title: Proteomic identification of VEGF-dependent protein enrichment to membrane caveolar-raft microdomains in endothelial progenitor cells.
abstract: Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.
authors.affiliation: Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Chillà
authors.forename: Francesca
authors.initials: F
authors.lastname: Magherini
authors.forename: Francesca
authors.initials: F
authors.lastname: Margheri
authors.forename: Anna
authors.initials: A
authors.lastname: Laurenzana
authors.forename: Tania
authors.initials: T
authors.lastname: Gamberi
authors.forename: Luca
authors.initials: L
authors.lastname: Bini
authors.forename: Laura
authors.initials: L
authors.lastname: Bianchi
authors.forename: Giovanna
authors.initials: G
authors.lastname: Danza
authors.forename: Benedetta
authors.initials: B
authors.lastname: Mazzanti
authors.forename: Simona
authors.initials: S
authors.lastname: Serratì
authors.forename: Alessandra
authors.initials: A
authors.lastname: Modesti
authors.forename: Mario
authors.initials: M
authors.lastname: Del Rosso
authors.forename: Gabriella
authors.initials: G
authors.lastname: Fibbi
mesh_terms: D021941:Caveolae / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A