BackgroundThe 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 FindingsImmunoblotting 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 (p38MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr1175. Phosphorylation of VEGFR2, p38MAPK 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 p38MAPK 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 p38MAPK 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/SignificanceWe conclude that a functional endothelial p38MAPK/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.

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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 ␤ 3 , which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer ␣ v ␤ 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. Key Words: angiogenesis Ⅲ endothelium Ⅲ platelets Ⅲ reactive oxygen species Ⅲ deoxyribose-1-phosphate B esides their well-established physiological role in hemostasis, platelets are known to release paracrine factors that facilitate vascular repair and angiogenesis, 1-3 and platelet-based preparations (eg, autologous platelet preparation, platelet-rich plasma, and platelet-derived wound healing formula) have been investigated for their proangiogenic potential and used for tissue regenerative purposes in clinical practice. 4,5 Platelet-derived angiogenic factors, including basic fibroblast growth factor and vascular endothelial growth factor (VEGF), have been shown to stimulate angiogenesis in vitro by enhancing both endothelial cell motility 1 and proliferation. 6 Similarly, heparanase, platelet-derived growth factor, and stromal cell-derived factor 1␣, all of which are released by activated platelets, can increase endothelial motility and angiogenesis, 1,7-9 and there is also evidence that platelet-derived stromal cell-derived factor 1␣ directly promotes migration and differentiation of endothelial progenitor cells. 10,11 With the exception of platelet-derived sphingosine-1-phosphate, which stimulates endothelial cell migration and angiogenesis by upregulating the expression of adhesion molecules, 12 the majority of research in this area has focused on platelet secretion of protein factors. Conclusion-Platelet-derivedHere, we present evidence that platelets release lowmolecular-mass molecules (Ͻ2 kDa) that are capable of stimulating endothelial cell migration, a key component of angiogenesis. 13 Small molecules released into platelet supernatants were characterized by gas chromatography-mass spectrometry (GC-MS) and direct injection-mass spectrometry (DIMS), which revealed the presence of deoxyribose-1-phosphate (dRP), a known proangiogenic molecule that stimulates endothelial cell migration. 14...