Fig 2: Proposed mechanism for the role of RCAN1.4 in regulating VEGF action on endothelial cells. VEGF binding to VEGFR-2 leads to autophosphorylation on a number of c-terminal tyrosine residues resulting in the activation of PLC-? and cleavage of PtdIns(4,5)P2 (PIP2) generating diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3). Following VEGFR-2 phosphorylation, RCAN1.4 transiently interacts with VEGFR-2 to regulate internalisation at the leading edge. The resulting Ins(1,4,5)P3-mediated release of Ca2+ from the endoplasmic reticulum (ER) results in activation of calcineurin/PP2B and dephosphorylation of the nuclear factor activated T-cells (NFAT) transcription factor which translocates to the nucleus and increases transcription of RCAN1.4 mRNA. The resulting increase in RCAN1.4 protein acts on calcineurin to decrease NFAT-mediated gene transcription. Increased RCAN1.4 protein also acts independently of calcineurin to regulate VEGFR-2-mediated cytoskeletal reorganisation, filopodia and lamellipodia formation resulting in endothelial cell polarisation and directed migration

Fig 3: Presence of perivascular NRP1 allows VEGFR2/NRP1 trans complex formation, reducing tumor angiogenesis and enhancing patient survival. Schematic illustration showing the impact of endothelial cell NRP1 presentation (left column) compared to tumor cell presentation of NRP1 to endothelial VEGFR2 (right column). Presence of NRP1 in endothelial cells or perivascular tumor cells allows complex formation in cis or trans, respectively. Cis VEGFR2/NRP1 complex formation leads to increased vessel area and size (left column). In contrast, trans VEGFR2/NRP1 complex formation leads to reduced vessel parameters (right column), and improved overall survival in RCC patients.

Fig 4: Depletion of clathrin heavy chain reduces basal VEGFR2 and mature VEGFR1 levels. (A) Endothelial cells were subjected to scrambled (Scr) or clathrin heavy chain (CHC17)-specific siRNA duplexes, prior to cell lysis and processing for immunoblot analysis. (B) Quantification of basal VEGFR2, VEGFR1, NRP1, transferrin receptor (TfR), TGN46 and CHC17 levels upon depletion of clathrin heavy chain (CHC17). Error bars indicate ±s.e.m. (n=3). **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5: Tpl2 modulates eNOS phosphorylation and endothelial cell permeability. (A) Immunoblot analysis of control, scrambled or Tpl2-specific siRNA-treated endothelial cells to assess VEGFR2, eNOS, Akt and Tpl2 status. Quantification of immunoblot data for levels of (B) Akt-pS473 and (C) eNOS-pS1177 levels. Error bars indicate ±s.e.m. (n=3). In panels B and C, statistical tests=one-way ANOVA; **P<0.01. (D–E) Assessment of endothelial cell permeability by measuring trans-endothelial electrical resistance (TEER) on control, scrambled or Tpl2-depleted endothelial cells under (D) basal conditions in serum-free MCDB131 media, or (E) with addition of VEGF-A (25 ng/ml). High TEER values correspond to reduced cell monolayer permeability indicative of more stable cell–cell junctions. Horizontal red dotted and blue dashed lines depict baselines for control/scrambled or Tpl2-depleted cells respectively. Error bars indicate ±s.e.m. (n=3). In panels D and E, statistical tests=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001.