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Specific cell-derived microvesicles: Linking endothelial function to carotid artery intima-media thickness in low cardiovascular risk menopausal women

Open AccessPublished:December 27, 2015DOI:https://doi.org/10.1016/j.atherosclerosis.2015.12.030

      Highlights

      • Conventional cardiovascular risk factors do not reflect early disease stages in menopausal women.
      • Cellular origin of microvesicles (MV) can be identified by flow cytometry.
      • Principal component analysis of MV reflects complex interactions among cells.
      • A set of six specific MV associate with increases in carotid intima-media thickness.
      • Analysis of specific MV may help to identify early cardiovascular disease processes.

      Abstract

      Background

      Decreases in endothelial function measured by reactive hyperemic index (RHI) correlated with increases in carotid intima-media thickness (CIMT) in recently menopausal women with a low risk cardiovascular profile. Factors linking this association are unknown.

      Objective

      Assess, longitudinally, markers of platelet activation and cell-derived, blood-borne microvesicles (MV) in relationship to RHI and CIMT in asymptomatic, low risk menopausal women.

      Methods

      RHI by digital pulse tonometry (n = 93), CIMT by ultrasound (n = 113), measures of platelet activation and specific cell-derived, blood-borne MV were evaluated in women throughout the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic.

      Results

      CIMT, but not RHI, increased significantly over 4 years. The average change in CIMT correlated significantly with the average follow-up values of MV positive for common leukocyte antigen [CD45; ρ = 0.285 (P = 0.002)] and VCAM-1 [ρ = 0.270 (P = 0.0040)]. Using principal components analysis (PC) on the aggregate set of average follow-up measures, the first derived PC representing numbers of MV positive for markers of vascular endothelium, inflammatory cells (leukocyte and monocytes), pro-coagulant (tissue factor), and cell adhesion molecules (ICAM-1 and VCAM-1) associated with changes in RHI and CIMT. Changes in RHI associated with another PC defined by measures of platelet activation (dense granular ATP secretion, surface expression of P-selectin and fibrinogen receptors).

      Conclusions

      MV derived from activated endothelial and inflammatory cells, and those expressing cell adhesion and pro-coagulant molecules may reflect early vascular dysfunction in low risk menopausal women. Assays of MV as non-conventional measures to assess cardiovascular risk in asymptomatic women remain to be developed.

      Keywords

      1. Introduction

      Vascular endothelial activation or dysfunction is a hallmark of atherosclerotic processes [
      • Vanhoutte P.M.
      Endothelial dysfunction and atherosclerosis.
      ,
      • Bonetti P.O.
      • Pumper G.M.
      • Higano S.T.
      • Holmes Jr., D.R.
      • Kuvin J.T.
      • Lerman A.
      Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia.
      ]. Conventional cardiovascular risk factors such as smoking, hypertension, dyslipidemia and elevated blood glucose are factors contributing to chronic or over-activation of the vascular endothelium. These factors collectively and synergistically stimulate production of inflammatory cytokines, lipid peroxidation, and oxidative stress which increase the adhesive surface of the endothelium and decrease production of endothelium-derived relaxing and anti-coagulant factors [
      • Feletou M.
      • Vanhoutte P.M.
      Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture).
      ].
      Working theories regarding progression of atherosclerosis follow these processes and involve adhesion and activation of platelets [
      • Ross R.
      Cell biology of atherosclerosis.
      ,
      • Kurrelmeyer K.
      • Becker L.
      • Becker D.
      • Yanek L.
      • Goldschmidt-Clermont P.
      • Bray P.F.
      Platelet hyperreactivity in women from families with premature atherosclerosis.
      ], activation and migration of inflammatory leukocytes, particularly monocytes, into the vascular wall [
      • Libby P.
      • Ridker P.M.
      • Hansson G.K.
      Progress and challenges in translating the biology of atherosclerosis.
      ]. In addition to secreting soluble vasoactive and mitogenic products, these activated cells release membrane-bound microvesicles (MV; also referred to in the earlier literature as microparticles) containing bioactive material that varies with sex hormonal status [
      • Gustafson C.M.
      • Shepherd A.J.
      • Miller V.M.
      • Jayachandran M.
      Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans.
      ,
      • Wijten P.
      • van Holten T.
      • Woo L.L.
      • Bleijerveld O.B.
      • Roest M.
      • Heck A.J.
      • et al.
      High precision platelet releasate definition by quantitative reversed protein profiling–brief report.
      ,
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Miller V.M.
      Circulating microparticles and endogenous estrogen in newly menopausal women.
      ,
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Heit J.A.
      • Behrenbeck T.R.
      • Mulvagh S.L.
      • et al.
      Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women.
      ,
      • Miller V.M.
      • Jayachandran M.
      • Hashimoto K.
      • Heit J.A.
      • Owen W.G.
      Estrogen, inflammation, and platelet phenotype.
      ,
      • Miller V.M.
      • Lahr B.D.
      • Bailey K.R.
      • Heit J.A.
      • Harman S.M.
      • Jayachandran M.
      Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.
      ]. MV have been associated with endothelial dysfunction in patients with pre-existing cardiovascular or renal disease [
      • Agouni A.
      • Lagrue-Lak-Hal A.H.
      • Ducluzeau P.H.
      • Mostefai H.A.
      • Draunet-Busson C.
      • Leftheriotis G.
      • et al.
      Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome.
      ,
      • Brogan P.A.
      • Shah V.
      • Brachet C.
      • Harnden A.
      • Mant D.
      • Klein N.
      • et al.
      Endothelial and platelet microparticles in vasculitis of the young.
      ,
      • Bulut D.
      • Maier K.
      • Bulut-Streich N.
      • Borgel J.
      • Hanefeld C.
      • Mugge A.
      Circulating endothelial microparticles correlate inversely with endothelial function in patients with ischemic left ventricular dysfunction.
      ,
      • Amabile N.
      • Heiss C.
      • Real W.M.
      • Minasi P.
      • McGlothlin D.
      • Rame E.J.
      • et al.
      Circulating endothelial microparticle levels predict hemodynamic severity of pulmonary hypertension.
      ,
      • Amabile N.
      • Guerin A.P.
      • Leroyer A.
      • Mallat Z.
      • Nguyen C.
      • Boddaert J.
      • et al.
      Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure.
      ,
      • Boulanger C.M.
      • Scoazec A.
      • Ebrahimian T.
      • Henry P.
      • Mathieu E.
      • Tedgui A.
      • et al.
      Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction.
      ,
      • Werner N.
      • Wassmann S.
      • Ahlers P.
      • Kosiol S.
      • Nickenig G.
      Circulating CD31+/annexin V+ apoptotic microparticles correlate with coronary endothelial function in patients with coronary artery disease.
      ,
      • Horstman L.L.
      • Jy W.
      • Jimenez J.J.
      • Ahn Y.S.
      Endothelial microparticles as markers of endothelial dysfunction.
      ]. However, MV most likely are involved early in disease processes as the number of thrombogenic MV and those of platelet origin correlated with development (over a period of 4 years) of cerebral white matter hyper-intensities in women enrolled in Kronos Early Estrogen Prevention Study (KEEPS) [
      • Raz L.
      • Jayachandran M.
      • Tosakulwong N.
      • Lesnick T.G.
      • Wille S.M.
      • Murphy M.C.
      • et al.
      Thrombogenic microvesicles and white matter hyperintensities in postmenopausal women.
      ]. The relationship of blood-borne MV to endothelial function early in progression of vascular disease as quantified by CIMT has not been examined in asymptomatic women.
      One non-invasive metric of endothelial function is pulse volume digital tonometry which provides a measure of a reactive hyperemia index (RHI) [
      • Bonetti P.O.
      • Pumper G.M.
      • Higano S.T.
      • Holmes Jr., D.R.
      • Kuvin J.T.
      • Lerman A.
      A noninvasive test for endothelial dysfunction.
      ]. In a cohort of non-smoking, low cardiovascular risk, recently postmenopausal women enrolled in KEEPS, RHI did not associate with conventional cardiovascular risk factors at baseline [
      • Mulvagh S.L.
      • Behrenbeck T.
      • Lahr B.A.
      • Bailey K.R.
      • Zais T.G.
      • Araoz P.A.
      • et al.
      Endothelial function and cardiovascular risk stratification in menopausal women.
      ] nor did changes in RHI vary among treatment groups four years after randomization [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ]. However, over the course of four years in the overall cohort, decreased RHI (indicative of endothelial dysfunction) associated with increased CIMT [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ]. A longitudinal assessment of measures of activated cells of the vascular compartment, as might be provided by the number and characteristics of specific cell-derived MV, may help to confirm mechanisms involved in the early stages of atherosclerotic processes and may provide a set of additional measures that could be used to assess cardiovascular risk in otherwise low risk persons. Therefore, the purpose of this study was to determine the relationship, if any, between platelet and blood borne MV with RHI as a measure of endothelial function, and CIMT as a measure of early atherosclerosis in asymptomatic but vulnerable (due to their menopausal status) women. It was hypothesized that the number and characteristics of blood-borne MV would provide a generalized assessment of early endothelial activation (dysfunction) leading to peripheral vascular remodeling. Specifically, it was hypothesized that the number of thrombogenic-, adhesive-, inflammatory- and endothelium-derived MV would correlate negatively with RHI (endothelial function) and positively with CIMT (early atherosclerosis) in this asymptomatic cohort of recently menopausal women. As neither changes in RHI nor CIMT were statistically different among treatment groups [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ,
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ], the analysis could address mechanisms of vascular activation independent of hormonal status.

      2. Methods

      2.1 Participants

      Study participants were recently menopausal women (42–59 years old, within 6 months to 3 years of their last menses) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS; NCT00154180) at the Mayo Clinic, Rochester, MN [
      • Harman S.M.
      • Brinton E.A.
      • Cedars M.
      • Lobo R.
      • Manson J.E.
      • Merriam G.R.
      • et al.
      KEEPS: the Kronos Early Estrogen Prevention Study.
      ]. Women were excluded from KEEPS if they had a history of, or were symptomatic for, cardiovascular disease; smoked more than ten cigarettes/day; had coronary artery calcification (i.e., ≥50 Agatston Units), body mass index >35 kg/m2, dyslipidemia (low-density lipoprotein cholesterol >190 mg/dL), hypertriglyceridemia (triglycerides, >400 mg/dL), 17β-estradiol >40 pg/mL; uncontrolled hypertension (systolic blood pressure >150 mm Hg and/or diastolic blood pressure >95 mm Hg) or fasting blood glucose >126 mg/dL [
      • Harman S.M.
      • Brinton E.A.
      • Cedars M.
      • Lobo R.
      • Manson J.E.
      • Merriam G.R.
      • et al.
      KEEPS: the Kronos Early Estrogen Prevention Study.
      ,
      • Miller V.M.
      • Black D.M.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • Hodis H.N.
      • et al.
      Using basic science to design a clinical trial: baseline characteristics of women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS).
      ]; or used lipid lowering drugs. This study was approved by the Mayo Clinic Institutional Review Board (IRB #2241-04 and #09-003464). All participants gave written informed consent.
      Each woman underwent a medical examination including body mass index, waist-hip ratio measurements, standard blood chemistries prior to (baseline) and during up to 4 years after randomization to either oral conjugated equine estrogens (0.45 mg/day, oCEE), transdermal 17β estradiol (50 μg/day, tE2), each with intermittent progesterone (200 mg/day for the first 12 days of the month), or placebo pills and patch (PL). Details regarding the collection of blood for blood chemistries, measurement of CIMT by B-mode ultrasound and digital tonometry as a measure of endothelial function by RHI have been reported in detail previously [
      • Mulvagh S.L.
      • Behrenbeck T.
      • Lahr B.A.
      • Bailey K.R.
      • Zais T.G.
      • Araoz P.A.
      • et al.
      Endothelial function and cardiovascular risk stratification in menopausal women.
      ,
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ,
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ]. Mean coefficient of variation of measures of CIMT at baseline ranged from 0.0% to 7.7% [
      • Harman S.M.
      • Brinton E.A.
      • Cedars M.
      • Lobo R.
      • Manson J.E.
      • Merriam G.R.
      • et al.
      KEEPS: the Kronos Early Estrogen Prevention Study.
      ].

      2.2 Blood collection for platelet reactivity and blood-borne MV

      Blood from an antecubital vein was collected from participants after an overnight fast at baseline (prior to randomization to treatment) and yearly after randomization for 4 years using a 19 gauge needle into tubes containing various anticoagulants: EDTA (7.5% ethylenediaminetetraacetic acid tripotassium [EDTA (k3)] salt) and 3.2% sodium citrate anticoagulants (calcium chelator anticoagulants) for platelet count and platelet microaggregation; a mixture of 1 μM hirudin (inhibits thrombin) plus 10 μM soybean trypsin inhibitor (inhibitor for factor Xa) for the measurement of dense-granular ATP secretion from platelets, basal expression of P-selectin and fibrinogen receptor (PAC-1) on the surface of platelets, and markers of pro-coagulant and intravascular cellular adhesion, and cellular-origin of blood-borne MV. To provide consistency and to not activate the platelets, all samples were maintained at 33 °C until each test was performed within 30 min of collection.

      2.3 Isolation, identification, and characterization of blood-borne MV by flow cytometry

      The detailed standardized method for the isolation, identification, separation, and quantification of blood-borne MV (>0.2 micron) has been published by our group [
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Miller V.M.
      Circulating microparticles and endogenous estrogen in newly menopausal women.
      ,
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Heit J.A.
      • Behrenbeck T.R.
      • Mulvagh S.L.
      • et al.
      Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women.
      ,
      • Jayachandran M.
      • Preston C.C.
      • Hunter L.W.
      • Jahangir A.
      • Owen W.G.
      • Korach K.S.
      • et al.
      Loss of estrogen receptor ß decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice.
      ,
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. Briefly, platelet-free plasma was separated from whole blood by double centrifugation at 3,000g for 15 min. Contamination of the plasma by platelets and other cells was monitored by Coulter counter and flow cytometry. After validation, the platelet free plasma was centrifuged at 20,000g for 30 min for MV isolation [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. The pellets of MV were washed and reconstituted with twice filtered (0.2 μm pore membrane filter) 20 mM HEPES/HANK’S buffer (pH 7.4) and then vortexed for 1–2 min before staining with antibodies.
      For identification MV, digital flow cytometry (FACSCanto™) was used to define MV by size calibration beads and positive annexin-V-fluorescence [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. Gates to define size are set using an internal standard of 0.2, 0.5, 1, and 2 μm latex or silicon beads [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. The lowest detection limit for the digital flow cytometer based on size calibration beads is 0.2 microns [
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Heit J.A.
      • Behrenbeck T.R.
      • Mulvagh S.L.
      • et al.
      Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women.
      ,
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]; therefore, MV detection was set at this limit.
      For quantification, samples included a known quantity of beads (TruCOUNT™) of 4.2 μm diameter. All antibodies were directly conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The FITC and PE conjugated rat anti-mouse IgG and mouse anti-rabbit IgG isotype control antibodies were used as controls and for threshold setting for fluorescence dot or scatter plot [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ,
      • Jayachandran M.
      • Lugo G.
      • Heiling H.
      • Miller V.M.
      • Rule A.D.
      • Lieske J.C.
      Extracellular vesicles in urine of women with but not without kidney stones manifest patterns similar to men: a case control study.
      ]. Our previous study verified the cellular origin of each blood-borne MV using two different fluorophores (FITC and PE) conjugated to two distinct antibodies considered to be specific for each cell type [
      • Gustafson C.M.
      • Shepherd A.J.
      • Miller V.M.
      • Jayachandran M.
      Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans.
      ]. MV were separated by fluorescence scatter or dot plot quadrants derived MV gate of light scatter plot in the presence PE (Q1+Q2) and FITC (Q4+Q2) or absence of both (Q3) of staining [
      • Gustafson C.M.
      • Shepherd A.J.
      • Miller V.M.
      • Jayachandran M.
      Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans.
      ,
      • Jayachandran M.
      • Litwiller R.D.
      • Owen W.G.
      • Heit J.A.
      • Behrenbeck T.R.
      • Mulvagh S.L.
      • et al.
      Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women.
      ,
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. The absolute numbers of fluorophores positive MV was calculated based on counts of calibration beads. The absolute count of specific fluorophore positive MV = number of counts in each fluorophore positive MV region/number of counts in TruCOUNT™ bead region x number of beads per test (spiked known count)/test volume [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. The same calculation applied to quantitation of MV positive or negative for annexin-V and each cell membrane specific antibody. The calculated absolute number of MV from the two cell surface markers for each cell type was similar (r2 = 0.97) in same person [
      • Gustafson C.M.
      • Shepherd A.J.
      • Miller V.M.
      • Jayachandran M.
      Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans.
      ]. Numbers of isolated blood-borne MV from 0.2 to 1 microns are reported in this study. The intra-assay variability of MV analysis is <10% [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ].

      2.4 Statistical analyses

      Descriptive statistics were used to summarize the cohort, including counts (percentages) for categorical variables and means (SD, standard deviation) for continuous variables. In general, paired differences were tested for a significant change over time based on the Wilcoxon signed rank test, and group differences in response patterns were assessed using the Kruskal–Wallis test. To estimate the strength of association between two continuous variables, the nonparametric Spearman's rank correlation coefficient was used. Since previous findings revealed no significant treatment effect on the two study response variables of interest, RHI and CIMT, data for these analyses were pooled across treatment groups. All analyses were performed using the statistical programming language SAS, version 9.3 (SAS Institute Inc., Cary, NC). An alpha level of 0.05 was used to establish statistical significance.
      For serially collected responses RHI and CIMT, multiple measurements per woman were reduced to a single measure of trend by computing separately, for each woman, the change in their average follow-up value from baseline, i.e. the difference between their average follow-up value and their baseline value. This method facilitated describing and analyzing trends and reduced the noise in RHI measures which have shown high within-person variability [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ]. A similar approach was taken for summarizing the highly variable serial measures of MV and platelet parameters, by using the average over each participant's follow-up values. For each MV and platelet parameter, the mean follow-up measure was then correlated with the mean change of RHI and CIMT.
      To assess the combined effects of the MV and platelet variables on outcomes, while accounting for the varying degree of inter-correlation and interaction among the 13 measured variables, we used the statistical data reduction method known as principal components analysis (PC). The objective of PC analysis is to attempt to account for the joint variability in the 13 variables on the basis of a smaller number of independent factors. Each factor, or PC, is represented as a weighted linear combination of the original variables (a score), with statistical software computing the optimal weights. The 1st PC is defined as the linear combination that accounts for the largest amount of variability in the data; succeeding components are uncorrelated with the first PC and with each other, and account for progressively less variance. Having defined these principal components without regard to outcome data, each principal component was then assessed for its association with the outcome measures for RHI and CIMT using Spearman's correlation analysis. This technique helps to reduce the problem of multiple comparisons, while potentially increasing the signal to noise ratio by combining multiple noisy measures into a smaller number of independent variables.

      3. Results

      3.1 Clinical characteristics of participants

      At Mayo Clinic, 118 Caucasian women enrolled in KEEPS; baseline and at least one follow-up measure (either 1, 2, 3, or 4 years) were available from 93 women for RHI and from 114 women for CIMT. Phenotypic and clinical characteristics of the Mayo cohort have been reported previously [
      • Miller V.M.
      • Lahr B.D.
      • Bailey K.R.
      • Heit J.A.
      • Harman S.M.
      • Jayachandran M.
      Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.
      ,
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ], but are shown again in Table 1. In general, baseline characteristics were similar across the three treatment groups. At baseline, conventional risk factors for cardiovascular disease (waist circumference, systolic blood pressure, blood lipids, triglycerides, fasting blood glucose and C-reactive protein) in the Mayo cohort were similar to those of the entire KEEPS cohort [
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ] and place these women in a “low” (<5%) 10 year risk category for cardiovascular disease as defined by the Framingham Risk Score [
      • Mulvagh S.L.
      • Behrenbeck T.
      • Lahr B.A.
      • Bailey K.R.
      • Zais T.G.
      • Araoz P.A.
      • et al.
      Endothelial function and cardiovascular risk stratification in menopausal women.
      ,
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ,
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ,
      • Miller V.M.
      • Black D.M.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • Hodis H.N.
      • et al.
      Using basic science to design a clinical trial: baseline characteristics of women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS).
      ].
      Table 1Summary of baseline characteristics.
      Baseline variableTotal (n = 118)Placebo (n = 43)tE2 (n = 36)oCEE (n = 39)
      Age, years53.1 ± 2.453.0 ± 2.453.1 ± 2.453.1 ± 2.4
      Menopausal age (months)19.2 ± 9.117.0 ± 9.020.9 ± 8.320.2 ± 9.7
      Body mass index (kg/m2)27.1 ± 4.227.1 ± 3.826.3 ± 4.227.7 ± 4.5
      Waist circumference (cm)84.3 ± 11.684.3 ± 11.883.2 ± 11.385.4 ± 12.0
      Systolic blood pressure (mm Hg)122.2 ± 14.0122.2 ± 12.5120.0 ± 16.1124.2 ± 13.7
      Diastolic blood pressure (mm Hg)75.4 ± 8.075.3 ± 7.573.8 ± 8.777.0 ± 7.7
      Fasting glucose (mg/dL)80.1 ± 9.379.8 ± 7.181.8 ± 12.378.8 ± 8.1
      Total cholesterol (mg/dL)205.4 ± 31.6200.8 ± 32.7213.7 ± 30.4202.9 ± 30.7
      HDL cholesterol (mg/dL)70.2 ± 13.369.2 ± 13.270.3 ± 11.471.1 ± 15.1
      LDL cholesterol (mg/dL)118.5 ± 28.1116.3 ± 27.7123.7 ± 31.8116.1 ± 24.9
      Triglycerides (mg/dL)87.4 ± 47.184.1 ± 46.093.3 ± 42.585.6 ± 52.9
      C-reactive protein (mg/L)
      Median (25th and 75th percentile).
      1.5 (0.5, 3.0)1.5 (0.5, 2.8)1.6 (0.7, 2.8)1.1 (0.4, 3.6)
      a Median (25th and 75th percentile).

      3.2 Longitudinal changes in RHI, CIMT, platelet parameters and blood-borne MV

      Measured on 93 participants, the mean (±SD) RHI was 2.40 (±0.69) at baseline and averaged 2.32 (±0.45) over available measurements during follow-up (Fig. 1). Based on the paired difference between each participant's average follow-up and baseline value, there was no significant mean change in RHI over the study period (- 0.07 ± 0.61; P = 0.211), nor was there a significant difference in these changes across treatment groups (P = 0.833). Consistent with published results of the entire KEEPS cohort [
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ], CIMT increased significantly in the KEEPS participants at Mayo over the study period (P < 0.001), and these changes did not differ statistically across treatment groups (P = 0.332). For 114 participants, CIMT increased on average from 0.678 mm (±0.079) at baseline to 0.690 (±0.087) mm at follow-up, corresponding to an average increase of 0.012 mm (±0.028) over 4 years (Fig. 1). None of the baseline clinical factors listed in Table 1 correlated significantly with changes in CIMT or in RHI [range of Spearman's correlation coefficients, (−0.12, 0.14)].
      Figure thumbnail gr1
      Fig. 1Absolute values of RHI (left panel) and CIMT (right panel) during follow-up from the subset of women enrolled in KEEPS at Mayo Clinic that had been randomly assigned to receive placebo (as there were no statistically significant differences in these trends among treatment groups, presentation of data was limited to this subgroup for ease of visualizing the trends). Each line connects serial measures from an individual, and the bolded curve estimates the overall group trend using a nonparametric loess smooth.
      In general, there was high intra-individual variability in measurements of platelet activation and the MV parameters during the four years of follow-up. Examples of the longitudinal variability for platelet-, endothelium- and monocyte-derived MV are shown in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Measures of platelet- (left panel), endothelium- (middle panel) and monocyte- (right panel) derived MV from the subset of women enrolled in KEEPS at Mayo Clinic that had been randomly assigned to receive placebo (as there were no statistically significant differences in these trends among treatment groups, presentation of data was limited to this subgroup for ease of visualizing the trends). Each line tracks values over time for an individual and the bolded curve estimates the overall group trend using a nonparametric loess smooth.

      3.3 Associations of activation of platelets and blood-borne MV with RHI and CIMT

      To account for the longitudinal, intra-individual variability in each parameter, the average of each follow-up value for the particular measure of platelet activation or cell-specific MV was obtained for each woman and assessed for correlation with the average change in RHI and CIMT (Table 2). Among those markers of platelet activation and MV showing a nominally significant result, the average follow-up values for basal expression of fibrinogen receptors on platelets (P=0.021) and monocyte-derived (P=0.023) MV were positively associated with the average change in RHI (Table 2). In addition, the average follow-up levels of both total leukocyte-derived MV (P=0.002) and MV positive for VCAM-1 (P=0.004) showed positive associations with changes in CIMT (Table 2; Fig. 3).
      Table 2Correlation of average follow-up value of blood platelet reactivity and blood-borne MV with average change in vascular reactivity (RHI) and structure (CIMT).
      Abbreviations: RHI, reactive hyperemic index; CIMT, carotid intima-media thickness.
      ParameterAvg change in RHI
      There was no significant change in RHI based on average change (p = 0.211).


      N = 93

      Spearman ρ
      Avg change in CIMT
      A significant change in CIMT was detected using the average (Avg) change since baseline (p < 0.001).


      N = 114

      Spearman ρ
      Platelets and measures of activation
       Platelet count−0.1570.064
       Platelet microaggregates0.0970.038
       ATP secretion0.0770.019
       Basal expression of P-selectin0.097−0.054
       Basal expression of fibrinogen receptor0.240*−0.095
      Cellular origin of microvesicles (MV)
       Endothelium (CD62E)-derived MV0.0900.055
       Leukocyte (CD45)-derived MV0.0680.285**
       Monocyte (CD14)-derived MV0.236*0.104
       Platelet (CD42a)-derived MV−0.032−0.107
      Thrombogenic microvesicles
       Phosphatidylserine positive MV0.008−0.164
       Tissue factor positive MV0.0470.021
      Microvesicles expressing adhesion molecules
       ICAM-1 positive MV0.1940.107
       VCAM-1 positive MV0.1620.270**
      Note: Spearman's ρ rank correlation coefficient for measuring the association between summary measure and response (*P < 0.05, **P < 0.01).
      a Abbreviations: RHI, reactive hyperemic index; CIMT, carotid intima-media thickness.
      b A significant change in CIMT was detected using the average (Avg) change since baseline (p < 0.001).
      c There was no significant change in RHI based on average change (p = 0.211).
      Figure thumbnail gr3
      Fig. 3Relationships between the average change in CIMT with the average follow-up values for leukocyte-derived MV (left panel) and MV positive for VCAM-1 (right panel). Each point represents an individual.
      To account for the varying degree of inter-correlation and interaction among the 13 measured variables for platelet and MV parameters, principal components (PC) analysis was performed. This analysis leads to a small number of independent dimensions (or principal components) while retaining most of the original information. Using this approach, five principal components were retained in the analysis, which altogether accounted for about 68% of the information in the original data (Table 3). In testing the association with each vascular outcome, two of the 5 independent principal components (PC1 and PC3) correlated significantly with changes in RHI, while one of the five (PC1) correlated significantly with changes in CIMT for a total of 3 (out of 10) nominally significant associations. Based on the null distribution of p-values from 10 separate correlations tested, the expected number of type I errors from these analyses is ∼0.5, which is to say that on average, zero to one associations detected as nominally significant at the 0.05 level would have occurred by chance alone. With a total of 3 such associations detected from the 10 essentially independent tests (i.e., well more than the expected chance finding of 0.5), it is unlikely that chance alone explains the associations and more likely that at least some of these findings were not type I errors.
      Table 3Principal components (PC) analysis of the average change in platelet and MV parameters with the average change in RHI and CIMT.
      VariableAverage follow-up value
      LoadingsPC1PC2PC3PC4PC5
      Platelets and measures of activation
      Platelet count−0.030.17−0.530.330.26
      Platelet microaggregates−0.030.190.060.630.38
      ATP secretion−0.020.040.380.59−0.44
      Basal expression of P-selectin−0.060.210.52−0.070.01
      Basal expression of fibrinogen receptor−0.020.030.46−0.190.67
      Cellular origin of microvesicles (MV)
      Endothelium (CD62-E)-derived MV0.440.130.06−0.030.11
      Leukocyte (CD45)-derived MV0.380.22−0.18−0.12−0.03
      Monocyte (CD14)-derived MV0.440.060.130.18−0.01
      Platelet (CD42a)-derived MV−0.130.63−0.06−0.15−0.14
      Thrombogenic microvesicles
      Phosphatidylserine positive MV−0.130.64−0.01−0.11−0.07
      Tissue factor positive MV0.32−0.050.11−0.12−0.30
      Microvesicles expressing adhesion molecules
      ICAM-1 positive MV0.430.120.080.010.00
      VCAM-1 positive MV0.37−0.05−0.150.030.15
      Proportion of variance24%16%11%10%8%
      Cumulative proportion24%40%50%60%68%
      Spearman's ρ rank correlation with
       Change in RHI0.211
      Denotes a significant correlation between PC and outcome measure (P < 0.05).
      0.0460.237
      Denotes a significant correlation between PC and outcome measure (P < 0.05).
      0.0260.119
       Change in CIMT0.199
      Denotes a significant correlation between PC and outcome measure (P < 0.05).
      −0.071−0.1180.0530.051
      Bolded values represent the variables with the strongest loadings on each of the 5 PCs.
      Abbreviations: RHI, reactive hyperemic index; CIMT, carotid intima-media thickness; MV, microvesicles.
      a Denotes a significant correlation between PC and outcome measure (P < 0.05).
      The first principal component (PC1) accounted for 24% of the variability in these data, and included MV derived from endothelial cells, leukocytes, and monocytes and those positive for tissue factor, and cellular adhesion molecules ICAM-1 and VCAM1 (bolded entries in Table 3). The third principal component (PC3), which accounted for 11% of the variance (that was not explained by the first two components), included measures of platelet activation, specifically the secretion of ATP, basal expression of P-selectin and fibrinogen receptor with platelet count.

      4. Discussion

      This study provides the first longitudinal assessment of platelet activation and specific cell-derived, blood borne MV reflecting a measure of global activation of the cellular vascular compartment in a cohort of asymptomatic recently menopausal women categorized as “low risk” by conventional cardiovascular risk factors. Specifically, a panel of MV parameters is identified that most likely reflects early stages of disease processes. Although each conventional cardiovascular risk factor was within normative ranges for these women at entry into the study, variables such as blood pressure, blood lipids and glucose, etc. are continuous variables the collective and synergistic effects of which may activate the cellular vascular compartment contributing to early stages and progression of atherosclerosis as measured by CIMT. This conclusion is consistent with previous studies demonstrating that individual components of the metabolic syndrome (waist circumference, blood pressure, blood glucose, triglycerides and high density lipoproteins) as well as the formulation of hormone treatments (oral conjugated equine estrogens or transdermal 17β-estradiol) associate with specific aspects of platelet activation in vitro [
      • Miller V.M.
      • Lahr B.D.
      • Bailey K.R.
      • Heit J.A.
      • Harman S.M.
      • Jayachandran M.
      Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.
      ,
      • Jayachandran M.
      • Litwiller R.D.
      • Lahr B.D.
      • Bailey K.R.
      • Owen W.G.
      • Mulvagh S.L.
      • et al.
      Alterations in platelet function and cell-derived microvesicles in recently menopausal women: relationship to metabolic syndrome and atherogenic risk.
      ,
      • Raz L.
      • Hunter L.W.
      • Jayachandran M.
      • Heit J.A.
      • Miller V.M.
      Differential effects of oral and transdermal menopausal hormone therapy on prostacyclin and thromboxane in platelets.
      ] which manifest as a profile of MV characteristics in vivo.
      In addition, the association of markers of immunological, inflammatory and pro-coagulant functions with CIMT is consistent with the working theories regarding contributions of platelets, leukocytes, and immune competence to vascular disease processes [
      • Mosca L.
      • Benjamin E.J.
      • Berra K.
      • Bezanson J.L.
      • Dolor R.J.
      • Lloyd-Jones D.M.
      • et al.
      Effectiveness-based guidelines for the prevention of cardiovascular disease in women–2011 update: a guideline from the American Heart Association.
      ,
      • Mason J.C.
      • Libby P.
      Cardiovascular disease in patients with chronic inflammation: mechanisms underlying premature cardiovascular events in rheumatologic conditions.
      ]. These observations are also consistent with the identified genetic variants within the innate immunity pathways and the pharmacogenomic interactions of treatment with those genetic variants that associated with increases in CIMT in the KEEPS cohort [
      • Miller V.M.
      • Petterson T.M.
      • Jeavons E.N.
      • Lnu A.S.
      • Rider D.N.
      • Heit J.A.
      • et al.
      Genetic polymorphisms associated carotid artery intima-media thickness and coronary artery calcification in women of the Kronos Early Estrogen Prevention Study.
      ,
      • Miller V.M.
      • Jenkins G.D.
      • Biernacka J.M.
      • Heit J.A.
      • Huggins G.S.
      • Hodis H.N.
      • et al.
      Pharmacogenomics of estrogens on changes in carotid artery intima-medial thickness and coronary arterial calcification – Kronos Early Estrogen Prevention Study.
      ].
      Global assessment of immune and thrombogenic activation identified by the MV shown to associate with CIMT in the present study may help to stratify cardiovascular risk for recently menopausal women. It must be cautioned that these associations were identified in a longitudinal analysis in which there was large biological variability in individual measures within each parameter over the four years. However, it should be noted that the longitudinal intra-individual variability in MV measures exceeded the intra-assay variability [
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ]. Some biological variability in longitudinal assessment of each parameter should be expected given the short circulating half-life of platelets, the dynamic nature of the interaction of various blood elements with sources of their stimulation (i.e. changes in soluble proteins, glucose and lipids and commensal and acute infection), and their interaction with each other, other intravascular cells and cells of the vascular wall. This variability raises a caution for the development of both diagnostic and prognostic “biomarkers” of cardiovascular disease measured at a single time point. Development of a panel of MV from different cell types as provided by the PC analysis, rather than assessment of single MV alone, may provide a better risk assessment for cardiovascular disease at a single time point. However such panels would need to be developed and validated in larger and more diverse groups of women than those represented by KEEPS.
      One unexpected finding of the present study was the positive correlation of PC1 components with RHI. It was hypothesized that increases in inflammatory processes would correlate with decreases rather than increases in the RHI. However, the RHI represents the response to mechanical shear stress across the surface of the endothelial cells as well as the autonomic tone of resistance arteries. RHI associated with the PC reflecting platelet activation which seems to be consistent with shorter-term, reactive functions of platelets. The average decreases in RHI did associate with increases in CIMT [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ] thus, perhaps reflecting, chronic cell–cell activation and interactions. In the future, MV reflecting endothelial activation should be investigated following arterial vasodilatation in response to intra-arterial injection of acetylcholine.
      The KEEPS cohort provides an ideal study population for investigation of mechanisms involved in the early stages of vascular remodeling as the inclusion criteria for KEEPS were rigorous. First, vascular structure quantified by CIMT and computed tomography of coronary arterial calcification prior to enrollment indicated that these women were without clinical classification of cardiovascular disease. In addition, all conventional cardiovascular risk factors were defined within clinically normative ranges [
      • Harman S.M.
      • Brinton E.A.
      • Cedars M.
      • Lobo R.
      • Manson J.E.
      • Merriam G.R.
      • et al.
      KEEPS: the Kronos Early Estrogen Prevention Study.
      ]. Finally, the lower doses and formulations of hormonal treatments used in KEEPS did not significantly affect either changes in CIMT or RHI [
      • Kling J.M.
      • Lahr B.
      • Bailey K.
      • Harman S.M.
      • Miller V.
      • Mulvagh S.L.
      Endothelial function in women of the Kronos Early Estrogen Prevention Study.
      ,
      • Harman S.M.
      • Black D.M.
      • Naftolin F.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • et al.
      Arterial imaging outcomes and cardiovascular risk factors in recently menopausal women: a randomized trial.
      ]. It should be noted that, in spite of significant hormonal effects on specific platelet functions [
      • Miller V.M.
      • Lahr B.D.
      • Bailey K.R.
      • Heit J.A.
      • Harman S.M.
      • Jayachandran M.
      Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.
      ,
      • Raz L.
      • Hunter L.W.
      • Jayachandran M.
      • Heit J.A.
      • Miller V.M.
      Differential effects of oral and transdermal menopausal hormone therapy on prostacyclin and thromboxane in platelets.
      ], the rate of change in CIMT observed in KEEPS is comparable to that reported in other studies [
      • Hodis H.
      • Mack W.
      • Lobo R.
      • Shoupe D.
      • Sevanian A.
      • Mahrer P.
      • et al.
      Estrogen in the prevention of atherosclerosis. A randomized, double-blind, placebo-controlled trial.
      ]. The absence a hormonal effect most likely reflects the “low risk” status of the participants [
      • Hodis H.
      • Mack W.
      • Lobo R.
      • Shoupe D.
      • Sevanian A.
      • Mahrer P.
      • et al.
      Estrogen in the prevention of atherosclerosis. A randomized, double-blind, placebo-controlled trial.
      ], doses of hormones used in the study [
      • Karim R.
      • Hodis H.N.
      • Stanczyk F.Z.
      • Lobo R.A.
      • Mack W.J.
      Relationship between serum levels of sex hormones and progression of subclinical atherosclerosis in postmenopausal women.
      ,
      • Miller V.M.
      Congress on women's health Trudy Bush lecture 2014: new insights into sex hormones and cardiovascular disease.
      ] and significant pharmacogenomics interactions [
      • Miller V.M.
      • Jenkins G.D.
      • Biernacka J.M.
      • Heit J.A.
      • Huggins G.S.
      • Hodis H.N.
      • et al.
      Pharmacogenomics of estrogens on changes in carotid artery intima-medial thickness and coronary arterial calcification – Kronos Early Estrogen Prevention Study.
      ].
      A limitation of the current study is that the MV were quantified without measurement of percentage of MV associated with other intravascular cells at the time of the blood collection. Evaluating the MV association with other cells by use of dual or triple marker identification (e.g., MV carrying a combination of platelet, leukocyte, adhesion molecule, and thrombogenic signals) may reduce individual variability in the data and provide a better indication of the interaction among the cells in the blood with the vascular wall.
      In summary, results of this study provide evidence that in women categorized as “low risk” by conventional cardiovascular risk assessment tools, markers of cellular activation, inflammation and pro-coagulant properties associate positively with increases in CIMT. These markers may reflect the synergistic activity of conventional cardiovascular risk factors that are on a continuum within normative ranges. Markers of platelet activation in conjunction with MV reflecting activation of cells involved with thrombogenic and inflammatory responses suggest that a more global assessment of activation of the vascular compartment may add value to establishing cardiovascular risk stratification for women.

      Study funding

      The Aurora Foundation to the Kronos Longevity Research Institute, NIH HL90639, HD65987, P50 AG033514, UL1 RR024150
      From the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research. Contents of this paper are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.
      and the Mayo Foundation. Study medications for KEEPS were supplied in part by Bayer Health Care and by Abbott Pharmaceuticals.

      Author disclosures

      None.

      Additional contributions

      We gratefully acknowledge the dedicated efforts of our study coordinator, Teresa G. Zais (deceased), Robert D. Litwiller for technical assistance collecting the blood and technical staff at the KEEPS CIMT reading center and data processing centers. Above all, we recognize and thank the KEEPS participants for their dedication and commitment to the programs of the Mayo Clinic Women's Health Research Center.

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