Limits...
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.

Show MeSH

Related in: MedlinePlus

Neurons cultured for 15 days in serum-free Maat Medium release extracellular vesicles that contain both VEGF and FGF-2. A(a): Typical neuronal culture, observed in bright field, showing many extracellular vesicles of different sizes, some of which are indicated by arrows for reference; A(b) immunofluorescent staining of nuclei with DAPI; same field as in A(a). B: Schematic drawing of the protocol used to prepare an extracellular vesicle fraction (according to Dolo et al., 1994). C: Western analysis of total cell lysates (lane 1) and vesicles (lane 2) prepared, as outlined in B, from neurons cultured for 15 days in Maat medium and then labelled with 35S-methionine for 24 hrs. Proteins were immunostained with either rabbit polyclonal anti-human VEGF antibodies (a) or mouse monoclonal anti-FGF-2 (b). After electrophoresis and western blot, the same membranes were also exposed to X-ray sensitive films for 3–5 days: an example of the radioactive protein pattern, 48 hrs after metabolic labelling and medium replacement, is shown in (c). Arrowheads in (a) and arrows in (b) indicate VEGF isoforms and FGF-2 isoforms, respectively. The arrow in (c) indicates the only band that could be interpreted as an FGF-2 isoform. The arrowhead in (c) indicates the only band that should represent a radioactive VEGF isoform.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4401300&req=5

fig01: Neurons cultured for 15 days in serum-free Maat Medium release extracellular vesicles that contain both VEGF and FGF-2. A(a): Typical neuronal culture, observed in bright field, showing many extracellular vesicles of different sizes, some of which are indicated by arrows for reference; A(b) immunofluorescent staining of nuclei with DAPI; same field as in A(a). B: Schematic drawing of the protocol used to prepare an extracellular vesicle fraction (according to Dolo et al., 1994). C: Western analysis of total cell lysates (lane 1) and vesicles (lane 2) prepared, as outlined in B, from neurons cultured for 15 days in Maat medium and then labelled with 35S-methionine for 24 hrs. Proteins were immunostained with either rabbit polyclonal anti-human VEGF antibodies (a) or mouse monoclonal anti-FGF-2 (b). After electrophoresis and western blot, the same membranes were also exposed to X-ray sensitive films for 3–5 days: an example of the radioactive protein pattern, 48 hrs after metabolic labelling and medium replacement, is shown in (c). Arrowheads in (a) and arrows in (b) indicate VEGF isoforms and FGF-2 isoforms, respectively. The arrow in (c) indicates the only band that could be interpreted as an FGF-2 isoform. The arrowhead in (c) indicates the only band that should represent a radioactive VEGF isoform.

Mentions: We previously found that, in a three-cell type system of culture, containing neurons, astrocytes and endothelial cells, both neurons and astrocytes influence the ability of endothelial cells to form a barrier with properties resembling those of BBB [3, 4]. As in that system cells were not in physical contact, we deduced that the effects discovered should be due, at least in part, to molecular mechanisms not based on cell-to-cell contacts. Moreover, independent experiments showed that oligodendroglioma cells shed membrane vesicles [6]. Thus, we asked whether extracellular vesicles could be also produced by neurons to address inductive signals to endothelial cells. A further question was which kind of signals neurons deliver to them. As two of the major factors acting on endothelial cells (i.e. FGF-2 and VEGF) have been reported to be released through extracellular vesicles in other cell systems, we looked for the possible existence of extracellular structures, produced by neurons, carrying the two factors. As shown in Fig. 1A, there is evidence that neuronal cultures contain vesicular structures with sizes ranging from 400 to even 2000 nm. Most of these vesicles are in the range of size (100–1000 nm) already reported for the extracellular structures produced by several kinds of viable cells [10–12] and called MVs. As some of the structures observed are very large, we also looked for the presence of whole nuclei or nuclei fragments in them. We found that only intact nuclei, but not the putative MVs, were stained by DAPI (Fig. 1Ab).


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)

Neurons cultured for 15 days in serum-free Maat Medium release extracellular vesicles that contain both VEGF and FGF-2. A(a): Typical neuronal culture, observed in bright field, showing many extracellular vesicles of different sizes, some of which are indicated by arrows for reference; A(b) immunofluorescent staining of nuclei with DAPI; same field as in A(a). B: Schematic drawing of the protocol used to prepare an extracellular vesicle fraction (according to Dolo et al., 1994). C: Western analysis of total cell lysates (lane 1) and vesicles (lane 2) prepared, as outlined in B, from neurons cultured for 15 days in Maat medium and then labelled with 35S-methionine for 24 hrs. Proteins were immunostained with either rabbit polyclonal anti-human VEGF antibodies (a) or mouse monoclonal anti-FGF-2 (b). After electrophoresis and western blot, the same membranes were also exposed to X-ray sensitive films for 3–5 days: an example of the radioactive protein pattern, 48 hrs after metabolic labelling and medium replacement, is shown in (c). Arrowheads in (a) and arrows in (b) indicate VEGF isoforms and FGF-2 isoforms, respectively. The arrow in (c) indicates the only band that could be interpreted as an FGF-2 isoform. The arrowhead in (c) indicates the only band that should represent a radioactive VEGF isoform.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401300&req=5

fig01: Neurons cultured for 15 days in serum-free Maat Medium release extracellular vesicles that contain both VEGF and FGF-2. A(a): Typical neuronal culture, observed in bright field, showing many extracellular vesicles of different sizes, some of which are indicated by arrows for reference; A(b) immunofluorescent staining of nuclei with DAPI; same field as in A(a). B: Schematic drawing of the protocol used to prepare an extracellular vesicle fraction (according to Dolo et al., 1994). C: Western analysis of total cell lysates (lane 1) and vesicles (lane 2) prepared, as outlined in B, from neurons cultured for 15 days in Maat medium and then labelled with 35S-methionine for 24 hrs. Proteins were immunostained with either rabbit polyclonal anti-human VEGF antibodies (a) or mouse monoclonal anti-FGF-2 (b). After electrophoresis and western blot, the same membranes were also exposed to X-ray sensitive films for 3–5 days: an example of the radioactive protein pattern, 48 hrs after metabolic labelling and medium replacement, is shown in (c). Arrowheads in (a) and arrows in (b) indicate VEGF isoforms and FGF-2 isoforms, respectively. The arrow in (c) indicates the only band that could be interpreted as an FGF-2 isoform. The arrowhead in (c) indicates the only band that should represent a radioactive VEGF isoform.
Mentions: We previously found that, in a three-cell type system of culture, containing neurons, astrocytes and endothelial cells, both neurons and astrocytes influence the ability of endothelial cells to form a barrier with properties resembling those of BBB [3, 4]. As in that system cells were not in physical contact, we deduced that the effects discovered should be due, at least in part, to molecular mechanisms not based on cell-to-cell contacts. Moreover, independent experiments showed that oligodendroglioma cells shed membrane vesicles [6]. Thus, we asked whether extracellular vesicles could be also produced by neurons to address inductive signals to endothelial cells. A further question was which kind of signals neurons deliver to them. As two of the major factors acting on endothelial cells (i.e. FGF-2 and VEGF) have been reported to be released through extracellular vesicles in other cell systems, we looked for the possible existence of extracellular structures, produced by neurons, carrying the two factors. As shown in Fig. 1A, there is evidence that neuronal cultures contain vesicular structures with sizes ranging from 400 to even 2000 nm. Most of these vesicles are in the range of size (100–1000 nm) already reported for the extracellular structures produced by several kinds of viable cells [10–12] and called MVs. As some of the structures observed are very large, we also looked for the presence of whole nuclei or nuclei fragments in them. We found that only intact nuclei, but not the putative MVs, were stained by DAPI (Fig. 1Ab).

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