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Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles.

Schiera G, Proia P, Alberti C, Mineo M, Savettieri G, Di Liegro I - J. Cell. Mol. Med. (2007 Nov-Dec)

Bottom Line: We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier.In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors.Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università degli Studi di Palermo, Palermo, Italy.

ABSTRACT
We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain beta1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

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Control- (A and C) and camptothecin-treated (B and D) neurons cultured in Maat Medium for 15 days. Cells were incubated for 24 hrs with 10-mM camptothecin. As camptothecin was dissolved in dimethyl sulfoxide (DMSO), control cells were treated for 24 hrs with an equivalent amount of DMSO alone. A, B: Control cells (A) and cells treated with camptothecin (B), stained with a combination of the fluorescent DNA-binding dyes acridine orange and ethidium bromide, 100 mg each/ml in PBS. C, D: Control cells (C) and cells treated with campthothecin (D), immunostained with anti- MAP2- (green fluorescence) and anti-FGF-2 antibodies (red fluorescence). In addition, all cells were also stained with DAPI. The figure shows the overlay of the three fluorescences. Putative apoptotic bodies in treated cells are indicated by arrows, while putative extracellular vesicles in untreated cells are indicated by an asterisk. Cells were observed in an Olympus BX-50 microscope, equipped with Vario Cam B/W camera.
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fig06: Control- (A and C) and camptothecin-treated (B and D) neurons cultured in Maat Medium for 15 days. Cells were incubated for 24 hrs with 10-mM camptothecin. As camptothecin was dissolved in dimethyl sulfoxide (DMSO), control cells were treated for 24 hrs with an equivalent amount of DMSO alone. A, B: Control cells (A) and cells treated with camptothecin (B), stained with a combination of the fluorescent DNA-binding dyes acridine orange and ethidium bromide, 100 mg each/ml in PBS. C, D: Control cells (C) and cells treated with campthothecin (D), immunostained with anti- MAP2- (green fluorescence) and anti-FGF-2 antibodies (red fluorescence). In addition, all cells were also stained with DAPI. The figure shows the overlay of the three fluorescences. Putative apoptotic bodies in treated cells are indicated by arrows, while putative extracellular vesicles in untreated cells are indicated by an asterisk. Cells were observed in an Olympus BX-50 microscope, equipped with Vario Cam B/W camera.

Mentions: Finally, as neurons in our cell culture system are post-mitotic and are fed with a serum-free medium, the possibility existed that vesicle production was the effect of cell death. This possibility was also suggested by the large size of vesicles. In order to compare vesicles produced by vital cells with vesicular structures (such as apoptotic bodies), produced by dying cells, we treated neurons with campothecin, an S-phase-specific topoisomerase I inhibitor that has been shown to induce significant, dose-dependent cell death of post-mitotic rat cortical neurons in vitro[42]. Camptothecin-induced neuronal death was already shown to be apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites [42]. Camptothecin induced massive cell death also in our cell culture system (Fig. 6): in most fields, neurons were fragmented into putative apoptotic bodies (indicated by arrows in Fig. 6B and D). These cell fragments appeared orange/red when cells were stained with a mixture of the fluorescent DNA-binding dyes AO and EO (Fig. 6B). On the contrary, nuclei of control neurons (Fig. 6A) appeared intact and bright green (as expected for alive cells). More important, vesicular structures deriving from cell death were also stained by DAPI (Fig. 6D: structures indicated by arrows), while the putative extracellular vesicles identified by us in normal cultures were not stained (Figs 1A and 6C).


Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles.

Schiera G, Proia P, Alberti C, Mineo M, Savettieri G, Di Liegro I - J. Cell. Mol. Med. (2007 Nov-Dec)

Control- (A and C) and camptothecin-treated (B and D) neurons cultured in Maat Medium for 15 days. Cells were incubated for 24 hrs with 10-mM camptothecin. As camptothecin was dissolved in dimethyl sulfoxide (DMSO), control cells were treated for 24 hrs with an equivalent amount of DMSO alone. A, B: Control cells (A) and cells treated with camptothecin (B), stained with a combination of the fluorescent DNA-binding dyes acridine orange and ethidium bromide, 100 mg each/ml in PBS. C, D: Control cells (C) and cells treated with campthothecin (D), immunostained with anti- MAP2- (green fluorescence) and anti-FGF-2 antibodies (red fluorescence). In addition, all cells were also stained with DAPI. The figure shows the overlay of the three fluorescences. Putative apoptotic bodies in treated cells are indicated by arrows, while putative extracellular vesicles in untreated cells are indicated by an asterisk. Cells were observed in an Olympus BX-50 microscope, equipped with Vario Cam B/W camera.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401300&req=5

fig06: Control- (A and C) and camptothecin-treated (B and D) neurons cultured in Maat Medium for 15 days. Cells were incubated for 24 hrs with 10-mM camptothecin. As camptothecin was dissolved in dimethyl sulfoxide (DMSO), control cells were treated for 24 hrs with an equivalent amount of DMSO alone. A, B: Control cells (A) and cells treated with camptothecin (B), stained with a combination of the fluorescent DNA-binding dyes acridine orange and ethidium bromide, 100 mg each/ml in PBS. C, D: Control cells (C) and cells treated with campthothecin (D), immunostained with anti- MAP2- (green fluorescence) and anti-FGF-2 antibodies (red fluorescence). In addition, all cells were also stained with DAPI. The figure shows the overlay of the three fluorescences. Putative apoptotic bodies in treated cells are indicated by arrows, while putative extracellular vesicles in untreated cells are indicated by an asterisk. Cells were observed in an Olympus BX-50 microscope, equipped with Vario Cam B/W camera.
Mentions: Finally, as neurons in our cell culture system are post-mitotic and are fed with a serum-free medium, the possibility existed that vesicle production was the effect of cell death. This possibility was also suggested by the large size of vesicles. In order to compare vesicles produced by vital cells with vesicular structures (such as apoptotic bodies), produced by dying cells, we treated neurons with campothecin, an S-phase-specific topoisomerase I inhibitor that has been shown to induce significant, dose-dependent cell death of post-mitotic rat cortical neurons in vitro[42]. Camptothecin-induced neuronal death was already shown to be apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites [42]. Camptothecin induced massive cell death also in our cell culture system (Fig. 6): in most fields, neurons were fragmented into putative apoptotic bodies (indicated by arrows in Fig. 6B and D). These cell fragments appeared orange/red when cells were stained with a mixture of the fluorescent DNA-binding dyes AO and EO (Fig. 6B). On the contrary, nuclei of control neurons (Fig. 6A) appeared intact and bright green (as expected for alive cells). More important, vesicular structures deriving from cell death were also stained by DAPI (Fig. 6D: structures indicated by arrows), while the putative extracellular vesicles identified by us in normal cultures were not stained (Figs 1A and 6C).

Bottom Line: We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier.In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors.Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università degli Studi di Palermo, Palermo, Italy.

ABSTRACT
We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain beta1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

Show MeSH
Related in: MedlinePlus