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Human Cardiovascular Tissue Extracellular Vesicles – Density Gradient

ABSTRACT: Fewer than 50% of patients develop vascular and valvular calcification, implying differential pathogenesis. Tissue-entrapped extracellular vesicles (EVs) are found in mineralization but their contents and functions are unstudied. Tissue EVs were isolated from diseased human carotid arteries and aortic valves by enzymatic digestion, serial (ultra)centrifugation and density-gradient separation. Mass spectrometry characterized the density-gradient separation and quantified protein cargoes of tissue-derived cardiovascular EVs.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Hathewaya Proteolytica Hathi

TISSUE(S): Aortic Valve, Carotid Atherosclerotic Plaque

SUBMITTER: Mark Blaser

LAB HEAD: Elena Aikawa

PROVIDER: PXD035580 | Pride | 2023-07-10

REPOSITORIES: Pride

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			Action	DRS
	02_FB_AoV296_ODG_1X_Tops_CID-IT_30min_fr01.raw	Raw
	02_FB_AoV296_ODG_1X_Tops_CID-IT_30min_fr02.raw	Raw
	02_FB_AoV296_ODG_1X_Tops_CID-IT_30min_fr03.raw	Raw
	02_FB_AoV296_ODG_1X_Tops_CID-IT_30min_fr04.raw	Raw
	02_FB_AoV296_ODG_1X_Tops_CID-IT_30min_fr05.raw	Raw

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Dataset Information

Human Cardiovascular Tissue Extracellular Vesicles – Density Gradient

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