Colonies of cells grown in fibronectin culture assays from peripheral blood mononuclear cells— believed to represent bone marrow–derived EPCs—have been associated with endothelial function in men,9
reduced in patients with coronary artery disease,21
and inversely associated with cardiovascular outcomes.26
However, markers of endothelial phenotype used by several groups to validate these assays— including CD31, CD105, CD144, lectin binding, and DiIacetyl LDL uptake—are shared by many subsets of mononuclear cells.11–17,27–29
Because of the uncertainty involved in phenotypic characterization using cellular markers, we considered whether genotypic characterization could serve as a more exact determinant of cellular phenotype. Our data suggest that cells derived from a commercially available EPC-CFU assay, based on the assay used by Hill et al,9
are primarily T lymphocytes by cell surface marker expression, by flow cytometry, and by transcriptome regression analysis with T lymphocytes. Although gene expression patterns correlated weakly with those of endothelial cells in our database, few endothelial cells (CD146+CD45−) were observed by flow cytometry.
Marginal expression of the transcript for CD146, a surface marker traditionally present on endothelial cells, was present in our microarray analysis. Flow cytometry of EPC-CFUs showed a small number of CD146+ cells, but the majority coexpressed CD3, suggesting that these cells are T lymphocytes and not endothelial cells. Coexpression of CD146 on lymphocytes is representative of an activated lymphocyte phenotype with increased capacity to bind to endothelial cells and extravasate to sites of inflammation.24,30,31
Blood samples from healthy subjects in this study showed that approximately 2% of CD3+ cells in circulation were also CD146+. Accordingly, cells from our assay likely represent an expansion of this unique population.
Our findings are consistent with reports from Hur et al13
and Rohde et al,15
who reported that colonies from mononuclear cells plated on fibronectin are predominately lymphocytes. Rohde et al15
used the same commercial assay as was used in our study and demonstrated predominance of T cells and monocytes in their colonies by flow cytometric determination of cell surface markers. Of interest, inclusion of both CD2+ lymphocytes and CD14+ monocytes in their starting population of blood mononuclear cells in culture was necessary for colony formation to be observed after five days of culture. Our data extends these findings by using a microarray approach to determining the phenotype of cells in this assay, demonstrating expression of genes encoding surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns closely matched T lymphocytes in our microarray database. Flow cytometric analysis of cells from colonies was also consistent with T lymphocytes, with few endothelial cells present after 5 days of plating on fibronectin-coated plates. We also investigated whether colonies in this assay may have relevance in subjects participating in a worksite wellness program, the majority of whom had risk factors for impaired endothelial function. Contrary to our original hypothesis and to the report of Hill et al,9
we found no correlations between EPC-CFUs and FMD at baseline. Several groups have suggested that cells in colonies, despite limited potential of endothelial differentiation, may have paracrine effects on endothelium via secretion of cytokines and growth factors. In this regard, our findings are consistent in part with data of Hur et al,13
who also reported a predominance of CD3+CD31+ cells in their colony assay. They claimed that these cells represent a unique population of T lymphocytes capable of secreting angiogenic cytokines and facilitating endothelial differentiation.
Exercise has been reported to mobilize bone marrow–derived EPCs into the circulation,18–22
with the potential of attachment to arteries in the circulation and replacement of dysfunctional endothelium. Although we saw an increase in EPC-CFUs in our culture assay after exercise, the cells remained predominately T lymphocytes by microarray analysis of cell surface markers and by transcriptome regression analysis with T lymphocytes. Gene expression profiling before and after exercise training revealed 25 differentially regulated genes, in a magnitude of change that has been considered relevant for human microarray studies.32
Gene ontology analysis of these genes revealed an enrichment of transcripts involved in signal transduction, metabolism, immune response, translation, transcription, and transport. Yet the majority of these genes were downregulated after exercise, thus the relevance to the observed increase in colonies is obscure. Despite absence of correlations with endothelial testing to our study, it remains possible that the observed T lymphocyte population recovered from the EPC-CFU assay has a functional relationship to the endothelium or EPC capacity. Alternatively, the observed improvement in endothelial function by FMD may be independent of these T lymphocytes and may be attributable to more conventional responses. For example, repetitive shear stress attributable to exercise has been shown to increase expression and activity of endothelial nitric oxide synthase (eNOS), the product of which (NO) improves endothelial function, and could account for the enhanced dilator response of the brachial artery to shear stress in our study participants.33,34
Although other assays than the one used in our study have been proposed for demonstrating endothelial differentiation potential of blood-derived progenitor cells,12
none to date has been shown to be relevant to endothelial function in humans. Further, the role of bone marrow–derived cells in postnatal angiogenesis or arterial repair has been questioned by some groups.35–39
Animal models of bone marrow transplant and parabiosis, with donor animals genetically engineered to permit cells tracking, have shown few-to-no cells incorporated into vasculature of recipient animals, even with stimulated mobilization and homing by VEGF administration or tumor induction.39
Accordingly, improvement in endothelial function with exercise training may depend more on upregulation of eNOS within endothelium or reparative effects of resident progenitor cells within vasculature rather than bone marrow–derived progenitor cells in the circulation.40–43