Owen J. MacEneaney

Endothelial progenitor cell number and colony-forming capacity in overweight and obese adults

Objective:To investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity.Design:Cross-sectional study of normal weight, overweight and obese adult humans.Participants:Sixty-seven sedentary adults (aged 45–65 years): 25 normal weight (body mass index (BMI) ⩽25 kg/m2; 12 males/13 females); 18 overweight (BMI=25–29.9 kg/m2; 12 males/6 females); and 24 obese (BMI ⩾30 kg/m2; 18 males/6 females). All participants were non-smokers and free of overt cardiometabolic disease.Measurements:Peripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit (CFU) assay.Results:Number of circulating putative EPCs (either CD45−/CD34+/VEGFR-2+/CD133+ or CD45−/CD34+ cells) was lower (P<0.05) in obese (0.0007±0.0001%; 0.050±0.006%) compared with overweight (0.0016±0.0004%; 0.089±0.019%) and normal weight (0.0015±0.0003%; 0.082±0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony formation was significantly less in the obese (6±1) and overweight (4±1) compared with normal weight (9±2) adults.Conclusion:These results indicate that: (1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and (2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk.

Aging and Endothelial Progenitor Cell Telomere Length in Healthy Men

Abstract Background: Telomere length declines with age in mature endothelial cells and is thought to contribute to endothelial dysfunction and atherogenesis. Bone marrow-derived circulating endothelial progenitor cells (EPCs) are critical to vascular health as they contribute to both reendothelialization and neovascularization. We tested the hypothesis that EPC telomere length decreases with age in healthy adult humans. Methods: Peripheral blood samples were collected from 40 healthy, non-obese, sedentary men: 12 young (age 21–34 years), 12 middle-aged (43–55 years) and 16 older (57–68 years). Putative EPCs were isolated from peripheral blood mononuclear cells and telomere length was determined using genomic DNA preparation and Southern hybridization techniques. Results: EPC telomere length (base pairs) was ∼20% (p=0.01) lower in the older (8492+523 bp) compared to the middle-aged (10,565+572 bp) and young (10,205+501 bp) men. Of note, there was no difference in EPC telomere length between the middle-aged and young men. Conclusions: These results demonstrate that EPC telomere length declines with age in healthy, sedentary men. Interestingly, telomere length was well preserved in the middle-aged compared to young men, suggesting that EPC telomere shortening occurs after the age of 55 years. Clin Chem Lab Med 2009;47:47–50.

Aging Is Associated with a Proapoptotic Endothelial Progenitor Cell Phenotype

The aim of this study was to determine if aging is associated with enhanced endothelial progenitor cell (EPC) sensitivity to apoptosis. Cells with phenotypic EPC characteristics were isolated from healthy, nonobese young (age 25 ± 1 years) and older (61 ± 1 years) men. Intracellular active caspase-3 concentrations in response to staurosporine stimulation were approximately 35% higher (p < 0.05) in EPCs from older (3.15 ± 0.29 pg/ml) compared with young (2.33 ± 0.24 pg/ml) men. Protein expression of Akt, p70 S6-kinase and Bcl-2 was markedly lower (approx. 35, 75 and 60%, respectively, all p < 0.05) in EPCs from older compared with young men, whereas there were no age-related differences in either 14-3-3Ε or Bax expression. Additionally, EPC telomerase activity was 57% lower (p < 0.05) in older (0.18 ± 0.11 AU) versus young (0.43 ± 0.11 AU) men. These results indicate that aging is associated with a proapoptotic EPC phenotype characterized by decreased expression of key antiapoptotic proteins associated with the PI-3-kinase signaling pathway and reduced telomerase activity. These age-related changes likely contribute, in part, to the diminished ability of EPCs to resist an apoptotic stimulus in older men. Increased susceptibility to apoptosis may contribute to the numerical and functional impairments observed in EPCs with aging.

Endothelial Progenitor Cell Function, Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle‐aged and older adults (42–67 years) were studied: 25 normal weight (normal weight; BMI: 18.5–24.9 kg/m2) and 42 overweight/obese (overweight/obese; BMI: 25.0–34.9 kg/m2). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony–stimulating factor (G‐CSF) was determined in the absence and presence of phytohemagglutinin (10 µg/ml). Intracellular active caspase‐3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell–derived factor‐1α (SDF‐1α; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin‐stimulated release of VEGF (90.6 ± 7.6 vs. 127.2 ± 11.6 pg/ml) and G‐CSF (896.1 ± 77.4 vs. 1,176.3 ± 126.3 pg/ml) was ∼25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ∼30% greater (P < 0.05) increase in active caspase‐3 in EPCs from overweight/obese (2.8 ± 0.2 ng/ml) compared with normal weight (2.2 ± 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF‐1α. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.

CD31+ T cells represent a functionally distinct vascular T cell phenotype

In contrast to CD3(+)/CD31(-) cells, CD3(+)/CD31(+) cells aid in endothelial repair and revascularization. There are limited data regarding the functional differences between circulating CD3(+)/CD31(+) and CD3(+)/CD31(-) cells that may contribute to their divergent cardiovascular effects. The aim of the present study was to characterize functional differences between CD3(+)/CD31(+) and CD3(+)/CD31(-) cells. To address this aim, migratory capacity, proangiogenic cytokine release and apoptotic susceptibility of CD3(+)/CD31(+) and CD3(+)/CD31(-) cells were determined. Human CD3(+)/CD31(+) and CD3(+)/CD31(-)cells from peripheral blood were isolated using magnetic-activated cell sorting. CD3(+)/CD31(+) cells demonstrated significantly higher ( approximately 60%) migratory capacity to the chemokines SDF-1alpha (655+/-99 vs. 273+/-54 AU) and VEGF (618+/-99 vs. 259+/-57 AU) vs. CD3(+)/CD31(-) cells. Release of angiogenic cytokines G-CSF, interleukin-8 and matrix metallopeptidase-9 were all approximately 100% higher (P<0.05) in CD3(+)/CD31(+) than CD3(+)/CD31(-) cells. CD3(+)/CD31(+) cells exhibited significantly higher intracellular concentrations of active caspase-3 (2.61+/-0.60 vs. 0.34+/-0.09 ng/mL) and cytochrome-c (21.8+/-1.4 vs. 13.7+/-1.0 ng/mL). In summary, CD3(+)/CD31(+) cells have greater migratory and angiogenic cytokine release capacity, but are more susceptible to apoptosis compared with CD3(+)/CD31(-) cells. Enhanced migratory capacity and angiogenic cytokine release may contribute to the vasculogenic properties of this unique T cell subpopulation.

Prehypertension and Endothelial Progenitor Cell Function.

Prehypertension is associated with significant damage to the coronary vasculature and increased rates of adverse cardiovascular events. Circulating endothelial progenitor cells (EPCs) are critical to vascular repair and the formation of new blood vessels. We tested the hypothesis that prehypertension is associated with EPC dysfunction. Peripheral blood samples were collected from 83 middle-aged and older adults (51 male and 32 female): 40 normotensive subjects (age 53±2 years; BP 111/74±1/1 mm Hg) and 43 prehypertensive subjects (age 54±2 years; 128/77±1/1 mm Hg). EPCs were isolated from peripheral blood, and EPC colony-forming capacity (colony-forming unit (CFU) assay), migratory activity (Boyden chamber) and apoptotic susceptibility (active caspase-3 concentrations) were determined. There were no significant differences in the number of EPC CFUs (10±2 vs 9±1), EPC migration (1165±82 vs 1120±84 fluorescent units) or active intracellular caspase-3 concentrations (2.7±0.3 vs 2.3±0.2 ng ml−1) between the normotensive and prehypertensive groups. When groups were stratified into low prehypertension (n=27; systolic blood pressure: 120–129 mm Hg) and high prehypertension (n=16; 130–139 mm Hg), it was found that EPCs from the high prehypertensive group produced fewer (∼65%, P<0.05) CFUs compared with the low prehypertensive (4±1 vs 12±2) and normotensive adults. In conclusion, EPC colony-forming capacity is impaired only in prehypertensive adults with systolic BP greater than 130 mm Hg. Prehypertension is not associated with migratory dysfunction or enhanced apoptosis of EPCs.

Ageing and endothelial progenitor cellrelease of proangiogenic cytokines

SIR—Circulating endothelial progenitor cells (EPCs) are widely recognised to contribute to the reparative process of the vascular endothelium and participate in angiogenesis [1]. Although declines in the circulating population of EPCs are associated with poor cardiovascular disease prognosis and are predictive of future adverse cardiovascular events [2], transplantation of ex vivo expanded EPCs into the coronary artery can rescue ischaemic tissue and significantly improve coronary function in patients with myocardial infarction [3]. The angiogenic potential of these cells can be explained, in part, through their ability to home to local sites of ischaemia and vascular damage and secrete potent proangiogenic factors, such as cytokines, chemokines and growth factors, which are integral in promoting new blood vessel formation and repair. For example, both vascular endothelial growth factor (VEGF) [4] and granulocyte-colony stimulating factor (G-CSF) [5] stimulate recruitment and migration of EPCs from the bone marrow, inhibit apoptosis and support the angiogenic capacity of mature endothelial cells [6, 7]. In addition, interleukin (IL)-8, a proangiogenic cytokine, has been shown to attract EPCs to infarcted tissue and enhance the effect of G-CSF to mobilise progenitor cells from the bone marrow [8–10]. In older adults, endothelial injury and compromised EPC-mediated vascular repair are thought to contribute to atherosclerosis [11]. We have previously reported that EPC colony-forming capacity, migration and telomere length decline with ageing [12, 13]. In the present study, we tested the hypothesis that the capacity of circulating EPCs to release proangiogenic cytokines declines with age in healthy adults.

Habitual short sleep duration and circulating endothelial progenitor cells

Chronic short sleep duration has been linked to endothelial dysfunction and increased risk of cardiovascular disease. Circulating endothelial progenitor cells (EPCs) are vital to endogenous vascular repair processes and cardiovascular health. We tested the hypothesis that habitual short sleep duration is associated with impairment in EPC number and function. Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 with normal sleep duration (13M/7F; age: 59±1 years; sleep duration: 7.7±0.1 h/night) and 17 with short sleep duration (9M/8F; 56±2 years; 6.0±0.2 h/night). EPC number was assessed by flow cytometric analysis of the percentage of peripheral blood mononuclear cells negative for CD45 and positive for CD34, VEGFR-2, and CD133 antigens. EPC colony-forming capacity was determined by colony-forming unit (CFU) assay; migration by Boyden chamber; and intracellular caspase-3 concentrations by immunoassay. There were no significant differences between groups in EPC number (0.001±0.0004 vs. 0.001±0.0003 %), colony-forming capacity (6.1±1.5 vs. 5.4±1.7 CFUs), or migration to VEGF (1410.1±151.2 vs. 1334.3±111.1 AU). Furthermore, there were no group differences in basal and staurosporine-stimulated intracellular concentrations of active caspase-3 (0.3±0.03 vs. 0.5±0.1 ng/mL; and 2.9±0.4 vs. 2.7±0.3 ng/mL), a marker of apoptotic susceptibility. Taken together, these data indicate that short sleep duration is not associated with EPC dysfunction in healthy adults. Numerical and functional impairment in circulating EPCs may not contribute to the increased cardiovascular risk with habitual short sleep duration.

Effects of endothelin-1 on endothelial progenitor cell function

Abstract Background: Circulating endothelial progenitor cells (EPCs) contribute to vascular endothelial repair. Endothelin (ET)-1 is associated with endothelial damage and atherogenesis. The experimental aim of this study was to determine, in vitro, the effects of ET-1 on the ability of EPCs to form colonies, migrate, release angiogenic growth factors and resist apoptosis. Methods: Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC colony-forming capacity (CFU assay), migratory activity (Boyden chamber), release of angiogenic growth factors (enzyme immunoassay) and apoptosis (TUNEL assay) were determined in the absence and presence of ET-1 (100 pmol). Results: EPC colony-forming units (42±12 vs. 39±11), migratory capacity (910±146 vs. 936±148 AU) and release of vascular endothelial growth factor (202.8±68.1 vs. 204.8±69.8 pg/mL) and granulocyte-colony stimulating factor (1294.4±378.3 vs. 1136.1±310.3 pg/mL) were not significantly affected by ET-1. EPCs treated with ET-1 demonstrated a 20% increase (p<0.05) in cellular apoptosis. The proapoptotic effect of ET-1 was abolished with ET receptor blockade as well as with apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) inhibitor. Conclusions: These results indicate that ET-1 does not affect EPC colony formation, migratory capacity or angiogenic growth factor release, but does increase EPC susceptibility to apoptosis through an NADPH-dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET-1.

Effects of HIV-1 gp120 and protease inhibitors on apoptotic susceptibility of CD34+ hematopoietic progenitor cells.

To the Editors: HIV-1 infection is associated with several immunological disturbances, including a reduction in circulating CD34 hematopoietic progenitor cells. Depletion of CD34 cells may have far-reaching consequences, given their role in immune reconstitution and their association with vascular repair and cardiovascular disease. Although CD34 cells are generally resistant to HIV-1 infection, uninfected CD34 cells from patients with AIDS demonstrate a commitment to apoptosis. This adverse effect is likely due in large part to direct cytotoxic interactions with HIV-1 and, in particular, the envelope glycoprotein gp120. The advent of highly active antiretroviral therapy (HAART) in the treatment of HIV-1 infection has dramatically slowed the rate of progression of HIV-1 to AIDS and improved patient outcomes. As HIV-1–infected patients are living longer, HIV-1–related metabolic and cardiovascular complications are growing in this population, which may be linked to numerical or functional deficits in circulating progenitors. Treatment with protease inhibitors (PIs), an important component of many HAART regimens, generally results in improved viability of CD34 cells from HIV-1– infected individuals. However, this may be largely driven by the antiviral activity of these medications, although the direct effects on CD34 cells are less clear. One prior investigation reported that ritonavir (RTV) had antiapoptotic effects on CD34 cells. Accordingly, we hypothesized that other common PIs would also reduce CD34 cell apoptosis at physiological concentrations (ie, typical maximum plasma levels) in vitro. Freshly isolated CD34 cells from healthy G-CSF–treated human donors (n = 3) were obtained from an independent supplier (Allcells, LLC, Berkeley, CA). Donors tested negative for HIV-1, hepatitis B, and hepatitis C. Viability of cells determined by trypan blue exclusion tests was 95.7% 6 0.4%. Purity, as assessed by flow cytometric analysis with propidium iodide, was 97.7% 6 0.4%. CD34 cells were cultured in serumfree medium (StemCell Technologies, Inc, Vancouver, Canada) supplemented with 100 ng/mL of thrombopoietin, Flt3 ligand, and stem cell factor. Cells were treated for 48 hours with 1 mg/mL RTV, 5.2 mg/mL atazanavir (ATV), or 9.8 mg/mL lopinavir (LPV) (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Positive control experiments were performed using 100 ng/mL HIV-1Bal gp120 (R5 trophic) (AIDS Research and Reference Reagent Program) and HIV-1Lav gp120 (X4 trophic) 12 (Protein Sciences Corporation, Meriden, CT). Activation of caspase-3 was induced in CD34 cells by incubating with staurosporine (1 mmol/L for 3 hours; Sigma Aldrich, St Louis, MO). The concentration of active caspase-3 large subunit in cell lysates was determined using enzyme immunoassays (R&D Systems, Minneapolis, MN). The intraand interassay coefficients of variation for this assay in our laboratory are <10%. Experimental points were performed in duplicate with 3 independent experiments. Differences between treatments were determined by analysis of variance. Where indicated by a significant F value, post hoc tests, with Bonferroni correction for multiple comparisons, were performed to determine differences between treatment groups. Results are expressed as mean 6 standard error of the mean. Statistical significance was set at P < 0.05. On stimulation with staurosporine, both R5 (6.0 6 0.4 ng/mL) and X4 (6.0 6 0.1 ng/mL) gp120 resulted in a 65% greater activation of caspase-3 compared with the control condition (3.6 6 0.4 ng/ mL; P < 0.05) (Fig. 1A). Within the PI group, the capacity of staurosporine to induce an apoptotic response was 40% higher in cells treated with ATV (4.9 6 0.7 ng/mL) and LPV (5.1 6 0.2 ng/mL) compared with the control condition (P < 0.05) (Fig. 1B). In contrast, treatment with RTV (4.1 6 0.5 ng/mL) did not significantly alter intracellular active caspase-3. The novel findings of the present study are that the PIs, ATV and LPV, reduce the resistance of CD34 cells to an apoptotic stimulus. To our knowledge, this is the first study to demonstrate the proapoptotic effects of PIs on CD34 cells from healthy adults. CD34 cells from patients with AIDS demonstrate a marked predisposition toward apoptosis. Zauli et al showed that virus isolates from HIV-1– seropositive patients induced a dosedependent inhibition on the growth of CD34 cells in vitro, an effect blocked by pretreatment with an anti-gp120 antibody. Subsequent investigations further implicated gp120 (in concentrations ranging from 0.01 to 20 mg/mL) in the induction of CD34 cell apoptosis. Our results extend these findings to broadly physiological concentrations of gp120 from 2 strains of the HIV-1 virus, HIV-1Bal and HIV-1Lav, and add to the growing evidence base that indicates a direct cytotoxic activity of HIV-1 gpl20 on human CD34 cells. HAART has been shown to restore CD34 cell function in HIV-1–infected patients. For example, increased CD34 cell colony formation has been documented after 3–6 months of HAART. In addition, the PI RTV lowered apoptosis in CD34 cells from HIV-1–infected patients in vitro but only at a concentration of 5 nmol/L; no differences were recorded at concentrations between 5 and 20 nmol and a decrease was observed at concentrations >20 nmol. Interestingly, these effects have only been documented in CD34 cells from HIV-1–infected patients and only after short-term HAART administration. Our results show that the PIs ATV and LPV reduce resistance to apoptosis in CD34 cells from healthy adults. Although RTV had no effect in the present investigation, it should be noted that the concentration of RTV used in this study was that which is achieved in vivo to boost LPV levels through inhibition of cytochrome P450