TAT-Ahx-AKAPis significantly reduced microvascular endothelial barrier function and reverted F/R- mediated barrier stabilization.

<p>TER measurements were carried out to monitor endothelial barrier alterations in response to different mediators and synthetic peptides. (A) displays the time course of TER measurements under various experimental conditions for HDMEC. The first arrow indicated the time point of TAT-Ahx-AKAPis and/or TAT-Ahx-mhK77 addition. 1 hour after the first application, F/R was added (second arrow). (B) summarizes the results after 600 min, the time point at which the monitored effects reached their peaks. TAT-Ahx-AKAPis significantly decreased TER compared to control/vehicle condition and treatment with scrambled peptide (TAT-Ahx-mhK77) starting at 80 min after application for HDMEC. F/R addition resulted in pronounced and continuous increase of TER after 1 hour. A similar effect was detected after pre-incubation with TAT-Ahx-mhK77 scrambled peptide. In contrast, 1 hour pre-treatment with TAT-Ahx-AKAPis initially reduced, but subsequently did not abolished the effect of F/R. (C) To further test the effect of TAT-Ahx-AKAPis on F/R- mediated enhancement of endothelial barrier function, HDMEC cells were exposed to F/R for 1 hour (first arrow) and post- incubated with TAT-Ahx-AKAPis inhibitory peptide (second arrow). (D) graphically represents the statistical outcome of the data presented in panel (C). Based on ANOVA multiple analysis, the most significant peaks of the monitored effects were determined at 1000 min. At that time point (1000 min), the similarly responding TAT-Ahx-AKAPis- and F/R+TAT-Ahx-AKAPis- cell monolayers displayed TER significantly lower than the one in control monolayers. In contrast, F/R- mediated enhancement in TER remained constant over time. Data were collected from more than three independent experiments (N ≥3, n≥4–10). *** p≤0.001, ** p≤0.01, * p≤0.05, indicate statistically significant difference between examined groups. n.s. – not significant.