Phosphorylation of VE-cadherin is modulated by haemodynamic forces and contributes to the regulation of vascular permeability in vivo

Abstract

Endothelial adherens junctions maintain vascular integrity. Arteries and veins differ in their permeability but whether organization and strength of their adherens junctions vary has not been demonstrated in vivo. Here we report that vascular endothelial cadherin, an endothelial specific adhesion protein located at adherens junctions, is phosphorylated in Y658 and Y685 in vivo in veins but not in arteries under resting conditions. This difference is due to shear stress-induced junctional Src activation in veins. Phosphorylated vascular endothelial-cadherin is internalized and ubiquitinated in response to permeability-increasing agents such as bradykinin and histamine. Inhibition of Src blocks vascular endothelial cadherin phosphorylation and bradykinin-induced permeability. Point mutation of Y658F and Y685F prevents vascular endothelial cadherin internalization, ubiquitination and an increase in permeability by bradykinin in vitro. Thus, phosphorylation of vascular endothelial cadherin contributes to a dynamic state of adherens junctions, but is not sufficient to increase vascular permeability in the absence of inflammatory agents.

Figure 1. pY658- and pY685-VEC antibodies specifically recognize phosphorylated VEC in vitro.

(a) The cytoplasmic tail of murine VEC and human VEC contains eight and nine tyrosine residues, respectively, as indicated. Antibodies to murine pYVEC have been developed using the sequences around residue Y658 and Y685 (red), respectively. The corresponding human (hVEC) sequences are reported. The sequence deleted in human VEC-Δ622-702 mutant is indicated. (b) Immunoprecipitation (IP) of wt human VEC followed by western blot (WB) showed that antigen recognition by pY658-VEC and pY685-VEC antibodies was increased upon 5 min VEGF treatment. Both pYVEC antibodies did not recognize the VEC-Δ622-702 mutant, conversely an unselective anti-phosphorylated tyrosine antibody (pY) could bind both VEC-wt and VEC-Δ622-702 mutant. (c) VEC-siRNA treatment of VEGF-stimulated HUVEC inhibited pY658-VEC staining (in red) of endothelial cells junctions, VEC is shown in green. Scale bar: 20 μm. (d) Antibody pY685-VEC did not decorate cell junctions of VEGF-stimulated mVEC-null endothelial cells (right panels). Staining with pY658-VEC gave comparable results (not shown). Scale bar: 20 μm. (e) Antibody pY658-VEC did not bind to VEC-Y658F mutant (left panels), but recognized VEC-Y685F mutant (central and right panels) in pervanadate-treated cells. (f) Conversely, antibody pY685-VEC did not recognize VEC-Y685F mutant (left panels) but could bind to VEC-Y658F mutant (central and right panels) in pervanadate treated cells. Pixels presenting the co-localization of pY658- or pY685-VEC with total VEC are highlited in white. Scale bar: 50 μm. (g) Antibody pY658-VEC did not bind to immunoprecipitated VEC-Y658F mutant (central lanes), but recognized VEC-wt or VEC-Y685F mutant (left and right lanes). Conversely, antibody pY685-VEC did not recognize immunoprecipitated VEC-Y685F mutant (right lanes), but could bind to VEC-wt or VEC-Y658F mutant in pervanadate-treated cells (left and central lanes). (h) IP of wt hVEC followed by WB showed that antigen recognition by the commercially available antityrosine-phosphorylated-(Y658)-VEC antibody was increased upon pervanadate treatment. However, the antibody tested was also equally able to recognize the VEC-Δ622-702 mutant, in which the indicated tyrosine is not present. Data shown are representative of at least three independent experiments.

Figure 2. In vivo phosphorylation of VEC in veins but not in arteries.

Staining of veins (V) and capillaries (C) of diaphragm and bladder with anti-pY658-VEC (a), anti-pY685-VEC (b) (both in red) and an antibody against total VEC (green). Both pYVEC antibodies label cell junctions. Scale bar: 50 μm. pY658-VEC (c) and pY685-VEC (d) antibodies stain endothelial junctions in veins but not in small arterioles (A). Scale bar: 50 μm. The boxes represent the area of magnification where pixels presenting VEC/pYVEC co-localization are highlighted in white. Scale bar: 20 μm. Data in a–d are representative of at least 10 independent mice per group. (e,f) Quantification of vessels positive or negative to either pY658-VEC (e) or pY685-VEC antibody (f); data are expressed as percentage of positive (red columns) and negative (blue columns) vessels grouped by diameter (μm), n is the total number of vessels considered for each group (P.C., postcapillaries).

Figure 3. Shear stress modulates VEC phosphorylation.

(a,b) Parametric study of cultured HUVEC exposed to different levels of shear stress as indicated. Low and medium shear stress (from 3,5 dynes cm⁻² up to 28 dynes cm⁻²) increased pY658-VEC junctional staining as compared with static conditions. Further increase of shear stress up to 50 dynes cm⁻² (corresponding to high arterial flow) induced a progressive reduction of pY658-VEC junctional staining until levels similar to control cells. Scale bar: 20 μm. Chart in (b) shows the co-localization (VEC/pY658-VEC) data as Pearson’s correlation coefficient. Ten to 20 stacks were collected on different fields (each containing an average of 10 cells) for each sample under analysis. The grey area shows the co-localization value for static control plus/minus two times its s.e. Statistical comparisons of Pearson’s coefficients measured in different fields from two to three independent specimens were performed with non-parametric two-tailed Mann–Whitney tests (α=0.05); see Methods for details. (c) Mean fluorescence intensity of co-localized pY658-VEC and total VEC pixels as in (d). Statistical significance was determined on four individual animals by independent two-tailed t-test assuming unequal variances. (b.p., bypass, n=4 rat per group). (d) Untreated rat carotid artery showed lower pY658-VEC antibody staining as compared with the jugular vein. Exposure of jugular vein endothelium to arterial flow for 30 min significantly decreased pY658-VEC antibody staining. Scale bar: 50 μm. Pixels showing VEC/pY658-VEC co-localization (white) are highlighted in magnifications (scale bar: 20 μm). *P<0.01 by the above specified test.

Figure 4. Shear stress induced VEC phosphorylation is mediated by Src activation.

(a) Src (green) and VEC (red) staining of arterial and venous endothelium in the mouse diaphragm. Staining presented in left panels shows junctional distribution of total Src in both vessel types (n=4 mice). Conversely, active Src (pY418-Src, right panels) is present at cell-to-cell contacts in veins only (n=4 mice). (b,c) In vivo staining for active Src (pY418-Src) of venous endothelium (mouse diaphragm) was abolished by treatment with a Src inhibitor (AZD0530). Mean fluorescence intensity of co-localized VEC and pY418-Src pixels is shown in (c). Data are mean±s.e.m. of five mice analysed. Statistical significance was determined by independent two-tail t-test assuming unequal variances. Scale bar: 50 μm. (d,e) VEC phosphorylation (pY658- or pY685-VEC in red, total VEC in green) of venous endothelium of mouse diaphragm was abrogated by AZD0530. Quantification in (e) reports mean fluorescence intensity of co-localized VEC/pY658-VEC (left) or pY685-VEC (right) pixels. Data are mean±s.e.m. of at least 10 veins analysed. Statistical significance was determined by independent two-tail t-test assuming unequal variances. Scale bar: 50 μm. (f,g) Flow-induced Y658-VEC phosphorylation in cultured HUVEC was abolished upon treatment with Src inhibitors PP1 or SU6656 by immunofluorescence (f). Co-localization (VEC/pY658-VEC) analysis (g) is reported as fold change versus static conditions. Statistical comparisons of Pearson’s coefficients measured in different fields from two to three independent specimens were performed with non-parametric two-tailed Mann–Whitney tests (α=0.05); see Methods for details. Scale bar: 20 μm. *P<0.05 and **P<0.01 by the above specified test.

Figure 5. pYVEC staining is decreased by bradykinin treatment.

(a) At 4 min, bradykinin (bradyk) treatment in vivo strongly increased venous permeability of the trachea vasculature, as measured by fluorescent microsphere extravasation (white), and markedly reduced pY685-VEC staining (red), VEC is shown in green. At 10 min permeability and pY685-VEC staining were restored. Permeability did not increase in small arteries (asterisk). Treatment with angiopoietin-1 (Ang-1) strongly inhibited the effect of bradykinin on both pY685-VEC staining and permeability. (b) Magnifications of the boxed areas for control and bradykinin-treated mice (4 min). Junctional pY685-VEC was strongly reduced in the sites of altered permeability while total VEC was still present with similar intensity than in control mice (lower panels and arrows). Middle panels show that in the areas unaffected by bradykinin treatment, pY685-VEC was only minimally altered. Staining with pY658-VEC antibody resulted in superimposable results (see Supplementary Fig. S2a). Scale bars: 50 μm (left images) and 20 μm in magnified fields. (c) Bradykinin-induced vascular permeability in the mouse trachea was blocked by Src inhibitor AZD0530 as shown by extravasated microspheres (red, upper panels). Higher magnifications are shown in the lower panels and show VEC staining (green) and extravasated microspheres (red). Scale bar: 500 μm. (d) Immunoprecipitation (IP) of VEC from lung extracts showed that in the presence of bradykinin, phosphorylation of both Y658 and Y685 was reduced and Ang-1 prevented this effect. (e) Quantification of normalized band intensity as in (d). Data are mean±s.e.m. of five separate experiments. (f) Quantification of the areas covered by extravasated beads as in (a) and (c). Data are mean±s.e.m. of at least three mice considering 10 separate fields per mice in each group. Statistical significance was determined by analysis of variance test (e) or by independent two-tailed t-test assuming unequal variances (f). (g) Bradykinin reduces the co-localization of VEC with pY658-VEC in flow conditioned HUVEC (see Fig. 3a) while chlorpromazine inhibits this effect. Statistical comparisons were performed as in Fig. 3b. *P<0.05 and **P<0.01 by the above specified test.

Figure 6. Phosphorylated VEC is internalized.

(a) Immunoprecipitation (IP) of surface VEC in HUVEC. Bradykinin treatment reduces surface VEC and its pY658 phosphorylated form. Treatment of the cells with siRNA targeting VE-PTP and Dep-1 increased resting VEC phosphorylation, but did not inhibit the decrease of surface amount of total and pYVEC induced by bradykinin. Western blot (WB) analysis of silencing efficiency (>70%) is shown in Supplementary Fig. S3e. (b) Quantification of surface VEC (left) and pY658-VEC (right) reduction in siRNA-treated cells (Control, Dep-1 or VE-PTP siRNA in blue, red or green, respectively) after bradykinin stimulus, fold change versus unstimulated cells. Right graph depicts the quantification (fold change versus control siRNA) of surface pY658-VEC in siRNA-treated cells (Dep-1 or VE-PTP siRNA in red or green, respectively). (c) 30 min bradykinin (bradyk) treatment of HUVEC, in the presence of chloroquine. Acid wash highlights the signal from internalized VEC (right panels and magnification). VEC internalization corresponded to partial disruption of endothelial junctions as can be observed in the absence of acid wash (left panels and magnification). Scale bars: 20 and 50 μm in magnifications. (d) Co-staining of VEC and pY658-VEC upon cell treatment with bradykinin and acid wash in the presence of chloroquine. Channels merge (bottom panel) shows that a fraction of internalized VEC is phosphorylated (arrows). Scale bars: 20 μm and 50 μm in magnified fields. (e) HUVEC showed a discontinuous junctional staining with pY658-VEC (in red), which was lost upon bradykinin treatment. This effect was prevented by chlorpromazine, a general inhibitor of endocytosis. VEC is shown in green. Scale bar: 50 μm. (f) IP of internalized VEC in HUVEC after 3 or 30 min bradykinin treatment. Internalized VEC was tyrosine (Y658) phosphorylated and still bound to p120. Quantification of normalized band intensities is shown in bottom panel. (g) IP of surface VEC. Bradykinin decreased the amount of total and pYVEC present on the surface. Quantification of normalized band intensities is shown in right panel. Data in (c,d and e) are representative of at least three independent experiments per group. Data in (b), (f, bottom panel) and (g, right panel) are mean±s.e.m. of three independent experiments. *P<0.05 and **P<0.01 by independent two-tailed t-test assuming unequal variances.

Figure 7. VEC phosphorylation is necessary for its internalization.

(a) VEC null murine endothelial cells were transduced with VEC-wt or non-phosphorylatable mutants (VEC-Y658F and VEC-Y685F). Treatment of the cells with 30 min bradykinin, in the presence of chloroquine, increased the number of cells showing internalized VEC-wt, but was ineffective on non-phosphorylatable mutants (lower panels, with acid wash). As observed for HUVEC, VEC internalization corresponded to partial disruption of endothelial junctions on cells expressing wt, but not mutant, VEC (upper panels, without acid wash). Scale bar: 50 μm. (b) Quantification of VEC internalization is reported as percentage of cells showing internalized VEC as in (a). Data are mean±s.e.m. of at least 100 cells analysed. (c) AZD0530 prevented bradykinin-induced VEC internalization. Data are shown as percentage of cells showing internalized VEC and are mean±s.e.m. of at least 100 cells analysed. (d) Three or 30 min bradykinin treatment increased paracellular permeability of cells expressing VEC-wt and, to a much lower extent, of cells expressing VEC-Y658F and VEC-Y685F (red diamonds, fold change over untreated cells). Dynasore (green squares) or chlorpromazine (blue triangles) were able to block the effect on cell permeability induced by bradykinin. Data are mean±s.d. of five replicates from a typical experiment out of three experiments performed. (e–g) FRAP experiment in HUVEC expressing fluorescent Y658F-cherry or wt-VEC-cherry revealed a reduced protein motility of Y658F-cherry mutant compared with wt-VEC-cherry. The strongest difference was at 300 s after bleach as shown in (f). Half-time of recovery (τ1/2) of wt-VEC-cherry protein was two fold higher upon chlorpromazine treatment (g). Y658F-cherry mutant presented a 2.5-fold higher basal τ1/2 than wt-VEC-cherry, and it was insensitive to chlorpromazine. Statistical significance was determined on at least 10 replicates. Scale bar is 4 μm. *P<0.05 and **P<0.01, by analysis of variance test (c,d) or by independent two-tailed t-test assuming unequal variances (b, e and g).

Figure 8. Bradykinin induced VEC internalization is coupled by ubiquitination.

(a) IP of lung extracts followed by WB revealed that, at 4 min after bradykinin, reduction of VEC recognition by pY-VEC antibodies was accompanied by a strong increase in ubiquitination. Quantification of band intensity is shown as absolute values on the right panel. (b) HUVEC were treated, in presence of chloroquine, with bradykinin or left untreated. Bradykinin-induced VEC internalization (red staining) and co-localization in intracellular vesicles with K63-linked ubiquitin (green staining) in HUVEC cells. Pixels presenting VEC and ubiquitin colocalization are highlighted in white in the magnified fields (bottom). Red arrowheads point to internalized VEC. Data are representative of at least three independent experiments. Scale bar: 20 μm. (c) IP of wt or point mutated VEC followed by WB for ubiquitin showed that bradykinin induces stronger ubiquitination of wt protein than of Y658F- or Y685F-VEC mutants. Data in (a) are mean±s.e.m. of three independent experiments. *P<0.01 by independent two-tailed t-test assuming unequal variances.