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. 2009 Jan;29(1):121-7.
doi: 10.1161/ATVBAHA.108.174573.

Microarray-based characterization of a colony assay used to investigate endothelial progenitor cells and relevance to endothelial function in humans

Aditi Desai  1 , Alexander Glaser, Delong Liu, Nalini Raghavachari, Arnon Blum, Gloria Zalos, Margaret Lippincott, J Philip McCoy, Peter J Munson, Michael A Solomon, Robert L Danner, Richard O Cannon 3rd
Affiliations

Microarray-based characterization of a colony assay used to investigate endothelial progenitor cells and relevance to endothelial function in humans

Aditi Desai et al. Arterioscler Thromb Vasc Biol. 2009 Jan.

Abstract

Objective: An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function.

Methods and results: Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45- endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=-0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7+/-2.0 to 8.7+/-1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87).

Conclusions: Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

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Figures

Figure 1
Figure 1
Expression of transcripts of 21 cell-surface markers from 12 study participants’ EPC-CFUs (A) at baseline and (B) after 3 months of exercise training, ranging from 0 (green) to 4.0 (red) in log10 transform. Highly expressed transcripts for cell-identification markers are consistent with lymphocytes (CD2, CD3, CD20, CD25, CD38, CD45, CD69, and CD71).
Figure 2
Figure 2
Comparison of transcript intensity, in logio scale, from study participants’ EPC-CFUs with chips from flow-sorted resting CD3+CD45+CD146− T lymphocytes and cultured HUVECs selected from our microarray gene expression database. Cells from the EPC-CFU assay display a high degree of similarity with T lymphocytes, both at baseline (A) and after 3 months of exercise training (C). Correlations using transcripts from EPC-CFUs and HUVECs are less robust, both at baseline (B) and after 3 months of exercise training (D).
Figure 3
Figure 3
Cultured cells analyzed by flow cytometry to confirm presence of T lymphocytes (CD45+CD3+) and endothelial cells (CD146+CD45−), after exclusion of dead cells. Top rows are cells from a healthy subject cultured in a commercial EPC-CFU assay (A) and from the assay used by Hill et al (B). Row C displays cells from cultured HUVECs, as a positive control.
See this image and copyright information in PMC

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