VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation via macropinocytosis

Endocytosis plays critical role in receptor signalling. VEGFR2 and its ligand VEGFA are fundamental in neovascularization. Yet, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway of VEGFR2 is the clathrin-mediated. Here, we show that this pathway is the predominant internalisation route of VEGFR2 only in the absence of ligand. Intriguingly, VEGF introduces a novel internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route of the receptor in the presence of ligand, while the route of clathrin becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated via the small GTPase CDC42, takes place via macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays critical role in VEGF-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, while clathrin-mediated endocytosis is not essential for VEGF signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGF-driven internalisation of VEGFR2 via macropinocytosis is essential for endothelial cell signalling and angiogenesis

The origins and evolution of macropinocytosis

In macropinocytosis, cells take up micrometre-sized droplets of medium into internal vesicles. These vesicles are acidified and fused to lysosomes, their contents digested and useful compounds extracted. Indigestible contents can be exocytosed. Macropinocytosis has been known for approaching 100 years and is described in both metazoa and amoebae, but not in plants or fungi. Its evolutionary origin goes back to at least the common ancestor of the amoebozoa and opisthokonts, with apparent secondary loss from fungi. The primary function of macropinocytosis in amoebae and some cancer cells is feeding, but the conserved processing pathway for macropinosomes, which involves shrinkage and the retrieval of membrane to the cell surface, has been adapted in immune cells for antigen presentation. Macropinocytic cups are large actin-driven processes, closely related to phagocytic cups and pseudopods and appear to be organized around a conserved signalling patch of PIP3, active Ras and active Rac that directs actin polymerization to its periphery. Patches can form spontaneously and must be sustained by excitable kinetics with strong cooperation from the actin cytoskeleton. Growth-factor signalling shares core components with macropinocytosis, based around phosphatidylinositol 3-kinase (PI3-kinase), and we suggest that it evolved to take control of ancient feeding structures through a coupled growth factor receptor. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Background and Purpose: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to NanoLuciferase-tagged VEGF Receptor 2 (VEGFR2) in living cells is that the bioluminescence resonance energy transfer (BRET) signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.Experimental Approach: Ligand binding kinetics were monitored in both intact HEK293T cells and membranes (expressing NanoLuciferase tagged VEGFR2) using BRET between the tagged receptor and fluorescent analogues of VEGF165a, VEGF165b and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.Key Results: Quantitiative analysis of the effect of fluorescent VEGF-A isoforms onVEGFR2 endocytosis in cells demonstrated that they produced a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand binding association curves were maintained for the duration of the 90 minute experiment. Measurement of koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to intracellular endosomes

Activation of the sweet taste receptor T1R3 by sucralose attenuates VEGF-induced vasculogenesis in a cell model of the retinal microvascular endothelium

Background: One of the most prevalent microvascular complications for patients with diabetes is diabetic retinopathy (DR) associated with increased retinal endothelial blood vessel formation. Treatments to reduce vascularisation in the retinal endothelium are linked to improved sight in patients with DR. Recently we have demonstrated the novel protective role of the artificial sweetener, sucralose, and the sweet taste receptor, T1R3, in the pulmonary endothelium to reduce vascular leak. In the present study, we examined the role of sucralose and sweet taste receptors on vasculogenic processes (proliferation, migration, adhesion and tube formation) in a cell model of the retinal endothelium . Methods: We exposed human retinal microvascular endothelial cells (RMVEC) to VEGF as an in vitro model of DR in the presence and absence of T1R3 agonist sucralose. Results: In RMVEC, we observed increased VEGF-induced cell proliferation, migration, adhesion and tube formation, which was significantly attenuated by exposure to the artificial sweetener sucralose. Following siRNA knockdown of the sweet taste receptor, T1R3, but not T1R2, the protective effect of sucralose on VEGF-induced RMVEC vasculogenic processes was blocked. We further demonstrate that sucralose attenuates VEGF-induced Akt phosphorylation to protect the retinal microvasculature. Conclusion: These studies are the first to demonstrate a protective effect of an artificial sweetener, through the sweet taste receptor T1R3, on VEGF-induced vasculogenesis in a retinal microvascular endothelial cell line

Methodology of IT development

65 σ.Εθνικό Μετσόβιο Πολυτεχνείο--Μεταπτυχιακή Εργασία. Διεπιστημονικό-Διατμηματικό Πρόγραμμα Μεταπτυχιακών Σπουδών (Δ.Π.Μ.Σ.) “Μαθηματική Προτυποποίηση σε Σύγχρονες Τεχνολογίες στην Οικονομία”Βάσει της συγκεκριμένης μεθοδολογίας, ο κύκλος ζωής ενός έργου διαιρείται στις φάσεις Έναρξης, Επεξεργασίας, Κατασκευής και Μετάβασης, ενώ οι προβλεπόμενες δραστηριότητες ομαδοποιούνται στους τομείς της Επιχειρησιακής Μοντελοποίησης, της Ανάλυσης Απαιτήσεων, της Ανάλυσης και του Σχεδιασμού, της Υλοποίησης, του Ελέγχου και της Ανάπτυξης. Ακολουθώντας μια επαναληπτική προσέγγιση για την ανάπτυξη του έργου, ο κύκλος ζωής αποτελείται από πολλές επαναλήψεις. Σε μια επανάληψη πραγματοποιείται ένα σύνολο δραστηριοτήτων από όλους τους τομείς σε διάφορες αναλογίες, ανάλογα με το σημείο του κύκλου ζωής στο οποίο βρίσκεται η επανάληψη. Με τον τρόπο αυτό οι κίνδυνοι ενός έργου πληροφορικής αντιμετωπίζονται νωρίτερα, με αποτέλεσμα να αποφεύγεται η πολυέξοδη επανάληψη εργασίας και, σε μερικές περιπτώσεις, ακόμη και η ακύρωση του έργου, εξαιτίας πιθανών λαθών του αρχικού σχεδιασμού.According to this methodology, the software lifecycle is decomposed over time into four sequential phases: Inception, Elaboration, Construction and Transition, while activities are grouped into disciplines: Business Modelling, Requirements, Analysis and Design, Implementation, Test and Deployment. Following an iterative development, the software lifecycle consists of several iterations. An iteration incorporates a loosely sequential set of activities in Business Modelling, Requirements, Analysis and Design, Implementation, Test, and Deployment, in various proportions depending on where in the development cycle the iteration is located. With this methodology, risks are mitigated earlier, because elements are integrated progressively. Late discovery of design defects results in costly over-runs and, in some cases, even project cancellation.Παναγιώτης Δ. Μπασαγιάννη

3D Fusion Echocardiography Improves Transoeosphageal LV Assessment

Accurate left ventricular (LV) assessment by transoesophageal echocardiography (TOE) can be clinically important particularly in intensive care or intraoperative settings. Assessment from TOE views is limited by foreshortened imaging planes and mitral valve pathology. Transgastric short-axis (SAX) views allow better alignment but may be limited by patient tolerability or patient-related factors (e.g. post gastric surgery). Fusion of multiple 3D volume datasets is now technically possible and improves LV visualisation from transthoracic windows. We hypothesized that fusion of full 3D TOE volumes would improve LV image quality and allow SAX image reconstruction from the oesophagus with improved image quality vs. transgastric SAX views. © 2011 Springer-Verlag Berlin Heidelberg