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

This prospectively designed study was aimed at determining the numbers and functional capacities of EPC before and after vasodilator treatment, and especially after treprostinil therapy. Blood samples were collected during outpatient visits from patients already on single- or dual-agent therapy. In the 8 treprostinil-treated patients, EPC were studied before treatment initiation, 2 and 5 days later and monthly thereafter.

The study was approved by the institutional ethics committee of Necker-Enfants Malades hospital, and the parents gave their signed informed consent. Fifty-two consecutive patients with CHD and PAH, and 27 consecutive patients with idiopathic PAH (Table 1) were enrolled between February 2008 and 2010. The 27 patients with idiopathic PAH had a median age of 6 years. Among the patients with CHD, 28 (median age 2 years) had reversible PAH, as defined by normal pulmonary pressure 6 months after surgery, while 24 (median age 8 years) had irreversible PAH, based on persistently elevated pulmonary artery pressure and pulmonary vascular resistance. Children with irreversible PAH were older than children with reversible and idiopathic PAH (P = 0.01 and P = 0.06, respectively) and had lower oxygen saturation values (P = 0.02 and P = 0.0008, respectively). Pulmonary artery pressure was similar in the three groups (Table 1). Patients with familial PAH associated with BMPR2 mutations were not included in this study, because of a possible impact of BMP on EPC angiogenic properties [6, 21, 22]. Indeed, BMPR2 mutations have been reported to affect both early [23] and late EPC, the latter showing a hyperproliferative phenotype and an impaired capacity to form vascular networks [22]. Because of adverse effects reported with IV epoprostenol and inhaled prostanoids, subcutaneous treprostinil was used as first-line add-on therapy for 8 young children who deteriorated while receiving combined oral therapy with an endothelin receptor agonist and a PDE5 inhibitor. Three of them had idiopathic PAH and five patients had PAH associated with a congenital heart defect. The decision to add subcutaneous tresprostinil was based on their clinical status (change in functional class), including right ventricular dysfunction (hepatomegaly, increase in tricuspid regurgitation volume, dilation of hepatic veins and the inferior vena cava) in seven patients. Two of them also had syncope. One patient in functional class II had severe complications associated with the central venous line used to deliver epoprostenol. All the patients had right heart catheterization prior to treprostinil treatment. Treprostinil administration was initiated at hospital, through a subcutaneous catheter in the outer leg. All the patients initially received a fixed dose of 1.25 ng/kg/min. The dose was then daily increased by 1.25 ng/kg/min, reaching an average of 20 ng/kg/min at hospital discharge. During treprostinil treatment, site pain was evaluated every 6 h using standard pain scales adapted to age. After hospital discharge the treprostinil dose was adjusted at monthly out-patient visits and reached an average of 40 ng/kg/min. Technical assistance was provided at home by specialized nurses trained in the management of parenteral prostanoid therapy in PAH patients. All patients had experienced a clinical improvement performed, for oldest children by 6MWT, and they all showed an improvement in oxygen saturation and functional status, as they were all in functional class I or II. In five patients who had right heart catheterization after treprostinil, pulmonary arterial pressure did not change but cardiac output increased and pulmonary vascular resistance decreased.

Circulating CD34+ cells were counted by flow cytometry (FC500 Cytometer; Beckman Coulter, Villepinte, France) after staining of whole blood with a fluorescein isothiocyanate (FITC)-labeled monoclonal mouse antihuman CD45 antibody, a phycoerythrin (PE)-labeled monoclonal mouse antihuman-CD34 antibody, and 7AAD (Stemkit^®; Beckman Coulter). Absolute numbers of CD34+ cells μl⁻¹ were determined by using calibration beads, as recommended by the manufacturer.

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.

After 16 h of serum and growth-factor privation, ECFC were incubated for 48 h in EBM medium, 5% FBS. Proliferation was examined after 48 h by measuring cell phosphatase activity based on the release of paranitrophenol (pNPP) (Sigma) measured at OD 405 nm (Fluostar optima; BMG labtech, Champigny-sur-Marne, France). Control cells were ECFC from cord blood [6, 25‐27].

After 16 h of serum and growth-factor privation, ECFC (3 × 10⁴ cells/well) were seeded on growth factor-reduced Matrigel (200 μl) (BD Biosciences) in EBM2 medium and cultured for 18 h at 37°C with 5% CO₂. Capillary-like structures were observed by phase-contrast microscopy and networks formed by ECFC were quantified with Videomet software version 5.4.0.

A reproducible circular “wound” was made with a tip in confluent monolayers of ECFC cultured in 12-well plates. The surface area of the wound was measured at ×20 magnification under an inverted microscope, then the medium was removed and the cells were further incubated for 4 or 18 h with EBM, 5% FBS and VEGF 50 ng/ml. Wound repair activity was calculated by expressing the area of the wound after 4 or 18 h of incubation as a percentage of the initial area.

Nude mice underwent surgery to induce unilateral hindlimb ischemia as described elsewhere [6, 28] and were randomized to receive an intravenous injection of 1 × 10⁵ ECFC derived from the two treated patient groups or 1 × 10⁵ ECFC derived from cord blood. Two independent groups of 20 mice were realized with, in each experiment, 5 mice injected with PBS, 5 mice with cord blood ECFC, 5 mice with ECFC from patients with oral therapy and 5 mice with ECFC from patients with SC-treprostinil. After 2 weeks, the ischemic/normal limb blood flow ratio was determined with a laser Doppler perfusion imaging system.