Extracellular vesicles derived from amniotic fluid stem cells as a novel strategy to prevent pulmonary hypertension secondary to congenital diaphragmatic hernia
Pediatric and Adolescent Medicine
Final Report Abstract
Fetal pulmonary hypoplasia is a devastating disease secondary to congenital anomalies such as congenital diaphragmatic hernia (CDH) or oligohydramnios. It is associated with high morbidity and mortality mainly related to postnatal pulmonary hypertension and impaired lung development. Since fetal lung hypoplasia is often diagnosed during the routine anatomy scan around 20 weeks of gestation, this early detection offers a critical window for potential antenatal intervention aimed at promoting normal lung development before birth. A potential novel therapy to treat fetal pulmonary hypoplasia are extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSC). EVs are membrane-bound particles secreted by all cells for paracrine signaling, carrying bioactive cargo such as RNAs, DNAs, and proteins. When isolated from AFSC, they are enriched with micro-RNAs (miR) which are known to regulate key pathways in fetal lung development. In experimental models of CDH, antenatal delivery of AFSC-EVs has been shown to rescue key aspects of lung hypoplasia, including improved airway branching and epithelial and mesenchymal cell maturation. Over the past 3 years, we have extensively investigated the regenerative potential of AFSC-EVs in fetal lung hypoplasia. Specifically, we examined 1) their regenerative mechanisms in CDH lungs, 2) their impact on vascular remodeling in CDH lungs 3) their effects on postnatal CDH lung function, and 4) their potential in treating pulmonary hypoplasia secondary to oligohydramnios. We were the first to identify a multi-lineage inflammatory signature with macrophage enrichment in both human and rodent CDH lungs. Interestingly, antenatal AFSC-EV administration in a rat model of CDH attenuated this inflammatory response, likely via the release of the EV cargo (e.g., miR-9, miR- 125, and miR-128). We also identified long non-coding RNAs (lncRNAs), such as TUG1, in the EV cargo that may drive vascular regeneration. Treatment with AFSC-EVs restored pulmonary vascular architecture and upregulated angiogenic markers in a rat model of CDH. These regenerative effects of AFSC-EVs on the lung vasculature were also confirmed in another model of CDH using mice. Beyond molecular and structural improvements, AFSC-EV treatment also led to enhanced postnatal lung function, with improved compliance, resistance, elastance, and tissue damping in CDH rat pups. Lastly, we also explored whether these regenerative effects of AFSC-EVs extend to non-CDH causes of pulmonary hypoplasia. Remarkably, antenatal AFSC-EV treatment promoted lung growth in a rat model of oligohydramnios, presumably through the delivery of miR-93-5p and the modulation of the TGF-β signaling pathway. In summary, AFSC-EVs offer a multifaceted, cell-free therapeutic strategy for fetal lung hypoplasia. Rather than targeting a single pathway, their complex cargo exerts broad regenerative effects, positioning them as a promising intervention for translational research. This knowledge will now be used to test AFSC-EVs in a large animal model of CDH prior to translating the application of AFSC-EVs in hypoplastic lungs in a clinical trial.
Publications
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Characterization of the congenital diaphragmatic hernia model in C57BL/6J fetal mice: a step toward lineage tracing experiments. Pediatric Surgery International, 39(1).
Doktor, Fabian; Figueira, Rebeca Lopes; Khalaj, Kasra; Ijaz, Aizah; Lacher, Martin; Blundell, Matisse; Antounians, Lina & Zani, Augusto
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Fetal lung vascular development is disrupted by mechanical compression and rescued by administration of amniotic fluid stem cell extracellular vesicles via regulation of the Hippo signaling pathway. openRxiv.
Figueira, Rebeca L.; Khalaj, Kasra; Antounians, Lina; Doktor, Fabian; Gaffi, Maria Sole; Obed, Mikal; Gandhi, Sree & Zani, Augusto
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Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia. Journal of Pediatric Surgery, 59(9), 1771-1777.
Figueira, Rebeca L.; Khoshgoo, Naghmeh; Doktor, Fabian; Khalaj, Kasra; Islam, Tasneem; Moheimani, Nazgol; Blundell, Matisse; Antounians, Lina; Post, Martin & Zani, Augusto
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Cellular origins and translational approaches to congenital diaphragmatic hernia. Seminars in Pediatric Surgery, 33(4), 151444.
Jank, Marietta; Doktor, Fabian; Zani, Augusto & Keijzer, Richard
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Fetal hypoplastic lungs have multilineage inflammation that is reversed by amniotic fluid stem cell extracellular vesicle treatment. Science Advances, 10(30).
Antounians, Lina; Figueira, Rebeca Lopes; Kukreja, Bharti; Litvack, Michael L.; Zani-Ruttenstock, Elke; Khalaj, Kasra; Montalva, Louise; Doktor, Fabian; Obed, Mikal; Blundell, Matisse; Wu, Taiyi; Chan, Cadia; Wagner, Richard; Lacher, Martin; Wilson, Michael D.; Post, Martin; Kalish, Brian T. & Zani, Augusto
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Sex-specific differences in the severity of pulmonary hypoplasia in experimental congenital diaphragmatic hernia and implications for extracellular vesicle-based therapy. Pediatric Surgery International, 40(1).
Doktor, Fabian; Lo, Emily; Fortuna, Victoria; Khalaj, Kasra; Garcia, Miguel; Figueira, Rebeca Lopes; Lacher, Martin; Antounians, Lina & Zani, Augusto
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Amniotic fluid stem cell extracellular vesicles as a novel fetal therapy for pulmonary hypoplasia: a review on mechanisms and translational potential. Stem Cells Translational Medicine, 14(1).
Doktor, Fabian; Antounians, Lina; Figueira, Rebeca Lopes; Khalaj, Kasra; Duci, Miriam & Zani, Augusto
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Amniotic fluid stem cell extracellular vesicles promote lung development via TGF-beta modulation in a fetal rat model of oligohydramnios. Journal of Controlled Release, 377, 427-441.
Doktor, Fabian; Figueira, Rebeca Lopes; Fortuna, Victoria; Biouss, George; Stasiewicz, Kaya; Obed, Mikal; Khalaj, Kasra; Antounians, Lina & Zani, Augusto
