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First published online May 6, 2009
doi: 10.1242/10.1242/jcs.040212

Journal of Cell Science 122, 1616-1625 (2009)
Published by The Company of Biologists 2009
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Endothelial barrier stabilization by a cyclic tandem peptide targeting VE-cadherin transinteraction in vitro and in vivo

Wolfgang-Moritz Heupel1,*, Athina Efthymiadis1,*, Nicolas Schlegel1, Thomas Müller2, Yvonne Baumer1, Werner Baumgartner3, Detlev Drenckhahn1,{ddagger} and Jens Waschke1,{ddagger}

1 University of Würzburg, Institute of Anatomy and Cell Biology, Koellikerstr. 6, D-97070 Würzburg, Germany
2 University of Würzburg, Department of Botany I, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
3 RWTH Aachen, Institute of Biology II, Kopernikusstr. 16, D-52056 Aachen, Germany


Figure 1
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  		Fig. 1. Structure of VE-cadherin, and peptide design. (A) Model of a trans-interacting VE-cadherin EC1 pair. Secondary structure elements and N- and C-termini are indicated. Side chains of the RVDAE motif are shown in stick format. (B) Docking of the RVDAE peptide onto the surface of a VE-cadherin EC1 module (grey with transparent surface). (C) Same as in B but with the surface of VE-cadherin EC1 colour-coded according to polarity: blue, nitrogen atoms; cyan, carbon atoms; red, oxygen atoms. (D) Magnification of the peptide binding site. Putative hydrogen bonds between the peptide and the EC1 domain of VE-cadherin are shown as dashed lines. (E) Model of the tandem peptide, which is cyclized by two cysteine residues at the N- and C-terminus and a 6-aminohexanoic acid linker.

 

Figure 2
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  		Fig. 2. Effect of SP and TP on single molecule VE-cadherin transinteraction. (A) Dose-response curves show action of SP and TP on VE-cadherin transinteraction as probed by AFM single molecule unbinding studies. SP reduced VE-cadherin binding activity at concentrations of 20 µM or higher, resulting in a reduction of about 70% at 200 µM. By contrast, TP displayed a shifted dose-response curve with inhibiting activity starting at 200 µM. The dashed line indicates VE-cadherin binding activity in Ca2+-free conditions. (B) Sample force-distance cycles in control, 20 µM TP or 200 µM SP conditions. Note the similar unbinding events in TP and control conditions but reduced interaction frequency in SP condition. (C) Frequency distribution of single molecule VE-cadherin-Fc unbinding events measured at retrace velocities of 600 nm/second. For each condition, >500 unbinding curves were evaluated and resulting probability densities corrected by interaction frequencies, which were 52.8% for control, 54.0% for TP and 20.3% for SP condition. In control conditions, three different force peaks could be differentiated (marked by grey columns). Note the frequency increase for the first unbinding force peak in TP condition (61% versus 50%; see also insets with Gaussian multiple peak fits of control and TP probability density curves) and reduced interaction frequency after SP treatment. (D) Binding activities of transinteracting VE-cadherin-Fc were strongly reduced after treatment with monoclonal VE-cadherin antibody 11D4.1. As already demonstrated in A, SP at 200 µM blocked VE-cadherin transinteraction whereas TP at 20 µM did not. Preincubation with 20 µM TP, however, prevented 11D4.1-induced loss of VE-cadherin adhesion. Sequence specificity of SP and TP action was demonstrated by a scrambled SP or control peptides CP1 and CP2, which did not have an effect on VE-cadherin binding activity, and by CP2 which was unable to block 11D4.1-mediated inhibition. Moreover, SP and TP at the concentrations used did not influence transinteraction of desmonglein-3-Fc (n=3–4 for each condition).

 

Figure 3
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  		Fig. 3. SP and TP peptides modified lateral diffusion of VE-cadherin-EYFP in FRAP studies. (A) In CHO-A1 cells, VE-cadherin-EYFP signals were continuously seen at sites of cell-cell contacts. In this sample post-bleach image, rectangles indicate region of interests where EYFP fluorescence had just been bleached at sites of cell-cell contacts. Scale bar: 20 µm. (B) Measurements of fluorescence intensities in these regions showed a biphasic, double-exponential recovery of EYFP signals after bleaching in all three conditions. After t{approx}9 seconds, however, VE-cadherin-EYFP recovery was enhanced in SP-pretreated cells, whereas it was reduced during nearly the whole time series in TP-pretreated cells. Note the overall slow recovery and high fraction of immobile molecules in all conditions.

 

Figure 4
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  		Fig. 4. TP prevented mAb 11D4.1- and A23187-induced loss of VE-cadherin-mediated adhesion but acted independently of F-actin or Rho GTPase function. (A) Principle of laser tweezer experiments. VE-cadherin-coated microbeads were allowed to settle on the surface of MyEnd cells to induce VE-cadherin-mediated homophilic adhesion. Afterwards, beads were trapped in a laser beam focus to discriminate bound from unbound beads. (B) On MyEnd cells, the number of bound beads was significantly increased after incubation with TP for 30 minutes, compared with controls. Incubation with mAb 11D4.1 led to a strong loss of VE-cadherin bead binding. Preincubation with TP (20 µM), however, completely prevented this reduction. Incubation with the Ca2+ ionophore A23187 (10 µM) for 45 minutes similarly reduced bead binding, whereas preincubation with TP (20 µM) again completely blocked this effect. Incubation with the F-actin-disrupting agent cytochalasin D (10 µM) for 30 minutes strongly reduced the number of surface-bound beads. Nevertheless, preincubation with TP in combination with cytochalasin D resulted in increased bead binding compared to cytochalasin D alone (n=6–8). (C) Quantification of F-actin demonstrated that only cytochalasin D but not TP treatment significantly decreased the F-actin content. (D) In GTPase activity assays, TP treatment of MyEnd cells did not lead to activation of the small GTPases Rac1 and RhoA.

 

Figure 5
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  		Fig. 5. TP prevented A23187-induced alterations of endothelial adherens junctions. (A-F) Confluent MyEnd monolayers were treated with A23187 (10 µM) for the indicated times. In parallel experiments (D-F), cells were preincubated for 30 minutes with TP (20 µM) before addition of A23187. In controls (0 minutes), VE-cadherin immunostaining was regularly distributed along cell borders (A). Incubation with A23187 led to frayed and fragmented immunostainings after 45 minutes (B,C). This effect was prevented by the presence of TP (D-F). (G) Quantification of VE-cadherin-positive immunosignals confirmed frayed and broadened VE-cadherin staining after 45 minutes incubation with A23187 and the protective effects of preincubation with TP. Images shown are representative of six experiments. Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. TP partially prevented loss of TER induced by mAb 11D4.1 and A23187 in endothelial cells. (A) A significant reduction in TER was observed 240 minutes after addition of mAb 11D4.1 (50 µg/ml). Starting after 300 minutes, treatment with TP in addition to mAb 11D4.1 resulted in a significant higher TER compared with mAb 11D4.1 alone (n=5). (B) Treatment of endothelial monolayers with A23187 (10 µM) led to a significant reduction in TER within 45 minutes compared with controls, reaching a plateau after 180 minutes. In endothelial cells preincubated for 30 minutes with 20 µM TP, the A23187-induced drop in TER was partially prevented (n=5).

 

Figure 7
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  		Fig. 7. TP treatment abolished TNF-{alpha}-induced increase of microvessel permeability in vivo. (A) Measurements of hydraulic conductivity (Lp) demonstrated that TNF-{alpha} strongly increased microvessel permeability, starting after 120 minutes, which was, however, completely prevented by cotreatment with TP. Note that at every time point three independent Lp measurements were made and plotted in this graph. (B) Mean Lp values for each condition show that after 120 minutes TNF-{alpha} significantly increased Lp compared with controls. Although having no effect when applied alone, TP completely abolished TNF-{alpha}-induced increase of endothelial permeability (n≥5 for each condition).

 

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