{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T04:23:30Z","timestamp":1791433410858,"version":"4.3.3"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,8,30]],"date-time":"2023-08-30T00:00:00Z","timestamp":1693353600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01HL140239 R01HL156526 R01HL159862 R01HL158076 R01HL161004 R01HL162120 R01HL161071 R01GM113838 5T32HL007698-26"],"award-info":[{"award-number":["R01HL140239 R01HL156526 R01HL159862 R01HL158076 R01HL161004 R01HL162120 R01HL161071 R01GM113838 5T32HL007698-26"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000968","name":"American Heart Association","doi-asserted-by":"publisher","award":["916512 915916"],"award-info":[{"award-number":["916512 915916"]}],"id":[{"id":"10.13039\/100000968","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Physiol."],"abstract":"<jats:p><jats:bold>Introduction:<\/jats:bold> Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs <jats:italic>in vitro<\/jats:italic> via shear stress and mechanosensitive piezo1 ion channel stimulation.<\/jats:p><jats:p><jats:bold>Methods:<\/jats:bold> RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation.<\/jats:p><jats:p><jats:bold>Results:<\/jats:bold> Our results showed that treating RBCs at 6% hematocrit with 10\u00a0\u00b5M yoda1 for 30\u00a0min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation.<\/jats:p><jats:p><jats:bold>Conclusion:<\/jats:bold> This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated <jats:italic>in vivo<\/jats:italic> under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.<\/jats:p>","DOI":"10.3389\/fphys.2023.1246910","type":"journal-article","created":{"date-parts":[[2023,8,31]],"date-time":"2023-08-31T05:39:26Z","timestamp":1693460366000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":29,"title":["Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles"],"prefix":"10.3389","volume":"14","author":[{"given":"Gurneet S.","family":"Sangha","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Callie M.","family":"Weber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan M.","family":"Sapp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saini","family":"Setua","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiruphagaran","family":"Thangaraju","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morgan","family":"Pettebone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen C.","family":"Rogers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allan","family":"Doctor","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul W.","family":"Buehler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alisa M.","family":"Clyne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,8,30]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"881","DOI":"10.1042\/bj1880881","article-title":"The isolation and characterization of 60 nm vesicles (\u2018nanovesicles\u2019) produced during ionophore A23187-induced budding of human erythrocytes","volume":"188","author":"Allan","year":"1980","journal-title":"Biochem. 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