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Treprostinil increases the number and angiogenic potential of endothelial progenitor cells in children with pulmonary hypertension.

Smadja DM, Mauge L, Gaussem P, d'Audigier C, Israel-Biet D, Celermajer DS, Bonnet D, Lévy M - Angiogenesis (2010)

Bottom Line: ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors.CD34+ cell and CFU-Hill counts were unaffected.ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Paris, France. david.smadja@egp.aphp.fr

ABSTRACT

Background: Pulmonary vasodilators in general and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). As endothelial dysfunction is a key feature of PAH, and as endothelial progenitor cells (EPC) may contribute to vascular repair in PAH, we suspected that prostacyclin therapy might enhance EPC numbers and functions. In the present study, objectives were to determine whether EPC may contribute to vasodilator treatment efficacy in PAH.

Methods: We quantified CD34+ cells, CFU-Hill and ECFC (endothelial colony forming cells) in peripheral blood from children with idiopathic PAH (n = 27) or PAH secondary to congenital heart disease (n = 52). CD34+ were enumerated by flow cytometry, CFU-Hill and ECFC by a culture assay. ECFC grown ex vivo were tested for their angiogenic capacities before and after prostacyclin analog therapy (subcutaneous treprostinil).

Results: ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors. CD34+ cell and CFU-Hill counts were unaffected. ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia.

Conclusions: ECFC may partly mediate the clinical benefits of prostanoids in pulmonary arterial hypertension.

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Related in: MedlinePlus

Basal HPC, CFU-Hill and ECFC counts in the children with PAH. a NUMBER of CD34+ hematopoietic progenitor cells (HPC) determined by FACS analysis. HPC is significantly higher in idiopathic PAH group. b Representative phase-contrast photomicrograph of CFU-Hill colony obtained with Endocult®. Note the central core of rounded cells with spindle-shaped cells sprouting through the periphery. c Number of CFU-Hill counted with cell culture Endocult® assay. No difference was observed between the different patient groups. d Representative phase-contrast photomicrograph of an ECFC colony (mag. ×20). e Number of ECFC by cell culture. ECFC is significantly higher in the reversible PAH group
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Fig1: Basal HPC, CFU-Hill and ECFC counts in the children with PAH. a NUMBER of CD34+ hematopoietic progenitor cells (HPC) determined by FACS analysis. HPC is significantly higher in idiopathic PAH group. b Representative phase-contrast photomicrograph of CFU-Hill colony obtained with Endocult®. Note the central core of rounded cells with spindle-shaped cells sprouting through the periphery. c Number of CFU-Hill counted with cell culture Endocult® assay. No difference was observed between the different patient groups. d Representative phase-contrast photomicrograph of an ECFC colony (mag. ×20). e Number of ECFC by cell culture. ECFC is significantly higher in the reversible PAH group

Mentions: Blood was diluted 1:1 with PBS, 0.2 M EDTA and overlaid on Histopaque-1077 (Sigma–Aldrich, Saint-Quentin Fallavier, France). Cells were centrifuged at 100g for 20 min. Mononuclear cells (MNC) were collected and washed 3 times in PBS, 0.2 M EDTA. CFU-Hill were cultured with the EndoCult® Liquid Medium kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Briefly, MNC were resuspended in complete EndoCult® medium and seeded at 5 × 106 cells/well in fibronectin-coated tissue culture plates (BD, Becton–Dickinson Biosciences). After 48 h, to obtain CFU-Hill, nonadherent cells were collected and plated in Endocult® buffer at 106 cells/well in 24-well fibronectin-coated plates. CFU-Hill colonies were counted after another 3 days, as recommended by the manufacturer. As previously described [7, 13, 24], these cells did not replicate in vitro and gradually disappeared 20 days after plating (Fig. 1b). To obtain ECFC, adherent cells at 48 h were kept in 6-well fibronectin-coated plates in EGM2 medium (Lonza, Saint-Beauzire, France) composed of endothelial cell basal medium-2 (EBM2), 5% fetal bovine serum (FBS) and growth factors. ECFC appeared between 7 and 30 days of culture and consisted of well-circumscribed cobblestone monolayers (Fig. 1d). Colonies were counted with an inverted microscope at ×20 magnification. The colonies were then harvested, trypsinized and reseeded in 6-well plates for complementary studies.Fig. 1


Treprostinil increases the number and angiogenic potential of endothelial progenitor cells in children with pulmonary hypertension.

Smadja DM, Mauge L, Gaussem P, d'Audigier C, Israel-Biet D, Celermajer DS, Bonnet D, Lévy M - Angiogenesis (2010)

Basal HPC, CFU-Hill and ECFC counts in the children with PAH. a NUMBER of CD34+ hematopoietic progenitor cells (HPC) determined by FACS analysis. HPC is significantly higher in idiopathic PAH group. b Representative phase-contrast photomicrograph of CFU-Hill colony obtained with Endocult®. Note the central core of rounded cells with spindle-shaped cells sprouting through the periphery. c Number of CFU-Hill counted with cell culture Endocult® assay. No difference was observed between the different patient groups. d Representative phase-contrast photomicrograph of an ECFC colony (mag. ×20). e Number of ECFC by cell culture. ECFC is significantly higher in the reversible PAH group
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040815&req=5

Fig1: Basal HPC, CFU-Hill and ECFC counts in the children with PAH. a NUMBER of CD34+ hematopoietic progenitor cells (HPC) determined by FACS analysis. HPC is significantly higher in idiopathic PAH group. b Representative phase-contrast photomicrograph of CFU-Hill colony obtained with Endocult®. Note the central core of rounded cells with spindle-shaped cells sprouting through the periphery. c Number of CFU-Hill counted with cell culture Endocult® assay. No difference was observed between the different patient groups. d Representative phase-contrast photomicrograph of an ECFC colony (mag. ×20). e Number of ECFC by cell culture. ECFC is significantly higher in the reversible PAH group
Mentions: Blood was diluted 1:1 with PBS, 0.2 M EDTA and overlaid on Histopaque-1077 (Sigma–Aldrich, Saint-Quentin Fallavier, France). Cells were centrifuged at 100g for 20 min. Mononuclear cells (MNC) were collected and washed 3 times in PBS, 0.2 M EDTA. CFU-Hill were cultured with the EndoCult® Liquid Medium kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Briefly, MNC were resuspended in complete EndoCult® medium and seeded at 5 × 106 cells/well in fibronectin-coated tissue culture plates (BD, Becton–Dickinson Biosciences). After 48 h, to obtain CFU-Hill, nonadherent cells were collected and plated in Endocult® buffer at 106 cells/well in 24-well fibronectin-coated plates. CFU-Hill colonies were counted after another 3 days, as recommended by the manufacturer. As previously described [7, 13, 24], these cells did not replicate in vitro and gradually disappeared 20 days after plating (Fig. 1b). To obtain ECFC, adherent cells at 48 h were kept in 6-well fibronectin-coated plates in EGM2 medium (Lonza, Saint-Beauzire, France) composed of endothelial cell basal medium-2 (EBM2), 5% fetal bovine serum (FBS) and growth factors. ECFC appeared between 7 and 30 days of culture and consisted of well-circumscribed cobblestone monolayers (Fig. 1d). Colonies were counted with an inverted microscope at ×20 magnification. The colonies were then harvested, trypsinized and reseeded in 6-well plates for complementary studies.Fig. 1

Bottom Line: ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors.CD34+ cell and CFU-Hill counts were unaffected.ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Paris, France. david.smadja@egp.aphp.fr

ABSTRACT

Background: Pulmonary vasodilators in general and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). As endothelial dysfunction is a key feature of PAH, and as endothelial progenitor cells (EPC) may contribute to vascular repair in PAH, we suspected that prostacyclin therapy might enhance EPC numbers and functions. In the present study, objectives were to determine whether EPC may contribute to vasodilator treatment efficacy in PAH.

Methods: We quantified CD34+ cells, CFU-Hill and ECFC (endothelial colony forming cells) in peripheral blood from children with idiopathic PAH (n = 27) or PAH secondary to congenital heart disease (n = 52). CD34+ were enumerated by flow cytometry, CFU-Hill and ECFC by a culture assay. ECFC grown ex vivo were tested for their angiogenic capacities before and after prostacyclin analog therapy (subcutaneous treprostinil).

Results: ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors. CD34+ cell and CFU-Hill counts were unaffected. ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia.

Conclusions: ECFC may partly mediate the clinical benefits of prostanoids in pulmonary arterial hypertension.

Show MeSH
Related in: MedlinePlus