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Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6).

Smadja DM, Bièche I, Helley D, Laurendeau I, Simonin G, Muller L, Aiach M, Gaussem P - J. Cell. Mol. Med. (2007 Sep-Oct)

Bottom Line: Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs.VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression.In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Service d'Hématologie Biologique A, Hôpital Européen Georges Pompidou, Paris, France.

ABSTRACT
In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34(+) cord blood cells and expanded for up to 5 weeks. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34(+) cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

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Influence of in vitro expansion on EPC proliferation and markers of apoptosis A. Effect of VEGF on EPC proliferation, as evaluated by the release of pNPP (OD at 405 nm) in EBM-2 medium containing 2% FBS (mean ± SEM). VEGF induced late EPC proliferation at week 3 (*P= 0.004) and week 5 (*P= 0.046) compared to control EPCs. No significant difference was observed between week 3 and week 5 of expansion (P= 0.701). The mean and SEM of three experiments are shown. B. Effect of in vitro expansion on mRNA levels of proliferative and anti-apoptotic factors. mRNA levels were normalized to TBP mRNA levels and to the sample with the lowest quantifiable level (i.e. 1 on the left ordinate, corresponding to a Ct value of 35). Values above 100 represent strong gene expression. Mean and SEM of three different colonies are shown at each point.
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fig04: Influence of in vitro expansion on EPC proliferation and markers of apoptosis A. Effect of VEGF on EPC proliferation, as evaluated by the release of pNPP (OD at 405 nm) in EBM-2 medium containing 2% FBS (mean ± SEM). VEGF induced late EPC proliferation at week 3 (*P= 0.004) and week 5 (*P= 0.046) compared to control EPCs. No significant difference was observed between week 3 and week 5 of expansion (P= 0.701). The mean and SEM of three experiments are shown. B. Effect of in vitro expansion on mRNA levels of proliferative and anti-apoptotic factors. mRNA levels were normalized to TBP mRNA levels and to the sample with the lowest quantifiable level (i.e. 1 on the left ordinate, corresponding to a Ct value of 35). Values above 100 represent strong gene expression. Mean and SEM of three different colonies are shown at each point.

Mentions: In order to explore the effect of VEGFR2 activation on EPC proliferation, cells were deprived of serum and growth factors (EBM-2 medium) for 16 hrs before adding 50 ng/ml human VEGF. In these conditions, VEGF induced an expected EPC proliferation, as quantified by pNPP release at week 3 of expansion (Fig. 4A). However, the increase in VEGFR2 expression observed at week 5 of expansion did not coincide with an increase in cell proliferation. We then determined at week 3 and 5 of expansion basal expression of mRNA levels of several proliferation or anti-apoptotic factors, including Ki67, Topoisomerase2A (TOP2A), anti-apoptotic members of the bcl-2 protein family bcl-2 and bcl-A1 and an anti-apoptotic protein shown to be a target of p53 GADD45 (Fig. 4B). In vitro expansion of EPCs induced an increase in proliferation markers Ki67 and TOP2A, compared to CD34+ cells. Moreover, we observe a decrease of 50% of these two proliferation markers after 5 weeks of culture. Contrary to proliferation markers, anti-apoptotic markers decrease between CD34+ cells and EPCs after 3 weeks of culture, this phenomenon is also observed between EPCs at week 3 and 5 of culture. Absence of increased proliferation during expansion could also be explained by a decrease of proliferation and anti-apoptotic potential of EPCs during expansion.


Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6).

Smadja DM, Bièche I, Helley D, Laurendeau I, Simonin G, Muller L, Aiach M, Gaussem P - J. Cell. Mol. Med. (2007 Sep-Oct)

Influence of in vitro expansion on EPC proliferation and markers of apoptosis A. Effect of VEGF on EPC proliferation, as evaluated by the release of pNPP (OD at 405 nm) in EBM-2 medium containing 2% FBS (mean ± SEM). VEGF induced late EPC proliferation at week 3 (*P= 0.004) and week 5 (*P= 0.046) compared to control EPCs. No significant difference was observed between week 3 and week 5 of expansion (P= 0.701). The mean and SEM of three experiments are shown. B. Effect of in vitro expansion on mRNA levels of proliferative and anti-apoptotic factors. mRNA levels were normalized to TBP mRNA levels and to the sample with the lowest quantifiable level (i.e. 1 on the left ordinate, corresponding to a Ct value of 35). Values above 100 represent strong gene expression. Mean and SEM of three different colonies are shown at each point.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401281&req=5

fig04: Influence of in vitro expansion on EPC proliferation and markers of apoptosis A. Effect of VEGF on EPC proliferation, as evaluated by the release of pNPP (OD at 405 nm) in EBM-2 medium containing 2% FBS (mean ± SEM). VEGF induced late EPC proliferation at week 3 (*P= 0.004) and week 5 (*P= 0.046) compared to control EPCs. No significant difference was observed between week 3 and week 5 of expansion (P= 0.701). The mean and SEM of three experiments are shown. B. Effect of in vitro expansion on mRNA levels of proliferative and anti-apoptotic factors. mRNA levels were normalized to TBP mRNA levels and to the sample with the lowest quantifiable level (i.e. 1 on the left ordinate, corresponding to a Ct value of 35). Values above 100 represent strong gene expression. Mean and SEM of three different colonies are shown at each point.
Mentions: In order to explore the effect of VEGFR2 activation on EPC proliferation, cells were deprived of serum and growth factors (EBM-2 medium) for 16 hrs before adding 50 ng/ml human VEGF. In these conditions, VEGF induced an expected EPC proliferation, as quantified by pNPP release at week 3 of expansion (Fig. 4A). However, the increase in VEGFR2 expression observed at week 5 of expansion did not coincide with an increase in cell proliferation. We then determined at week 3 and 5 of expansion basal expression of mRNA levels of several proliferation or anti-apoptotic factors, including Ki67, Topoisomerase2A (TOP2A), anti-apoptotic members of the bcl-2 protein family bcl-2 and bcl-A1 and an anti-apoptotic protein shown to be a target of p53 GADD45 (Fig. 4B). In vitro expansion of EPCs induced an increase in proliferation markers Ki67 and TOP2A, compared to CD34+ cells. Moreover, we observe a decrease of 50% of these two proliferation markers after 5 weeks of culture. Contrary to proliferation markers, anti-apoptotic markers decrease between CD34+ cells and EPCs after 3 weeks of culture, this phenomenon is also observed between EPCs at week 3 and 5 of culture. Absence of increased proliferation during expansion could also be explained by a decrease of proliferation and anti-apoptotic potential of EPCs during expansion.

Bottom Line: Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs.VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression.In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Service d'Hématologie Biologique A, Hôpital Européen Georges Pompidou, Paris, France.

ABSTRACT
In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34(+) cord blood cells and expanded for up to 5 weeks. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34(+) cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

Show MeSH
Related in: MedlinePlus