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Cells respond to soluble and membrane-bound factors to activate signalling cascades that control cell proliferation and cell death. Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that modulates a variety of cellular responses including cell proliferation and apoptosis. It is not well understood how VEGF-A signalling pathways regulate cell proliferation and cell death. To address this, we examined VEGF-A-regulated signalling pathways in the cytosol and nucleus and functional requirement for such cellular responses. The VEGF-A-regulated transcription factor, ATF-2, is required for cell cycle proteins such as p53, p21 and Cyclin D1. A cytosolic serine/threonine protein kinase (Tpl2) modulates ATF-2-regulated effects on the endothelial cell cycle. Such regulatory effects impact on endothelial cell proliferation, cell viability and apoptosis. These cellular effects influence complex cell-based organisation such as endothelial tubulogenesis. Our study now provides a framework for incorporating VEGF-A-stimulated signalling events from the cytosol to the nucleus which helps to understand how cell proliferation and apoptosis are controlled., (Copyright © 2019 Elsevier Inc. All rights reserved.)

New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells. Notably, stimulation with exogenous VEGF-A increased Tpl2 mRNA levels and consequently de novo protein synthesis. Depletion of Tpl2 levels reveals a role in both basal and VEGF-A-stimulated endothelial cell responses, including endothelial-leukocyte interactions, monolayer permeability and new blood vessel formation. Under basal conditions, Tpl2 modulates a signal transduction cascade resulting in phosphorylation of a nuclear transcription factor (ATF-2) and altered endothelial gene expression, a pathway previously identified as crucial in VEGF-dependent vascular responses. Loss of Tpl2 expression or activity impairs signal transduction through Akt, eNOS and ATF-2, broadly impacting on endothelial function. Our study now provides a mechanism for Tpl2 as a central component of signal transduction pathways in the endothelium., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The mammalian vascular endothelial growth factor receptor tyrosine kinases (VEGFRs) bind circulating growth factors and regulate the process of angiogenesis. The discovery of new small molecules that target the enzymatic activity of the VEGFR family as potential antiangiogenic drugs is of much commercial interest in the pharmaceutical sector. Here, we describe the use of a combined cell surface biotinylation and affinity isolation procedure to monitor ligand-stimulated VEGFR trafficking in endothelial cells, in which novel VEGFR inhibitors from chemical libraries can be identified by their ability to inhibit receptor internalization. Unlike a traditional cell-free enzyme activity assay, such a cell-based approach provides a physiologically relevant readout of inhibitor activity. In this example, we use the VEGF-A-VEGFR-2 axis and the well-characterized tyrosine kinase inhibitor sunitinib as a working model; however this technique is highly applicable for the identification of inhibitors to other receptor tyrosine kinases.

Background: Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues., Methodology: We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis., Conclusions: We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways., (© 2014 Fearnley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function., (© 2014 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Purpose: Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target for the treatment of diseases such as cancer. Small-molecule VEGFR2 inhibitors of a variety of chemical classes are currently under development or in clinical use. In this study, we describe the de novo design of a new generation pyrazole-based molecule (JK-P3) that targets VEGFR2 kinase activity and angiogenesis., Experimental Approach: JK-P compound series were designed using de novo structure-based identification methods. Compounds were tested in an in vitro VEGFR2 kinase assay. Using primary endothelial cells, JK-P compounds were assessed for their ability to inhibit VEGF-A-stimulated VEGFR2 activation and intracellular signalling. We tested these compounds in cell migration, proliferation and angiogenesis assays., Key Results: JK-P3 and JK-P5 were predicted to bind the VEGFR2 kinase domain with high affinity, and both compounds showed pronounced inhibition of endogenous VEGFR2 kinase activity in primary human endothelial cells. Only JK-P3 inhibited VEGF-A-stimulated VEGFR2 activation and intracellular signalling. Interestingly, JK-P3 inhibited endothelial monolayer wound closure and angiogenesis but not endothelial cell proliferation. Both compounds inhibited fibroblast growth factor receptor kinase activity in vitro, but not basic fibroblast growth factor-mediated signalling in endothelial cells., Conclusions and Implications: This is the first report that describes an anti-angiogenic inhibitor based on such a pyrazole core. Using a de novo structure-based identification approach is an attractive method to aid such drug discovery. These results thus provide an important basis for the development of multi-tyrosine kinase inhibitors for clinical use in the near future., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration in vitro. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair in vivo consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.

Background and Purpose: The potent pro-angiogenic growth factors VEGF-A and basic fibroblast growth factor (bFGF) exert their effects by binding VEGF receptor 2 and FGF receptor tyrosine kinases, respectively. Indolinones (e.g. SU5416 and Sutent) and anilinophthalazines (e.g. PTK787) are potent small molecule inhibitors of VEGFR2 and other tyrosine kinases, but their effects on VEGF-A- and bFGF-stimulated endothelial responses are unclear. Here we assess the ability of these compounds to inhibit pro-angiogenic responses through perturbation of receptor activity and endothelial function(s)., Experimental Approach: We used in silico modelling, in vitro tyrosine kinase assays, biochemistry and microscopy to evaluate the effects of small molecules on receptor tyrosine kinase activation and intracellular signalling. Primary human endothelial cells were used to assess intracellular signalling, cell migration, proliferation and tubulogenesis., Key Results: We predicted that the anilinophthalazine PTK787 binds the tyrosine kinase activation loop whereas indolinones are predicted to bind within the hinge region of the split kinase domain. Sutent is a potent inhibitor of both VEGFR2 and FGFR1 tyrosine kinase activity in vitro. The compounds inhibit both ligand-dependent and -independent VEGFR2 trafficking events, are not selective for endothelial cell responses and inhibit both VEGF-A- and bFGF-mediated migration, wound healing and tubulogenesis at low concentrations. CONCLUSIONS AND IMPLICATIONS; We propose that these compounds have novel properties including inhibition of bFGF-mediated endothelial responses and perturbation of VEGFR2 trafficking. Differential inhibitor binding to receptor tyrosine kinases translates into more potent inhibition of bFGF- and VEGF-A-mediated intracellular signalling, cell migration and tubulogenesis. Indolinones and anilinophthalazines thus belong to a class of multi-kinase inhibitors that show clinical efficacy in disease therapy., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Vascular endothelial growth factor A (VEGF-A) and its cognate receptors are central to the regulation of angiogenesis in both physiological and pathological states. In cancer, local tumour hypoxia stimulates VEGF-A synthesis and VEGF-A levels are subsequently elevated in a wide variety of cancers. VEGF-A thus has enormous potential as a diagnostic and prognostic biomarker of disease status. The justification of VEGF-A as a biomarker has not yet been achieved, primarily due to our lack of understanding of its multiple splice variants and its spatio-temporal distribution. Here we highlight how recent technological advancements and kinetic-dynamic modelling could be used towards validating VEGF-A as a biomarker for clinical use in human disease management.