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. 2007 Sep-Oct;11(5):1149-61.
doi: 10.1111/j.1582-4934.2007.00090.x.

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

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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)

David M Smadja et al. J Cell Mol Med. 2007 Sep-Oct.

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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Figures

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Characterization of late EPCs. A. Morphological aspect and confocal immuno-fluorescence analysis showing VWF expression (green) and Dil-Ac-LDL incorporation (red) of late EPC colonies emerging from CD34+ cord blood cells cultured for 2 weeks in endothelial conditions. Confocal images were acquired with a x63/1.32 PL APO objective. Photomicrographs of EPCs derived from cord blood CD34+ cells are representative of at least three observations. B. Representative histograms based on flow cytometric analysis of detached EPCs after immunolabelling with a control antibody (black line) and specific antibodies (red line) to endothelial markers (CD146, CD31, CD144, KDR and Tie-2), haematopoietic markers (CD34 and CD133) and leukocyte markers (CD45 and CD14). Histograms of EPCs derived from cord blood CD34+ cells are representative of at least three observations.
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VEGFR1, 2 and 3 and CD31 mRNA quantification in CD34+ cells, late EPCs, and HUVECs. 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 a strong gene expression. Mean and SEM values of three different colonies are shown at each point.
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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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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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In vitro expansion modulates VEGF-induced EPC migration VEGF-induced EPC chemotaxis was tested in a Boyden chamber migration assay. Cell expansion increased late EPC migration towards VEGF (10 ng/ml). Data are the numbers of migrating EPCs. The mean and SEM of three experiments are shown (*P= 0.023).
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In vitro expansion modulates EPC tube formation in Matrigel by up-regulating integrin α6 expression. EPCs were stimulated with VEGF (50 ng/ml) for 36 hrs before being used in the tubule formation assay on Matrigel for 18 hrs. EPCs were plated on Matrigel in the presence or absence of a monoclonal antibody against human α6 (clone GoH3; R&D systems, 10 μg/ml). Results are means ± SEM of three determinations.*: P < 0.05. A. Photographs show pseudotube formation by untreated EPCs and EPCs treated with 50 ng/ml VEGF. Bottom:EPCs treated with VEGF were incubated with 10 μg/ml anti-α6 antibody. Photos (original magnification, x20) are representative of three independent experiments of EPCs after 3 weeks of culture. B. Quantitative analysis of network length of untreated EPCs, EPCs treated with 50 ng/ml VEGF with or without 10 μg/ml anti-α6 antibody at week 3 and week 5 of expansion. Quantitative analysis of network length with Videomet software. (network length of control W3 versus VEGF W3 and VEGF W3 versus VEGF W5, respectively, P= 0.011 and P= 0.009); (network length of VEGF W3 versus VEGF W3 with anti- α6 and VEGF W5 versus VEGF W5 with anti- α6, respectively, P= 0.024 and P= 0.005). C. Effect of VEGF on EPC integrin α6 subunit expression. EPCs were analysed by flow cytometry before and after treatment with VEGF (50 ng/ml). Geometric mean fluorescence intensities are expressed in percentages, 100% corresponding to the control value obtained with VEGF treatment at week 3 of expansion.(Geometric mean fluorescence intensities of control W3 versus VEGF W3 and VEGF W3 versus VEGF W5 respectively P= 0.002 and P= 0.028).

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