Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E/S) and IL-11 (G/S) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitution of active Hyper-cytokines, which were efficiently secreted from transfected cells. Our strategy comprises the development of a background-free cytokine signaling system from reversibly inactivated precursor cytokines.
Obesity is one of the major risk factors that can lead to a myocardial infarction and can negatively influence subsequent cardiac remodeling. The onset of obesity is related to different genetic variants of the fat mass and obesity associated gene (Fto). Fto deficient mice were protected from obesity and showed an improved glucose tolerance under a high fat diet. In addition, it is known that Fto acts as an m6A RNA demethylase, whereby it can influence mRNA stability and translation. In a first approach, we used Fto deficient mice to perform an ischemic/reperfusion (I/R) model. At 24 h after reperfusion, Fto deficient mice already had a smaller infarct size compared to their wild type littermates. Over a time period of three weeks, the heart function was investigated by echocardiography. Three weeks after reperfusion, Fto deficient mice showed a preserved heart function and had reduced collagen scar formation compared to wild‐type control mice. Initial molecular studies indicated a reduction of the mTORC1 pathway and consequently a down regulation of S6K phosphorylation. For a first approach of therapeutic intervention, we used the Fto inhibitor rhein, which binds the active center of Fto and thereby inhibits its demethylase activity. Wild type mice were pre‐treated with rhein or the solvent over three weeks before the I/R procedure was performed. The rhein‐treated mice showed a significant reduction in the infarct size compared to the solvent treated littermates. In this study, we demonstrated that Fto deficiency can modulate the outcome of a myocardial infarction by improving left ventricular function and reduction of the infarct size.Support or Funding InformationSupported by the Deutsche Forschung Gesellschaft (DFG) as Part of the SFB 1116 (project number 236177352)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fibrosis, which occurs in various heart diseases like acute myocardial ischemia and pressure overload, is triggered by the differentiation of fibroblasts into myofibroblasts. Dysregulation of this reparative mechanism results in excessive collagen accumulation leading to cardiac stiffness and impaired heart function. The aim of this study was to determine whether the rhubarb anthraquinone Rhein, a drug already used as treatment for chondroarthritis, prevents the transdifferentiation of cardiac fibroblasts. We observed that Rhein pre-treatment ameliorates the cardiac function and reduces adverse remodeling after acute myocardial infarction in mice, in vivo . In primary human cardiac fibroblasts, Rhein incubation dose-dependently inhibited the TGF-β-mediated upregulation of α-SMA, the master marker for myofibrolasts, and prevented the contraction of fibroblast-populated collagen gel lattices upon TGF-β stimulation. Further, Rhein reduced TGFβ-R1 expression in primary human cardiac fibroblast, resulting in decreased SMAD2 phosphorylation and blunting of the fibrogenic response. Furthermore, Rhein stabilized protein levels of SMAD7, a key inhibitor of TGF-β signaling. Collectively, these data show for the first time that Rhein administration prevents cardiac fibrosis in vivo and in vitro by blunting the TGF-β signaling pathway, and identify Rhein as potential therapeutic treatment to prevent excessive fibrosis and adverse remodeling in cardiac pathologies.
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