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CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs).

Tasev D, Konijnenberg LS, Amado-Azevedo J, van Wijhe MH, Koolwijk P, van Hinsbergh VW - Angiogenesis (2016)

Bottom Line: Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells.Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression.Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center Amsterdam, De Boelelaan 1118, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT
Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(-) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(-) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(-): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells. However, during cell culture CD34(-) cells re-expressed CD34. Cell-seeding density, cell-cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(-) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function.

No MeSH data available.


Related in: MedlinePlus

Effect of serum supplements on CD34 expression in PB-ECFCs. a Time course of CD34 induction in PB-ECFCs upon incubation with media supplemented with different serum supplements during 24-h period. Open circle depicts 10 % NBCS, open square depicts 10 % NBCS + 10 % PL, open pointed up triangle depicts 10 % PL, open diamond depicts 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 mRNA levels compared to the mRNA levels in the cells incubated with M199 + 3 % HSA at each time point (HSA, p < 0.05: # to NBCS, * to NBCS + PL, & to PL; PL, p < 0.05: $ to NBCS, ∞ to NBCS + PL). b Flow cytometry assessment of CD34 expression in PB-ECFCs cultured in the presence of NBCS or PL. Data are expressed as a mean ± SEM percentage of cells positive for CD34 in the cell cultures incubated with CMi medium which contains 10 % NBCS (open bar) or M199 + 10 % PL (closed bar). c qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), FV (10 ng/mL FGF-2 + 25 ng/mL VEGF-A), HGF (10 ng/mL), and 10 % PL for 24 h prepared in M199 + 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in M199 + 3 % HSA (open bar). d qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in EBM-2 + 5 % PL (open bar). e Flow cytometry evaluation of the effect of VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL on CD34 expression in PB-ECFCs. Closed bars represent the effect of each of soluble factors prepared in control medium (open bar) on CD34 expression. Data are expressed as mean ± SEM of mean fluorescence intensity (MFI) of CD34 antibody fluorescence intensity minus autofluorescence of matched isotype antibody
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Fig3: Effect of serum supplements on CD34 expression in PB-ECFCs. a Time course of CD34 induction in PB-ECFCs upon incubation with media supplemented with different serum supplements during 24-h period. Open circle depicts 10 % NBCS, open square depicts 10 % NBCS + 10 % PL, open pointed up triangle depicts 10 % PL, open diamond depicts 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 mRNA levels compared to the mRNA levels in the cells incubated with M199 + 3 % HSA at each time point (HSA, p < 0.05: # to NBCS, * to NBCS + PL, & to PL; PL, p < 0.05: $ to NBCS, ∞ to NBCS + PL). b Flow cytometry assessment of CD34 expression in PB-ECFCs cultured in the presence of NBCS or PL. Data are expressed as a mean ± SEM percentage of cells positive for CD34 in the cell cultures incubated with CMi medium which contains 10 % NBCS (open bar) or M199 + 10 % PL (closed bar). c qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), FV (10 ng/mL FGF-2 + 25 ng/mL VEGF-A), HGF (10 ng/mL), and 10 % PL for 24 h prepared in M199 + 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in M199 + 3 % HSA (open bar). d qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in EBM-2 + 5 % PL (open bar). e Flow cytometry evaluation of the effect of VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL on CD34 expression in PB-ECFCs. Closed bars represent the effect of each of soluble factors prepared in control medium (open bar) on CD34 expression. Data are expressed as mean ± SEM of mean fluorescence intensity (MFI) of CD34 antibody fluorescence intensity minus autofluorescence of matched isotype antibody

Mentions: We subsequently evaluated which serum supplement(s) might modulate CD34 expression in PB-ECFCs. To that end we compared platelet lysate and serum used in CMi, in particular 10 % NBCS and 10 % HS. Confluent monolayers of PB-ECFCs grown in CMi were transferred into M199 medium supplemented with 10 % NBCS, 10 % PL, 10 % NBCS/10 % PL, or 3 % HSA and incubated for 9, 12, and 24 h. In the presence of only 3 % HSA, CD34 mRNA expression increased fivefold within 9 h and remained constant thereafter (Fig. 3a).Fig. 3


CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs).

Tasev D, Konijnenberg LS, Amado-Azevedo J, van Wijhe MH, Koolwijk P, van Hinsbergh VW - Angiogenesis (2016)

Effect of serum supplements on CD34 expression in PB-ECFCs. a Time course of CD34 induction in PB-ECFCs upon incubation with media supplemented with different serum supplements during 24-h period. Open circle depicts 10 % NBCS, open square depicts 10 % NBCS + 10 % PL, open pointed up triangle depicts 10 % PL, open diamond depicts 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 mRNA levels compared to the mRNA levels in the cells incubated with M199 + 3 % HSA at each time point (HSA, p < 0.05: # to NBCS, * to NBCS + PL, & to PL; PL, p < 0.05: $ to NBCS, ∞ to NBCS + PL). b Flow cytometry assessment of CD34 expression in PB-ECFCs cultured in the presence of NBCS or PL. Data are expressed as a mean ± SEM percentage of cells positive for CD34 in the cell cultures incubated with CMi medium which contains 10 % NBCS (open bar) or M199 + 10 % PL (closed bar). c qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), FV (10 ng/mL FGF-2 + 25 ng/mL VEGF-A), HGF (10 ng/mL), and 10 % PL for 24 h prepared in M199 + 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in M199 + 3 % HSA (open bar). d qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in EBM-2 + 5 % PL (open bar). e Flow cytometry evaluation of the effect of VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL on CD34 expression in PB-ECFCs. Closed bars represent the effect of each of soluble factors prepared in control medium (open bar) on CD34 expression. Data are expressed as mean ± SEM of mean fluorescence intensity (MFI) of CD34 antibody fluorescence intensity minus autofluorescence of matched isotype antibody
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Fig3: Effect of serum supplements on CD34 expression in PB-ECFCs. a Time course of CD34 induction in PB-ECFCs upon incubation with media supplemented with different serum supplements during 24-h period. Open circle depicts 10 % NBCS, open square depicts 10 % NBCS + 10 % PL, open pointed up triangle depicts 10 % PL, open diamond depicts 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 mRNA levels compared to the mRNA levels in the cells incubated with M199 + 3 % HSA at each time point (HSA, p < 0.05: # to NBCS, * to NBCS + PL, & to PL; PL, p < 0.05: $ to NBCS, ∞ to NBCS + PL). b Flow cytometry assessment of CD34 expression in PB-ECFCs cultured in the presence of NBCS or PL. Data are expressed as a mean ± SEM percentage of cells positive for CD34 in the cell cultures incubated with CMi medium which contains 10 % NBCS (open bar) or M199 + 10 % PL (closed bar). c qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), FV (10 ng/mL FGF-2 + 25 ng/mL VEGF-A), HGF (10 ng/mL), and 10 % PL for 24 h prepared in M199 + 3 % HSA. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in M199 + 3 % HSA (open bar). d qRT-PCR evaluation of CD34 expression in cells incubated with VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL. Data are expressed as mean ± SEM of n-fold difference of CD34 gene levels in stimulated cells (closed bars) normalized to control cells incubated only in EBM-2 + 5 % PL (open bar). e Flow cytometry evaluation of the effect of VEGF-A (25 ng/mL), FGF-2 (10 ng/mL), HGF (10 ng/mL), and TNF-α (10 ng/mL) for 24 h prepared in EBM-2 + 5 % PL on CD34 expression in PB-ECFCs. Closed bars represent the effect of each of soluble factors prepared in control medium (open bar) on CD34 expression. Data are expressed as mean ± SEM of mean fluorescence intensity (MFI) of CD34 antibody fluorescence intensity minus autofluorescence of matched isotype antibody
Mentions: We subsequently evaluated which serum supplement(s) might modulate CD34 expression in PB-ECFCs. To that end we compared platelet lysate and serum used in CMi, in particular 10 % NBCS and 10 % HS. Confluent monolayers of PB-ECFCs grown in CMi were transferred into M199 medium supplemented with 10 % NBCS, 10 % PL, 10 % NBCS/10 % PL, or 3 % HSA and incubated for 9, 12, and 24 h. In the presence of only 3 % HSA, CD34 mRNA expression increased fivefold within 9 h and remained constant thereafter (Fig. 3a).Fig. 3

Bottom Line: Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells.Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression.Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center Amsterdam, De Boelelaan 1118, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT
Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(-) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(-) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(-): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells. However, during cell culture CD34(-) cells re-expressed CD34. Cell-seeding density, cell-cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(-) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function.

No MeSH data available.


Related in: MedlinePlus