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.
Highlights
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in infancy and results from prolonged ventilator and oxygen treatment
Human- and rat-specific vascular endothelial growth factor (VEGF) protein and mRNA expression levels were measured by enzyme-linked immunosorbent assay (ELISA) and PCR, Fig. 2 Induction of VEGF expression in rat lung epithelial L2 cells by the VEGF from MSC-derived extracellular vesicles (EVs) rescues oxidative injury in vitro. a The VEGF levels were measured in the EVs derived from naive mesenchymal stem cells (MSCs), the EVs from scramble siRNA-transfected MSCs, the EVs from VEGF siRNA-transfected MSCs, and the EVs from fibroblasts
The human VEGF protein levels in the H2O2-exposed rat L2 cell supernatant were highest when co-cultured with MSCs, high when cocultured with non-transfected or scramble siRNAtransfected MSC-derived EVs, and lowest when cocultured with VEGF siRNA-transfected MSC-derived EVs, respectively
Summary
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 treatments[1,2]. We and others have reported that mesenchymal stem cell (MSC) transplantation or MSCconditioned medium significantly attenuates neonatal hyperoxic lung injuries in preclinical animal BPD models, and this protective effect was predominantly mediated by paracrine rather than regenerative mechanisms[3,4,5,6,7,8,9,10]. 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 infants[11,12]. Concerns remain regarding the tumorigenicity and other side effects of transplanting viable MSCs13
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