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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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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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fig01: 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.

Mentions: When cultured in the presence of specific endothelial growth factors (EGM-2 medium), human cord blood CD34+ cells yielded small colonies after between 7 and 14 days of culture. At confluence, EPCs exhibited a cobblestone morphology and a monolayer growth pattern typical of the endothelial lineage, expressed VWF and were able to incorporate diL-low-density lipoprotein (LDL) (Fig. 1A) [8, 9, 26]. The endothelial phenotype of expanded EPCs (so-called late EPCs) [6] was further characterized by measuring the expression of endothelial markers, such as CD31 (PECAM), CD146 (S-Endo1) and VEGFR2 (Fig. 1B). Late EPCs expressed CD34 antigen, but not the haematopoietic stem cell antigen CD133 or the leukocyte markers CD45 and CD14.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401281&req=5

fig01: 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.
Mentions: When cultured in the presence of specific endothelial growth factors (EGM-2 medium), human cord blood CD34+ cells yielded small colonies after between 7 and 14 days of culture. At confluence, EPCs exhibited a cobblestone morphology and a monolayer growth pattern typical of the endothelial lineage, expressed VWF and were able to incorporate diL-low-density lipoprotein (LDL) (Fig. 1A) [8, 9, 26]. The endothelial phenotype of expanded EPCs (so-called late EPCs) [6] was further characterized by measuring the expression of endothelial markers, such as CD31 (PECAM), CD146 (S-Endo1) and VEGFR2 (Fig. 1B). Late EPCs expressed CD34 antigen, but not the haematopoietic stem cell antigen CD133 or the leukocyte markers CD45 and CD14.

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