ABSTRACT Our long term goals are to understand the mechanisms by which pulmonary vascular smooth muscle cell (PASMC) proliferation is regulated in the context of vascular remodeling in pulmonary hypertension (PH). In the lung, the pulmonary vascular endothelial cell (PVEC) regulates the function and proliferation of the underlying smooth muscle cell (PASMC) by various bioactive agents that get to the PASMC via into the circulation, myoendothelial junctions and/or extracellular vesicles (EV). The central hypothesis is that in hypoxia, PVEC- derived EV contain microRNA that not only promote PASMC proliferation but also those that inhibit proliferation and it is the imbalance between these two types of microRNAs that leads to disease. The objective of this grant is to characterize the role of EV and their microRNA cargo in hypoxia, in modulating the progression of pulmonary vascular remodeling and PH. Our specific aims are 1) To determine the role of PVEC-derived EV and the roles of two of their miRNA cargo, miR-212-5p and miR-210-3p, in modulating vascular remodeling in hypoxia-induced PH. 2) To determine the mechanisms by which miR-212-5p inhibits PASMC proliferation; specifically the roles of TNF Receptor Associated Factor 3 (TRAF3) and the TGF-? pathway and KLF4 in the inhibitory effect of miR- 212-5p on PASMC proliferation. In addition, novel targets of miR-212-5p and miR-210-3p will be identified using iTRAQ mass spectrometry/proteomics analysis. 3) To provide proof of concept that engineered EV can be used to treat severe PH. We will engineer EV to carry miR-212-5p and anti-miR-210-3p and determine if they can ameliorate PH in two rodent models of PH. This contribution is significant since it will establish a critical role for PVEC-derived EV and their microRNA cargo in the pathogenesis of PH; will delineate the role of miR-212-5p as an important negative regulator of pulmonary vascular remodeling and PH and will test the possibility of utilizing engineered EV for treatment of severe PH. The proposed research is innovative and knowledge derived from the proposal should open new areas for further exploration and enable us to find new treatment strategies for severe PH.