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Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential.

Lopatina T, Bruno S, Tetta C, Kalinina N, Porta M, Camussi G - Cell Commun. Signal (2014)

Bottom Line: Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC).The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Sciences and Molecular Biotechnology Center, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy. giovanni.camussi@unito.it.

ABSTRACT

Background: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process.

Results: Herein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.

Conclusions: Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

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Related in: MedlinePlus

EV effect on HMEC. A: representative confocal microphotography of 100k total EVs labelled with PKH26 dye (red), and HMEC (nuclei are blue) incubated with EVs for 30 minutes, 3 and 6 hours; four experiments were done with similar results; B: diagram of HMEC proliferation in response to b-EV or PDGF-EV addition; 10% FBS was used as positive control (mean ± SEM, * p < 0.05 vs. non-stimulated control HMEC, n = 6); C: diagram of HMEC invasiveness into Matrigel after b-EV or PDGF-EV stimulation in presence (red column) or absence (blue column) of the MMPs inhibitor Batimastat (mean ± SEM, * p < 0.05 vs. non-stimulated HMEC, # p < 0.05 stimulation in the presence of Batimastat vs. absence of Batimastat, n = 12); D: representative zymography of b-EVs and PDGF-EVs; intensity of MMP2 and MMP9 bands are indicated; E: representative zymography of conditioned media of HMEC 24 hours after b-EV or PDGF-EV addition; proMMP-9 (92 kD), active MMP-9 (82 kD), proMMP-2 (72 kD) and active MMP-2 (62 kD) are present; the intensity (itn.) of bands is indicated.
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Figure 2: EV effect on HMEC. A: representative confocal microphotography of 100k total EVs labelled with PKH26 dye (red), and HMEC (nuclei are blue) incubated with EVs for 30 minutes, 3 and 6 hours; four experiments were done with similar results; B: diagram of HMEC proliferation in response to b-EV or PDGF-EV addition; 10% FBS was used as positive control (mean ± SEM, * p < 0.05 vs. non-stimulated control HMEC, n = 6); C: diagram of HMEC invasiveness into Matrigel after b-EV or PDGF-EV stimulation in presence (red column) or absence (blue column) of the MMPs inhibitor Batimastat (mean ± SEM, * p < 0.05 vs. non-stimulated HMEC, # p < 0.05 stimulation in the presence of Batimastat vs. absence of Batimastat, n = 12); D: representative zymography of b-EVs and PDGF-EVs; intensity of MMP2 and MMP9 bands are indicated; E: representative zymography of conditioned media of HMEC 24 hours after b-EV or PDGF-EV addition; proMMP-9 (92 kD), active MMP-9 (82 kD), proMMP-2 (72 kD) and active MMP-2 (62 kD) are present; the intensity (itn.) of bands is indicated.

Mentions: DNA was not detected in 10k and 100k EV fractions after DNA extraction and analysis using spectrophotometry (NanoDrop) and by agarose gel electrophoresis (Bioanalyser). EVs were stained with PKH26, which labelled membrane phospholipids, indicating that particles detected both in 10K and 100K fractions were not protein aggregates (Figure 2A). Both 10k and 100k fractions expressed several mesenchymal surface markers characteristic of cell origin as seen by GUAVA FACS analysis. EVs expressed mesenchymal surface markers (CD73, CD29, CD90, CD105, CD44), endothelial markers (CD105, CD31), and marker of exosomes (CD63, CD81). The expression did not change in different fractions of EVs or EVs obtained after stimulation with PDGF with the exception of CD81 expression, which was increased in 100 k fraction of PDGF-EVs in respect to b-EVs (Table 1). Similar results were obtained by FACS analysis performed on vesicles pre-absorbed on beads (not shown).


Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential.

Lopatina T, Bruno S, Tetta C, Kalinina N, Porta M, Camussi G - Cell Commun. Signal (2014)

EV effect on HMEC. A: representative confocal microphotography of 100k total EVs labelled with PKH26 dye (red), and HMEC (nuclei are blue) incubated with EVs for 30 minutes, 3 and 6 hours; four experiments were done with similar results; B: diagram of HMEC proliferation in response to b-EV or PDGF-EV addition; 10% FBS was used as positive control (mean ± SEM, * p < 0.05 vs. non-stimulated control HMEC, n = 6); C: diagram of HMEC invasiveness into Matrigel after b-EV or PDGF-EV stimulation in presence (red column) or absence (blue column) of the MMPs inhibitor Batimastat (mean ± SEM, * p < 0.05 vs. non-stimulated HMEC, # p < 0.05 stimulation in the presence of Batimastat vs. absence of Batimastat, n = 12); D: representative zymography of b-EVs and PDGF-EVs; intensity of MMP2 and MMP9 bands are indicated; E: representative zymography of conditioned media of HMEC 24 hours after b-EV or PDGF-EV addition; proMMP-9 (92 kD), active MMP-9 (82 kD), proMMP-2 (72 kD) and active MMP-2 (62 kD) are present; the intensity (itn.) of bands is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4022079&req=5

Figure 2: EV effect on HMEC. A: representative confocal microphotography of 100k total EVs labelled with PKH26 dye (red), and HMEC (nuclei are blue) incubated with EVs for 30 minutes, 3 and 6 hours; four experiments were done with similar results; B: diagram of HMEC proliferation in response to b-EV or PDGF-EV addition; 10% FBS was used as positive control (mean ± SEM, * p < 0.05 vs. non-stimulated control HMEC, n = 6); C: diagram of HMEC invasiveness into Matrigel after b-EV or PDGF-EV stimulation in presence (red column) or absence (blue column) of the MMPs inhibitor Batimastat (mean ± SEM, * p < 0.05 vs. non-stimulated HMEC, # p < 0.05 stimulation in the presence of Batimastat vs. absence of Batimastat, n = 12); D: representative zymography of b-EVs and PDGF-EVs; intensity of MMP2 and MMP9 bands are indicated; E: representative zymography of conditioned media of HMEC 24 hours after b-EV or PDGF-EV addition; proMMP-9 (92 kD), active MMP-9 (82 kD), proMMP-2 (72 kD) and active MMP-2 (62 kD) are present; the intensity (itn.) of bands is indicated.
Mentions: DNA was not detected in 10k and 100k EV fractions after DNA extraction and analysis using spectrophotometry (NanoDrop) and by agarose gel electrophoresis (Bioanalyser). EVs were stained with PKH26, which labelled membrane phospholipids, indicating that particles detected both in 10K and 100K fractions were not protein aggregates (Figure 2A). Both 10k and 100k fractions expressed several mesenchymal surface markers characteristic of cell origin as seen by GUAVA FACS analysis. EVs expressed mesenchymal surface markers (CD73, CD29, CD90, CD105, CD44), endothelial markers (CD105, CD31), and marker of exosomes (CD63, CD81). The expression did not change in different fractions of EVs or EVs obtained after stimulation with PDGF with the exception of CD81 expression, which was increased in 100 k fraction of PDGF-EVs in respect to b-EVs (Table 1). Similar results were obtained by FACS analysis performed on vesicles pre-absorbed on beads (not shown).

Bottom Line: Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC).The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Sciences and Molecular Biotechnology Center, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy. giovanni.camussi@unito.it.

ABSTRACT

Background: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process.

Results: Herein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.

Conclusions: Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

Show MeSH
Related in: MedlinePlus