Characterization of Extracellular Vesicle Cargo in the Experimental Rat Model of Pulmonary Hypertension Mitochondrial Components and Post-Translationally Modified Proteins

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; however, whether EVs transmit 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, S.C.)- 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. EVs from SU/Hx rats contained specific proteins involved in cytoskeletal structure, metabolism, immune signaling, and cell adhesion that were preferentially enriched and contained post-translational modifications. These molecular changes are possibly linked to underlying PH progression. Importantly, EVs from SU/Hx rats contained a significantly greater abundance of mitochondrial protein components, detected by mass spectrometry and a more hyperpolarized mitochondrial membrane potential 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, the presence of mitochondrial content and protein cargo in circulating EV-enriched preparations from an experimental PH model.