Abstract P166: Paracrine Endothelial Microrna214 Exosomes Modulate Smooth Muscle Cell Function in Pulmonary Arterial Hypertension

Abstract

Background: Communication among various cell types is known to play a pivotal role in PAH. Endothelial cell (EC)-derived exosomes have emerged as important mediators of intercellular communication and are known to influence the function of recipient cells. A role for miR-214 in EC-derived exosomes in PAH, however, is entirely unknown. In this study, we hypothesize that hypoxia-induced EC injury releases exosomes enriched in miR-214 that mediates phenotype switching of smooth muscle cells (SMCs) resulting in increased SMC proliferation in PAH. Methods and Results: We subjected human pulmonary artery endothelial cells (hPAECs) to hypoxia vs. normoxia (norm) to recapitulate the PAH phenotype in vitro . Quantitative RT-PCR of hPAECs showed that hypoxia upregulated miR-214 by 1.51±0.24-fold (p<0.05 vs. norm). Moreover, exosomes derived from hypoxic hPAECs were found to be enriched in miR-214 (1.73±0.16-fold vs. norm, p<0.05). TEM and Nanotracking Analysis System determined the size of EC-derived exosomes to be within a characteristic 30-100nm. Furthermore, Z-stack confocal imaging demonstrated that FITC-PHK67-labeled EC-derived exosomes were taken up by hPASMCs. Western blot of hPASMCs treated with hypoxia-induced EC-derived exosomes showed upregulation of phospho-Akt (1.65±0.29-fold vs. norm, p <0.05), phospho-ERK1/2 (1.68±0.27-fold vs. norm, p <0.05), and Bcl2 (1.63±0.19-fold vs. norm, NS). Similar treatment also decreased SMC-specific genes, such as MYH11, calponin and smoothelin, indicating that EC-derived hypoxic exosomes can induce switching of PASMCs to a synthetic, pro-proliferative phenotype. Moreover, scratch assay demonstrated that application of EC-derived exosomes laden with exogenous miR-214 increased SMC migration (1.28±0.08-fold vs. control, p =0.05). Conclusions & Discussion: We found that miR-214 expression was induced in hypoxia both in ECs and in exosomes released from them. Furthermore, EC-derived hypoxic or miR-214-laden exosomes were able to transduce changes in SMC, which result in a proliferative and synthetic phenotype. These findings demonstrate that EC-derived miR-214 can direct a proliferative fate for smooth muscle cells, and may contribute to SMC-associated pathophysiological changes in PAH.