Abstract Background Extracellular vesicles (EVs) are emerging as key mediators of intercellular communication in the pulmonary vasculature, influencing endothelial, smooth muscle, fibroblast, and immune cells, including macrophage interactions that drive vascular remodelling. Recent studies have implicated both soluble and matrix-bound vesicles (MBVs) in propagation of inflammatory and fibrotic signals in pulmonary hypertension (PH), yet most work has focused on soluble EVs isolated from biofluids or cultured cells. However, their spatial localization, cellular origins, and molecular complexity within the intact pulmonary artery wall remain poorly defined. Methods We combined precision-cut lung slices (PCLS) with transmission electron microscopy TEM to visualize EVs directly within human, bovine, and mouse pulmonary arteries. A new isolation workflow was developed to extract and compare soluble EVs and MBVs from the same tissue source. Vesicle populations were analyzed using nanoparticle tracking analysis (NTA), TEM, and quantitative proteomics. Results High-resolution TEM revealed abundant EVs distributed throughout the pulmonary artery wall, with the greatest density in the adventitia, indicating this region as a major signalling niche. Both soluble EVs and MBVs showed similar size profiles but distinct proteomic signatures. MBVs were enriched in extracellular matrix and fibroblast-associated proteins, whereas soluble EVs contained canonical exosome markers. Proteomic analysis of EVs revealed significant disease-associated changes. Pathway analysis demonstrated that EVs were enriched for pathways linked to apical surface organization and Wnt/β-catenin signaling, consistent with secretory activity, whereas MBVs were enriched in IL6-JAK-STAT3, TNFα-NFκB, complement, and TGFβ signaling, reflecting inflammatory and remodeling-associated processes. Conclusions This study provides the first spatial and molecular analysis of extracellular vesicles in intact pulmonary artery tissue. MBVs represent a matrix-embedded vesicle population that sustains inflammatory and remodeling signals in pulmonary hypertension, distinguishing them from transient SEV-mediated communication. These findings reveal new dimensions of extracellular signaling within the vascular wall and identify MBVs as potential targets for biomarker discovery and therapeutic intervention in PH. This abstract is funded by: HT9425-25-1-0008(DoD), P01HL152961
Kumar et al. (Fri,) studied this question.