This project aims to characterize the proteomic landscape of extracellular vesicles (EVs) released by human pulmonary microvascular endothelial cells (hPMVECs) following Sema6A gene knockdown. Using lentiviral-mediated RNA interference, we generated stable Sema6A-knockdown cell lines (*sh-Sema6A*) alongside negative control cells (sh-NC). Exosomes were isolated from conditioned media of both groups via ultracentrifugation and validated for EV-specific markers (e.g., CD63, TSG101). Subsequent high-resolution tandem mass spectrometry (LC-MS/MS)-based proteomic analysis will quantitatively compare protein cargoes between *sh-Sema6A*-EVs and sh-NC-EVs. This study seeks to identify Sema6A-regulated exosomal proteins implicated in endothelial cell communication, angiogenesis, or vascular pathophysiology, providing mechanistic insights into semaphorin signaling pathways in the pulmonary vasculature.