Amniotic Fluid Stem Cell Derived Extracellular Vesicles Attenuate the Severity of Fetal Lung Vasculature Remodeling in Experimental Congenital Diaphragmatic Hernia

Background/Purpose:
The lungs of fetuses with congenital diaphragmatic hernia (CDH) undergo vascular remodeling, a phenomenon characterized by the presence of fewer and hypermuscularized pulmonary vessels that lead to postnatal pulmonary hypertension. We previously showed that administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) improves growth and maturation of hypoplastic lungs of rat fetuses with CDH. Herein, we assessed whether in vivo administration of AFSC-EVs could also attenuate vascular remodeling.

Methods:
AFSC-EVs were isolated from AFSC conditioned medium by ultracentrifugation and characterized for size (nanoparticle tracking analysis), morphology (transmission electron microscopy), and expression of protein markers (Western blot). AFSC-EVs were fluorescently labeled to track their presence in fetal lungs. At E9.5, dams were gavaged with nitrofen to induce CDH or olive oil as control. At E19.5, fetuses received an intra-amniotic injection of saline (control+saline group, n=7; nitrofen+saline group, n=6) or AFSC-EVs (nitrofen+AFSC-EV group, n=7). Lungs were harvested at E21.5. Groups were blindly compared for number of vessels/mm2, identified with immunofluorescence co-staining (endothelial cells, vWF; smooth muscle cells, SMA; Fig.A), and medial wall thickness, using Kruskal–Wallis test.

Results:
Following intra-amniotic injection, AFSC-EVs were found in fetal lungs. Nitrofen exposure reduced the number of pulmonary vessels, which had increased medial wall thickness (Fig.B-C). Conversely, AFSC-EV treated lungs had an increased number of vessels, which also had a medial wall thickness similar to normal (Fig.B-C).

Conclusions:
This is the first study to show that intra-amniotic injection of AFSC-EVs reduces the severity of lung vascular remodeling in fetuses with CDH. Further studies are underway to evaluate whether this promising antenatal treatment could also prevent postnatal pulmonary hypertension.