A novel minimally-invasive method to sample human endothelial cells for molecular profiling

• Published in: PloS one Vol. 10; no. 2; p. e0118081
• Main Authors: Waldo, Stephen W, Brenner, Daniel A, McCabe, James M, Dela Cruz, Mark, Long, Brian, Narla, Venkata A, Park, Joseph, Kulkarni, Ameya, Sinclair, Elizabeth, Chan, Stephen Y, Schick, Suzaynn F, Malik, Namita, Ganz, Peter, Hsue, Priscilla Y
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13-02-2015; Public Library of Science (PLoS)
• Subjects: Adult; Analysis; Atherosclerosis; Biopsy; Cardiology; Cardiovascular diseases; Catheterization - methods; CD105 antigen; CD11b antigen; CD34 antigen; CD45 antigen; Cell adhesion; Cell adhesion & migration; Cell adhesion molecules; Cell Separation - methods; Contamination; Diabetes; Endothelial cells; Endothelial Cells - chemistry; Endothelial Cells - metabolism; Endothelin; Endothelium; Environmental health; Exercise; Female; Flow Cytometry; Fluorescence; Gene expression; Gene Expression Profiling; Homeostasis; Hospitals; Humans; Leukocytes; Male; Markers; Medicine; Methods; MicroRNA; MicroRNAs; MicroRNAs - analysis; Middle Aged; miRNA; Molecular chains; Molecular modelling; Nitric oxide; Nitric-oxide synthase; NOS3 protein; Pathophysiology; Polymerase chain reaction; Population; Purity; Ribonucleic acid; RNA; Surface markers; Therapeutic applications; Vascular cell adhesion molecule 1; Veins & arteries; Von Willebrand factor; White blood cells; United States > US; San Francisco California; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0118081

The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.
Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR).
A median of 4,212 (IQR: 2161-6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001).
This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.