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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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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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fig06: 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).

Mentions: Finally, we used a Matrigel model to examine the capacity of VEGF-activated EPCs to differentiate into capillary-like structures. After 16 hrs of culture in unsupplemented EBM-2 medium, EPCs formed few capillary-like structures (Fig. 6A, top panel). At week 3 of expansion, treatment with VEGF (50 ng/ml) promoted EPC organization into branched structures and pseudotubes with enclosed areas (network length: 1532 ± 41 μm in untreated controls versus 3005 ± 743 μm in VEGF-treated cells, (P= 0.011)) (Fig. 6A and B). At week 5 of expansion, VEGF treatment increased the density of this network 1.5-fold compared to week 3 of expansion (Fig. 6B). To explain this result, we explored the expression of surface proteins potentially involved in tube formation, such as integrin subunits α6 and β1[28], and the adhesion molecule CD31 (PECAM-1) [29]. No significant increase was found in β1 or CD31 expression upon VEGF activation (data not shown). By contrast, integrin α6 subunit expression was up-regulated 1.5-fold upon VEGF treatment after 3 weeks of expansion (P= 0.002, Fig. 6C). After 5 weeks of expansion, VEGF treatment increased integrin α6 subunit expression 3-fold compared to week 3 (P= 0.025), in line with the increase in VEGFR2 expression. An anti-α6 blocking antibody completely inhibited VEGF-induced tube formation (Fig. 6A, bottom), whereas an irrelevant isotype-matched antibody had no effect (data not shown).


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)

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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Related In: Results  -  Collection

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fig06: 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).
Mentions: Finally, we used a Matrigel model to examine the capacity of VEGF-activated EPCs to differentiate into capillary-like structures. After 16 hrs of culture in unsupplemented EBM-2 medium, EPCs formed few capillary-like structures (Fig. 6A, top panel). At week 3 of expansion, treatment with VEGF (50 ng/ml) promoted EPC organization into branched structures and pseudotubes with enclosed areas (network length: 1532 ± 41 μm in untreated controls versus 3005 ± 743 μm in VEGF-treated cells, (P= 0.011)) (Fig. 6A and B). At week 5 of expansion, VEGF treatment increased the density of this network 1.5-fold compared to week 3 of expansion (Fig. 6B). To explain this result, we explored the expression of surface proteins potentially involved in tube formation, such as integrin subunits α6 and β1[28], and the adhesion molecule CD31 (PECAM-1) [29]. No significant increase was found in β1 or CD31 expression upon VEGF activation (data not shown). By contrast, integrin α6 subunit expression was up-regulated 1.5-fold upon VEGF treatment after 3 weeks of expansion (P= 0.002, Fig. 6C). After 5 weeks of expansion, VEGF treatment increased integrin α6 subunit expression 3-fold compared to week 3 (P= 0.025), in line with the increase in VEGFR2 expression. An anti-α6 blocking antibody completely inhibited VEGF-induced tube formation (Fig. 6A, bottom), whereas an irrelevant isotype-matched antibody had no effect (data not shown).

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