VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

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.

Velocigene technology was used to generate the exchange of amino acid Y949 (TAC) for Phe (TTC) in the 5´sequence corresponding to exon 21 flanking a LoxP-restricted Neomycin cassette. The sequence exchange was introduced through homologous recombination into F1H4 embryonic stem cells of background 129S6SvEv/C57Bl6F1. The cassette was removed by treatment with Cre recombinase, resulting in an 82 bp deletion in intron 21. The correct introduction of the mutation and the otherwise unaffected sequence of exon 21 and 22 was verified by nucleotide sequencing on both strands on DNA from tail biopsy from Vegfr2 Y949F/Y949F mice. Mice were back-crossed onto C57Bl6 background for more than 10 generations before being used for analyses described in the accompanying study by Li, Padhan et al. a. WT and Vegfr2 Y949F/Y949F (Y949F) embryos harvested at embryonic day (E) 11.5 were subjected to immunostaining for isolectin B4. b-d. Quantification for junction density (b), vascular area (c) and vessel width (d). Y949F data were normalized to WT. n=6-7 mice/genotype from 5 matched litters. Student's test, ns; not significant. Data in b-d are presented as mean ± SEM. Scale bar, 50 μm.
f. Transmission electron microscopy (TEM) ultrastructural analysis of WT and Y949F pancreas showing fenestrated vessels (insets) with similar morphology in the two genotypes. Similar results were obtained in 3 independent analyses. Scale bars, 500 nm (left) and 2 μm (right).
g. Vessel perfusion in WT and Vegfr2 Y949F/Y949F (Y949F) mice. Mice were tail vein-injected with FITC-lectin and tracheas harvested after 20 min circulation followed by CD31 immunostaining. The lectin distributed similarly in the two genotypes. n= 2; performed twice. Scale bar; 50 μm. a. Perfusion in RipTag insulinomas was estimated by lectin perfusion followed by immunostaining for CD31. There was no difference in the extent of perfusion in insulinomas from WT and Vegfr2 Y949F/Y949F RipTag mice. n=6-7 mice/genotype. Student's t-test, ns; not significant, performed once. b. Pericytes surrounding insulinoma vessels in WT and Vegfr2 Y949F/Y949F RipTag tumors was determined by immunostaining for nerve-glia2 (NG2). The NG2-positive area was normalized to CD31 area. There was no difference between the genotypes. n=8-12 mice/genotype, multiple tumors/mouse. Student's t-test, ns; not significant, performed once.
Supplementary Fig. 4. Effect of Y949F mutation on B16F10 properties. a. Effect of Temozolomide (TMZ) on volumes of primary B16F10 tumors at day 18 (D18). Mice were treated with TMZ or vehicle (DMSO) between D4 and D8 after inoculation. Student's test, ns; not significant. Data are presented as mean ± SEM.
b. Correlation analysis shows no relationship between primary B16F10 tumor growth and spontaneous lung metastasis; analysis of data shown in main Fig. 5h  a. VEGFA-induced activation of VEGFR2 and phosphorylation of Y949 and Y1173. WT and Vegfr2 Y949F/Y949F (Y949F) mice were injected with VEGFA in the tail vein and lungs were harvested after 1 min circulation, followed by lysis and immunoblotting for VEGFR2 phosphotyrosines Y949 and Y1173. Immunoblotting was performed for VEGFR2 and GAPDH to control for equal loading. Representative blot is shown. Note that Y949 was phosphorylated only in the WT. Background in the Y949F lysate may be due to crossreactivity of the anti-pY949 antibody with unphosphorylated receptor. Performed 3 independent times. b. VEGFA-induced complex formation between VEGFR2 and VEPTP. VEGFR2 was immunoprecipitated from WT and Vegfr2 Y949F/Y949F mouse lungs harvested from mice injected with PBS (P) followed by circulation for 1, 2 or 5 minutes, or VEGFA (V) followed by different circulation periods (1, 2, 5, 10, 15 and 60 min). Lung lysates were used for immunoprecipitation of VEGFR2 followed by immunoblotting for VEGFR2 (as shown in main Fig. 6a) or VEPTP. The quantification shows VEGFR2/VEPTP complexes normalized to mean values in the PBS samples from all 4 independent repeats (12 samples). Two-way ANOVA p(genotype)=0.0658, p(time)=0.0037.
c. VEPTP expression levels in B16F10 tumors. VEPTP expression was estimated by immunoblotting on B16F10 tumor lysates harvested at D12 from WT and Vegfr2 Y949F/Y949F (Y949F) mice; band intensities were normalized to tubulin, analyzed in parallel. There was no change in VEPTP expression levels with tumor progression. n=19, pooled from 3 independent studies. Student's t-test, ns; not significant. Panels used in Fig. 6b are marked with a red square. Mice (WT or Vegfr2 Y949F/Y949F ) were tail vein-injected with PBS or VEGFA followed by circulation for different time periods and preparation of total lung lysates that were used for immunoblotting for c-Src pY418 (uppermost), c-Src (middle upper), Akt pT308 (middle lower) and Akt (lower). Molecular weight markers are indicated to the left. See Fig. 6b  Panels used in Fig. 6c are marked with a red square. B16F10 melanomas from WT or Vegfr2 Y949F/Y949F mice were harvested at day 12 (D12) or D18 after inoculation, lysed and used for immunoprecipitation of VEGFR2 followed by immunoblotting for VEGFR2 pY1173 (D12, uppermost), VEGFR2 (D12, upper middle), VEGFR2 pY1173 (D18, lower middle) and VEGFR2 (D18, lowermost). Molecular weight markers are indicated to the left. See Fig. 6c in Li, Padhan et al. for details.