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bioRxiv · 10.1101/2022.10.04.510887

Mechanical Stimuli such as Shear Stress and Piezo1 Stimulation Generate Red Blood Cell Extracellular Vesicles

Sangha, G. S.·Weber, C. M.·Sapp, R. M.·Setua, S.·Thangaraju, K.·Pettebone, M.·Doctor, A.·Buehler, P. W.·Clyne, A. M.
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Abstract

Circulating red blood cell extracellular vesicles (RBC-EVs) are a promising biomarker for vascular health. However, generating, isolating, and characterizing physiologically relevant RBC-EVs with sufficient yield and purity for biological studies is non-trivial. Here, we present and rigorously characterize an in vitro model to mimic RBC-EV production during shear stress via mechanosensitive piezo1 ion channel stimulation. We optimize our RBC-EV isolation protocol to minimize hemolysis, maximize RBC-EV yield and purity, and improve the ease of EV characterization. RBC-EV purity was measured by quantifying protein (e.g., particles/g), large particle (e.g., protein aggregates), and platelet EV contamination. This study compared RBC-EV isolation performance using membrane-based affinity (e.g., exoEasy), ultrafiltration (e.g., Amicon Ultra-15), and ultracentrifugation, with and without size exclusion chromatography purification. We found that treating 6% hematocrit with 10 M piezo1-agonist yoda1 for 30 minutes and isolating RBC-EVs using ultracentrifugation minimized RBC hemolysis and maximized RBC-EV yield (~1012 particles/mL) and purity, provided the most consistent RBC-EV preparations, and improved ease of RBC-EV characterization. Our pressure myography experiments suggest that co-isolated protein contaminants, but not piezo1 RBC-EVs, induce rapid mouse carotid artery vasodilation. These results underscore the importance of characterizing EV purity for biological experiments. The standardized methods outlined here enable mechanistic studies of how RBC-EVs generated in physiological flow affect vascular response.

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Sangha, G. S., Weber, C. M., Sapp, R. M., Setua, S., Thangaraju, K., Pettebone, M., Doctor, A., Buehler, P. W., Clyne, A. M.. 2022-10-07. Mechanical Stimuli such as Shear Stress and Piezo1 Stimulation Generate Red Blood Cell Extracellular Vesicles. https://doi.org/10.1101/2022.10.04.510887

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