RESUMEN
OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.
Asunto(s)
Plaquetas/metabolismo , Movimiento Celular , Membrana Corioalantoides/irrigación sanguínea , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Comunicación Paracrina , Ribosamonofosfatos/metabolismo , Animales , Plaquetas/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Cromatografía de Gases y Espectrometría de Masas , Silenciador del Gen , Humanos , Integrina alfaV/metabolismo , Integrina alfaVbeta3/metabolismo , Integrina beta3/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Trombina/metabolismo , Timidina Fosforilasa/antagonistas & inhibidores , Timidina Fosforilasa/genética , Timidina Fosforilasa/metabolismo , Factores de Tiempo , Uridina Fosforilasa/antagonistas & inhibidores , Uridina Fosforilasa/genética , Uridina Fosforilasa/metabolismoRESUMEN
COX (cyclo-oxygenase)-2 and members of the PAR (protease-activated receptor) family (PARs 1-4) are highly overexpressed in a number of angiogenesis-dependent pathologies, including advanced atherosclerosis and cancer. An appreciation of the potential role(s) of PARs and COX enzymes in physiological angiogenesis is, however, currently lacking. Exposure of human endothelial cells to serine proteases (e.g. thrombin) or to PAR-selective agonist peptides leads to a wide range of cellular responses, including enhanced expression of COX-2, and we have shown that this induction depends on activation of classic pro-inflammatory signalling elements [e.g. MAPKs (mitogen-activated protein kinases) and NF-kappaB (nuclear factor kappaB)]. Our current studies suggest that COX-2-derived mediators are important autocrine regulators of PAR-stimulated angiogenesis. This mechanism could help us to explain how this novel family of receptors couple vascular inflammation with repair and angiogenesis in health and disease.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Células Endoteliales/fisiología , Neovascularización Fisiológica/fisiología , Receptores Proteinasa-Activados/metabolismo , Transducción de Señal/fisiología , Animales , Células Endoteliales/citología , Epoprostenol/metabolismo , Humanos , Inflamación/metabolismo , Ratones , Prostaglandinas/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE(2) on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE(2) increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF(1alpha) release and EC proliferation. In contrast, PGE(2) attenuated VEGF(165)-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE(2) restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH(2) (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.
Asunto(s)
Comunicación Celular/fisiología , Diferenciación Celular , Ciclooxigenasa 2/metabolismo , Células Endoteliales/fisiología , Osteoblastos/fisiología , Prostaglandinas/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa/metabolismo , Dinoprostona/metabolismo , Células Endoteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Nitrobencenos/metabolismo , Osteoblastos/citología , Sulfonamidas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0018823.].
RESUMEN
BACKGROUND: The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs. METHODOLOGY/PRINCIPAL FINDINGS: Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38(MAPK)) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr(1175). Phosphorylation of VEGFR2, p38(MAPK) and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38(MAPK) abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2'-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38(MAPK) and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo. CONCLUSIONS/SIGNIFICANCE: We conclude that a functional endothelial p38(MAPK)/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Endotelio Vascular/citología , Neovascularización Fisiológica/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Células Cultivadas , Humanos , Inmunoprecipitación , Fosforilación , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endothelial dysfunction is characterised by pro-inflammatory/pro-coagulant changes in the endothelium and supports leukocyte adhesion and transmigration, key steps in early atherogenesis. There is emerging evidence that triacylglycerol-rich lipoproteins (TGRLs) present in the circulation during the postprandial phase influence vascular inflammation but the specific contribution of the remnant lipoprotein component of TGRLs is largely unexplored and the mechanistic basis of their actions poorly defined. This article provides a brief overview of the evidence supporting direct actions of these particles on endothelial cells and highlights the importance of their fatty acid composition and oxidative state as determinants of their cellular actions.