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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

CD34 modulates sprouting response of PB-ECFCs in fibrin matrices. a-d Representative phase contrast pictures of sprout formation in fibrin matrices. a unstimulated cells; b control, non-transfected cells; c cells transfected with non-targeting siRNA; d cells transfected with CD34 siRNA. e Effect of silencing of CD34 in PB-ECFCs on sprout formation in fibrin matrices. Data are expressed as a mean ± SEM of mean length of formed sprouts of four independent experiments each performed with different donor. f mRNA levels of tip-cell-associated and fibrinolytic genes in PB-ECFCs after silencing of CD34. Data are expressed as mean ± SEM of n-fold difference of mRNA levels of depicted genes in the siRNA CD34 transfected cells (closed bars) compared to the cells transfected with non-targeting siRNA (open bars) which served as control
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Fig6: CD34 modulates sprouting response of PB-ECFCs in fibrin matrices. a-d Representative phase contrast pictures of sprout formation in fibrin matrices. a unstimulated cells; b control, non-transfected cells; c cells transfected with non-targeting siRNA; d cells transfected with CD34 siRNA. e Effect of silencing of CD34 in PB-ECFCs on sprout formation in fibrin matrices. Data are expressed as a mean ± SEM of mean length of formed sprouts of four independent experiments each performed with different donor. f mRNA levels of tip-cell-associated and fibrinolytic genes in PB-ECFCs after silencing of CD34. Data are expressed as mean ± SEM of n-fold difference of mRNA levels of depicted genes in the siRNA CD34 transfected cells (closed bars) compared to the cells transfected with non-targeting siRNA (open bars) which served as control

Mentions: During the course of 2-day stimulation period, the siRNA NT cells exhibited similar sprouting response as the control, non-transfected cells indicating that the transfection procedure did not alter the sprouting response of PB-ECFCs (Fig. 6a–c), while the siRNA CD34-transfected cells exhibited significantly reduced sprout formation (Fig. 6d, e).Fig. 6


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)

CD34 modulates sprouting response of PB-ECFCs in fibrin matrices. a-d Representative phase contrast pictures of sprout formation in fibrin matrices. a unstimulated cells; b control, non-transfected cells; c cells transfected with non-targeting siRNA; d cells transfected with CD34 siRNA. e Effect of silencing of CD34 in PB-ECFCs on sprout formation in fibrin matrices. Data are expressed as a mean ± SEM of mean length of formed sprouts of four independent experiments each performed with different donor. f mRNA levels of tip-cell-associated and fibrinolytic genes in PB-ECFCs after silencing of CD34. Data are expressed as mean ± SEM of n-fold difference of mRNA levels of depicted genes in the siRNA CD34 transfected cells (closed bars) compared to the cells transfected with non-targeting siRNA (open bars) which served as control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930476&req=5

Fig6: CD34 modulates sprouting response of PB-ECFCs in fibrin matrices. a-d Representative phase contrast pictures of sprout formation in fibrin matrices. a unstimulated cells; b control, non-transfected cells; c cells transfected with non-targeting siRNA; d cells transfected with CD34 siRNA. e Effect of silencing of CD34 in PB-ECFCs on sprout formation in fibrin matrices. Data are expressed as a mean ± SEM of mean length of formed sprouts of four independent experiments each performed with different donor. f mRNA levels of tip-cell-associated and fibrinolytic genes in PB-ECFCs after silencing of CD34. Data are expressed as mean ± SEM of n-fold difference of mRNA levels of depicted genes in the siRNA CD34 transfected cells (closed bars) compared to the cells transfected with non-targeting siRNA (open bars) which served as control
Mentions: During the course of 2-day stimulation period, the siRNA NT cells exhibited similar sprouting response as the control, non-transfected cells indicating that the transfection procedure did not alter the sprouting response of PB-ECFCs (Fig. 6a–c), while the siRNA CD34-transfected cells exhibited significantly reduced sprout formation (Fig. 6d, e).Fig. 6

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