 in presence or absence of 10 nM E2 for 24 h. A, shown are representative western blots for mTOR, rictor and raptor and B,C,D, quantitative analysis with mean ± SEM of fold stimulation by IGF-1 of at least 3 independently performed experiments (B,C,D). * p < 0.05, **p < 0.0095. Exposure to rapamycin lead to downregulation of mTOR and rictor, which were more pronounced under culture conditions without E2. E, Western blots for GSK3β-pS9, GSK3β, rictor pS1235 and rictor and F,G,H,I quantitative analysis of at least 3 independently performed experiments indicate that rictor phosphorylation at S1235 by GSK-3β which has been reported to interfere with Akt-substrate binding to mTORC2, thereby downregulating mTORC2 activity. IGF-1 induced strong phosphorylation of GSK-3β at Ser9 in the absence and presence of E2, however, rapamycin pretreatment only reduced this increased phosphorylation in E2 co-treated cardiomyocytes, indicating increased activity of GSK-3β. This higher activity was not associated with increased phosphorylation of rictor at S1235.</p

Sexual dimorphism of mTORC2 activation in response to rapamycin <i>in vivo</i>.

<p>Male and female C57Bl/6J mice were treated with rapamycin (“low concentration” 1.5 mg/kg, administered i.p. every third day) or vehicle (control) for 42 days (n = 6-8/group). mTORC2 activity was assessed by assessment of Akt phosphorylation at S473 and its nuclear localization. A, representative western blots are shown for Akt-pS473, Akt and GAPDH of mice treated as described above. B shows results from quantitative analysis of western blots for Akt-pS473 normalized to Akt (mean ± SEM; * p < 0.035) from female and male mouse hearts. C, Immunostaining for Akt-pS473 of male and female cardiac tissue sections treated with either vehicle (control) or rapamycin for 42 days and D, quantification of % cardiomyocyte nuclei stained for Akt-pS743 (mean ± SEM; *** p < 0.0001). Male mice had lower basal mTORC2 activity, yet responded to rapamycin with an increase in phosphorylation of Akt at S473, associated with increased nuclear localization important for induction of cardioprotective mechanisms. In contrast, female mice responded to rapamycin with reduced phosphorylation of Akt at S473 and loss of cardioprotective nuclear Akt.</p

Analysis of the Transforming growth factor-β1 signaling network.

<p><b>A,</b> Expression of TGF-β1 mRNA and <b>B,</b> secreted TGF-β1 protein for assessment of the autocrine TGF-β feedback loop. Bars represent the mean+SEM of 6 independent experiments, 3 replicates each. <b>C,</b> Expression of CTGF mRNA. Quantitative real-time PCR analysis of mRNA transcripts. Bars represent the mean+SEM of 6 independent experiments quantified with the ΔΔCt method in duplicates. <b>D,</b> Expression of TGF-β receptor 1 protein. <b>E-G,</b> Induction of canonic and non-canonic TGF-β signaling. <b>E,</b> Phosphorylation of SMAD3 at Ser423/425. <b>F,</b> Phosphorylation of AKT at Ser473. <b>G,</b> Phosphorylation of ERK1/2 at Thr202/Tyr204. Analysis of all experiments was performed after 6 days treatment with 10 ng/ml transformin growth factor -β1. Representative western blots of 6 independent experiments are shown. Bars represent the mean+SEM of densitometrically determined band intensities after normalization to GAPDH (TGFBR1, pSMAD3) or total kinase (pAKT, pERK). Control was set to 1. *P<0.05, **P<0.01, ***P<0.001.</p

Measurement of functional L-type Cav1.2 Ca2+ channels in response to growth factors.

<p>Functional L-type Cav1.2 Ca²⁺ channels measured as nimodipine sensitive Ca²⁺-influx upon depolarisation with 60 mmol/L KCl in response to treatment with systemic sclerosis microenvironment defining growth factors for 6 days. <b>A,</b> Bars represent the mean+SEM of nimodipine sensitive Ca²⁺-influx from 3 independent experiments. Control was set to 1. *P<0.05, **P<0.01, ***P<0.001. <b>B-F,</b> Representative recordings of Ca²⁺ dependent intracellular fluorescence intensities in response to depolarisation with KCl. Mean±SEM F/F0 of 30–40 cells per growth factor are plotted against time. Black lines represent tracings without the specific L-type calcium channel blocker nimodipine, grey lines those obtained after preincubation with 1 mmol/L nimodipine.</p

Rapamycin does not impair E2 induced ERK activation.

<p>A, ERK phosphorylation was assessed by immunoblots from lysates of HL-1 cells cultured in the presence of 10 nM E2 and treated with 20 nM rapamycin and IGF-1 for 24 h. B, Results from densitometric analysis of blots and determination of IGF-1 induced increase in protein phosphorylation levels from at least 3 independently performed experiments. C, Immunoblots and D, E, densitometric analyses of cardiomyocytes with MEK1/2 inhibition by 1μM PD 184352 1 h prior to IGF-1 stimulation resulted in increased mTORC2 activity as indicated by increased Akt-pS473 in E2 cotreated cells. * p < 0.04,** p < 0.007, *** p < 0.0007. F, Inhibition of Erk phosphorylation by MEK1/2 inhibitor PD 184352 did not inverse rapamycin effect on Akt-pS473 in E2 cultured cardiomyocytes as investigated by western blotting and G-I, Densitometric analyses; mean ± SEM of fold stimulation by IGF-1 is shown of at least 3 independently performed experiments. * p < 0.05.</p

Rapamycin disturbs adaptive cardiomyocyte responses in E2 and disrupts E2-induced increase in SERCA2A expression.

<p>A, Cells underwent cell volume measurements by FACS analysis. Bars indicate mean GeoMean of flow cytometry forward scatter (FSC-H) from vital cells stimulated with 20 nM IGF-1 for 48 h as indicated from at least 3 independently performed experiments. * p < 0,00, ** p < 0,00. Rapamycin had no significant influence on cardiomyocyte cell size when E2 was absent. However, in presence of E2, rapamycin significantly decreased cardiomyocyte cell size at basal conditions and in response to IGF-1. B, representative histograms for FSC-H from cardiomyocytes cultured with 10 nM E2 and stimulated for 24 h with IGF-1 with or without prior 30 min incubation with 20 nM rapamycin showing left shift of cell volume distribution in rapamycin pretreated cells indicating decrease in cell size of all cardiomyocytes under this treatment. C, D and E, HL-1- cells were cultured with or without E2 and stimulated for 24 h with 10 nM IGF-1 with or without preincubation with 20 nM rapamycin. C-E, SERCA2A expression was assessed on C, mRNA level by qRT-PCR (values were normalized to GAPDH) and D, protein expression of cell lysates stimulated as described above. E, Densitometric analysis of immunoblots from 3 independently performed experiments shown as mean ± SEM. * p < 0.02, ** p < 0.006 for analyses as indicated; # p < 0.04, ## p < 0.006 for comparison of IGF-1 stimulated cells with E2 compared to IGF-1 stimulated cells without E2. IGF-1 alone did not significantly affect SERCA2A expression in female cardiomyocytes. However, rapamycin significantly increased SERCA2A gene expression followed by minor increases in SERCA2A protein predominantly in the IGF-1 treated cells without E2. E2 itself significantly induced SERCA2A gene expression irrespective of additional IGF-1 treatment compared to control cells without E2 (# p< 0.05). However, co-treatment with rapamycin abrogated these increases.</p

Phenotypic modulation upon long-term stimulation (6 days) with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Representative phase contrast photomicrographs from a total of 6 independent experiments are shown. (original magnification x200, scale bar = 50 μm) <b>B,</b> Representative immunofluorescence images from a total of 6 independent experiments are shown. (green = smooth muscle-α-actin, blue = nuclear DNA stained with 4',6-diamidino-2-phenylindole (DAPI), original magnification x400, scale bar = 50 μm).</p

Characterization of mesenchymal stromal cells from healthy controls and patients with systemic sclerosis.

<p><b>A,</b> Representative FACS analysis of an MSC defining surface marker panel confirming homogeneity of isolated cells. <b>B,</b> Osteoblastic differentiation of MSCs after incubation for 21 days with osteoblast-induction medium. (Alizarin red S staining, original magnification x100, scale bars = 50 μm) <b>C,</b> Adipocytic differentiation of MSCs after incubation for 21 days with adipocyte-induction medium. (Oil-Red-O staining, original magnification x200, scale bars = 50 μm) <b>D,</b> Chondroblastic differentiation of MSCs after incubation for 32 days with chondroblast induction medium. Western blot analysis for chondrocyte specific type II collagen.</p