Respiratory extracellular vesicle isolation optimization through proteomic profiling of equine samples and identification of candidates for cell-of-origin studies.
Authors: Hickman Elise, Carberry Victoria, Carberry Celeste, Cooper Bethanie, Mordant Angie L, Mills Allie, Sokolsky Marina, Herring Laura E, Alexis Neil E, Rebuli Meghan E, Jaspers Ilona, Sheats Katie, Rager Julia E
Journal: PloS one
Summary
# Editorial Summary Extracellular vesicles (EVs) released from respiratory tract cells are emerging as important signalling molecules in equine airway disease, yet standardised methods for isolating and analysing them from respiratory samples remain underdeveloped. Researchers compared four isolation techniques—ultracentrifugation, microcentrifugation, and two size exclusion chromatography (SEC) variants—using bronchoalveolar lavage fluid (BALF) from horses, measuring particle yield, purity, protein content, and proteomic composition with particular attention to surface markers that indicate cellular origin. Size exclusion chromatography substantially outperformed centrifugation approaches, producing higher particle counts, superior purity markers, and markedly better expression of vesicle surface proteins; proteomic analysis revealed distinct clustering between SEC and centrifugation methods, suggesting fundamentally different EV populations were being recovered. For equine practitioners and researchers, these findings establish SEC as the preferred isolation method for characterising respiratory EVs from BALF samples, enabling more reliable identification of which airway cells (epithelial, immune, endothelial) are actively secreting vesicles and therefore providing clearer insight into inflammatory or pathological processes underlying conditions like equine asthma. This methodological foundation opens the door to longitudinal EV studies in horses—leveraging their naturally occurring respiratory diseases, large sample volumes, and physiological similarity to humans—offering a powerful translational model for understanding how cell-to-cell communication via EVs drives respiratory pathology in both equine and human medicine.
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Practical Takeaways
- •For respiratory research using equine samples, size exclusion chromatography should be preferred over centrifugation methods when investigating extracellular vesicle composition and cellular origins
- •The equine model offers practical advantages for longitudinal respiratory disease studies due to naturally occurring asthma, sample volume availability, and physiological similarities to human respiratory pathology
- •This methodological optimization may improve future diagnostic or prognostic applications of extracellular vesicles in equine respiratory disease assessment
Key Findings
- •Size exclusion chromatography yielded the highest particle counts and greatest EV purity markers compared to ultracentrifugation and microcentrifugation methods
- •Proteomic profiles differed significantly across isolation methods, with size exclusion chromatography clustering separately from centrifugation approaches
- •Size exclusion chromatography demonstrated elevated vesicle surface marker expression, better characterizing EV cell-of-origin information
- •Equine bronchoalveolar lavage fluid provides large sample volumes and cell yields suitable for extracellular vesicle research translation to human respiratory studies