Characterization of Extracellular Vesicle Cargo in the Experimental Rat Model of Pulmonary Hypertension

Extracellular vesicles (EVs) released by cells carry a diverse molecular cargo, including proteins, lipids, nucleic acids, and mitochondria. In addition, EVs carry antioxidants and free radical producing enzymes like NADPH oxidases or other oxidases. Growing evidence suggests that EV cargo plays a critical role in intercellular communication and disease pathogenesis. In pulmonary hypertension (PH), circulating EV levels are elevated and can alter the function of recipient cells; however, whether EVs transmit functional mitochondria and the composition of their protein cargo remain poorly understood. To address this knowledge gap, we characterized the cargo of EVs in a rat model of Sugen5416 (SU; 20 mg/kg, i.p.)- and hypoxia (10% O₂)-induced PH. EVs were isolated from blood collected from the left ventricles of SU/hypoxia (SU/Hx) and SU/normoxia (SU/Nx) rats and analyzed using quantitative mass spectrometry–based proteomics and flow cytometry. Proteomic profiling revealed that EVs contain proteins involved in coagulation, complement and immune signaling, metabolism, receptor signaling, and cytoskeletal organization. Although EVs from SU/Hx rats exhibited an overall decrease in protein abundance, specific proteins involved in cytoskeletal structure, metabolism, immune signaling, and cell adhesion were preferentially enriched and contained reactive oxygen species-dependent and redox-sensitive post-translational modifications, implicating these molecular changes in mechanisms underlying PH progression. Importantly, EVs from SU/Hx rats contained a significantly greater abundance of functionally active mitochondria, as determined by the MitoTracker Red-to-MitoTracker Green signal ratio, while exhibiting lower levels of mitochondrial superoxide anions, as determined by MitoSOX fluorescence. Together, these findings demonstrate, for the first time, that EVs released from the pulmonary circulation in PH can transmit functional mitochondria and biologically active protein cargo capable of modulating vascular and systemic cellular responses.