[Show abstract][Hide abstract] ABSTRACT: Current therapies for common types of cancer such as renal cell cancer are often ineffective and unspecific, and novel pharmacological targets and approaches are in high demand. Here we show the unexpected possibility for the rapid and selective killing of renal cancer cells through activation of calcium-permeable nonselective transient receptor potential canonical (TRPC) calcium channels by the sesquiterpene (−)-englerin A. This compound was found to be a highly efficient, fast-acting, potent, selective, and direct stimulator of TRPC4 and TRPC5 channels. TRPC4/5 activation through a high-affinity extracellular (−)-englerin A binding site may open up novel opportunities for drug discovery aimed at renal cancer.
[Show abstract][Hide abstract] ABSTRACT: Background: Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans, and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. Methods: In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. Results: SA OEC exhibited impaired colony formation, migration and in vitro angiogenesis, associated with decreased expression of the pro-angiogenic molecules Akt1 and eNOS. Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis which was abrogated by a nitric oxide synthase antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with non-manipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Conclusions: Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men. Stem Cells 2014.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic1, 2, 3, 4, 5. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact6, 7, 8. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.
[Show abstract][Hide abstract] ABSTRACT: 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 is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression that 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, ATF-2 was shown 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 only essential 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.
Molecular Biology of the Cell 06/2014; 25(16). DOI:10.1091/mbc.E14-05-0962 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 11/2012; 7(11):e48539. DOI:10.1371/journal.pone.0048539 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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).
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.
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.
British Journal of Pharmacology 06/2011; 165(1):245-59. DOI:10.1111/j.1476-5381.2011.01545.x · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian endothelium expresses two related but distinct receptor tyrosine kinases, VEGFR1 and VEGFR2 [VEGF (vascular endothelial growth factor) receptor 1 and 2], that regulate the vascular response to a key cytokine, VEGF-A. In the present review, we suggest a model for integrating the signals from these receptor tyrosine kinases by co-ordinating the spatial and temporal segregation of these membrane proteins linked to distinct signalling outputs associated with each intracellular location. Activation of pro-angiogenic VEGFR2 stimulates a programme of tyrosine phosphorylation, ubiquitination and proteolysis. This is linked to ESCRT (endosomal sorting complex required for transport)-mediated recognition of activated VEGFR2 and sorting in endosomes before arrival in lysosomes for terminal degradation. In addition, Rab GTPases regulate key events in VEGFR2 trafficking between the plasma membrane, early and late endosomes, with distinct roles for Rab4a, Rab5a and Rab7a. Manipulation of GTPase levels affects not only VEGFR2 activation and intracellular signalling, but also functional outputs such as VEGF-A-stimulated endothelial cell migration. In contrast, VEGFR1 displays stable Golgi localization that can be perturbed by cell stimuli that elevate cytosolic Ca(2+) ion levels. One model is that VEGFR1 translocates from the trans-Golgi network to the plasma membrane via a calcium-sensitive trafficking step. This allows rapid and preferential sequestration of VEGF-A by the higher-affinity VEGFR1, thus blocking further VEGFR2 activation. Recycling or degradation of VEGFR1 allows resensitization of the VEGFR2-dependent signalling pathway. Thus a dual VEGFR system with a built-in negative-feedback loop is utilized by endothelial cells to sense a key cytokine in vascular tissues.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor receptor 1 (VEGFR1) is an essential receptor tyrosine kinase that regulates mammalian vascular development and embryogenesis but its function is not well understood. Herein, we present evidence whereby endothelial VEGFR1 is largely resident within the Golgi apparatus but translocates to the plasma membrane via a calcium-regulated process. Primary human endothelial cells reveal differing VEGFR1 and VEGFR2 intracellular distribution and dynamics. The major proportion of the full-length VEGFR1 membrane protein was resident within the Golgi apparatus in primary endothelial cells. Whereas VEGFR2 displayed down-regulation in response to VEGF-A, VEGFR1 was not significantly affected arguing for a significant intracellular pool that was inaccessible to extracellular VEGF-A. This intracellular VEGFR1 pool showed significant co-distribution with key Golgi residents. Brefeldin A caused VEGFR1 Golgi fragmentation consistent with redistribution to the endoplasmic reticulum. Metabolic labeling experiments and microscopy using domain-specific VEGFR1 antibodies indicated that the mature processed VEGFR1 species and an integral membrane protein was resident within Golgi apparatus. Cytosolic calcium ions play a key role in VEGFR1 trafficking as treatment with either VEGF-A, histamine, thrombin, thapsigargin or A23187 ionophore caused VEGFR1 redistribution from the Golgi apparatus to small punctate vesicles and plasma membrane. We thus propose a model whereby the balance of VEGFR1 and VEGFR2 plasma membrane levels dictate either negative or positive endothelial signaling to influence vascular physiology.
Experimental Cell Research 02/2009; 315(5):877-89. DOI:10.1016/j.yexcr.2008.12.020 · 3.25 Impact Factor