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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.

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Vegfr2Y949F/Y949F vessel parameters.(a) Isolectin-B4 (IB4, red) and ERG (green) immunostaining on WT and Vegfr2Y949F/Y949F (Y949F) P6 retinal vasculature. Scale bar, 100 μm. (b) Tip cells numbers. n=3–4 retinas per genotype. (c) IB4-positive vessel area normalized to WT. n=3–4 retinas per genotype. (d) Radial vessel outgrowth normalized to WT. n=4–5 retinas per genotype. (e) Heatmap of VE-cadherin morphology in WT and Vegfr2Y949F/Y949F (Y949F) P6 retinas. Scale bar, 50 μm. (f) Retinal images in e processed for VE-cadherin morphology, lying within the vascular mask, into ‘patches' of 100 × 100 pixels (× 63, n=6 per group). The VE-cadherin morphology in each patch was hand-classified according to the classification key in g. (g) Adherens junctions classification from ‘active' (red; irregular/serrated morphology with diffuse/vesicular regions) to ‘inactive' (blue; straighter morphology with less vesicular staining). (h) Transmission electron microscopy analysis of kidney glomerulus and peritubular capillary (cap) show normal appearance of junctions (red arrows) and fenestrae (insets) in the two genotypes. (i) PV1 protein immunoblot in lung lysates after injection of PBS (P) or VEGFA (V) and circulation for 1–10 min (P1; injection of PBS and 1 min circulation and so on). GAPDH; equal loading. Molecular weight markers to the right. (j). Blood pressure 10 min after saline or VEGFA infusion, monitored in the ascending aorta. Values (positive or negative) show diastolic blood pressure (BP) normalized to BP before VEGFA/PBS injection (70 mm Hg; NS between genotypes). n=6–8 mice per genotype. Student's t-test, *P<0.05. Two-way ANOVA: P(treatment)=0.0001; P(genotype)=0.738; P(interaction)=0.5321. (k) CD31-positive (red) tracheal endothelium and desmin-positive (green) pericytes. Scale bar; 50 μm. (l) Pericyte area per total area from k. n=4 mice per genotype. (m) CD31-positive (red) tracheal endothelium and collagen IV-positive (green) basement membrane. Scale bar; 50 μm. (n) Collagen IV area per total area in m. n=3 mice per genotype. (o) RNAseq analysis of total lung polyA+ RNA. Data for selected endothelial genes as log10-scaled fold-change based on the frequency of normalized expression. For Vegfr2 (KDR) the Y949F mutant showed a −0.24 (DESeq2log2) fold decrease compared with WT. Data presented as mean±s.e.m. Student's t-test (b–d) and Wilcoxon rank test (f); NS, not significant. Experiments were performed three independent times or performed once with at least three independent data sets (j,o).
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f2: Vegfr2Y949F/Y949F vessel parameters.(a) Isolectin-B4 (IB4, red) and ERG (green) immunostaining on WT and Vegfr2Y949F/Y949F (Y949F) P6 retinal vasculature. Scale bar, 100 μm. (b) Tip cells numbers. n=3–4 retinas per genotype. (c) IB4-positive vessel area normalized to WT. n=3–4 retinas per genotype. (d) Radial vessel outgrowth normalized to WT. n=4–5 retinas per genotype. (e) Heatmap of VE-cadherin morphology in WT and Vegfr2Y949F/Y949F (Y949F) P6 retinas. Scale bar, 50 μm. (f) Retinal images in e processed for VE-cadherin morphology, lying within the vascular mask, into ‘patches' of 100 × 100 pixels (× 63, n=6 per group). The VE-cadherin morphology in each patch was hand-classified according to the classification key in g. (g) Adherens junctions classification from ‘active' (red; irregular/serrated morphology with diffuse/vesicular regions) to ‘inactive' (blue; straighter morphology with less vesicular staining). (h) Transmission electron microscopy analysis of kidney glomerulus and peritubular capillary (cap) show normal appearance of junctions (red arrows) and fenestrae (insets) in the two genotypes. (i) PV1 protein immunoblot in lung lysates after injection of PBS (P) or VEGFA (V) and circulation for 1–10 min (P1; injection of PBS and 1 min circulation and so on). GAPDH; equal loading. Molecular weight markers to the right. (j). Blood pressure 10 min after saline or VEGFA infusion, monitored in the ascending aorta. Values (positive or negative) show diastolic blood pressure (BP) normalized to BP before VEGFA/PBS injection (70 mm Hg; NS between genotypes). n=6–8 mice per genotype. Student's t-test, *P<0.05. Two-way ANOVA: P(treatment)=0.0001; P(genotype)=0.738; P(interaction)=0.5321. (k) CD31-positive (red) tracheal endothelium and desmin-positive (green) pericytes. Scale bar; 50 μm. (l) Pericyte area per total area from k. n=4 mice per genotype. (m) CD31-positive (red) tracheal endothelium and collagen IV-positive (green) basement membrane. Scale bar; 50 μm. (n) Collagen IV area per total area in m. n=3 mice per genotype. (o) RNAseq analysis of total lung polyA+ RNA. Data for selected endothelial genes as log10-scaled fold-change based on the frequency of normalized expression. For Vegfr2 (KDR) the Y949F mutant showed a −0.24 (DESeq2log2) fold decrease compared with WT. Data presented as mean±s.e.m. Student's t-test (b–d) and Wilcoxon rank test (f); NS, not significant. Experiments were performed three independent times or performed once with at least three independent data sets (j,o).

Mentions: The Vegfr2Y949F/Y949F mice showed no developmental vascular abnormality, for example, in the embryonic day 11.5 (E11.5) hindbrain (Supplementary Fig. 2a–d) and were born at the expected Mendelian ratios. There was also no apparent effect of the mutation on the development and organization of lymphatics (see Supplementary Fig. 2e for tracheal lymphatics). The postnatal retinal vasculature develops in a strictly regulated, VEGFA-dependent manner to establish the junctional barrier14. There was no difference in retinal vascular development between WT and Vegfr2Y949F/Y949F mice as judged by the overall morphology of the vascular network, the abundance of endothelial tip cells and filopodia, and the total vascular area (Fig. 2a–d). Thus, development of the retinal vasculature does not depend on VEGFR2-Y949 signalling. However, the morphology of the VE-cadherin immunostaining differed between the two genotypes. The VE-cadherin pattern at individual junctions was evaluated using established image analysis software15, classifying junctions in a graded scale from ‘active'—that is, being irregular/serrated with vesicular/diffuse appearance—to ‘inactive' ones, presenting a straighter and less vesicular morphology (Fig. 2e, see Fig. 2f,g for quantification and schematic outline). By this criterion, the WT retinal vasculature displayed a significantly higher prevalence of active adherens junctions compared with the Vegfr2Y949F/Y949F retinal vasculature (Fig. 2f; active junction patches was 58.9% (WT) versus 48.1% (Vegfr2Y949F/Y949F); P=0.0367, calculated using median% in one-tailed Wilcoxon rank sum test.


VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
Vegfr2Y949F/Y949F vessel parameters.(a) Isolectin-B4 (IB4, red) and ERG (green) immunostaining on WT and Vegfr2Y949F/Y949F (Y949F) P6 retinal vasculature. Scale bar, 100 μm. (b) Tip cells numbers. n=3–4 retinas per genotype. (c) IB4-positive vessel area normalized to WT. n=3–4 retinas per genotype. (d) Radial vessel outgrowth normalized to WT. n=4–5 retinas per genotype. (e) Heatmap of VE-cadherin morphology in WT and Vegfr2Y949F/Y949F (Y949F) P6 retinas. Scale bar, 50 μm. (f) Retinal images in e processed for VE-cadherin morphology, lying within the vascular mask, into ‘patches' of 100 × 100 pixels (× 63, n=6 per group). The VE-cadherin morphology in each patch was hand-classified according to the classification key in g. (g) Adherens junctions classification from ‘active' (red; irregular/serrated morphology with diffuse/vesicular regions) to ‘inactive' (blue; straighter morphology with less vesicular staining). (h) Transmission electron microscopy analysis of kidney glomerulus and peritubular capillary (cap) show normal appearance of junctions (red arrows) and fenestrae (insets) in the two genotypes. (i) PV1 protein immunoblot in lung lysates after injection of PBS (P) or VEGFA (V) and circulation for 1–10 min (P1; injection of PBS and 1 min circulation and so on). GAPDH; equal loading. Molecular weight markers to the right. (j). Blood pressure 10 min after saline or VEGFA infusion, monitored in the ascending aorta. Values (positive or negative) show diastolic blood pressure (BP) normalized to BP before VEGFA/PBS injection (70 mm Hg; NS between genotypes). n=6–8 mice per genotype. Student's t-test, *P<0.05. Two-way ANOVA: P(treatment)=0.0001; P(genotype)=0.738; P(interaction)=0.5321. (k) CD31-positive (red) tracheal endothelium and desmin-positive (green) pericytes. Scale bar; 50 μm. (l) Pericyte area per total area from k. n=4 mice per genotype. (m) CD31-positive (red) tracheal endothelium and collagen IV-positive (green) basement membrane. Scale bar; 50 μm. (n) Collagen IV area per total area in m. n=3 mice per genotype. (o) RNAseq analysis of total lung polyA+ RNA. Data for selected endothelial genes as log10-scaled fold-change based on the frequency of normalized expression. For Vegfr2 (KDR) the Y949F mutant showed a −0.24 (DESeq2log2) fold decrease compared with WT. Data presented as mean±s.e.m. Student's t-test (b–d) and Wilcoxon rank test (f); NS, not significant. Experiments were performed three independent times or performed once with at least three independent data sets (j,o).
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f2: Vegfr2Y949F/Y949F vessel parameters.(a) Isolectin-B4 (IB4, red) and ERG (green) immunostaining on WT and Vegfr2Y949F/Y949F (Y949F) P6 retinal vasculature. Scale bar, 100 μm. (b) Tip cells numbers. n=3–4 retinas per genotype. (c) IB4-positive vessel area normalized to WT. n=3–4 retinas per genotype. (d) Radial vessel outgrowth normalized to WT. n=4–5 retinas per genotype. (e) Heatmap of VE-cadherin morphology in WT and Vegfr2Y949F/Y949F (Y949F) P6 retinas. Scale bar, 50 μm. (f) Retinal images in e processed for VE-cadherin morphology, lying within the vascular mask, into ‘patches' of 100 × 100 pixels (× 63, n=6 per group). The VE-cadherin morphology in each patch was hand-classified according to the classification key in g. (g) Adherens junctions classification from ‘active' (red; irregular/serrated morphology with diffuse/vesicular regions) to ‘inactive' (blue; straighter morphology with less vesicular staining). (h) Transmission electron microscopy analysis of kidney glomerulus and peritubular capillary (cap) show normal appearance of junctions (red arrows) and fenestrae (insets) in the two genotypes. (i) PV1 protein immunoblot in lung lysates after injection of PBS (P) or VEGFA (V) and circulation for 1–10 min (P1; injection of PBS and 1 min circulation and so on). GAPDH; equal loading. Molecular weight markers to the right. (j). Blood pressure 10 min after saline or VEGFA infusion, monitored in the ascending aorta. Values (positive or negative) show diastolic blood pressure (BP) normalized to BP before VEGFA/PBS injection (70 mm Hg; NS between genotypes). n=6–8 mice per genotype. Student's t-test, *P<0.05. Two-way ANOVA: P(treatment)=0.0001; P(genotype)=0.738; P(interaction)=0.5321. (k) CD31-positive (red) tracheal endothelium and desmin-positive (green) pericytes. Scale bar; 50 μm. (l) Pericyte area per total area from k. n=4 mice per genotype. (m) CD31-positive (red) tracheal endothelium and collagen IV-positive (green) basement membrane. Scale bar; 50 μm. (n) Collagen IV area per total area in m. n=3 mice per genotype. (o) RNAseq analysis of total lung polyA+ RNA. Data for selected endothelial genes as log10-scaled fold-change based on the frequency of normalized expression. For Vegfr2 (KDR) the Y949F mutant showed a −0.24 (DESeq2log2) fold decrease compared with WT. Data presented as mean±s.e.m. Student's t-test (b–d) and Wilcoxon rank test (f); NS, not significant. Experiments were performed three independent times or performed once with at least three independent data sets (j,o).
Mentions: The Vegfr2Y949F/Y949F mice showed no developmental vascular abnormality, for example, in the embryonic day 11.5 (E11.5) hindbrain (Supplementary Fig. 2a–d) and were born at the expected Mendelian ratios. There was also no apparent effect of the mutation on the development and organization of lymphatics (see Supplementary Fig. 2e for tracheal lymphatics). The postnatal retinal vasculature develops in a strictly regulated, VEGFA-dependent manner to establish the junctional barrier14. There was no difference in retinal vascular development between WT and Vegfr2Y949F/Y949F mice as judged by the overall morphology of the vascular network, the abundance of endothelial tip cells and filopodia, and the total vascular area (Fig. 2a–d). Thus, development of the retinal vasculature does not depend on VEGFR2-Y949 signalling. However, the morphology of the VE-cadherin immunostaining differed between the two genotypes. The VE-cadherin pattern at individual junctions was evaluated using established image analysis software15, classifying junctions in a graded scale from ‘active'—that is, being irregular/serrated with vesicular/diffuse appearance—to ‘inactive' ones, presenting a straighter and less vesicular morphology (Fig. 2e, see Fig. 2f,g for quantification and schematic outline). By this criterion, the WT retinal vasculature displayed a significantly higher prevalence of active adherens junctions compared with the Vegfr2Y949F/Y949F retinal vasculature (Fig. 2f; active junction patches was 58.9% (WT) versus 48.1% (Vegfr2Y949F/Y949F); P=0.0367, calculated using median% in one-tailed Wilcoxon rank sum test.

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