We set a three-cell type coculture system in which neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological blood-brain barrier (BBB) (Schiera et al., 2003; Schiera et al., 2005). On the basis of immunofluorescence, scanner electron microscopy and western blot analyses, we also suggested that both astrocytes and neurons in culture shed extracellular vesicles that contain FGF-2 and VEGF, as well as beta1-integrin, a membrane protein that can be considered a marker of shedding (Schiera et al, 2007; Proia et al., 2008).In addition, it was already known that transformed glial cells (oligodendroglioma cells ) are able to shed extracellular vesicles (D'Agostino et al., 2006). We supposed that shedding could be a general cell-to-cell communication mode among brain cells and that other factors might be released through this pathway. Thus we looked at the transforming growth factors beta (TGFbeta), a well known secreted protein that exists in different isoforms, some of which are already known to be produced in the brain. We also searched for the presence, in the extracellular vesicular fraction, of the Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells, but not in normal cells. Here we report that TRAIL, a protein that is recently emerging as a potential key inductor of apoptosis also in the brain, seems indeed to be present in vesicles shed by many kinds of tumor cells, among which oligodendroglioma cells. On the basis of immunofluorescence, as well as of western blot analyses, we can conclude that TGFbeta can be found in vesicles too. To clarify this special putative pathway of secretion, we are analyzing the events involved in the synthesis and release of the angiogenic and pro-apoptotic factors apparently present in the extracellular vesicles. One of the approaches we have been using has been to clone the coding portion of TGFbeta1 mRNA into the pEGFP-N2 plasmid, to be then transferred into mammalian cells, in order to produce a fluorescent recombinant protein. We are now selecting stably transfected fluorescent cell lines that will be used to study the fate of the protein during the process of secretion and/or shedding of extracellular vesicles.Cestelli A. et al., 2001, J. Controll. Rel. 76: 139-47.D’Agostino S. et al., 2006, Int J Oncol, 29: 1075-85.Savettieri G. et al., 2000, NeuroReport 11: 1081-4.Schiera G. et al, 2003, J. Cell. Mol. Med. 7: 165-70. Schiera G. et al., 2005, J. Cell. Mol. Med. 9: 373-9.Schiera G et al. 2007, J. Cell. Mol. Med. 11: 1384-94.Proia P. et al (2008), Int. J. Mol. Med. 21: 63-7.
|Numero di pagine||1|
|Stato di pubblicazione||Published - 2008|