A Novel Minimally-Invasive Method to Sample Human Endothelial Cells for Molecular Profiling

Abstract

ObjectiveThe 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.MethodsNine 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).ResultsA 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).ConclusionThis 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.

Endovascular Device for Endothelial Cell Sampling

“…Previously reported sampling of ECs utilized devices designed for other purposes, such as stents and coils, and generated a limited or highly variable cell yield. [8][9][10][11][12][13][14][15][16] Previous approaches are further limited in navigability or require cell sample enrichment due to blood contamination. Our device yields samples suitable for transcriptomic analysis without the need for cell sample enrichment.…”

“…[2,3] Previous attempts to sample endothelial cells through endovascular sampling strategies, e.g., with coils, stents, stent retrievers, or guidewires, suffer from poor selectivity and a low or highly variable cell yield. [8][9][10][11][12][13][14][15][16] When harvesting ECs from stent retrievers used for mechanical thrombectomy, there was a low EC yield or high contamination of thrombotic material. [15,16] In these studies, fluorescence-activated single-cell sorting (FACS) was used to sort the sample, and in one study, ECs were cultured to allow cytological analysis and single-cell RNA sequencing.…”

“…This method, however, required external mechanical pressure on the arm to be successful. [11] Current approaches lack a dedicated sampling mechanism for the endothelium, are too large or invasive to be navigated to sub-millimeter-sized peripheral DOI: 10.1002/anbr.202200023 Endothelial cells play an important role in several vascular diseases, and molecular analysis of these cells could provide valuable information on underlying tissue status. However, no clinically established procedure for harvesting endothelial cells exists.…”

Generation of Endothelial Cells From Human Pluripotent Stem Cells

“…Primary human ECs obtained by endothelial biopsy [14][15][16] have provided invaluable insight into the molecular mechanisms of endothelial dysfunction in a number of disease states. [17][18][19][20] However, primary human ECs are a suboptimal source of ECs for cell therapy and tissue engineering applications because they are difficult to obtain, have limited replicative potential, 21 and are allogenic unless used in the same individual from which they were obtained.…”

Comparative Transcriptomics of Ex Vivo, Patient-Derived Endothelial Cells Reveals Novel Pathways Associated With Type 2 Diabetes Mellitus

“…Other blood cells, especially leukocytes, contaminate these preparations, thereby complicating analysis using conventional microscopy. To overcome these issues, fluorescence-activated cell sorting (FACS) (6) or magnetic microbeads (7) have been used to enrich for ECs during the procedure. However, FACS is time intensive and can damage fragile cells.…”