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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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Membrane expression of CD31 and VEGFR2 on late EPCs A and B. CD31 and VEGFR2 surface density on EPCs during a 5-week ex vivo expansion period (corresponding to 45 to 60 days of culture), by comparison to HUVECs. A mean of 15 colonies were tested at each time point. Boxes represent the median values with the 25th and 75th percentiles, and the bar chart shows the 90th and 10th percentiles. EPC surface CD31 and VEGFR2 expression were quantified by flow cytometry using a calibrator (Qifikit, Dako) containing a mixture of five calibration beads coated with increasing densities of mouse IgG (approximately 3000 to 600,000 molecules). The staining reagent was a polyclonal FITC-conjugated f(ab’)2 fragment of a goat antimouse antibody. Surface molecule numbers were derived from the calibration curve, after subtracting the negative isotype control value. C. Time course of VEGFR2 density on EPCs derived from four different colonies of late EPCs. VEGFR2 was quantified by flow cytometry every week during a 5-week expansion period.
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fig03: Membrane expression of CD31 and VEGFR2 on late EPCs A and B. CD31 and VEGFR2 surface density on EPCs during a 5-week ex vivo expansion period (corresponding to 45 to 60 days of culture), by comparison to HUVECs. A mean of 15 colonies were tested at each time point. Boxes represent the median values with the 25th and 75th percentiles, and the bar chart shows the 90th and 10th percentiles. EPC surface CD31 and VEGFR2 expression were quantified by flow cytometry using a calibrator (Qifikit, Dako) containing a mixture of five calibration beads coated with increasing densities of mouse IgG (approximately 3000 to 600,000 molecules). The staining reagent was a polyclonal FITC-conjugated f(ab’)2 fragment of a goat antimouse antibody. Surface molecule numbers were derived from the calibration curve, after subtracting the negative isotype control value. C. Time course of VEGFR2 density on EPCs derived from four different colonies of late EPCs. VEGFR2 was quantified by flow cytometry every week during a 5-week expansion period.

Mentions: Given the strong expression of the VEGFR2 gene during EPC expansion, we quantified the receptor density on the EPC membrane. As a control, we quantified CD31, reported to be the first endothelial antigen to be expressed by progenitor cells committed to the endothelial lineage [27]. EPCs were expanded for 5 weeks after the first passage, corresponding to a total of 45 to 60 days of culture. During the expansion phase, CD31 and VEGFR2 density was measured by means of quantitative flow cytometry, as previously described [9]. As shown in Fig. 3A, CD31 density varied widely among late EPC colonies but was of the same order as that found on HUVECs and remained constant during the 5-week expansion period. In keeping with gene expression analysis, the VEGFR2 protein level increased gradually and significantly throughout the expansion period (trend test = 0.02). VEGFR2 density rose from a mean value of 9200 sites per cell at week 3 to 25,800 sites at week 5 (Fig. 3B). This 3-fold increase from week 3 to week 5 was in line with the concomitant increase in mRNA (Fig. 2). VEGFR2 density was significantly higher on EPCs than on HUVECs at week 4 (P= 0.001) and week 5 (P= 0.03) of expansion, again in keeping with the concomitant 5-fold increase in the mRNA level. This increase was EPC colony-independent, as shown by the kinetics of VEGFR2 expression on cells expanded from single EPC colonies (four typical examples are shown in Fig. 3C). The consequences of this increase in VEGFR2 receptor expression were then evaluated in vitro on the three theoretical steps of angiogenesis, i.e., proliferation, migration and differentiation.


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)

Membrane expression of CD31 and VEGFR2 on late EPCs A and B. CD31 and VEGFR2 surface density on EPCs during a 5-week ex vivo expansion period (corresponding to 45 to 60 days of culture), by comparison to HUVECs. A mean of 15 colonies were tested at each time point. Boxes represent the median values with the 25th and 75th percentiles, and the bar chart shows the 90th and 10th percentiles. EPC surface CD31 and VEGFR2 expression were quantified by flow cytometry using a calibrator (Qifikit, Dako) containing a mixture of five calibration beads coated with increasing densities of mouse IgG (approximately 3000 to 600,000 molecules). The staining reagent was a polyclonal FITC-conjugated f(ab’)2 fragment of a goat antimouse antibody. Surface molecule numbers were derived from the calibration curve, after subtracting the negative isotype control value. C. Time course of VEGFR2 density on EPCs derived from four different colonies of late EPCs. VEGFR2 was quantified by flow cytometry every week during a 5-week expansion period.
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Related In: Results  -  Collection

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fig03: Membrane expression of CD31 and VEGFR2 on late EPCs A and B. CD31 and VEGFR2 surface density on EPCs during a 5-week ex vivo expansion period (corresponding to 45 to 60 days of culture), by comparison to HUVECs. A mean of 15 colonies were tested at each time point. Boxes represent the median values with the 25th and 75th percentiles, and the bar chart shows the 90th and 10th percentiles. EPC surface CD31 and VEGFR2 expression were quantified by flow cytometry using a calibrator (Qifikit, Dako) containing a mixture of five calibration beads coated with increasing densities of mouse IgG (approximately 3000 to 600,000 molecules). The staining reagent was a polyclonal FITC-conjugated f(ab’)2 fragment of a goat antimouse antibody. Surface molecule numbers were derived from the calibration curve, after subtracting the negative isotype control value. C. Time course of VEGFR2 density on EPCs derived from four different colonies of late EPCs. VEGFR2 was quantified by flow cytometry every week during a 5-week expansion period.
Mentions: Given the strong expression of the VEGFR2 gene during EPC expansion, we quantified the receptor density on the EPC membrane. As a control, we quantified CD31, reported to be the first endothelial antigen to be expressed by progenitor cells committed to the endothelial lineage [27]. EPCs were expanded for 5 weeks after the first passage, corresponding to a total of 45 to 60 days of culture. During the expansion phase, CD31 and VEGFR2 density was measured by means of quantitative flow cytometry, as previously described [9]. As shown in Fig. 3A, CD31 density varied widely among late EPC colonies but was of the same order as that found on HUVECs and remained constant during the 5-week expansion period. In keeping with gene expression analysis, the VEGFR2 protein level increased gradually and significantly throughout the expansion period (trend test = 0.02). VEGFR2 density rose from a mean value of 9200 sites per cell at week 3 to 25,800 sites at week 5 (Fig. 3B). This 3-fold increase from week 3 to week 5 was in line with the concomitant increase in mRNA (Fig. 2). VEGFR2 density was significantly higher on EPCs than on HUVECs at week 4 (P= 0.001) and week 5 (P= 0.03) of expansion, again in keeping with the concomitant 5-fold increase in the mRNA level. This increase was EPC colony-independent, as shown by the kinetics of VEGFR2 expression on cells expanded from single EPC colonies (four typical examples are shown in Fig. 3C). The consequences of this increase in VEGFR2 receptor expression were then evaluated in vitro on the three theoretical steps of angiogenesis, i.e., proliferation, migration and differentiation.

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