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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.

SUBMITTER: Chilla A

PROVIDER: S-EPMC3708176 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Proteomic identification of VEGF-dependent protein enrichment to membrane caveolar-raft microdomains in endothelial progenitor cells.

Chillà Anastasia A Magherini Francesca F Margheri Francesca F Laurenzana Anna A Gamberi Tania T Bini Luca L Bianchi Laura L Danza Giovanna G Mazzanti Benedetta B Serratì Simona S Modesti Alessandra A Del Rosso Mario M Fibbi Gabriella G

Molecular & cellular proteomics : MCP 20130409 7

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 ...[more]

PMID: 23572564

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Project description:BACKGROUND:The subcellular distribution of prorenin receptor and adaptor protein ATP6AP2 may affect neurogenesis. In this study, we hypothesized that ATP6AP2 expression and subcellular relocalization from caveolae/lipid raft microdomains (CLR-Ms) to intracellular sites may correlate with neuronal differentiation (Neu-Dif) of adipose-derived mesenchymal stem cells (ADSCs). METHODS:Human ADSCs isolated from 24 healthy donors and 24 patients with neurological disorders (ND) were cultured and induced for Neu-Dif. The mechanism of action of ATP6AP2 and the impact of its localization within the plasma membrane (particularly CLR-Ms) and intracellular sites on several pathways (mitogen-activated protein kinase, Wnt(s) signaling and others) and intracellular calcium and exosome release were evaluated. The impact of CLR-Ms on ATP6AP2 or vice versa was determined by pharmacological disruption of CLR-Ms or siATP6AP2 assays. RESULTS:In patients with ND, loss of ATP6AP2 from CLR-Ms correlated with an inhibition of Neu-Dif and signaling. However, its relocalization in CLR-Ms was positively correlated to induction of Neu-Dif in healthy subjects. An apparent switch from canonical to noncanonical Wnt signaling as well as from caveolin to flotillin occurs concurrently with the increases of ATP6AP2 expression during neurogenesis. Stimulation by renin activates ERK/JNK/CREB/c-Jun but failed to induce ?-catenin. Wnt5a enhanced the renin-induced JNK responsiveness. G? proteins crosslink ATP6AP2 to caveolin where a switch from G?i to G?q is necessary for Neu-Dif. In ATP6AP2-enriched CLR-Ms, the release of exosomes was induced dependently from the intracellular Ca2+ and G?q. Pharmacological disruption of CLR-M formation/stability impairs both ATP6AP2 localization and Neu-Dif in addition to reducing exosome release, indicating an essential role of ATP6AP2 enrichment in CLR-Ms for the induction of Neu-Dif. The mechanism is dependent on CLR-M dynamics, particularly the membrane fluidity. Knockdown of ATP6AP2 inhibited Neu-Dif but increased astrocytic-Dif, depleted ATP6AP2/flotillin/G?q but accumulated caveolin/G?i in CLR-Ms, and blocked the activation of JNK/ERK/c-Jun/CREB/exosome release. siATP6AP2 cells treated with sphingomyelinase/methyl-?-cyclodextrin reversed the levels of caveolin/flotillin in CLR-Ms but did not induce Neu-Dif, indicating the crucial relocalization of ATP6AP2 in CLR-Ms for neurogenesis. Treatment of ND-derived cells with nSMase showed reversibility in ATP6AP2 abundance in CLR-Ms and enhanced Neu-Dif. CONCLUSIONS:This study gives evidence of the determinant role of CLR-M ATP6AP2 localization for neuronal and oligodendrocyte differentiation involving mechanisms of switches from G?i/caveolin/canonical to G?q/flotillin/PCP, the ERK/JNK pathway and Ca2+-dependent release of exosomes and as a potential target of drug therapy for neurodegenerative disorders.

Dataset Information

Proteomic identification of VEGF-dependent protein enrichment to membrane caveolar-raft microdomains in endothelial progenitor cells.

Publications

Proteomic identification of VEGF-dependent protein enrichment to membrane caveolar-raft microdomains in endothelial progenitor cells.

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