Vascular endothelial growth factor mediates the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles against neonatal hyperoxic lung injury

Abstract

We previously reported the role of vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSCs) in protecting against neonatal hyperoxic lung injuries. Recently, the paracrine protective effect of MSCs was reported to be primarily mediated by extracellular vesicle (EV) secretion. However, the therapeutic efficacy of MSC-derived EVs and the role of the VEGF contained within EVs in neonatal hyperoxic lung injury have not been elucidated. The aim of the study was to determine whether MSC-derived EVs attenuate neonatal hyperoxic lung injury and, if so, whether this protection is mediated via the transfer of VEGF. We compared the therapeutic efficacy of MSCs, MSC-derived EVs with or without VEGF knockdown, and fibroblast-derived EVs in vitro with a rat lung epithelial cell line challenged with H2O2 and in vivo with newborn Sprague-Dawley rats exposed to hyperoxia (90%) for 14 days. MSCs (1 × 105 cells) or EVs (20 µg) were administered intratracheally on postnatal day 5. The MSCs and MSC-derived EVs, but not the EVs derived from VEGF-knockdown MSCs or fibroblasts, attenuated the in vitro H2O2-induced L2 cell death and the in vivo hyperoxic lung injuries, such as impaired alveolarization and angiogenesis, increased cell death, and activated macrophages and proinflammatory cytokines. PKH67-stained EVs were internalized into vascular pericytes (22.7%), macrophages (21.3%), type 2 epithelial cells (19.5%), and fibroblasts (4.4%) but not into vascular endothelial cells. MSC-derived EVs are as effective as parental MSCs for attenuating neonatal hyperoxic lung injuries, and this protection was mediated primarily by the transfer of VEGF.

Introduction

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in infancy and results from prolonged ventilator and oxygen treatment. Despite recent advances in neonatal intensive care medicine, BPD remains a major cause of mortality and morbidity in premature infants, with few clinically effective treatments1,2. Therefore, new effective therapies for BPD are urgently needed.

Previously, we and others have reported that mesenchymal stem cell (MSC) transplantation or MSC-conditioned medium significantly attenuates neonatal hyperoxic lung injuries in preclinical animal BPD models, and this protective effect was predominantly mediated by paracrine rather than regenerative mechanisms3,4,5,6,7,8,9,10. Moreover, the feasibility and short- and long-term safety of allogenic MSC transplantation in preterm neonates have been reported in a recent phase I clinical trial of MSC administration for BPD prevention with a 2-year follow-up in infants11,12. However, concerns remain regarding the tumorigenicity and other side effects of transplanting viable MSCs13.

Extracellular vesicles (EVs) are a nuclear membrane vesicles secreted by a variety of cells, 40–100 nm in diameter that contain numerous proteins, lipids, and RNAs, similar to those present in the originating cells; these EVs transport extracellular messages and mediate cell-to-cell communication14,15,16,17,18. Recently, MSC-derived EVs were shown to mediate the therapeutic efficacy of MSCs in various disorders, such as cardiovascular disease19, lung injury13,20, acute kidney injury21, fetal hypoxic ischemic brain injury22, and hypoxic pulmonary hypertension20,22, through the transfer of mRNA, miRNA, and proteins20,21,23,24. The use of MSC-derived EVs is a promising new therapeutic modality for BPD, since this therapy is cell-free and thus may bypass concerns associated with viable MSC treatment. Nevertheless, the therapeutic efficacy of MSC-derived EVs for BPD is unclear.

In this study, we evaluated whether the intratracheal transplantation of MSC-derived EVs is as effective as MSCs alone in a newborn rat model of hyperoxic lung injuries and, if so, whether this protection is mediated primarily through mRNA and protein transfer from the EVs to the injured lung tissue. We specifically examined the transfer of vascular endothelial growth factor (VEGF), as we previously identified a critical role for MSC-secreted VEGF in attenuating hyperoxic lung injuries in neonatal rats9.

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