Figure 1.
TAT-Ahx-AKAPis significantly reduced microvascular endothelial barrier function and reverted F/R- mediated barrier stabilization.
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.
Figure 2.
Effects of PKA compartmentalization on adherens junctions, actin cytoskeleton and AKAP organization.
To determine the AKAPs protein expression profile, equal amounts of MyEnd and HDMEC cell lysates (20 µg per line) were subjected to WB analysis. The expression patterns of AKAP220 (A) and AKAP12 (B) were analyzed. Loading was controlled by alpha tubulin expression. Data are representative of three or more independent experiments (N≥3). In addition, HDMEC monolayers were treated for 1 hour either with vehicle, TAT-Ahx-AKAPis, TAT-Ahx-mhK77, F/R or with a combination of 1 hour TAT-Ahx-AKAPis pretreatment followed by 1 hour F/R application. The distributions of VE-cadherin, PKA, AKAP220 and AKAP12 were assayed by immunofluorescence. Additionally, ALEXA-488-conjugated phalloidin was used for visualization of F-actin. (C) Under control condition, VE-cadherin displayed slightly interdigitated but continuous staining along cell borders, and the actin cytoskeleton was preferentially organized cortically. The staining of AKAP220, AKAP12 and PKA was detectable at cell borders (arrows). (D) In clear contrast, exposure to TAT-Ahx-AKAPis increased interdigitations and significantly reduced the intensity of VE-cadherin staining. Profound reorganization of the actin cytoskeleton was paralleled by substantial reduction of AKAP220 and PKA, but not of AKAP12 membrane staining. (E) However, cell monolayers incubated with TAT-Ahx-mhK77 showed immunofluorescence staining similar to control for all proteins under investigation. (F) Not surprisingly, F/R treatment resulted in pronounced and linearized VE-cadherin appearance, intensified cortical actin cytoskeleton, and pronounced membrane staining for AKAP220, AKAP12 and PKA compared to control conditions. (G) 1 hour pre-incubation with TAT-Ahx-AKAPis followed by 1 hour treatment with F/R resulted in monolayers largely similar to controls, but not to F/R incubation alone. Images are representative of three or more independent experiments (N ≥3). Scale bar = 20 µm. The above presented data were confirmed by quantification of signal intensity distribution at cell borders. (H–K) demonstrates the mean intensity peak observed at cell borders (n≥15). *** p≤0.001, ** p≤0.01, * p≤0.05, indicate statistically significant difference between examined groups; n.s., not significant.
Figure 3.
PKA and AKAP220 form a complex with junctional-associated proteins VE-cadherin and ß-catenin.
A complex consisting of PKA, ß-catenin, VE-cadherin and AKAP220 was detected in MyEnd either by pulling down VE-cadherin (A) or PKA (B). Immunoprecipitated proteins and their binding partners were detected in the lysates used for immunoprecipitation. The lysates were loaded at the first lane and denoted as an “Input”. Immunoprecipitation without antibody (IP: Beads, no Ab) was used as a negative control. Every other lane represents proteins in complex under different experimental conditions. (C) To test the role of PKA compartmentalization on the stability of the determined complex, PKA pull down was performed in cells treated either with inhibitory or with the respective scrambled peptide. Additionally, the effect of F/R on complex stability was analyzed. (D) summarizes data collected from three independent immunoprecipitation experiments. Firstly, intensity readings for each band were background corrected. Secondly, data was modified by substraction of “non-specific background intensities” detected in IP without Ab (negative control). Since PKA was used to pull-down the complex, signal intensity of the immunoprecipitated proteins was normalized to the band intensity of PKA (represented as fold changes of PKA staining intensity). As a final, the data was presented as percent of IP: PKA, TAT-Ahx-mhK77. The analysis revealed decreased complex association of ß-catenin, VE cadherin and AKAP220 after TAT-Ahx-AKAPis treatment. In contrast, F/R application led to stabilization of the complex indicated by more prominent intensities for bands representing ß-catenin, VE-cadherin and AKAP220. Noncontiguous bands run on the same gel are separated by a black line. Images are representative of three independent experiments (N = 3).
Figure 4.
siRNA-mediated AKAP12 and AKAP220 knockdown significantly impaired endothelial barrier function.
(A) Barrier function of subconfluent MyEnd cells was monitored by TER measurements following transfection with siRNA specific for AKAP12 and AKAP220. Non-targeting siRNA was used as a control. The results were compared to cells treated with TAT-Ahx-AKAPis inhibitory peptide. AKAP12 and AKAP220 depletion lead to a significant decrease in TER compared to monolayers transfected with n.t. siRNA. Similar, but more prominent, was the effect obtained after TAT-Ahx-AKAPis application. Data were collected from three or more independent experiments (N ≥3, n ≥12). (B) To test the role of specific AKAPs on cAMP-mediated endothelial barrier formation, TER measurements in cells treated with F/R, 48 hours after successful AKAP-depletion were performed; (n≥10). (C) AKAP12 and AKAP220 knockdown was confirmed by Western blot, 48 hours after transfection. ß-actin was used as an internal gel-loading control. (D) Western blot data were analyzed densitometrically. Normalized intensities for AKAP signals are presented as a bar graph. Data were collected from more than three independent experiments (N ≥3). * p≤0.05, **p≤0.01 and ***p≤0.001 indicate statistically significant difference between the examined group and n.t. siRNA; # p≤0.05 shows statistically significant difference between AKAP12 siRNA and AKAP220 siRNA upon treatment with F/R. The difference was significant starting at 1500 min.
Figure 5.
TAT-Ahx-AKAPis reduced Rac1 activity.
HDMEC monolayers grown on glass coverslips were subjected to immunostaining with anti-Rac1 antibody. (A) In controls, Rac1 was partially present along cell borders (arrows). (B) Similar Rac1 staining was observed after application of TAT-Ahx-mhK77. (C) In contrast, treatment with TAT-Ahx-AKAPis strongly reduced Rac1 membrane staining and induced re-localization of the molecule to the cytoplasm. (D) However, the cAMP enhancers F/R led to pronounced and intensified Rac1 membrane staining. Images are representative of three or more experiments (N≥3). Scale bar = 20 µm. (E) Quantification of the signal intensity distributed along cell-cell junctions supported the above mentioned observations. In order to summarize all experiments, data were presented as percent of control (N≥3¸n≥10). (F) In HDMEC, ELISA-based measurements revealed a significant decrease of Rac1 activity in response to 1 hour treatment with TAT-Ahx-AKAPis. Application of F/R also significantly increased Rac1 activity when compared to control. As expected, TAT-Ahx-mhK77 administration had no effect on Rac1 activity. (G) The effects observed in MyEnd cells were similar. In agreement with TER, the effect of TAT-Ahx-AKAPis inhibitory peptide was evident later. Therefore, the MyEnd samples were collected 6 hours after peptide application. The results are representative of three independent experiments (N = 3, n ≥6). * defines statistically significant difference; * p≤0.05, ** p≤0.05, *** p≤0.001. n.s. not significant.
Figure 6.
Simultaneous knockdown of AKAP12 and AKAP220 mRNA impaired cAMP-mediated Rac1 activation.
(A) Subconfluent monolayers of MyEnd cells were transfected either with single AKAP siRNA or with a pool of AKAP12 and AKAP220 specific siRNAs. Basal Rac1 activity was tested 48 hours after initial siRNA depletion. For evaluation of cAMP-mediated Rac1 activation, transfected cells were subjected to F/R for 1 hour. Non-target siRNA (n.t. siRNA) alone or in combination with F/R (1 hour) was used as a respective control. Bars represent mean values ± SEM. Basal activity of Rac1 was unaffected after single or combined silencing of AKAPs. In contrast, cAMP-mediated Rac1 activation was significantly impaired following simultaneous AKAP12 and AKAP220 mRNA knockdown. (B) Successful knockdown was verified by Western blot 48 hours after initial siRNA transfection; ß-actin was used as an internal gel-loading control. ** indicates p≤0.01 versus cells transfected with n.t. siRNA treated for an hour with F/R.
Figure 7.
TAT-Ahx-AKAPis application increased hydraulic conductivity (Lp) in rat mesenteric microvessels in vivo.
Hydraulic conductivity (Lp) of isolated post-capillary microvessels from rat mesentery was monitored over time. (A) displays data from a representative vessel constantly perfused either with vehicle or with TAT-Ahx-AKAPis. (B) Bar diagram summarizes the mean values of all five independent experiments. 120 min after TAT-Ahx-AKAPis perfusion, Lp was significantly increased to 4.57±0.91×10−7 [cm/(s×cm H20)] compared to the corresponding vehicle condition (2.53±0.47×10−7 [cm/(s×cm H20)]). *indicates p≤0.05 versus control condition; N = 5.