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VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

View Article: PubMed Central - PubMed

ABSTRACT

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.

No MeSH data available.


Related in: MedlinePlus

Arrest in VEGFA-induced vascular leakage in Vegfr2Y949F/Y949F mice.(a) Tracheal vessel leakage in WT (left) and Vegfr2Y949F/Y949F (Y949F; right) mice after tail vein injection of VEGFA or PBS combined with 30 nm fluorescent microspheres (green). Arrows: microsphere leakage from CD31+ vessels (red). Scale bar, 50 μm. (b) Microsphere area per CD31-positive area fraction in VEGFA/PBS samples from a. n=3 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.001; P(interaction)=0.0008. (c) Tracheal vessel leakage in response to tail vein-injected histamine (as in a), in WT and Vegfr2Y949F/Y949F (Y949F) mice. (d) Microsphere area per CD31-positive area fraction in histamine/PBS samples from c. n=3–6 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.84; P(interaction)=0.80. (e) Evans' blue extravasation induced by VEGFA/PBS in the dermis of WT and Vegfr2Y949F/Y949F (Y949F) mice. Values were normalized to tissue weight. n=3 mice per genotype. Two-way ANOVA: P(treatment)=0.0053; P(genotype)=0.0206; P(interaction)=0.0482. (f) VEGFR2/TSAd complex formation in isolated mouse lung endothelial cells from WT and Vegfr2Y949F/Y949F (Y949F) mice, treated or not with VEGFA. Immunoprecipitation (IP) for VEGFR2 and immunoblotting for TSAd and VEGFR2. Total lysates were immunoblotted for TSAd and tubulin. Positive control, total lysate. Molecular weight markers to the right. (g) VE-cadherin immunostaining (yellow–black heatmap) on trachea from WT and Vegfr2Y949F/Y949F (Y949F) mice, tail vein-injected with VEGFA/PBS and microspheres (pink–red heatmap). Images were rendered using structured illumination microscopy. Note VE-cadherin rearrangement in VEGFA-injected WT trachea. Scale bar, 10 μm. (h) VE-cadherin fragments per μm vessel length from g. n=3–6 individual tissues per genotype. Total vessel length assessed: WT/PBS, 236 μm; WT/VEGFA, 308 μm; Y949F/PBS, 150 μm; and Y949F/VEGFA, 145 μm. Two-way ANOVA: P(treatment)=0.0022; P(genotype)=0.0065; P(interaction)=0.0164. (i) VE-cadherin immunostaining (red) on endothelial cells isolated from WT and Vegfr2Y949F/Y949F (Y949F) mouse lungs and then treated or not with VEGFA (100 ng ml−1). Hoechst 33342 (blue) staining shows nuclei. Scale bar, 20 μm. (j) VE-cadherin-positive staining per cell; samples generated as in i. n=30 cells per condition. Two-way ANOVA: P(treatment)=0.0189; P(genotype)=0.099; P(interaction)=0.0741. Data presented as mean±s.e.m. Two-way ANOVA with Tukey's post hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Experiments were performed at least three independent times.
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f1: Arrest in VEGFA-induced vascular leakage in Vegfr2Y949F/Y949F mice.(a) Tracheal vessel leakage in WT (left) and Vegfr2Y949F/Y949F (Y949F; right) mice after tail vein injection of VEGFA or PBS combined with 30 nm fluorescent microspheres (green). Arrows: microsphere leakage from CD31+ vessels (red). Scale bar, 50 μm. (b) Microsphere area per CD31-positive area fraction in VEGFA/PBS samples from a. n=3 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.001; P(interaction)=0.0008. (c) Tracheal vessel leakage in response to tail vein-injected histamine (as in a), in WT and Vegfr2Y949F/Y949F (Y949F) mice. (d) Microsphere area per CD31-positive area fraction in histamine/PBS samples from c. n=3–6 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.84; P(interaction)=0.80. (e) Evans' blue extravasation induced by VEGFA/PBS in the dermis of WT and Vegfr2Y949F/Y949F (Y949F) mice. Values were normalized to tissue weight. n=3 mice per genotype. Two-way ANOVA: P(treatment)=0.0053; P(genotype)=0.0206; P(interaction)=0.0482. (f) VEGFR2/TSAd complex formation in isolated mouse lung endothelial cells from WT and Vegfr2Y949F/Y949F (Y949F) mice, treated or not with VEGFA. Immunoprecipitation (IP) for VEGFR2 and immunoblotting for TSAd and VEGFR2. Total lysates were immunoblotted for TSAd and tubulin. Positive control, total lysate. Molecular weight markers to the right. (g) VE-cadherin immunostaining (yellow–black heatmap) on trachea from WT and Vegfr2Y949F/Y949F (Y949F) mice, tail vein-injected with VEGFA/PBS and microspheres (pink–red heatmap). Images were rendered using structured illumination microscopy. Note VE-cadherin rearrangement in VEGFA-injected WT trachea. Scale bar, 10 μm. (h) VE-cadherin fragments per μm vessel length from g. n=3–6 individual tissues per genotype. Total vessel length assessed: WT/PBS, 236 μm; WT/VEGFA, 308 μm; Y949F/PBS, 150 μm; and Y949F/VEGFA, 145 μm. Two-way ANOVA: P(treatment)=0.0022; P(genotype)=0.0065; P(interaction)=0.0164. (i) VE-cadherin immunostaining (red) on endothelial cells isolated from WT and Vegfr2Y949F/Y949F (Y949F) mouse lungs and then treated or not with VEGFA (100 ng ml−1). Hoechst 33342 (blue) staining shows nuclei. Scale bar, 20 μm. (j) VE-cadherin-positive staining per cell; samples generated as in i. n=30 cells per condition. Two-way ANOVA: P(treatment)=0.0189; P(genotype)=0.099; P(interaction)=0.0741. Data presented as mean±s.e.m. Two-way ANOVA with Tukey's post hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Experiments were performed at least three independent times.

Mentions: Vegfr2Y949F/Y949F knock-in mice were created using Velocigene technology11 and used in this study after selection cassette removal, sequence verification and extensive backcrossing onto the C57Bl/6 background (Supplementary Fig. 1). The mutant mice were phenotypically normal and expressed similar levels of VEGFR2 protein in the vasculature as wild-type (WT) mice (see below). To determine the consequence of Vegfr2Y949F/Y949F mutation on VEGFA-induced vascular leakage in vivo, we employed two well-established assays, microsphere extravasation in the tracheal vasculature and Miles extravasation assay in the dermis. To induce microsphere extravasation, VEGFA or vehicle was administered by tail vein injection together with fluorescent 30-nm microspheres. VEGFA-induced extravasation of microspheres, lodged beneath the basement membrane in postcapillary venules, was significantly reduced at 60 min of circulation in the Vegfr2Y949F/Y949F trachea compared with WT (Fig. 1a,b). In contrast, tail vein-administered histamine induced similar levels of microsphere extravasation in WT and Vegfr2Y949F/Y949F trachea venules (Fig. 1c,d). Moreover, VEGFA induced a significant increase of Evans' blue leakage in the skin (Miles assay) in the WT but not in the Vegfr2Y949F/Y949F mouse (Fig. 1e).


VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
Arrest in VEGFA-induced vascular leakage in Vegfr2Y949F/Y949F mice.(a) Tracheal vessel leakage in WT (left) and Vegfr2Y949F/Y949F (Y949F; right) mice after tail vein injection of VEGFA or PBS combined with 30 nm fluorescent microspheres (green). Arrows: microsphere leakage from CD31+ vessels (red). Scale bar, 50 μm. (b) Microsphere area per CD31-positive area fraction in VEGFA/PBS samples from a. n=3 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.001; P(interaction)=0.0008. (c) Tracheal vessel leakage in response to tail vein-injected histamine (as in a), in WT and Vegfr2Y949F/Y949F (Y949F) mice. (d) Microsphere area per CD31-positive area fraction in histamine/PBS samples from c. n=3–6 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.84; P(interaction)=0.80. (e) Evans' blue extravasation induced by VEGFA/PBS in the dermis of WT and Vegfr2Y949F/Y949F (Y949F) mice. Values were normalized to tissue weight. n=3 mice per genotype. Two-way ANOVA: P(treatment)=0.0053; P(genotype)=0.0206; P(interaction)=0.0482. (f) VEGFR2/TSAd complex formation in isolated mouse lung endothelial cells from WT and Vegfr2Y949F/Y949F (Y949F) mice, treated or not with VEGFA. Immunoprecipitation (IP) for VEGFR2 and immunoblotting for TSAd and VEGFR2. Total lysates were immunoblotted for TSAd and tubulin. Positive control, total lysate. Molecular weight markers to the right. (g) VE-cadherin immunostaining (yellow–black heatmap) on trachea from WT and Vegfr2Y949F/Y949F (Y949F) mice, tail vein-injected with VEGFA/PBS and microspheres (pink–red heatmap). Images were rendered using structured illumination microscopy. Note VE-cadherin rearrangement in VEGFA-injected WT trachea. Scale bar, 10 μm. (h) VE-cadherin fragments per μm vessel length from g. n=3–6 individual tissues per genotype. Total vessel length assessed: WT/PBS, 236 μm; WT/VEGFA, 308 μm; Y949F/PBS, 150 μm; and Y949F/VEGFA, 145 μm. Two-way ANOVA: P(treatment)=0.0022; P(genotype)=0.0065; P(interaction)=0.0164. (i) VE-cadherin immunostaining (red) on endothelial cells isolated from WT and Vegfr2Y949F/Y949F (Y949F) mouse lungs and then treated or not with VEGFA (100 ng ml−1). Hoechst 33342 (blue) staining shows nuclei. Scale bar, 20 μm. (j) VE-cadherin-positive staining per cell; samples generated as in i. n=30 cells per condition. Two-way ANOVA: P(treatment)=0.0189; P(genotype)=0.099; P(interaction)=0.0741. Data presented as mean±s.e.m. Two-way ANOVA with Tukey's post hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Experiments were performed at least three independent times.
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f1: Arrest in VEGFA-induced vascular leakage in Vegfr2Y949F/Y949F mice.(a) Tracheal vessel leakage in WT (left) and Vegfr2Y949F/Y949F (Y949F; right) mice after tail vein injection of VEGFA or PBS combined with 30 nm fluorescent microspheres (green). Arrows: microsphere leakage from CD31+ vessels (red). Scale bar, 50 μm. (b) Microsphere area per CD31-positive area fraction in VEGFA/PBS samples from a. n=3 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.001; P(interaction)=0.0008. (c) Tracheal vessel leakage in response to tail vein-injected histamine (as in a), in WT and Vegfr2Y949F/Y949F (Y949F) mice. (d) Microsphere area per CD31-positive area fraction in histamine/PBS samples from c. n=3–6 mice per genotype. Two-way ANOVA: P(treatment)<0.0001; P(genotype)=0.84; P(interaction)=0.80. (e) Evans' blue extravasation induced by VEGFA/PBS in the dermis of WT and Vegfr2Y949F/Y949F (Y949F) mice. Values were normalized to tissue weight. n=3 mice per genotype. Two-way ANOVA: P(treatment)=0.0053; P(genotype)=0.0206; P(interaction)=0.0482. (f) VEGFR2/TSAd complex formation in isolated mouse lung endothelial cells from WT and Vegfr2Y949F/Y949F (Y949F) mice, treated or not with VEGFA. Immunoprecipitation (IP) for VEGFR2 and immunoblotting for TSAd and VEGFR2. Total lysates were immunoblotted for TSAd and tubulin. Positive control, total lysate. Molecular weight markers to the right. (g) VE-cadherin immunostaining (yellow–black heatmap) on trachea from WT and Vegfr2Y949F/Y949F (Y949F) mice, tail vein-injected with VEGFA/PBS and microspheres (pink–red heatmap). Images were rendered using structured illumination microscopy. Note VE-cadherin rearrangement in VEGFA-injected WT trachea. Scale bar, 10 μm. (h) VE-cadherin fragments per μm vessel length from g. n=3–6 individual tissues per genotype. Total vessel length assessed: WT/PBS, 236 μm; WT/VEGFA, 308 μm; Y949F/PBS, 150 μm; and Y949F/VEGFA, 145 μm. Two-way ANOVA: P(treatment)=0.0022; P(genotype)=0.0065; P(interaction)=0.0164. (i) VE-cadherin immunostaining (red) on endothelial cells isolated from WT and Vegfr2Y949F/Y949F (Y949F) mouse lungs and then treated or not with VEGFA (100 ng ml−1). Hoechst 33342 (blue) staining shows nuclei. Scale bar, 20 μm. (j) VE-cadherin-positive staining per cell; samples generated as in i. n=30 cells per condition. Two-way ANOVA: P(treatment)=0.0189; P(genotype)=0.099; P(interaction)=0.0741. Data presented as mean±s.e.m. Two-way ANOVA with Tukey's post hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Experiments were performed at least three independent times.
Mentions: Vegfr2Y949F/Y949F knock-in mice were created using Velocigene technology11 and used in this study after selection cassette removal, sequence verification and extensive backcrossing onto the C57Bl/6 background (Supplementary Fig. 1). The mutant mice were phenotypically normal and expressed similar levels of VEGFR2 protein in the vasculature as wild-type (WT) mice (see below). To determine the consequence of Vegfr2Y949F/Y949F mutation on VEGFA-induced vascular leakage in vivo, we employed two well-established assays, microsphere extravasation in the tracheal vasculature and Miles extravasation assay in the dermis. To induce microsphere extravasation, VEGFA or vehicle was administered by tail vein injection together with fluorescent 30-nm microspheres. VEGFA-induced extravasation of microspheres, lodged beneath the basement membrane in postcapillary venules, was significantly reduced at 60 min of circulation in the Vegfr2Y949F/Y949F trachea compared with WT (Fig. 1a,b). In contrast, tail vein-administered histamine induced similar levels of microsphere extravasation in WT and Vegfr2Y949F/Y949F trachea venules (Fig. 1c,d). Moreover, VEGFA induced a significant increase of Evans' blue leakage in the skin (Miles assay) in the WT but not in the Vegfr2Y949F/Y949F mouse (Fig. 1e).

View Article: PubMed Central - PubMed

ABSTRACT

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.

No MeSH data available.


Related in: MedlinePlus