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  • In this study we investigated the role


    In this study we investigated the role 17-phenyl tinor (pt)-PGE2 on endothelial barrier function and the underlying molecular mechanism in HMVEC-Ls as well as in a murine model of LPS-induced acute pulmonary inflammation. We found that 17-pt-PGE2 concentration-dependently enhanced endothelial barrier function, whereas a more specific EP1 receptor agonist ONO DI-004 [16] or the EP3 receptor agonist sulprostone did not mimic this effect. Surprisingly, the effect induced by 17-pt-PGE2 was mediated by the EP4 receptor and not by EP1 or EP3 receptors. Furthermore, we show that 17-pt-PGE2 strengthens the endothelial junctions of HMVEC-Ls and reduces stress fiber formation upon treatment with thrombin. Conversely to our findings in endothelial cells, 17-pt-PGE2 promotes platelet aggregation via EP3 receptors. In a murine model of acute pulmonary inflammation, 17-pt-PGE2 caused a decrease in pulmonary extravasation and a reduction of infiltrating neutrophils, which was mediated by EP4 receptor activation. Our results demonstrate that 17-pt-PGE2 - in addition to its described effects on EP1 and EP3 receptors - also acts as an EP4 agonist and thereby enhances vascular barrier function.
    Materials and methods
    Discussion In this study we demonstrate that the purported EP1/EP3 receptor agonist 17-pt-PGE2 concentration-dependently increases the endothelial barrier function of HMVEC-L as determined by an increase in electrical resistance, and protects against thrombin-induced disruption of endothelial junctions and stress fiber formation. These effects were comparable to PGE2[14]. Interestingly, the more specific EP1 receptor agonist, ONO DI-004 and the EP3 receptor agonist sulprostone failed to mimic this effect. The lack of involvement of EP1 and EP3 receptors was confirmed by EP1 and EP3 antagonists. In contrast, the barrier-strengthening effect of 17-pt-PGE2 was reversed by EP4 antagonists as well as by silencing of the EP4 receptor. Monolayers of endothelial mavacamten are accomplished by junctional structures, where VE-cadherin plays a major role in adherens junction formation. We found a protective effect of 17-pt-PGE2 on thrombin-induced disruption of the endothelial junctions and stress fiber formation, which was recently demonstrated to be mediated via EP4 receptor activation [14]. Strengthening of vascular barrier function in vitro was shown for PGI2 and PGE2 via cAMP mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation [6]. Recently, we demonstrated that EP4 receptor activation is responsible for the barrier-enhancing effect of PGE2, which was independent of cAMP/PKA, PKC, NO or Rac signaling but was mediated by cytoskeletal rearrangements [14]. This is in line with our findings, as 17-pt-PGE2 did not induce cAMP synthesis in HMVEC-L. In addition, wound healing was facilitated by EP4 receptor activation but was not influenced by EP2 and EP1/3 agonists [14]. Here we demonstrate that the barrier enhancing effect or 17-pt-PGE2 is mediated solely by EP4 but not by EP1 or EP3 receptor activation. Inflammatory stimuli cause disruption of the endothelial barrier by dissociation of cell-cell junctions, leading to plasma extravasation and infiltration of inflammatory cells. Our second main finding was that in a murine model of LPS-induced acute lung inflammation, 17-pt-PGE2 reduced pulmonary plasma extravasation and ameliorated the recruitment of neutrophils into the BAL fluid, which account as characteristic clinical complications of acute lung injury [31]. This effect was mediated by EP4 receptor activation, which is in agreement with our in vitro findings on the barrier function. PGE2 is well known to reduce the accumulation of neutrophils within the BAL fluid in LPS-induced acute lung inflammation [32], [33]. We showed recently that the effect of PGE2 on LPS-induced neutrophil infiltration and LPS- as well as oleic acid-induced plasma extravasation is mediated mainly via activation of the EP4 receptor [15]. In addition it was demonstrated in EP4-, but not in EP1-, EP2- and EP3-deficient mice that only the EP4 receptor seems to be responsible for the anti-inflammatory activity in mouse models of lung inflammation [12].