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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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Angiogenic potential of ECFC isolated from patients with pulmonary hypertension receiving vasodilator treatment. Each patient-derived ECFC were individually analyzed for proliferation and angiogenic properties in vitro and in vivo. At least 4 different patients derived-ECFC were used in each case. a Cells from patients receiving oral therapy and SC treprostinil showed an increased proliferative potential (in EBM, 5% FBS) compared with those from patients receiving oral therapy. Results are presented normalized to ECFC from cord blood (100%). The mean and SEM of three experiments are shown P < 0.05. b, c ECFC isolated from patients receiving oral therapy, with or without SC treprostinil, form intact vascular networks with similar efficiency. The mean and SEM of three experiments are shown. d, e VEGF-stimulated migration of ECFC in vitro in a wound healing assay was similar in patients receiving oral therapy with or without SC treprostinil. The mean and SEM of three experiments are shown. f, g ECFC from patients receiving oral therapy, with or without SC treprostinil, were injected intravenously into nude mice having undergone femoral artery ligature. Injection of ECFC from patients receiving oral therapy only improved foot perfusion on day 14 to the same extent as control ECFC isolated from cord blood. ECFC from patients receiving oral therapy plus SC treprostinil improved foot perfusion on day 14 by 25% more than ECFC from patients receiving oral therapy alone (P = 0.012) and ECFC isolated from cord blood (P = 0.019)
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Fig4: Angiogenic potential of ECFC isolated from patients with pulmonary hypertension receiving vasodilator treatment. Each patient-derived ECFC were individually analyzed for proliferation and angiogenic properties in vitro and in vivo. At least 4 different patients derived-ECFC were used in each case. a Cells from patients receiving oral therapy and SC treprostinil showed an increased proliferative potential (in EBM, 5% FBS) compared with those from patients receiving oral therapy. Results are presented normalized to ECFC from cord blood (100%). The mean and SEM of three experiments are shown P < 0.05. b, c ECFC isolated from patients receiving oral therapy, with or without SC treprostinil, form intact vascular networks with similar efficiency. The mean and SEM of three experiments are shown. d, e VEGF-stimulated migration of ECFC in vitro in a wound healing assay was similar in patients receiving oral therapy with or without SC treprostinil. The mean and SEM of three experiments are shown. f, g ECFC from patients receiving oral therapy, with or without SC treprostinil, were injected intravenously into nude mice having undergone femoral artery ligature. Injection of ECFC from patients receiving oral therapy only improved foot perfusion on day 14 to the same extent as control ECFC isolated from cord blood. ECFC from patients receiving oral therapy plus SC treprostinil improved foot perfusion on day 14 by 25% more than ECFC from patients receiving oral therapy alone (P = 0.012) and ECFC isolated from cord blood (P = 0.019)

Mentions: ECFC from patients receiving oral therapy, with or without SC treprostinil, were grown to confluence from single colonies. They retained the characteristic cobblestone aspect through numerous passages and stained positive for typical endothelial markers CD34, CD146, CD144, PAR-1 thrombin receptor and von Willebrand factor (data not shown). During the first 30 days of culture, cells cultured from patients receiving oral therapy plus SC treprostinil showed enhanced proliferation in EBM, 5% FBS compared to cells from patients receiving only oral therapy (Fig. 4a). In vitro vascular network formation in Matrigel was similar with ECFC from patients treated with or without treprostinil (Fig. 4b, c), as was VEGF-induced migration in the wound healing assay (Fig. 4d, e).Fig. 4


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)

Angiogenic potential of ECFC isolated from patients with pulmonary hypertension receiving vasodilator treatment. Each patient-derived ECFC were individually analyzed for proliferation and angiogenic properties in vitro and in vivo. At least 4 different patients derived-ECFC were used in each case. a Cells from patients receiving oral therapy and SC treprostinil showed an increased proliferative potential (in EBM, 5% FBS) compared with those from patients receiving oral therapy. Results are presented normalized to ECFC from cord blood (100%). The mean and SEM of three experiments are shown P < 0.05. b, c ECFC isolated from patients receiving oral therapy, with or without SC treprostinil, form intact vascular networks with similar efficiency. The mean and SEM of three experiments are shown. d, e VEGF-stimulated migration of ECFC in vitro in a wound healing assay was similar in patients receiving oral therapy with or without SC treprostinil. The mean and SEM of three experiments are shown. f, g ECFC from patients receiving oral therapy, with or without SC treprostinil, were injected intravenously into nude mice having undergone femoral artery ligature. Injection of ECFC from patients receiving oral therapy only improved foot perfusion on day 14 to the same extent as control ECFC isolated from cord blood. ECFC from patients receiving oral therapy plus SC treprostinil improved foot perfusion on day 14 by 25% more than ECFC from patients receiving oral therapy alone (P = 0.012) and ECFC isolated from cord blood (P = 0.019)
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Fig4: Angiogenic potential of ECFC isolated from patients with pulmonary hypertension receiving vasodilator treatment. Each patient-derived ECFC were individually analyzed for proliferation and angiogenic properties in vitro and in vivo. At least 4 different patients derived-ECFC were used in each case. a Cells from patients receiving oral therapy and SC treprostinil showed an increased proliferative potential (in EBM, 5% FBS) compared with those from patients receiving oral therapy. Results are presented normalized to ECFC from cord blood (100%). The mean and SEM of three experiments are shown P < 0.05. b, c ECFC isolated from patients receiving oral therapy, with or without SC treprostinil, form intact vascular networks with similar efficiency. The mean and SEM of three experiments are shown. d, e VEGF-stimulated migration of ECFC in vitro in a wound healing assay was similar in patients receiving oral therapy with or without SC treprostinil. The mean and SEM of three experiments are shown. f, g ECFC from patients receiving oral therapy, with or without SC treprostinil, were injected intravenously into nude mice having undergone femoral artery ligature. Injection of ECFC from patients receiving oral therapy only improved foot perfusion on day 14 to the same extent as control ECFC isolated from cord blood. ECFC from patients receiving oral therapy plus SC treprostinil improved foot perfusion on day 14 by 25% more than ECFC from patients receiving oral therapy alone (P = 0.012) and ECFC isolated from cord blood (P = 0.019)
Mentions: ECFC from patients receiving oral therapy, with or without SC treprostinil, were grown to confluence from single colonies. They retained the characteristic cobblestone aspect through numerous passages and stained positive for typical endothelial markers CD34, CD146, CD144, PAR-1 thrombin receptor and von Willebrand factor (data not shown). During the first 30 days of culture, cells cultured from patients receiving oral therapy plus SC treprostinil showed enhanced proliferation in EBM, 5% FBS compared to cells from patients receiving only oral therapy (Fig. 4a). In vitro vascular network formation in Matrigel was similar with ECFC from patients treated with or without treprostinil (Fig. 4b, c), as was VEGF-induced migration in the wound healing assay (Fig. 4d, e).Fig. 4

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