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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

Reduced B16F10 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F.(a) Subcutaneous B16F10 tumour volumes for WT (blue) and Vegfr2Y949F/Y949F (Y949F; magenta) mice at different days after inoculation. n=14–15 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (b) CD31+ area per field in B16F10 tumours from WT and Y949F mice. n=21 mice per genotype at D12 and 11–16 mice per genotype at D18. (c) Area of tomato lectin-perfused vessels normalized to CD31+ area. n=21 mice per genotype, D12; and 11–16 mice per genotype at D18. *P<0.05. (d) Tumour vasculature (upper panels; CD31+ vessels; white) and perivascular fibrinogen deposition (lower panels, heatmap from red, high, to blue, low) in B16F10 tumours from WT and Vegfr2Y949F/Y949F mice at D12. Scale bars, 50 μm. (e) Fibrinogen+ area normalized to CD31+ area. n=22–27 mice per genotype at D12 and 13–14 mice per genotype at D18. Student's t-test, P=0.0310. (f) Oedema in B16F10 tumours estimated by weighing tumours before and after drying. Data show (wet weight−dry weight)/wet weight in % at D12 and D18. n=16-18 mice per genotype at D12 and 19–24 mice per genotype at D18. Student's t-test, P=0.0385. (g) Tumour volumes at D12 of TMZ or vehicle (dimethylsulfoxide)-treated mice with B16F10 tumours, receiving treatment between D4 and D8 after inoculation. n=12–15 mice per group. Kruskal–Wallis test, P=0.0289. (h) B16F10 tumour volumes in mouse ear. n=12–13 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (i) Spontaneous B16F10 lung metastasis spreading to the lungs from primary B16F10 tumours in the ear. dsred-B16F10 metastases in four longitudinal sections of each left main lung lobe were counted. n=12 mice per genotype. Mann–Whitney U, P=0.0028. Data shown as mean±s.e.m. Dashed lines in b,c,e and f indicate that data sets from D12 and D18 were not compared statistically. *P<0.05, **P<0.01, NS, not significant. Experiments were performed three independent times (a–e,h–j) or two times (f,g), and data were pooled.
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f5: Reduced B16F10 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F.(a) Subcutaneous B16F10 tumour volumes for WT (blue) and Vegfr2Y949F/Y949F (Y949F; magenta) mice at different days after inoculation. n=14–15 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (b) CD31+ area per field in B16F10 tumours from WT and Y949F mice. n=21 mice per genotype at D12 and 11–16 mice per genotype at D18. (c) Area of tomato lectin-perfused vessels normalized to CD31+ area. n=21 mice per genotype, D12; and 11–16 mice per genotype at D18. *P<0.05. (d) Tumour vasculature (upper panels; CD31+ vessels; white) and perivascular fibrinogen deposition (lower panels, heatmap from red, high, to blue, low) in B16F10 tumours from WT and Vegfr2Y949F/Y949F mice at D12. Scale bars, 50 μm. (e) Fibrinogen+ area normalized to CD31+ area. n=22–27 mice per genotype at D12 and 13–14 mice per genotype at D18. Student's t-test, P=0.0310. (f) Oedema in B16F10 tumours estimated by weighing tumours before and after drying. Data show (wet weight−dry weight)/wet weight in % at D12 and D18. n=16-18 mice per genotype at D12 and 19–24 mice per genotype at D18. Student's t-test, P=0.0385. (g) Tumour volumes at D12 of TMZ or vehicle (dimethylsulfoxide)-treated mice with B16F10 tumours, receiving treatment between D4 and D8 after inoculation. n=12–15 mice per group. Kruskal–Wallis test, P=0.0289. (h) B16F10 tumour volumes in mouse ear. n=12–13 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (i) Spontaneous B16F10 lung metastasis spreading to the lungs from primary B16F10 tumours in the ear. dsred-B16F10 metastases in four longitudinal sections of each left main lung lobe were counted. n=12 mice per genotype. Mann–Whitney U, P=0.0028. Data shown as mean±s.e.m. Dashed lines in b,c,e and f indicate that data sets from D12 and D18 were not compared statistically. *P<0.05, **P<0.01, NS, not significant. Experiments were performed three independent times (a–e,h–j) or two times (f,g), and data were pooled.

Mentions: To further test the effect of the VEGFR2-Y949F mutation on vascular integrity in cancer, we chose to study subcutaneous B16F10 melanoma22. B16F10 tumour growth in the back skin was monitored continuously showing similar growth curves for the two genotypes (Fig. 5a). To monitor dynamic changes in tumour and vascular parameters, tumours were collected at two different time points after inoculation, at D12 (average tumour volume 200 mm3) and at D18 (average tumour volume 1.5 cm3) from WT and Vegfr2Y949F/Y949F mice. Vascular area (Fig. 5b) and pericyte density (Supplementary Fig. 3f) were similar in WT and Vegfr2Y949F/Y949F B16F10 tumours at D12 and D18. Vessel functionality as assessed by lectin perfusion was more efficient in the Vegfr2Y949F/Y949F tumour vasculature at D12 (Fig. 5c). At D18, vessel perfusion had improved in the WT and was similar to that of Vegfr2Y949F/Y949F tumours. In spite of the improved perfusion, necrosis in the tumour was similar in the two genotypes (Supplementary Fig. 3g). Importantly, there were no differences in infiltration of F4/80+, CD45+ and MMR-positive inflammatory leukocytes in the tumours at D12 (Supplementary Fig. 3h–j).


VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
Reduced B16F10 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F.(a) Subcutaneous B16F10 tumour volumes for WT (blue) and Vegfr2Y949F/Y949F (Y949F; magenta) mice at different days after inoculation. n=14–15 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (b) CD31+ area per field in B16F10 tumours from WT and Y949F mice. n=21 mice per genotype at D12 and 11–16 mice per genotype at D18. (c) Area of tomato lectin-perfused vessels normalized to CD31+ area. n=21 mice per genotype, D12; and 11–16 mice per genotype at D18. *P<0.05. (d) Tumour vasculature (upper panels; CD31+ vessels; white) and perivascular fibrinogen deposition (lower panels, heatmap from red, high, to blue, low) in B16F10 tumours from WT and Vegfr2Y949F/Y949F mice at D12. Scale bars, 50 μm. (e) Fibrinogen+ area normalized to CD31+ area. n=22–27 mice per genotype at D12 and 13–14 mice per genotype at D18. Student's t-test, P=0.0310. (f) Oedema in B16F10 tumours estimated by weighing tumours before and after drying. Data show (wet weight−dry weight)/wet weight in % at D12 and D18. n=16-18 mice per genotype at D12 and 19–24 mice per genotype at D18. Student's t-test, P=0.0385. (g) Tumour volumes at D12 of TMZ or vehicle (dimethylsulfoxide)-treated mice with B16F10 tumours, receiving treatment between D4 and D8 after inoculation. n=12–15 mice per group. Kruskal–Wallis test, P=0.0289. (h) B16F10 tumour volumes in mouse ear. n=12–13 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (i) Spontaneous B16F10 lung metastasis spreading to the lungs from primary B16F10 tumours in the ear. dsred-B16F10 metastases in four longitudinal sections of each left main lung lobe were counted. n=12 mice per genotype. Mann–Whitney U, P=0.0028. Data shown as mean±s.e.m. Dashed lines in b,c,e and f indicate that data sets from D12 and D18 were not compared statistically. *P<0.05, **P<0.01, NS, not significant. Experiments were performed three independent times (a–e,h–j) or two times (f,g), and data were pooled.
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f5: Reduced B16F10 vascular leakage and metastatic spread in Vegfr2Y949F/Y949F.(a) Subcutaneous B16F10 tumour volumes for WT (blue) and Vegfr2Y949F/Y949F (Y949F; magenta) mice at different days after inoculation. n=14–15 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (b) CD31+ area per field in B16F10 tumours from WT and Y949F mice. n=21 mice per genotype at D12 and 11–16 mice per genotype at D18. (c) Area of tomato lectin-perfused vessels normalized to CD31+ area. n=21 mice per genotype, D12; and 11–16 mice per genotype at D18. *P<0.05. (d) Tumour vasculature (upper panels; CD31+ vessels; white) and perivascular fibrinogen deposition (lower panels, heatmap from red, high, to blue, low) in B16F10 tumours from WT and Vegfr2Y949F/Y949F mice at D12. Scale bars, 50 μm. (e) Fibrinogen+ area normalized to CD31+ area. n=22–27 mice per genotype at D12 and 13–14 mice per genotype at D18. Student's t-test, P=0.0310. (f) Oedema in B16F10 tumours estimated by weighing tumours before and after drying. Data show (wet weight−dry weight)/wet weight in % at D12 and D18. n=16-18 mice per genotype at D12 and 19–24 mice per genotype at D18. Student's t-test, P=0.0385. (g) Tumour volumes at D12 of TMZ or vehicle (dimethylsulfoxide)-treated mice with B16F10 tumours, receiving treatment between D4 and D8 after inoculation. n=12–15 mice per group. Kruskal–Wallis test, P=0.0289. (h) B16F10 tumour volumes in mouse ear. n=12–13 mice per genotype. Repeated measures ANOVA: P(time)<0.0001; P(genotype)=NS. (i) Spontaneous B16F10 lung metastasis spreading to the lungs from primary B16F10 tumours in the ear. dsred-B16F10 metastases in four longitudinal sections of each left main lung lobe were counted. n=12 mice per genotype. Mann–Whitney U, P=0.0028. Data shown as mean±s.e.m. Dashed lines in b,c,e and f indicate that data sets from D12 and D18 were not compared statistically. *P<0.05, **P<0.01, NS, not significant. Experiments were performed three independent times (a–e,h–j) or two times (f,g), and data were pooled.
Mentions: To further test the effect of the VEGFR2-Y949F mutation on vascular integrity in cancer, we chose to study subcutaneous B16F10 melanoma22. B16F10 tumour growth in the back skin was monitored continuously showing similar growth curves for the two genotypes (Fig. 5a). To monitor dynamic changes in tumour and vascular parameters, tumours were collected at two different time points after inoculation, at D12 (average tumour volume 200 mm3) and at D18 (average tumour volume 1.5 cm3) from WT and Vegfr2Y949F/Y949F mice. Vascular area (Fig. 5b) and pericyte density (Supplementary Fig. 3f) were similar in WT and Vegfr2Y949F/Y949F B16F10 tumours at D12 and D18. Vessel functionality as assessed by lectin perfusion was more efficient in the Vegfr2Y949F/Y949F tumour vasculature at D12 (Fig. 5c). At D18, vessel perfusion had improved in the WT and was similar to that of Vegfr2Y949F/Y949F tumours. In spite of the improved perfusion, necrosis in the tumour was similar in the two genotypes (Supplementary Fig. 3g). Importantly, there were no differences in infiltration of F4/80+, CD45+ and MMR-positive inflammatory leukocytes in the tumours at D12 (Supplementary Fig. 3h–j).

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