Fig 1.
Comparison of ECFCs colony outgrowth in fetal bovine serum and platelet lysate.
Representative images of ECFC colonies that appeared in FBS-EGM (A) and PL-EGM (B,C) between 10–30 days. (D): The average number of colonies that grew in FBS (open triangle) and PL (black triangle) from 10 isolations at day 10. (E): The number of colonies yields in FBS-EGM-2 (open triangle) or PL-EGM (black triangle) during the isolation period in all donors relative to the number of mL of peripheral blood. Data are expressed as mean±SEM. Statistical analysis was performed with non-parametric Wilcoxon matched pairs test (* p<0.05).
Fig 2.
Phenotypical characterization of PB-ECFCs.
PB-ECFCs monolayers expanded in EGM-2 medium supplemented with 10%PL were assessed for the presence of endothelial cell markers by immunofluorescence cytochemistry (A-C) and flow cytometry as well as for uptake of Dil-Ac-LDL (D). For immunofluorescence cytochemistry staining, cells were seeded on glass cover slips, fixed and stained with antibody against CD31, VE-cadherin or vWF. Cell nuclei were visualized with DAPI staining. Cells were positive for CD31 (A, green), VE-cadherin (B, green), and vWF (C, green). Cell nuclei appear blue. (D): Incorporation of Dil-Ac-LDL by PB-ECFCs (red spots, cell nuclei stained blue with DAPI). Panel E:Flow cytometry characterization of PB-ECFCs for CD31, CD34, CD309, CD144, CD146, CD105, CD14, CD45, and CD133. Plots depict control isotype IgG staining (black histograms) versus specific antibody staining (empty histograms).
Fig 3.
Proliferative potential of PB-ECFCs in FBS and PL medium.
For growth kinetics as well as cumulative population doubling determination experiments, cells were serially expanded by seeding 5000 cells/cm2 in EGM-2 supplemented with either 10%FBS or 10%PL during period of 40 days. (A): Comparison of proliferative potential of PB-ECFCs maintained in FBS-EGM (open bar) or PL-EGM (closed bar) during 7 days. Results represent the mean ± SEM of counted number of cells relative to cm2 of plating surface of 3 independent experiments of three different donors. *p < 0.05 by Student paired t test. (B): Total number of cells yielded during long-term expansion of PB-ECFCs in FBS-EGM (open symbol, n:3) or PL-EGM (closed symbols, n:3). Each symbol indicates total number of cells at each passaging step. (C): Cumulative population doubling levels (CPDL) of PB-ECFCs in PL-EGM (closed bars) or FBS-EGM (open bars) after 10, 20, and 40 days of expansion. Results represent the mean ± SEM of CPDL at three different time points of 3 independent experiments of 3 different donors. Two-way ANOVA with Bonferroni post hoc test.
Fig 4.
Comparison of proliferation-related features in PB-ECFCs during long-term cell expansion.
During long-term expansion of PB-ECFCs in PL-EGM, a set of experiments at different CPDLs (white bar 6CPDL, grey bar 18CPDL, black bar 31CPDL) were performed to investigate proliferation rate, telomere length and expression of the progenitor-cell marker CD34. (A) Comparison of population doubling time (PDT). (B): Telomere (T) and single copy gene (S) were amplified by quantitative real-time PCR, with the T/S ratio proportional to telomere length at different stages of cells’ age. (C): Flow cytometry data of percentage of cells positive for CD34. Results represent the mean ± SEM of 3 independent experiments each performed with different donor at indicated CPDL. Comparison between each CPDL was performed by one-way ANOVA with Bonferroni post hoc test (*p<0.05).
Fig 5.
Comparison of tube-forming capacity of PB-ECFCs at different time-points of ex vivo expansion upon stimulation with VEGF-A and FGF-2.
PB-ECFCs obtained from 3 different donors were serially expanded in medium supplemented with PL and the sprouting ability of cells in fibrin matrices was assessed at 6, 18, and 31 CPDL. Cells at indicated CPDL (white bar 6 CPDL, grey bar 18 CPDL, black bar 31 CPDL) were either unstimulated (A) or stimulated with FGF-2(B), and VEGF-A(C), FGF-2+VEGF-A (D),TNF-α. Results represent the mean ± SEM of mean tube length of tube-like structures of the 3 donors each performed at indicated CPDL. Comparison between each CPDLs was performed using one-way ANOVA with Bonferroni post hoc test.(*p < 0.05).
Fig 6.
Expression of genes and soluble antigens involved during sprouting in fibrin matrix in PB-ECFCs at different maturation stages.
Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from PB-ECFCs at different CPDL (open bar 6 CPDL, grey bar 18 CPDL, black bar 31 CPDL). Gene expression levels of uPA (A), uPAR (B), tPA (C), and PAI-1 (D) in PB-ECFCs. Data are expressed as n-fold difference of expression of genes in cells at 6 CPDL. One-way ANOVA with Bonferroni post hoc test (p<0.05).Evaluation of the production of uPA (E) and PAI-1 (F) antigens in CM by PB-ECFCs was performed at 6 CPDL (open bar), 18 CPDL (grey bar), and 31 CPDL (black bar) using ELISA. Results represent the mean ± SEM of uPA or PAI-1 concentration in ng relative to mL of conditioned medium of 3 independent experiments each performed at indicated CPDL. Comparison between different groups was performed one-way ANOVA with Bonferroni post hoc test (p<0.05).
Fig 7.
Effect of uPA, uPAR and PAI-1 knockdown on angiogenic response of PB-ECFCs in fibrin matrices.
The involvement of uPA/uPAR/PAI-1 system during sprouting in fibrin matrices was assessed in PB-ECFCs obtained from 3 different donors at 18 CPDL. A and B: diminished angiogenic ability of PB-ECFCs transfected with siRNA-uPA or siRNA-uPAR in fibrin matrices. C: sprout formation by cells transfected with non-targeting, control siRNA (si-RNA-NT). D: sprout formation by cells transfected with siRNA-PAI-1 (si-RNA-PAi-1). E: Comparison of angiogenic response of PB-ECFCs transfected with non-targeting siRNA (siRNA-NT) and siRNA targeting uPA (siRNA-uPA), uPAR (siRNA-uPAR) or siRNA-PAI-1 (si-RNA-PAi-1) expressed as mean ± SEM of mean tube length of tube-like structures of 3 independent experiments of 3 different donors (open bars: unstimulated cells, black bars: cells stimulated with 10ng/mL TNF-α + 10ng/mL FGF-2). Comparison between different groups was performed using two-way ANOVA with Bonferroni post hoc test (p<0.05).
Fig 8.
Expression of inflammatory activation-related markers.
Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from 3 donors at 6, 18, and 31 CPDL. Gene expression levels of ICAM-1 (A), VCAM-1 (B), and uPA (C) in individual donors PB15 (◆), PB84 (●), and PB224(▲), during long-term expansion. Analysis of uPA expression was performed with the same data set as depicted in Fig 7A. Data are expressed as n-fold difference of expression of same genes in cells at 6 CPDL.