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Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Pannekoek WJ, Linnemann JR, Brouwer PM, Bos JL, Rehmann H - PLoS ONE (2013)

Bottom Line: In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance.Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance.This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

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Rap1 controls the barrier resistance.HUVEC monolayers transfected with siScrambled or siRap1 were grown on electrodes and analyzed by ECIS. (A) Western blot shows Rap1 depletion efficiency. (B) Impedance was recorded over time at 4000 Hz (here plotted as absolute value). (C) Frequency scans were performed before (pre) and after (post) stimulation with 007-AM as indicated in Fig. 1B. The real part of the impedance is displayed on the right axis (open symbols) and the imaginary part on the left axis (closed symbols). A single electrode of siScrambled (left) and siRap right is shown. The data were fitted (shown as lines) in relation to the reference electrode by the model of Lo et al. (D) The obtained values for the barrier resistance (Rb), α and Cm are represented in bar diagrams. Different colors represent individual independent experiments (n = 8). Averages are indicated by black lines.
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pone-0057903-g001: Rap1 controls the barrier resistance.HUVEC monolayers transfected with siScrambled or siRap1 were grown on electrodes and analyzed by ECIS. (A) Western blot shows Rap1 depletion efficiency. (B) Impedance was recorded over time at 4000 Hz (here plotted as absolute value). (C) Frequency scans were performed before (pre) and after (post) stimulation with 007-AM as indicated in Fig. 1B. The real part of the impedance is displayed on the right axis (open symbols) and the imaginary part on the left axis (closed symbols). A single electrode of siScrambled (left) and siRap right is shown. The data were fitted (shown as lines) in relation to the reference electrode by the model of Lo et al. (D) The obtained values for the barrier resistance (Rb), α and Cm are represented in bar diagrams. Different colors represent individual independent experiments (n = 8). Averages are indicated by black lines.

Mentions: ECIS measurements were essentially performed as described previously [13]. 48 hours after siRNA transfection and/or 24 hours after lentiviral infection, HUVECs were plated onto L-cysteine reduced, Fibronectin-coated 8W10E electrodes (Applied Biophysics) at a density of 1×105 cells/well and grown to confluency for another 24 hours. The time-dependent impedance was measured at 37°C and 6% CO2 using a 1600R Electrical Cell Impedance Sensing (ECIS) system (Applied Biophysics) at 4000 Hz. Frequency scans were performed before and after the timelapse recording, as indicated in figure 1B. These frequency scans were used to calculate α, Rb and Cm with ECIS software (v1.2.50.0 PC) from Applied Biophysics. For this analysis frequencies were limited to the range from 62.5 Hz to 16000 Hz, as a strong inductive component was observed at higher frequencies, which can not be processed by the used algorithms. Each data point in the graph represents the average of individual electrodes (N = 4) within an experiment. Multiple independent experiments are shown using differently colored data points. Averages are indicated by a black line. Statistical analysis of whole data set variance was performed by One-way ANOVA, which is depicted as A(F-score;P-value). Statistical analysis of variance between two groups within one experiment was calculated by Student’s t-test (two-tailed, paired).


Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Pannekoek WJ, Linnemann JR, Brouwer PM, Bos JL, Rehmann H - PLoS ONE (2013)

Rap1 controls the barrier resistance.HUVEC monolayers transfected with siScrambled or siRap1 were grown on electrodes and analyzed by ECIS. (A) Western blot shows Rap1 depletion efficiency. (B) Impedance was recorded over time at 4000 Hz (here plotted as absolute value). (C) Frequency scans were performed before (pre) and after (post) stimulation with 007-AM as indicated in Fig. 1B. The real part of the impedance is displayed on the right axis (open symbols) and the imaginary part on the left axis (closed symbols). A single electrode of siScrambled (left) and siRap right is shown. The data were fitted (shown as lines) in relation to the reference electrode by the model of Lo et al. (D) The obtained values for the barrier resistance (Rb), α and Cm are represented in bar diagrams. Different colors represent individual independent experiments (n = 8). Averages are indicated by black lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585282&req=5

pone-0057903-g001: Rap1 controls the barrier resistance.HUVEC monolayers transfected with siScrambled or siRap1 were grown on electrodes and analyzed by ECIS. (A) Western blot shows Rap1 depletion efficiency. (B) Impedance was recorded over time at 4000 Hz (here plotted as absolute value). (C) Frequency scans were performed before (pre) and after (post) stimulation with 007-AM as indicated in Fig. 1B. The real part of the impedance is displayed on the right axis (open symbols) and the imaginary part on the left axis (closed symbols). A single electrode of siScrambled (left) and siRap right is shown. The data were fitted (shown as lines) in relation to the reference electrode by the model of Lo et al. (D) The obtained values for the barrier resistance (Rb), α and Cm are represented in bar diagrams. Different colors represent individual independent experiments (n = 8). Averages are indicated by black lines.
Mentions: ECIS measurements were essentially performed as described previously [13]. 48 hours after siRNA transfection and/or 24 hours after lentiviral infection, HUVECs were plated onto L-cysteine reduced, Fibronectin-coated 8W10E electrodes (Applied Biophysics) at a density of 1×105 cells/well and grown to confluency for another 24 hours. The time-dependent impedance was measured at 37°C and 6% CO2 using a 1600R Electrical Cell Impedance Sensing (ECIS) system (Applied Biophysics) at 4000 Hz. Frequency scans were performed before and after the timelapse recording, as indicated in figure 1B. These frequency scans were used to calculate α, Rb and Cm with ECIS software (v1.2.50.0 PC) from Applied Biophysics. For this analysis frequencies were limited to the range from 62.5 Hz to 16000 Hz, as a strong inductive component was observed at higher frequencies, which can not be processed by the used algorithms. Each data point in the graph represents the average of individual electrodes (N = 4) within an experiment. Multiple independent experiments are shown using differently colored data points. Averages are indicated by a black line. Statistical analysis of whole data set variance was performed by One-way ANOVA, which is depicted as A(F-score;P-value). Statistical analysis of variance between two groups within one experiment was calculated by Student’s t-test (two-tailed, paired).

Bottom Line: In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance.Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance.This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

Show MeSH
Related in: MedlinePlus