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.