Background/Purpose: Amniotic fluid stem cells (AFSC) promote lung growth in experimental congenital diaphragmatic hernia (CDH) via the release of extracellular vesicles (EV) containing microRNAs. Further, a unique study has shown that autophagy is a crucial cellular mechanism for lung development. We aim to assess whether hypoplastic lungs from experimental CDH have abnormal autophagy and if AFSC-EVs can restore autophagy.
Methods: EVs were extracted from rat AFSC conditioned medium using ultracentrifugation. An experimental CDH model was utilized via nitrofen administration to pregnant rat dams at E9.5. Following nitrofen administration, fetal lungs were harvested at E14.5 and explants were established. Hypoplastic lung explants were treated with AFSC-EV or medium alone (72h). Fetal lungs from untreated dams served as control. Gene and protein levels of autophagy markers (Activator: ATG5,BECN1; Impairment: SQSTM1) were analyzed via RT-qPCR and western blotting (WB), respectively. Purified EV-RNA was sequenced using RNA sequencing (Illumina NextSeq). We compared nitrofen with nitrofen+AFSC-EV treated samples for microRNAs associated with autophagy. Data were analyzed statistically using One-way (qPCR and WB) and Two-way (RNAseq) ANOVAs (Tukey post-tests).
Results: Nitrofen-exposed hypoplastic lungs had significantly lower levels of Atg5 and Becn1 and higher levels of impaired autophagy marker, SQSTM1 (Fig.A). Conversely, administration of AFSC-EV to hypoplastic lungs restored expression levels of these markers back to normal levels (Fig.A). RNA-sequencing identified 3 microRNAs (miRs-17,-20a,-93) that are specifically associated with autophagy (in relation to Sqstm1) and were enriched in the AFSC-EV administration group (Fig.B).
Conclusion: This is the first study demonstrating impaired autophagy in CDH. Moreover, we discovered that autophagy mechanisms are restored with AFSC-EVs. Together, targeting autophagy with AFSC-EVs could represent a promising cell-free approach to treat CDH.