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Figures
- Fig. 1. VEGF-A isoform-specific stimulated endothelial cell signalling.
(A) Schematic depicting VEGF-A stimulated VEGFR2 phosphorylation, activation of downstream signalling pathways linking to cellular responses. Abbreviations: p38, p38 mitogen-activated protein kinase; HSP27, heat-shock protein of 27 kDa; Akt, Protein kinase B; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; MEK1, mitogen-activated protein kinase 1; ERK1/2, p42/44 mitogen-activated protein kinase. (B) Endothelial cells subjected to different VEGF-A165 or VEGF-A121 concentrations (0, 0.025, 0.25 or 1.25 nM) for 5 or 15 min were lysed and processed for immunoblot analysis using phospho-specific antibodies against p-VEGFR2, p-eNOS, p-p38 and p-ERK1/2. (C–J) Quantification of VEGF-A isoform-specific signal transduction events. Quantification of (C,D) VEGFR2-pY1175, (E,F) eNOS-pS1177, (G,H) p38 MAPK-pT180/pY182 or (I,J) ERK1/2-pT202/Y204 upon (C,E,G,I) VEGF-A165 or (D,F,H,J) VEGF-A121 stimulation. Error bars indicate ±SEM (n≥3). p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).
- Fig. 2. VEGF-A isoform-specific stimulated PLCγ1 activation.
(A) Schematic depicting VEGF-A-stimulated VEGFR2-pY1175 recruitment of PLCγ1, subsequent phosphorylation and activation. (B) Endothelial cells subjected to different VEGF-A165 or VEGF-A121 concentrations (0, 0.025, 0.25 or 1.25 nM) for 5, 15, 30 or 60 min were lysed and probed for phospho-PLCγ1. (C,D) Quantification of PLCγ1-pY783 levels upon (C) VEGF-A165 or (D) VEGF-A121 stimulation. Error bars indicate ±SEM (n≥3). p<0.05 (*), p<0.001 (***), p<0.0001 (****).
- Fig. 3. VEGF-A isoform-specific release of intracellular Ca2+.
(A,B) Real-time monitoring of cytosolic Ca2+ flux with (A) VEGF-A165 or (B) VEGF-A121 titration; graphs show a representative plot of multiple experiments. Error bars indicate ±SEM (n = 1, N = 4). (C–E) Quantification of (C) peak magnitude, (D) time taken to reach peak magnitude or (E) the total curve area upon stimulation with VEGF-A165 and VEGF-A121. Error bars indicate ±SEM (n = 3). (F) Schematic depicting VEGF-A-stimulated second messenger targeting of InsP3 receptors (InsP3R) and subsequent cytosolic calcium ion rise. PtdIns(4,5)P2 hydrolysis to generate diacylglycerol (DAG) and Ins(1,4,5)P3 is depicted. p<0.05 (*), p<0.01 (**), p<0.0001 (****).
- Fig. 4. VEGF-A isoform-specific Ca2+ flux promotes differential NFATc2 activation.
(A) Schematic depicting VEGF-A-stimulated cytosolic calcium ion rise and effects on NFATc2 dephosphorylation and nuclear translocation. (B) Endothelial cells subjected to stimulation with 0.25 nM VEGF-A165 or VEGF-A121 for 5, 15, 30 and 60 min were lysed and process for immunoblot analysis to detect phospho- (inactive) and dephospho- (active) NFATc2 species. (C) Quantification of relative active versus inactive NFATc2 levels upon stimulation with VEGF-A165 and VEGF-A121. Error bars indicate ±SEM (n = 3). p<0.05 (*), p<0.001 (***).
- Fig. 5. VEGF-A isoforms promote differential NFATc2 nuclear translocation.
(A) Endothelial cells stimulated with 0.25 nM VEGF-A165 or VEGF-A121 for 5, 15 and 30 min were fixed and processed for immunofluorescence microscopy using rabbit anti-NFATc2 (green); nuclei stained using DAPI (blue). Scale bar, 1000 µm. (B) Quantification of NFATc2 nuclear co-distribution at 0, 5, 15 and 30 min after stimulation with VEGF-A165 or VEGF-A121. Error bars indicate ±SEM (n = 3). p<0.0001 (****).
- Fig. 6. NFATc2 regulates VEGF-A isoform-specific endothelial cell migration but not tubulogenesis.
(A–F) Control, scrambled or NFATc2-specific siRNA duplex-treated endothelial cells were seeded into assays to assess endothelial cell (A–C) migration or (D–F) tubulogenesis. (A) Endothelial cells seeded into Transwell filters were stimulated with 0.25 nM VEGF-A165 or VEGF-A121 for 24 h before being fixed and stained with 20% (v/v) crystal violet. Scale bar, 1000 µm. (B,C) Quantification of endothelial cell migration compared to (B) non-transfected or (C) individual controls. Error bars indicate ±SEM (n≥3). (D) Endothelial cells subjected to different siRNA treatments were co-cultured on a bed of primary human fibroblast for 7 days and stimulated with 0.25 nM VEGF-A165 or VEGF-A121. Co-cultures were fixed and endothelial tubules were stained and visualised using an anti-PECAM1 antibody followed by fluorescent secondary antibody. Scale bar, 1000 µm. (E,F) Quantification of endothelial cell tubulogenesis including total (E) tubule length or (F) number of branch points. Error bars indicate ±SEM (n≥3). p<0.05 (*), p<0.01 (**), NS = non-specific.
- Fig. 7. Mechanism for VEGF-A isoform-specific regulation of endothelial cell migration.
Schematic depicting VEGF-A isoform-specific regulation of NFATc2 activation and regulation of endothelial cell migration. Numbered steps denote: (1) VEGF-A isoforms promote differential recruitment and activation of PLCγ1 through interaction with VEGFR2; (2) due to increased PLCγ1 activation, VEGF-A165 promotes an increased rise in intracellular Ca2+, versus VEGF-A121; (3) Increased VEGF-A165 stimulated intracellular Ca2+ flux results in increased NFATc2 dephosphorylation, versus VEGF-A121; (4) dephosphorylated NFATc2 translocates into the nucleus where it regulates endothelial gene transcription; (5) VEGF-A-stimulated NFATc2 regulated gene expression promotes endothelial cell migration.
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