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Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis.

Wyler von Ballmoos M, Yang Z, Völzmann J, Baumgartner I, Kalka C, Di Santo S - PLoS ONE (2010)

Bottom Line: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01).All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01).This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors.

Objective: The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells.

Methods and results: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone.

Conclusion: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

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EPC-CM induces PDGFRβ expression in HUVEC.mRNA expression of PDGFRβ was determined by real-time PCR of total RNA obtained from HUVEC cultured in EPC-CM or control medium. EPC-CM incubation promoted a 2.8-fold over-expression of PDGFRβ mRNA in HUVEC compared to control medium incubation. Incubation with HUVEC-CM did not change mRNA expression levels of PDGFRβ as compared to control medium. (A). Protein level of PDGFRβ was analyzed by Western blot (B). The expressions of total PDGFRβ (C) as well as its phosphorylated form (D) were significantly up-regulated upon incubation with EPC-CM. *, P<0.05.
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pone-0014107-g004: EPC-CM induces PDGFRβ expression in HUVEC.mRNA expression of PDGFRβ was determined by real-time PCR of total RNA obtained from HUVEC cultured in EPC-CM or control medium. EPC-CM incubation promoted a 2.8-fold over-expression of PDGFRβ mRNA in HUVEC compared to control medium incubation. Incubation with HUVEC-CM did not change mRNA expression levels of PDGFRβ as compared to control medium. (A). Protein level of PDGFRβ was analyzed by Western blot (B). The expressions of total PDGFRβ (C) as well as its phosphorylated form (D) were significantly up-regulated upon incubation with EPC-CM. *, P<0.05.

Mentions: To further investigate the signaling pathway underlying the above findings suggesting that PDGFRβ plays a major role in the EPC-CM stimulated angiogenic response of differentiated EC, we measured the transcriptional and translational levels of PDGFRβ in HUVEC. Upon EPC-CM stimulation, the mRNA level of PDGFRβ was up-regulated 2.8±0.6 -fold compared to control medium (P<0.05, Figure 4A). At the same time, conditioned medium obtained from differentiated endothelial cells (HUVEC-CM) did not change the expression levels of PDGFRβ. Western blotting also showed a significantly increased amount of both total PDGFRβ (2.31±0.56 -fold, P<0.05) and its phosphorylated form (3.67±0.66 -fold, P<0.05) expressed by HUVEC when incubated with EPC-CM (Figure 4B-D). Moreover, FACS analyses revealed a clear shift of HUVEC from a vWF+/PDGFRβ− towards a vWF+/PDGFRβ+ double positive phenotype after EPC-CM incubation (Figure 5A-B, E; P<0.001). Neutralizing the PDGFRβ bioactivity with AF385 had only marginal effect on blocking the stimulation by EPC-CM, and resulted in a similar upregulation of the PDGFRβ (Figure 5C, E; P<0.01). However, such an increased expression of PDGFRβ in HUVEC could not be elicited by adding 100 pg/ml or 100 ng/ml of rhPDGF-BB to the control medium (Figure 5C-D, E).


Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis.

Wyler von Ballmoos M, Yang Z, Völzmann J, Baumgartner I, Kalka C, Di Santo S - PLoS ONE (2010)

EPC-CM induces PDGFRβ expression in HUVEC.mRNA expression of PDGFRβ was determined by real-time PCR of total RNA obtained from HUVEC cultured in EPC-CM or control medium. EPC-CM incubation promoted a 2.8-fold over-expression of PDGFRβ mRNA in HUVEC compared to control medium incubation. Incubation with HUVEC-CM did not change mRNA expression levels of PDGFRβ as compared to control medium. (A). Protein level of PDGFRβ was analyzed by Western blot (B). The expressions of total PDGFRβ (C) as well as its phosphorylated form (D) were significantly up-regulated upon incubation with EPC-CM. *, P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991332&req=5

pone-0014107-g004: EPC-CM induces PDGFRβ expression in HUVEC.mRNA expression of PDGFRβ was determined by real-time PCR of total RNA obtained from HUVEC cultured in EPC-CM or control medium. EPC-CM incubation promoted a 2.8-fold over-expression of PDGFRβ mRNA in HUVEC compared to control medium incubation. Incubation with HUVEC-CM did not change mRNA expression levels of PDGFRβ as compared to control medium. (A). Protein level of PDGFRβ was analyzed by Western blot (B). The expressions of total PDGFRβ (C) as well as its phosphorylated form (D) were significantly up-regulated upon incubation with EPC-CM. *, P<0.05.
Mentions: To further investigate the signaling pathway underlying the above findings suggesting that PDGFRβ plays a major role in the EPC-CM stimulated angiogenic response of differentiated EC, we measured the transcriptional and translational levels of PDGFRβ in HUVEC. Upon EPC-CM stimulation, the mRNA level of PDGFRβ was up-regulated 2.8±0.6 -fold compared to control medium (P<0.05, Figure 4A). At the same time, conditioned medium obtained from differentiated endothelial cells (HUVEC-CM) did not change the expression levels of PDGFRβ. Western blotting also showed a significantly increased amount of both total PDGFRβ (2.31±0.56 -fold, P<0.05) and its phosphorylated form (3.67±0.66 -fold, P<0.05) expressed by HUVEC when incubated with EPC-CM (Figure 4B-D). Moreover, FACS analyses revealed a clear shift of HUVEC from a vWF+/PDGFRβ− towards a vWF+/PDGFRβ+ double positive phenotype after EPC-CM incubation (Figure 5A-B, E; P<0.001). Neutralizing the PDGFRβ bioactivity with AF385 had only marginal effect on blocking the stimulation by EPC-CM, and resulted in a similar upregulation of the PDGFRβ (Figure 5C, E; P<0.01). However, such an increased expression of PDGFRβ in HUVEC could not be elicited by adding 100 pg/ml or 100 ng/ml of rhPDGF-BB to the control medium (Figure 5C-D, E).

Bottom Line: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01).All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01).This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Surgery, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors.

Objective: The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells.

Methods and results: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone.

Conclusion: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

Show MeSH
Related in: MedlinePlus