There is considerable basic research and clinical interest in the relevance of bone marrow-derived cells to vascular homeostasis and repair. Initial reports focused on hematopoietic progenitor cells with CD34 marker, but subsequently included a population of immature progenitor cells indentified by the CD133 marker. The relevance of these progenitor cells in CAD was suggested by Werner et al17
, who reported an inverse relation between CD34+ and CD133+ cells and subsequent cardiac events at 12 months in a series of 519 patients with disease confirmed at coronary angiography. Currently, the common clinical concept claims a protective role of bone marrow-derived progenitor cells, which may be compromised in CAD due to reduced number and impaired function18,19
. Considering these reported findings, we undertook the current study with the aim of characterizing CD133+ cells from CAD patients at the genomic level.
Our analysis showed significant differences in expression of several genes in CD133+ cells from CAD patients that may be important to survival and differentiation potential of cells compared with gene expression in CD133+ cells isolated from healthy controls. Thus we identified 82 differentially expressed genes in CAD patients compared with controls, of which 59 genes, such as HOX A920
, RIPK1, NARF, ANAPC1322
, were found to be significantly up-regulated, and 23 genes, such as MAP3K24–26
, FNBP4 and RBM6, were found to be down-regulated. By Ingenuity Pathway analysis, these genes were found to be involved in multiple cellular functions, especially carbohydrate metabolism and molecular transport. After 3 months of rehabilitation associated with significant improvement in effort tolerance and increase in the number of CD133+ cells, gene expression patterns of the CD133+ cells resembled those of controls in 7 out of 10 patients in our study. In 4 CAD patients, exercise reversed expression levels towards those measured in controls in both up- and down-regulated genes determined at baseline. Correlation analysis identified only SP 100 expression to have statistically significant association to baseline CD133 cell number. The relevance of SP 100 gene product to stem cells of vascular endothelium and cardio vascular disease remains unknown.
To identify transcripts that are highly abundant in CD133+ cells from our study, positively detected transcripts from healthy controls were ranked in order of their abundance from high to low based on their log-transformed median signal intensity. The top 500 ranking probe sets representing 351 genes identified to be of high abundance in CD133+ cells by Ingenuity pathway analysis at p<0.05 indicated them to be involved in multiple cellular functions such as protein synthesis, post-transcriptional modification and cell death, and this list provides a novel expanded transcriptome of CD 133+ cells.
A comparative analysis of previously published 257 CD133+ highly abundant transcripts from cord blood and peripheral blood30–32
and CD133+ transcripts from the current study showed overlap of 42 highly expressed transcripts such as growth factor binding proteins, nucleosporin, transcription factors, sideroflexin, anaphase promoting complex, and map kinases. The non-overlapping genes included several transcripts with unknown functions and several zinc finger proteins. It should be noted that the methodology to sort the CD133+ cells and RNA processing for gene chip hybridization differ considerably among these studies.
Of particular interest among these gene expression modulations is the significant alteration in the expression of HOXA9 and histone acetyl transferase (HAT1) genes in our study. It is believed that the interplay between HAT, histone deacetyalse (HDAC) and HOXA9 genes determines the endothelial differentiation of progenitor cells23
. HOXA9 has been shown to be crucial for hematopoietic stem cell development and stem cell expansion33
. HOXA9 acts as a homeobox transcription factor expressed in mature endothelial cells and its expression is downregulated by inflammatory signals. HOXA9 overexpression is associated with an increase in the number of circulating EPCs while in HOXA deficient mice, there was a decrease in the number of EPCs and impaired postnatal neovasculaization23,34
. Studies on hypertensive patients with significant endothelial injury by Pirro et al35
revealed decrease in the number of peripheral EPCs and significantly lowered expression of HOXA9 gene in those cells. HAT1 is involved in fundamental processes that regulate the expression of multiple genes that play an important role in the multifactorial processes that lead to atherosclerosis and re stenosis36,37
. It is hypothesized that HOXA9 functions by modulating downstream target genes that contributes in physiological conditions to endothelial commitment of EPCs, postnatal neovascularization and injured endothelium repair20
. The relevance of expression of these genes in CAD patients at baseline or follow up is unclear as we observed no association with CD133 cell numbers and we did not measure the endothelial function in our patients.
Our findings may have clinical implications, as CD133+ cells may have limited capacity to repair vascular disease in patients with CAD and thus compromise cell-based treatment approaches, including autologous administration of CD133+ cells isolated from the circulation and injected in large numbers into the heart (17). Although CD34+ selected cells or nonselected bone marrow mononuclear cells are more commonly used for this purpose, at least 4 trials using CD133+ cells are registered in www.ClinicalTrials.gov
. We also report that exercise might reverse some of the gene expression patterns towards levels seen in cells from healthy subjects, thus improving their potential for vascular repair or new vessel growth. Further studies are needed to determine whether changes in CD133+ gene expression patterns towards that seen in healthy subjects are accompanied by changes in phenotype and differentiation potential that may be relevant to cardiovascular repair.