JCDR Original Article Habitual short sleep duration and circulating endothelial progenitor cells Brian R. Weil1, Owen J. MacEneaney1, Brian L. Stauffer1,2,3, Christopher A. DeSouza1,2 Integrative Vascular Biology Laboratory, 1Department of Integrative Physiology, University of Colorado, Boulder, 2Department of Medicine, University of Colorado at Denver, Aurora, 3Denver Health Medical Center, Denver, CO, USA Address for correspondence: Dr. Christopher DeSouza, Integrative Vascular Biology Laboratory, Department of Integrative Physiology,354 UCB, University of Colorado, Boulder, CO 80309, USA. E-mail: [email protected] ABSTRACT 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. Key words: Endothelium, progenitors, sleep INTRODUCTION cardiovascular morbidity and mortality.[1] However, the biological mechanisms linking habitual short sleep duration It has become increasingly apparent that chronic short and the development of cardiovascular diseases (CVDs) sleep duration adversely affects cardiovascular health. are not well-defined. Several recent epidemiological studies have reported that short sleep duration is associated with increased Endothelial damage and dysfunction is an important early event in the pathogenesis of atherosclerotic vascular disease and is independently associated with an increased risk of Access this article online cardiovascular events.[2] Recent data from our laboratory[3] Quick Response Code: Website: and others[4] suggest that acute and chronic sleep restriction www.jcdronline.com is associated with pathogenic alterations in endothelial cell function. The capacity to repair the endothelium and restore DOI: 10.4103/0975-3583.83039 its function after injury also plays a vital role in determining cardiovascular risk.[5] Circulating endothelial progenitor 110 Journal of Cardiovascular Disease Research Vol. 2 / No 2 Weil, et al.: Sleep duration and endothelial progenitor cells cells (EPCs) have recently been identified as an integral Body composition and metabolic measurements component of the endogenous vascular repair system.[5] Importantly, declines in both EPC number and function Body mass was measured to the nearest 0.1 kg using a have been linked with accelerated atherosclerotic disease medical beam balance. Percent body fat was determined progression and poor CVD prognosis.[6-8] Thus, a potential by dual-energy X-ray absorptiometry (Lunar, Madison, mechanism contributing to the heightened cardiovascular WI). Body mass index was calculated as mass (kilograms) risk with short sleep duration may be a reduction in EPC divided by height (meters) squared. Fasting plasma lipid bioavailability and/or function. Accordingly, we tested the and lipoprotein, glucose, and insulin concentrations were hypothesis that habitual short sleep duration is associated determined using conventional methods by the clinical with impairment in EPC number and function. laboratory affiliated with the University of Colorado at Boulder Clinical and Translational Research Center. MATERIALS AND METHODS EPC isolation, characterization, and numeration Subjects Circulating putative EPCs were isolated from peripheral blood by Ficoll density–gradient centrifugation (Histopaque Thirty-seven adults (age range: 43–65 years) were studied: 20 1077, Sigma Aldrich, St Louis, MO, USA) as previously with normal habitual sleep duration (13 males/7 females) and 17 with short habitual sleep duration (9 males/8 females). described by our laboratory.[11] Circulating EPC number was All subjects were free of overt cardiometabolic disease as determined by fluorescence-activated cell sorting (FACS) assessed by medical history, physical examination, and fasting analysis. Briefly, 2 × 106 cells were incubated at 4°C for blood chemistries. All female subjects were at least one year 30 min with monoclonal antibodies for PC7-conjugated postmenopausal and had never taken or had discontinued CD45 (Beckman Coulter, Fullerton, CA, USA), FITC- use of hormone replacement therapy at least one year prior conjugated CD34 (Beckman Coulter), PE-conjugated to the start of the study. None of the subjects smoked, were VEGFR-2 (R&D Systems, Minneapolis, MN, USA) and taking medications, or performed regular physical exercise APC-conjugated CD133 (Miltenyi Biotech, Auburn, CA, for at least 6 months before the start of the study. Prior to USA). Cells were gated for low expression of CD45 and participation, all of the subjects had the research study and its then CD34+ cells were analyzed for events double-positive potential risks and benefits explained fully before providing for VEGFR-2 and CD133 and presented as a percent of written informed consent according to the guidelines of the total viable mononuclear cells. University of Colorado at Boulder. Colony-forming assay Sleep duration EPC colony-forming capacity was determined as previously Habitual sleep duration was measured as a component of described.[11] Briefly, freshly isolated mononuclear cells were the Stanford Physical Activity Questionnaire, as previously plated on six-well plates coated with human fibronectin (BD described.[3] Consistent with previous studies,[9] nightly Biosciences, San Jose, CA, USA) for 48 h at 37ºC. Thereafter, average reported sleep duration was calculated as the non-adherent cells were collected and 5 × 105 cells were weighted average of weeknights and weekend values [(5 × replated onto 24-well fibronectin-coated plates (BD weekday sleep duration) + (2 × weekend sleep duration)/7]. Biosciences). Growth medium was changed every three days, Subjects were divided into two groups based upon their and the EPC colony-forming units (CFUs) were counted reported sleep duration: 7 to 9 h/night or “normal habitual manually in four random wells on the seventh day by two sleep duration” and <7 h/night or “short habitual sleep independent investigators blinded to sample identification. duration”. These criteria were chosen based on published reports indicating that habitual sleep duration shorter Migration assay than 7 h/night is associated with increased health risks, including hypertension, coronary artery disease, and EPC migratory capacity was measured using a modified stroke.[1] Although assessment of habitual sleep duration Boyden chamber.[12] Non-adherent cells (4 × 105) were by self-report has the potential to introduce reporting resuspended in serum-free culture medium (Medium 199, bias and experimental error, previous investigations have GIBCO) and loaded in the upper compartment of a 24- demonstrated a robust correlation between self-reported well modified Boyden chamber coated with fibronectin sleep duration and that measured objectively through (FluoroBlok, BD Biosciences). The upper chamber was actigraphic monitoring.[10] placed in the lower chamber containing culture medium Journal of Cardiovascular Disease Research Vol. 2 / No 2 111 Weil, et al.: Sleep duration and endothelial progenitor cells supplemented with vascular endothelial growth factor (2 ng/ml) and incubated for 22 h at 37°C. Following incubation, cells were labeled with calcein AM (Molecular Probes, Eugene, Oregon), and relative fluorescent units (RFUs) of migrated cells were determined in triplicate. Caspase-3 activation Activation of caspase-3 was induced by incubating EPCs with staurosporine (1 μmol/L) for 3 h; Sigma Aldrich, St. Louis, MO). Following staurosporine stimulation, cells were washed with PBS and incubated with 10 mmol biotin- ZVKD-fmk inhibitor for 1 h at 37°C. Thereafter, cells were washed and lysed with extraction buffer at a concentration a of 1 × 107 cells/mL. The concentration of active caspase-3 in the supernatant was determined by enzyme immunoassay (R&D Systems, Minneapolis, MN). Statistical analysis Differences in subject baseline characteristics and the primary outcome variables were determined by analysis of variance (ANOVA). There were no significant gender interactions in any of the primary outcome variables; therefore, the data were pooled and presented together. Relations between sleep duration and EPC measures were assessed by linear regression analysis. Data are reported as b mean ± SEM. Statistical significance was set at P<0.05. RESULTS Selected subject characteristics are presented in Table 1. Anthropometric characteristics were similar between the groups. The short sleep duration group reported a significantly lower average sleep duration (6.0 ± 0.2 h/night; range: 4.0 to 6.9 h/night) compared with the normal sleep duration group (7.7 ± 0.1 h/night; range: 7.0 to 9.0 h/night). There were no differences between the groups in plasma lipid and lipoprotein, glucose, and insulin concentrations. c The number of CD45-/CD34+/VEGFR-2+/CD133+ cells Figure 1: EPC colony-forming units (a), migratory activity (b), and intracellular concentrations of active caspase-3 under basal conditions was not different between the normal sleep (0.0011 ± 0.0003%) and in response to staurosporine (c) in the normal sleep and short sleep and short sleep duration (0.0014 ± 0.0004%) groups. There duration groups. Values are mean ± SEM were no significant differences in the number of EPC CFUs between the normal sleep (6 ± 2) and short sleep duration with the pro-apoptotic stimulus staurosporine, intracellular (5 ± 2) groups [Figure 1]. EPC migratory activity was similar caspase-3 concentrations significantly increased in both between adults reporting normal sleep duration (1410 ± 151 groups. However, there was no significant difference in RFU) and adults reporting short sleep duration (1334 ± 111 stimulated intracellular caspase-3 concentrations between RFU; Figure 1). Under basal conditions, there was no difference in intracellular active caspase-3 concentrations the normal sleep duration (2.8 ± 0.3 ng/mL) and short sleep between the normal sleep (0.3 ± 0.03 ng/mL) and short sleep (2.7 ± 0.3 ng/mL) groups [Figure 1]. There were no significant (0.4 ± 0.11 ng/mL) duration groups. Following treatment correlations between sleep duration and any EPC measure. 112 Journal of Cardiovascular Disease Research Vol. 2 / No 2 Weil, et al.: Sleep duration and endothelial progenitor cells In the present study, contrary to our hypothesis, habitual Table 1: Selected subject characteristics short sleep duration was not associated with impairments Variable Normal Sleep Short Sleep Duration Duration in EPC number or function. Circulating numbers of (n=20) (n=17) EPCs as well as their ability to form colonies, migrate and Males/Females 13/7 9/8 resist apoptosis was almost identical between middle-aged Age, year 59 ± 1 56 ± 2 adults who chronically sleep ~6 h/night compared with Sleep Duration (hrs/night) 7.7 ± 0.1 6.0 ± 0.2* those who sleep ~8 h/night. Although we observed no Body mass, kg 86.4 ± 3.6 85.4 ± 3.8 significant relation between sleep duration and any of the BMI, kg/m2 28.7 ± 1.0 29.1 ± 0.9 EPC measures, we studied a subpopulation (n=7) of adults Body fat, % 32.9 ± 1.9 36.1 ± 1.9 within the short sleep duration group who reported ≤5 h Waist circumference, cm 97.8 ± 2.8 95.3 ± 3.5 of sleep per night to dismiss the possibility that the mean Waist-to-hip ratio 0.90 ± 0.05 0.89 ± 0.03 habitual sleep duration was not low enough in the short sleep Systolic BP, mmHg 125 ± 2 123 ± 2 group to induce EPC abnormalities. Similar to our findings Diastolic BP, mmHg 77 ± 1 79 ± 1 in the overall study population, EPC number and functional Total cholesterol, mmol/L 5.0 ± 0.3 5.3 ± 0.2 characteristics were not different in the subpopulation with LDL-cholesterol, mmol/L 3.1 ± 0.2 3.4 ± 0.1 shorter chronic sleep duration compared with the normal HDL-cholesterol, mmol/L 1.3 ± 0.1 1.4 ± 0.1 sleep group. However, it remains possible that more severe Triglycerides, mmol/L 1.3 ± 0.1 1.2 ± 0.1 chronic sleep restriction is associated with impairments in Glucose, mmol/L 5.0 ± 0.3 5.0 ± 0.1 EPC number and/or function. This notion is supported Insulin, pmol/L 26.4 ± 6.3 33.9 ± 6.9 by data indicating that ten days of severe sleep restriction HOMA-IR 1.1 ± 0.3 1.3 ± 0.3 (<4 h/night) elicits increased production of inflammatory Values are mean±SEM. BMI: Body mass index; BP: Blood pressure; LDL: Low- density lipoprotein; HDL: High-density lipoprotein; HOMA-IR: Homeostasis model mediators that have been shown to attenuate EPC survival, of insulin resistance; *P < 0.05 vs. normal sleep duration. differentiation, and function.[13-15] DISCUSSION To our knowledge, this is the first study to examine the influence of habitual short sleep duration on EPC The primary new findings of the present study are that number and function. It is important to note, however, EPC number, capacity to form colonies, migratory ability, that we studied healthy individuals without co-existing and apoptotic susceptibility are not impaired in healthy cardiovascular risk factors. Considering obesity, type-2 adults who sleep less than 7 h/night. Collectively, these data diabetes, and the metabolic syndrome, common morbidities suggest that a decrement in EPC number or function is in middle-aged adults are associated with both short not involved in the increased cardiovascular risk observed sleep duration[1] and EPC dysfunction;[11,16] the presence in this population. of these and other risk factors may alter our results. Nevertheless, these data suggest that numerical and Endothelial dysfunction has recently been identified as a functional impairment in EPCs, independent of other risk potential link between short sleep duration and CVD.[3,4] factors do not contribute to the increased cardiovascular risk associated with habitual short sleep duration. Maintenance and repair of the endothelium is essential in preserving function and preventing the development Although there is currently no clear consensus on the and progression of atherosclerotic disease. EPCs play an most appropriate criteria for the isolation, culture or important role in vascular repair and can promote both re- quantification of EPCs, in the present study we isolated endothelialization and neovascularization.[5] Several studies and identified a putative EPC cell population that has been have linked reduced number and function of circulating consistently shown to participate in vasculogenesis and EPCs to endothelial dysfunction and CVD risk.[6-8] For vascular repair,[17] and is associated with both endothelial example, Schmidt-Lucke and colleagues reported that dysfunction and cardiovascular risk.[8] These cells were lower number of circulating EPCs independently predicts isolated with minimal time in culture to reduce phenotypic atherosclerotic disease progression.[6] Moreover, reduced drift and exhibited several endothelial phenotypic EPC colony-forming capacity and migratory ability have characteristics.[17] In contrast, other investigators have each been independently linked to an increased risk of argued that “outgrowth endothelial cells”, a proliferative CVD,[7,8] while heightened EPC susceptibility to apoptosis endothelial phenotype derived from mononuclear cells can underlie functional impairments and contribute to following an extended period of in vitro culture (2–4 weeks), endothelial dysfunction and atherogenesis.[5] should be considered as true endothelial progenitors, Journal of Cardiovascular Disease Research Vol. 2 / No 2 113 Weil, et al.: Sleep duration and endothelial progenitor cells given their morphological similarity to mature endothelial 6. Schmidt-Lucke C, Rossig L, Fichtlscherer S, Vasa M, Britten M, Kamper U, cells.[18] However, the culture of these cells involves et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: Proof of concept for the clinical importance untold phenotypic drift from the cells in circulation and of endogenous vascular repair. Circulation 2005;111:2981-7. their physiological and clinical relevance are unclear. 7. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, et al. Number Nevertheless, our results must be viewed within the context and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89:E1-7. of our isolation, culture, and characterization methodology. 8. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and In conclusion, short sleep duration is not associated with cardiovascular risk. N Engl J Med 2003;348:593-600. impairment in either EPC number or function in otherwise 9. Hall MH, Muldoon MF, Jennings JR, Buysse DJ, Flory JD, Manuck SB. Self-reported sleep duration is associated with the metabolic syndrome in healthy adults. Although recent data implicate endothelial midlife adults. Sleep 2008;31:635-43. damage and dysfunction as potential mechanisms 10. Lockley SW, Skene DJ, Arendt J. Comparison between subjective and underlying the increased cardiovascular risk associated actigraphic measurement of sleep and sleep rhythms. J Sleep Res 1999;8:175-83. with chronic short sleep duration, our results indicate that 11. MacEneaney OJ, Kushner EJ, Van Guilder GP, Greiner JJ, Stauffer BL, DeSouza CA. Endothelial progenitor cell number and colony-forming deficits in EPC number and function are not apparent capacity in overweight and obese adults. Int J Obes (Lond) 2009;33:219-25. in healthy individuals who sleep between four and seven 12. MacEneaney OJ, Kushner EJ, Westby CM, Cech JN, Greiner JJ, Stauffer BL, hours per night. Future research is necessary to determine et al. Endothelial Progenitor Cell Function, Apoptosis, and Telomere Length in Overweight/Obese Humans. Obesity (Silver Spring) 2010;18:1677-82. if more severe chronic sleep restriction is associated with 13. Meier-Ewert HK, Ridker PM, Rifai N, Regan MM, Price NJ, Dinges DF, impairment of endothelial repair processes. et al. Effect of sleep loss on C-reactive protein, an inflammatory marker of cardiovascular risk. J Am Coll Cardiol 2004;43:678-83. 14. Verma S, Kuliszewski MA, Li SH, Szmitko PE, Zucco L, Wang CH, ACKNOWLEDGMENTS et al. 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