Am J Respir Crit Care Med

. 2022 Jun 7.

 doi: 10.1164/rccm.202109-2168OC. Online ahead of print. https://pubmed.ncbi.nlm.nih.gov/35671495/

Autophagy is Impaired in Fetal Hypoplastic Lungs and Rescued by Administration of Amniotic Fluid Stem Cell Extracellular Vesicles

Kasra Khalaj ¹, Lina Antounians ^2 3, Rebeca Lopes Figueira ^4 5, Martin Post ⁶, Augusto Zani ^7 3 8

Affiliations expand

• PMID: 35671495
• DOI: 10.1164/rccm.202109-2168OC

Abstract

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development.

Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms.

Methods: EVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized by size, morphology, and EV marker expression. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy.

Measurements and main results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy at different developmental stages. AFSC-EV administration restored autophagy levels in both pulmonary hypoplasia models by transferring miR-17~92 cluster members contained in the EV cargo.

Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Exosomes; fetal medicine; lung development; miRNA; regenerative medicine.