The isolation of VCAM-1⁺ endothelial cell-derived extracellular vesicles using microfluidics

Abstract

Background: Vascular cell adhesion molecule-1 (VCAM-1⁺) endothelial cell-derived extracellular vesicles (EC-EVs) are augmented in cardiovascular disease, where they can signal the deployment of immune cells from the splenic reserve. Endothelial cells in culture activated with pro-inflammatory tumor necrosis factor-α (TNF-a) also release VCAM-1⁺ EC-EVs. However, isolating VCAM-1⁺ EC-EVs from conditioned cell culture media for subsequent in-depth analysis remains challenging.

Aim: We utilized the extracellular vesicles (EV) microfluidics herringbone chip (^EVHB-Chip), coated with anti-VCAM-1 antibodies, for selective capture of VCAM-1⁺ cells and EC-EVs.

Methods and Results: Engineered EA.hy926 endothelial cells overexpressing VCAM-1 (P < 0.001 versus control) showed increased binding to the VCAM-1- ^EVHB-Chip versus an IgG device. TNF-α-stimulated human umbilical cord vein endothelial cells (HUVECs) exhibited elevated VCAM-1 protein levels (P < 0.001) and preferential binding to the VCAM-1- ^EVHB-Chip versus the IgG device. HUVECs stimulated with TNF-α showed differential gene expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.001) and VCAM-1 (P < 0.001) by digital droplet PCR versus control cells. HUVEC-derived EC-EVs were positive for CD9, CD63, HSP70, and ALIX and had a modal size of 83.5 nm. Control and TNF-α-stimulated HUVEC-derived EC-EV cultures were captured on the VCAM-1- ^EVHB-Chip, demonstrating selective capture. VCAM-1⁺ EC-EV were significantly enriched for ICAM-1 (P < 0.001) mRNA transcripts.

Conclusion: This study presents a novel approach using the ^EVHB-Chip, coated with anti-VCAM-1 antibodies and digital droplet PCR for the study of VCAM-1⁺ EC-EVs. Isolation of VCAM-1⁺ EC-EV from heterogeneous sources such as conditioned cell culture media holds promise for subsequent detailed characterization, and may facilitate the study of VCAM-1⁺ EC-EVs in cardiovascular and metabolic diseases, for disease monitoring and therapeutic insights.

Keywords

Cardiovascular / vascular / biomarker / inflammation / atherosclerosis / endothelium

References

[1] Cybulsky MI.Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.Science1991;251:788-91

[2] Akbar N,Corr EM.Oxford Acute Myocardial Infarction Study (OxAMI)Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.Cardiovasc Res2023;119:236-51 PMCID:PMC10022859

[3] Guo TM,Ke L.Prognostic value of neutrophil to lymphocyte ratio for in-hospital mortality in elderly patients with acute myocardial infarction.Curr Med Sci2018;38:354-9

[4] Akbar N,Choudhury RP.Extracellular vesicles in metabolic disease.Diabetologia2019;62:2179-87 PMCID:PMC6861353

[5] Hazelton I,Dale A.Exacerbation of acute traumatic brain injury by circulating extracellular vesicles.J Neurotrauma2018;35:639-51

[6] Couch Y,Roodselaar J.Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation.Sci Rep2017;7:9574 PMCID:PMC5575066

[7] Akbar N,Cahill TJ.Oxford Acute Myocardial Infarction (OxAMI) StudyEndothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.JCI Insight2017;2:93344 PMCID:PMC5621885

[8] Paget D,Zöhrer B.Oxford Acute Myocardial Infarction Study (OxAMI)Comparative and integrated analysis of plasma extracellular vesicle isolation methods in healthy volunteers and patients following myocardial infarction.J of Extracellular Bio2022;1:e66

[9] Rabe DC,Choudhury A.Aryl-diazonium salts offer a rapid and cost-efficient method to functionalize plastic microfluidic devices for increased immunoaffinity capture.Adv Mater Technol2023;8:2300210 PMCID:PMC10812904

[10] Lai CP,Badr CE.Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.Nat Commun2015;6:7029 PMCID:PMC4435621

[11] Théry C,Aikawa E.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles2018;7:1535750 PMCID:PMC6322352

[12] Fernández-rhodes M,Williams S.Defining the influence of size-exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model.J of Extracellular Bio2023;2:e85

[13] Aya-Bonilla CA,Freeman JB.Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device.Oncotarget2017;8:67355-68 PMCID:PMC5620178

[14] de Jong OG,Chen Y.Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes.J Extracell Vesicles2012;1:18396 PMCID:PMC3760650

[15] Seibold T,Weeber F.Small extracellular vesicles propagate the inflammatory response after trauma.Adv Sci2021;8:e2102381 PMCID:PMC8693079

[16] Reátegui E,Lai CP.Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles.Nat Commun2018;9:175 PMCID:PMC5766611