Endothelial Progenitor Cell Quantification in Young Adult Non-Smokers and Smokers by a Novel Gating Strategy

PURPOSE: Endothelial progenitor cells (EPC) are an accepted predictor of cardiovascular disease risk. Accurate identification of EPC is debatable, and to date identifying markers and isolation techniques remain to be clearly established. Primitive EPC are conventionally defined as CD34+CD133+KDR+. Few studies have investigated CD34+ EPC that are endothelial in nature, and their phenotype and relation to subsets of hematopoietic cells remain elusive. Thus, reliable flow cytometry acquisition and analysis strategies are needed as EPC comprise \u3c1% of peripheral blood mononuclear cells. Previously, it has been shown that long-term smoking decreases circulating CD34+ cells in older individuals. We designed and optimized a novel cytometry protocol for accurate enumeration of EPC in recreationally-active, non-smoking young adults (n=36, 23.3±0.6yr) compared to chronic smokers (n=28,25.0±0.8yr) as part of a randomized-controlled trial (RCT) to investigate the effects of resistance training (RT) on vascular function in young, adult chronic smokers. METHODS: Using a whole blood lysing method, 2×107 cells were stained with a 5-color antibody panel and propidium iodide for dead cell exclusion. 4×106 cells were acquired to achieve sufficient events to classify PC reliably as viable, side scatterlow, CD45-/dim, CD3-CD19-CD33-CD34+ and subdivide them into CD133+ and KDR+. To improve the accuracy of rare EPC analysis in samples with varied red cell contamination of the gating region, we based our calculation of EPC frequency not on lymphocyte scatter gating, but on a novel strategy of a combination gate of CD45bright and CD34+. RESULTS: Preliminary data show that compared to non-smokers, smokers have a higher percentage of CD133+ (50±2.1% vs. 42±2.9%, p=0.03), but lower KDR+ within the CD34+subset of cells (1.9±0.44% vs. 4.8±0.94%, p=\u3c0.01). Interestingly though, smokers had a higher proportion of CD45bright lymphocytes than non-smokers (88±2.4% vs. 72±4.6%, p=\u3c0.01). CONCLUSION: We demonstrated the ability to detect specific circulating EPC subsets using our novel gating strategy. In young smokers, compensatory mechanisms may prevent the decline in CD34+ cells before true endothelial dysfunction exists. Our ongoing RCT will determine if RT has an effect on the number of EPC and EPC subsets in chronic smokers