A Quantitative Real-Time PCR Approach for the Detection and Characterization of Endothelial Cells in Whole Blood

Abstract

Pathological inflammation and endothelial dysfunction in atherosclerosis causes endothelial cell detachment from affected vasculature giving rise to circulating endothelial cells (CECs). A blood-based assay that can detect and characterize CECs in atherosclerosis could serve as a valuable diagnostic. Thus, we sought to develop a prototypic assay that detects and characterizes the inflammatory state of endothelial cells present in blood. For this purpose, we spiked resting and inflamed human umbilical vein endothelial cells (HUVEC) into separate samples of whole blood. RNA was harvested and analyzed via quantitative real-time PCR (qPCR) using melanoma cell adhesion molecule (MCAM), as an endothelial marker, and vascular cell adhesion molecule (VCAM-1), which is increased on inflamed endothelium. We found that MCAM mRNA levels correlated with the number of HUVEC spiked into the blood. VCAM-1 mRNA levels were elevated, and correlated with the number of HUVEC, in blood spiked with inflamed HUVEC but not in blood spiked with resting HUVEC. VCAM-1 and MCAM mRNA levels were converted into numerical indices that indicate the inflammatory state of the HUVEC. Combined, the blood spiking studies demonstrate that a VCAM-1/MCAM qPCR assay can successfully detect inflamed endothelial cells in whole blood thus providing proof-of-concept for a diagnostic based on a coupled-phenotypic qPCR assay.