Effects of NN-DMT and 5-MeO-DMT treatment on the gene expression and secretion of cytokines and chemokines in polyI:C-activated moDCs.

<p><b>A:</b> MoDCs were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106533#pone-0106533-g002" target="_blank">Figure 2</a>. In this case, cell activation was induced with 20 µg/ml polyI:C for 8 h (alone, or subsequently to an 1 h 100 µM NN-DMT or 5-MeO-DMT pre-treatment). The expression of IL-1β, TNFα, IL-6, IL8, and IL-10 was assessed by real-time Q-PCR and shown as relative mRNA expression. Results represent the Mean ± SEM of 4 independent experiments. <b>B:</b> Cytokine profile of cells treated as in Fig3A. Supernatants of DC cultures were collected after 24 h and were measured by ELISA. Concentration of the secreted cytokines and chemokines are shown as Mean ± SEM values of 4 independent donors. (*) means statistical significance as <i>p</i><0.05.</p

NN-DMT and 5-MeO-DMT pre-treatment of pathogen-activated human dendritic cells effectively inhibit their capacity to prime autologous naive T helper 1 and T helper 17 cells.

<p>Dendritic cells were activated either by heat-killed <i>E. coli</i> (E. coli) or inactivated influenza virus (IV) for 24 h, washed, and then co-cultured with naive autologous CD4⁺ T lymphocytes for 4 days. The number of primed, IFNγ or IL-17 secreting T cells was assessed by ELISPOT assay. T cells alone (T-cell ctrl), and non-activated DCs treated with DMT and co-cultured with autologous naive CD4⁺ T lymphocytes (NN-DMT, 5-MeO-DMT) were used as controls. <b>A–B:</b> Induction of IFNγ (A) or IL-17 (B) production of autologous naive CD4⁺ T cells induced by moDC loaded by <i>E. coli</i>. Bacteria alone (E. coli; red bars) or bacteria in combination with 1 h DMT pre-treatment (E. coli + NN-DMT, E. coli+5-MeO-DMT; empty bars) were used to activate moDCs as written above. <b>C–D:</b> Cells were activated as in Fig4A–B; in this case inactivated influenza virus (IV) was added to the moDCs alone for 24 h (blue bars), or in combination with an 1 h NN-DMT or 5-MeO-DMT pre-treatment (IV + NN-DMT, IV + 5-MeO-DMT; empty bars). Data represent Mean + SEM values of triplicate measurements of three independent donors. Asterisk indicates statistical significance (<i>p</i><0.05).</p

Expression of the Sigma-1 receptor in human monocytes, and differentiated monocyte-derived macrophages (moMACs) and dendritic cells (moDCs).

<p><b>A–B</b>: Expression of sigmar-1 protein in monocytes, and differentiated moMACs and moDCs measured by Western blot. In Fig1A a typical experiment out of four is demonstrated. Densitometry data of Fig1B show the Mean ± SEM values of four independent donors. C: Time kinetics of sigmar-1 gene expression in non-activated control (ctrl), 20 µg/ml polyI:C (polyI:C) or 500 ng/ml LPS (LPS) treated cells. Data of triplicates of four independent measurements are show as Mean ± SEM. D–E: Sigmar-1 protein expression in non-activated control, 20 µg/ml polyI:C or 500 ng/ml LPS treated cells following 24 hours of activation. In Fig1D, results of a typical experiment out of four is shown. Densitometry data of Fig1E show the Mean ± SEM values of four independent donors. (*) represents <i>p</i> values<0.05. (n.s.: “non-significant”).</p

Effects of NN-DMT and 5-MeO-DMT treatment on the gene expression and secretion of cytokines and chemokines in LPS-stimulated moDCs.

<p><b>A:</b> MoDCs were incubated with 100 µM NN-DMT (NN-DMT) or 5-MeO-DMT (5-MeO-DMT) for 8 h or left untreated (ctrl) were used as controls. Additionally, cells were either activated with 500 ng/ml LPS alone for 8 h, or pre-treated with tryptamines for 1 hour and subsequently were activated with LPS for 8 hours (LPS+NN-DMT; LPS+5-MeO-DMT). The expression of IL-1β, TNFα, IL-6, IL8, and IL-10 was assessed by real-time Q-PCR and shown as relative mRNA expression. Results represent the Mean ± SEM of 4 independent experiments. <b>B:</b> Cytokine profile of cells treated as above. Supernatants of DC cultures were collected after 24 h and were subjected to ELISA measurements. Concentration of the secreted cytokines and chemokines are shown as Mean ± SEM values of 4 independent donors. (*) means statistical significance as <i>p</i><0.05.</p

Effect of sigmar-1 gene silencing on the DMT-modified cytokine profile of LPS or polyI:C-activated moDCs.

<p><b>A:</b> Validation of siRNA knockdown by Western blot. MoDCs were transfected with negative control siRNA (ctrl siRNA) or with gene-targeting siRNA (sigmar-1 siRNA), or left untreated (non-transfected, NT). <b>B–C:</b> Non-treated, 24 h ctrl siRNA-only, and 24 h targeting siRNA-only treated cells were used as negative controls (black bars). Red bars represent 24 h 500 ng/ml LPS-treated cells, while white bars show ctrl siRNA and 1 h DMT pre-treated cells activated with LPS for one day. Grey (NN-DMT) and checkered bars (5-MeO-DMT) demonstrate 1 h DMT pre-treated and then 24 h LPS activated sigmar-1 knockdown cells. <b>D–E:</b> MoDCs were treated as in Fig5B–C. Here, cell activation was performed with a 24 h 20 µg/ml polyI:C treatment. Blue bars represent polyI:C-only stimulation as positive control. Results are shown as Mean ± SEM of three independent donors. (*) represents <i>p</i> values <0.05. Differences are significant (<i>p</i><0.05) in all cases of specific activation (LPS or polyI:C) versus control cells (no treatment, ctrl siRNA, sigmar-1 siRNA).</p

Image_1_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.JPEG

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

Video_2_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.MOV

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

Video_1_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.MOV

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

PET measures of myocardial fatty acid metabolism and blood flow.

<p>(<b>A</b>) Extrinsic myocardial fatty acid oxidation rate (MFAO) (<b>B</b>) Extrinsic myocardial fatty acid esterification rate (MFAE), (<b>C</b>) Extrinsic myocardial fatty acid utilization rate (MFAU), (<b>D</b>) myocardial Extraction Fraction (EF) in GK and control rats. *denotes that GK rats are significantly different (P<0.05) than Wistars for that measurement. All results are presented as mean ± 1 SEM with N=4/group.</p

Clustering and functional grouping of genes involved in fatty acid metabolism.

<p>(<b>A</b>-<b>D</b>) Heat map and Gene correlation clusters of genes involved in FA Metabolism including; fatty acid catabolism(<b>A</b>), fatty acid transport (<b>B</b>), triglyceride biosynthesis (<b>C</b>), and ketogenesis (<b>D</b>). Gene correlation clusters were calculated using the Spearman Correlation Distances and complete linkage for hierarchical clustering. Data for individual biological replicates are shown at the 95% or above confidence level. (<b>E</b>) Pie chart illustrating functional distribution of genes involved in FA metabolism in heart and varies between GK and Wistar rats. (N=4/group). .</p