Abstract

Endothelial progenitor cells (EPCs) originate in bone marrow and participate in revascularization and angiogenesis. EPCs can be differentiated from bone marrow, umbilical cord, or peripheral blood cells. In order to better understand the genetic regulation of EPC differentiation, we have characterized genes that are differentially expressed by human EPCs after their development from peripheral blood mononuclear cells, and compared them to genes expressed by endothelial cells from arteries, veins, and microvessels.

Methods: Peripheral blood mononuclear cells (MNC) were isolated from venous blood of healthy human volunteers by density fractionation. MNC were differentiated into EPCs by culturing on fibronectin-coated plates with EMG2-MV (microvascular endothelial) media (Lonza). Total RNA was collected from freshly isolated MNC and EPCs after 7 days of culture. Gene expression profiling was performed using 44k 60-mer Human Whole Genome oligo array (Agilent). Data were analyzed using GeneSpring and Ingenuity Pathways Analysis Enterprise Edition. MNC- and EPC-expressed genes were compared to gene expression profiles of 53 samples of human artery, vein, and microvessels obtained from the Stanford Microarray database. Data were filtered to identify genes that were more than 2-fold differentially expressed between EPCs or endothelial cells when compared to MNCs. Multiple testing correction was applied to achieve statistical significance p<0.05. Ingenuity Pathways Analysis was used to identify functional categories and canonical networks. Gene expression was validated by quantitative RT-PCR.

Results: Six samples of MNC and 4 samples of EPCs were analyzed by microarray. A total of 356 genes were differentially expressed in EPCs in comparison to MNCs (>2-fold, p<0.05). The highest expressed was Glycoprotein nmb (osteoactivin, +1718-fold) and the lowest was the platelet factor 4 (−100-fold). Combined results of EPCs, MNCs, and Stanford endothelial cell samples revealed 6,051 commonly expressed genes. We found 236 genes that were at least 2-fold differentially expressed in EPCs and all endothelial samples when compared to MNC. These genes fall into the following categories: cell growth and proliferation (68 genes), cell-cell signaling and interaction (60), cell death (55), cell signaling (68), hematologic system development and function (50), cardiovascular function (18). As expected, expression of genes with hematopoietic or immune functions, such as platelet factor 4, nuclear factor erythroid-derived (NFE) 2, CD8, and T-cell receptor, were lower in EPCs or endothelial cells versus MNC. EPCs and endothelial cells had higher expression of a number of genes related to cell-cell interaction, including chemokine ligand 18, selenoprotein P (SEPP1), and matrix metalloproteinases (MMP) 7 and 14. Hierarchical clustering of EPCs and all endothelial cell samples showed EPCs to be most similar to skin microvessels (correlation coefficient 0.848). RT-PCR results confirmed differential expression in EPCs versus MNCs in 8 out of 9 genes tested.

Conclusions: We have used gene expression profiling to identify genes that may be important in endothelial cell lineage commitment during EPC differentiation from peripheral blood mononuclear cells. These findings may provide valuable insight for the future development of treatment modalities for vessel repair and homeostasis.

Author notes

Disclosure: No relevant conflicts of interest to declare.