Ilg Dr. Marcus M, Ralph Prof. David J, Cellek Prof. Selim

(110) EXTRACELLULAR VESICLES ISOLATED FROM PEYRONIE’S DISEASE-DERIVED CELLS CAN ALTER TGF-BETA 1 SIGNALLING CASCADES

  • Urology
  • Reproductive Medicine
  • Endocrinology
  • Endocrinology, Diabetes and Metabolism
  • Psychiatry and Mental health
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Abstract Objectives The importance of extracellular vesicles (EVs) as key mediators of cell-cell communication has been highlighted in recent years. However, their role in the pathophysiology of Peyronie’s disease (PD) and other fibrotic disorders is still unclear. Fibroblasts and myofibroblasts are key effectors in PD and this study seeks to further elucidate a previously described role of EVs in myofibroblast transformation in PD. Methods Primary fibroblasts were isolated from tunica albuginea of PD patients. Cells were cultured in DMEM with 10% Knockout Serum Replacement and EVs were isolated from the conditioned media in presence and absence of transforming growth factor beta 1 (TGF-β1). Isolation of the EVs was performed using ultrafiltration with subsequent size exclusion chromatography. Fibroblast and myofibroblast transcriptome was analysed using Nanostring nCounter. Classical EV markers were confirmed using Western blot. EV quantity, size, and shape was analysed using Tunable resistive pulse sensing (TRPS). Expression of α-smooth muscle actin (α-SMA), pSmad2/3 and pErk1/2 in presence and absence of TGF-β1 was assessed using In-Cell ELISA (ICE). Results Myofibroblasts produced more EVs and smaller EVs than fibroblasts and classical EV marker expression was confirmed. Nanostring nCounter revealed that fatty acid metabolism, cholesterol metabolism, and de novo lipogenesis were differentially regulated in cells treated with TGF-β1, in line with increased EV production. Myofibroblast EVs prevented TGF-β1-induced myofibroblast transformation which could be prevented with an inhibitor of vesicle uptake (10 μM dynasore). Myofibroblast-derived EVs but not fibroblast-derived EVs were able to affect downstream effectors of TGF-β1, by preventing the phosphorylation of pErk1/2. No effect on pSmad2/3 phosphorylation was observed. Conclusions This is the first study to demonstrate that PD-cell-derived EVs can affect signalling cascades downstream of TGF-β1. Only non-canonical TGF-β1 signalling is influenced and the effect appears dependent on cellular uptake of the vesicles. Further study is needed to decipher the cargo responsible for this effect. Conflicts of Interest This study is partly funded by ESSM Research Grant (ESSM RG-19 04).

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