EPC mobilization after erythropoietin treatment in acute ST-elevation myocardial infarction: the REVEAL EPC substudy.

Erythropoietin (EPO) was hypothesized to mitigate reperfusion injury, in part via mobilization of endothelial progenitor cells (EPCs). The REVEAL trial found no reduction in infarct size with a single dose of EPO (60,000 U) in patients with ST-segment elevation myocardial infarction. In a substudy, we aimed to determine the feasibility of cryopreserving and centrally analyzing EPC levels to assess the relationship between EPC numbers, EPO administration, and infarct size. As a prespecified substudy, mononuclear cells were locally cryopreserved before as well as 24 and 48-72 h after primary percutaneous coronary intervention. EPC samples were collected in 163 of 222 enrolled patients. At least one sample was obtained from 125 patients, and all three time points were available in 83 patients. There were no significant differences in the absolute EPC numbers over time or between EPO- and placebo-treated patients; however, there was a trend toward a greater increase in EPC levels from 24 to 48-72 h postintervention in patients receiving ≥30,000 U of EPO (P = 0.099 for CD133(+) cells, 0.049 for CD34(+) cells, 0.099 for ALDH(br) cells). EPC numbers at baseline were inversely related to infarct size (P = 0.03 for CD133(+) cells, 0.006 for CD34(+) cells). Local whole cell cryopreservation and central EPC analysis in the context of a multicenter randomized trial is feasible but challenging. High-dose (≥30,000 U) EPO may mobilize EPCs at 48-72 h, and baseline EPC levels may be inversely associated with infarct size.

Calorie restriction and dwarf mice in gerontological research.

What aging process is delayed by calorie restriction (CR) and mutations that produce long-lived dwarf mice? From 1935 until 1996, CR was the only option for increasing the maximum lifespan of laboratory rodents. In 1996, the mutation producing the Ames dwarf mouse (Prop-1(-/-)) was reported to increase lifespan. Since 1996, other gene mutations that cause dwarfism or lower body weight have been reported to increase the lifespan of mice. The recent discovery of long-lived mutant dwarf mice provides an opportunity to investigate common features between CR and dwarf models. Both CR and dwarf mutations increase insulin sensitivity. Elevated insulin sensitivity reduces oxidative stress, a potential cause of aging. The elevation of liver insulin sensitivity by the hormone adiponectin in CR and long-lived dwarf mice can lower endogenous glucose production and raise fatty acid oxidation. Adiponectin reduction of plasma glucose in CR and long-lived dwarf mice can thereby lower age-related increases in oxidative damage and cancer.

Common endothelial progenitor cell assays identify discrete endothelial progenitor cell populations.

BACKGROUND: Multiple measures of endothelial progenitor cells (EPCs) have been described, but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies. METHODS: We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients, EPCs were also identified on the basis of cell surface marker expression (CD133, CD34, and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity. RESULTS: Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however, endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133, CD34, CD133/CD34, and ALDH activity correlated with each other, but not with VEGFR-2(+) cells. CONCLUSIONS: Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells, which give rise to endothelial CFUs, and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research.

Aldehyde dehydrogenase activity allows reliable EPC enumeration in stored peripheral blood samples.

BACKGROUND: Interest in the biology of endogenous progenitor cells (EPCs) continues to grow as evidence of their role in vascular repair mounts. EPC enumeration requires specialized laboratory techniques and is performed immediately after sample acquisition, limiting the clinical contexts in which EPC enumeration can be performed and the ability to increase sample sizes through multi-center participation. METHODS: We compared the numbers of EPCs enumerated in samples processed immediately after acquisition (n = 36) with EPCs enumerated in specimens stored for 24 hours or after cryopreservation of mononuclear cells (MNC) using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH(br) cells). RESULTS: EPCs assessed in fresh samples correlated with EPCs enumerated after whole blood storage (r = 0.699 for CD133(+)CD34(+) cells, r = 0.880 for ALDH(br) cells, P < 0.005 and P < 0.0001, respectively) or mononuclear cryopreservation (r = 0.590 for CD133(+)CD34(+) cells, r = 0.894 for ALDH(br) cells, P < 0.0001 for each); however, correlation based on assessment of ALDH(br) cells was higher (P < 0.0003 for comparison of correlation coefficients). Initial results from a multi-site clinical trial suggest that EPC enumeration after mononuclear cell cryopreservation is feasible. CONCLUSION: EPC analysis based on ALDH activity is reproducible, even after extended whole blood storage or MNC cryopreservation.

Circulating progenitor cells can be reliably identified on the basis of aldehyde dehydrogenase activity.

OBJECTIVES: Our objective was to develop and assess a novel endogenous progenitor cell (EPC) assay based on aldehyde dehydrogenase (ALDH) activity, and to define the relationship of ALDH-bright (ALDH(br)) cells with previously defined EPCs, patient age, and extent of coronary artery disease. BACKGROUND: Accurate assessment of circulating EPCs is of significant interest, yet current assays have limitations. Progenitor cells display high levels of ALDH activity. An assay based on ALDH activity may offer a simple means for enumerating EPCs. METHODS: We simultaneously determined the numbers of EPCs based on ALDH activity and cell surface expression of CD133, CD34, and vascular endothelial growth factor receptor-2 in 110 patients undergoing cardiac catheterization. We assessed the reproducibility of these estimates, correlation among EPC assays, and the association of ALDH(br) numbers with age and disease severity. RESULTS: Aldehyde dehydrogenase-bright cells were easily identified in nonmobilized peripheral blood with median and mean frequencies of 0.041% and 0.074%, respectively. Aldehyde dehydrogenase-bright cells expressed CD34 or CD133 cell surface markers (57.0% and 27.1%, respectively), correlated closely with CD133+CD34+ cells (r = 0.72; p < 0.001), and differentiated into endothelial cells with greater efficiency than CD133+CD34+ cells. Aldehyde dehydrogenase-bright cell numbers were inversely associated with patient age and coronary disease severity. CONCLUSIONS: Aldehyde dehydrogenase activity represents a novel simplified method for quantifying EPCs. The correlation of ALDH(br) cells with clinical factors and outcomes warrants further study.