BOB Winner Presentation - Dr. Rebeca Figueira - EUPSA

Chairpersons, ladies and gentlemen. Fetal lung vascular remodeling predisposes fetuses with CDH to post-natal pulmonary hypertension, which plays a central role in the poor outcomes of these babies. There's consensus that the pre-natal period offers a window of opportunity to promote normal lung development. However, no anti-natal treatments tested to date have been successful. In search for a therapy that could fully rescue normal lung development, our lab has started studying extracellular vesicles derived from amniotic fluid stem cells. We reported that AFSCVs promote growth and maturation in fetal hypoplastic lungs. And that these beneficial effects were exerted via the release of RNA cargo. With these studies, we have demonstrated that AFSCVs improve fetal lung growth and maturation. But what about vascularization? The aim of this study was to investigate whether anti-natal administration of AFSCVs would also rescue fetal lung vascular development. We isolated EVs using ultracentrifugation. and characterized them in accordance with the International Society of Extracellular Vesicles guidelines. We used a well-established nitrophen model of CDH in rats and performed the anti-natal approach at E18, where either saline or AFSCVs are injected into the amniotic sac. At E21, fetal rat lungs were harvested and allocated to control and CDH plus saline or CDH plus AFSCV treated groups. Fetal lungs were assessed for vascular density, mean wall thickness of pulmonary arteries and the gene expression of angiogenic factors. Moreover, we calculated the Fulton index, which expresses the degree of right ventricular hypertrophy, and indirect marker of pulmonary hypertension. We observed that vascular density was rescued back to normal levels after AFSCV administration. And that vascular remodeling was also attenuated in CDH treated lungs. Gene expression analysis revealed that angiogenic factors were rescued back to normal levels after AFSCV treatment. Moreover, AFSCVs attenuated the severity of right ventricular hypertrophy in CDH hearts. But are these findings relevant to human fetal lungs? To address this question, we first isolated and characterized EVs from GMP grade human AFSCs and used our human fetal explant model of pulmonary hypoplasia. Lung explants were divided into three groups, normal, hypoplasia and hypoplasia treated groups. At 96 hours lungs were harvested and assessed for markers of vascular development. We again observed that vascular density was restored by AFSCV treatment, as well as vascular remodeling was attenuated in hypoplastic treated lungs. Similarly, gene expression analysis showed that angiogenic factors were rescued back to normal levels after AFSCV treatment. After these very exciting findings, we then asked, what are the lung endothelial cell specific responses to AFSCV administration? To answer this question, we performed single nucleus RNA sequencing on the left lung of fetal rats. When we analyzed the endothelial cells by condition, we observed a striking difference between control and CDH in the pattern and distribution of endothelial cells, which was rescued to normal distribution following AFSCV treatment. We identified 245 differentially expressed genes. And we observed that these genes regulate vasculogenesis and angiogenesis and were down regulated in CDH lungs, and upregulated in CDH treated lungs. The rescue of endothelial cellular interactions in the treated lungs can be explained by AFSCV cargo that contained some known micro RNAs that control processes like angiogenesis and proliferation and migration of vascular endothelial cells. In conclusion, AFSCVs rescue vascular development and attenuate vascular remodeling rat and human fetal hypoplastic lungs. They also modulate angiogenic processes and rescue the expression of genes involved in lung vascular development. AFSCV cargo contains some microRNAs that regulate lung vascular development, pointing to a mechanism of action. Therefore, anti-natal AFSCV treatment is a promising avenue to restore normal vascular development in hypoplastic lungs and potentially prevent post-natal pulmonary hypertension in babies with CDH. I would like to thank the lab and all the sponsors listed, and you, for your attention.