Extracellular Vesicle-Mediated Delivery of microRNA-206 Antagomir Attenuates Lung Ischemia-Reperfusion Injury via Targeting Type II Epithelial Cell-Secreted CXCL1

Abstract

Our hypothesis is that human adipose mesenchymal stem cell (MSC)-derived extracellular vesicle (EV) can be used as a delivery platform for microRNAs to effectively attenuate lung ischemia-reperfusion injury (IRI). C57Bl/6 (WT) mice underwent sham or lung IRI (1hr of ischemia followed by 2hr reperfusion using left lung hilar-ligation model) with or without treatment (1hr prior to ischemia) with EVs or antagomiR-206 enriched EVs (3x106 EVs given intratracheally; n=5/group). MSCs were transfected with antagomiR-206 using Lipofectamine reagent for enrichment of purified EVs. Lung tissue was used for Affymetrix GeneChip microRNA microarray hybridization. Cytokine expression in BAL fluid by Luminex array, and lung injury by measuring neutrophil infiltration (immunohistochemistry) and activation (myeloperoxidase levels), was evaluated. Murine type II epithelial (MLE12 cells) were co-cultured with EVs and exposed to hypoxia/reoxygenation (3hr/1hr) followed by analysis of supernatants by ELISA. Groups were compared using ANOVA and data is shown as mean±S.E. miRNA microarray analysis of lung tissue demonstrated a significant upregulation of miR-206 (∼50-fold) after lung IRI compared to sham. Treatment with antagomiR-206-enriched EVs displayed significantly better lung function [decreased airway resistance (0.6±0.03 vs. 1.1±0.05 cm H2O/μl/sec; p<0.05), pulmonary artery pressure (7.3±0.3 vs. 9.7±0.3 cm H2O; p<0.05) and increased pulmonary compliance (5.8±0.2 vs. 3.7±0.3 μl/cmH2O; p<0.05)] and mitigation of lung injury (neutrophil infiltration and myeloperoxidase levels) compared to treatment with EVs alone, respectively, after lung IRI. Treatment with EVs or antagomiR-206-enriched EVs significantly decreased proinflammatory cytokines (IL-17, TNF-α, MCP-1, HMGB1 and CXCL1) compared to IR alone. AntagomiR-206 enriched EVs displayed a significant decrease in CXCL1 expression, a hallmark of lung epithelial activation, compared to EVs alone. Co-culture of MLE12 cells with antagomiR-206 enriched EVs showed a significant attenuation of HR-induced CXCL1 expression compared to co-culture with EVs. MSC-derived EVs can be used as biomimetic nanovehicles for protective immunomodulation against lung IRI by enriching them with antagomiR-206.