Nebulization of Hypoxic hUCMSC-EVs Attenuates Airway Epithelial Barrier Defects in Chronic Asthma Mice by Transferring CAV-1

Xinkai Luo,1,* Ying Wang,2,* Yufei Mao,3,* Xiaowei Xu,1,4 Weifeng Gu,1 Wen Li,1 Chaoming Mao,1 Tingting Zheng,1 Liyang Dong1 1Department of Nuclear Medicine, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, People’s Republic of China; 2Department of Respiratory Diseases, The Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an, Jiangsu, People’s Republic of China; 3Department of Ultrasound Medicine, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, People’s Republic of China; 4Department of Laboratory Medicine, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Liyang Dong; Tingting Zheng, Email [email protected]; [email protected]: Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions.Methods: The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs.Results: Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells.Conclusion: Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases.Keywords: nebulization, Hypo-EVs, asthma, airway epithelial barrier, caveolin-1