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Am J Physiol Heart Circ Physiol 295: H931-H938, 2008. First published July 11, 2008; doi:10.1152/ajpheart.00193.2008
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TRANSLATIONAL PHYSIOLOGY

Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women

Muthuvel Jayachandran,1 Robert D. Litwiller,2 Whyte G. Owen,2 John A. Heit,2,3 Thomas Behrenbeck,4 Sharon L. Mulvagh,4 Philip A. Araoz,5 Matthew J. Budoff,6 S. Mitchell Harman,7 and Virginia M. Miller1

Departments of 1Surgery and Physiology and Biomedical Engineering, 2Biochemistry and Molecular Biology and Hematology, 3Internal Medicine, Divisions of Hematology, 4Cardiovascular Diseases, and 5Radiology, Mayo Clinic College of Medicine, Rochester, Minnesota; 6Los Angeles Biomedical Research Institute at Harbor University of California at Los Angeles, Torrance, California; and the 7Kronos Longevity Research Institute, Phoenix, Arizona

Submitted 22 February 2008 ; accepted in final form 3 July 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC) was scored using 64-slice computed tomography scanners. The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score < 10%, range 1–3%), and high-sensitivity C-reactive protein did not differ significantly among women with low (0 < 35; range, 0.3–32 Agatston units) or high (>50; range, 93–315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis.

phosphatidylserine; procoagulant activity; thrombosis

CARDIOVASCULAR DISEASE INCREASES in women after menopause, but standard markers of risk such as hypertension, circulating lipids, and smoking status, used to calculate the Framingham risk score (FRS), do not predict risk in many women (14, 25, 26, 41). Furthermore, the concentrations of circulating proteins and peptides to assess risk provide little insight into intravascular processes leading to the formation of an arterial lesion, since biochemical markers turn over rapidly and their source usually cannot be identified (5, 38, 39). Alternatively, circulating platelets and leukocytes have defined lifetimes and bear marks of interactions with each other or the vascular wall (20, 28, 39, 40). Therefore, an alternative approach that takes advantage of these cellular interactions may provide a way to link vascular pathophysiology and thrombosis and to stratify risk early in disease processes.

During cellular activation and apoptosis, vesicles of <1 µm in diameter are shed from membranes into the circulation and by convention are referred to as microparticles (29). The outer surfaces of microparticles may bear phosphatidylserine, which binds coagulation factors for the generation of thrombin. Thus microparticles bearing phosphatidylserine may be procoagulant and, as such, may contribute to the pathophysiology of thrombosis and early vascular disease (7, 15, 33, 43, 45). Because microparticles can arise from interactions between blood-formed elements and the vascular lining, they may also provide information on the nature and magnitude of ongoing intravascular processes (34). Although alterations in concentrations of circulating microparticles have been described in symptomatic cardiovascular disease and have been proposed as prognostic markers (2, 9, 37, 44), there is little information on the characteristics of microparticle pools in apparently healthy, asymptomatic, but at-risk populations such as newly menopausal women. Thus newly menopausal women with no known health problems offer an opportune population or model system for biomarker discovery of early disease processes. In particular, changes in the microparticle pools that accompany a natural process, menopause, may identify intravascular pathophysiology that imparts the steep increase in risk for ischemic vascular disease.

This study was designed to test the hypothesis that cellular sources and characteristics of circulating microparticles can provide insight into early cardiovascular disease processes in newly menopausal women.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. This was a cross-sectional study of a subset (n = 33) of apparently healthy, newly menopausal women (between 6 mo and 3 yr from their last menses; chronological age 42–58 yr) screened to participate (n = 146) in the Kronos Early Estrogen Prevention Study (KEEPS; NCT000154180) at Mayo Clinic (Rochester, MN) (18). KEEPS is a randomized, double-blind trial designed to test the hypothesis that menopausal hormone therapy started early in menopause will reduce the progress of atherosclerotic disease as defined by the progression of carotid intimal-medial thickness and coronary calcification. Because one exclusion criterion for the study is a coronary artery calcification (CAC) score of >50 Agatston units (AU), all women undergo a coronary calcium scan at screening. Of the 146 women screened at Mayo, 5 had CAC > 50 AU (range, 93–315 AU), 18 women had CAC between 0 and 50 AU (range, 0.3–32 AU), and 123 women had a score of 0 AU. For this ancillary study, all women with a CAC score > 0 were included, and 10 women were randomly selected from the 123 women with a CAC score = 0. Among the 33 women included in this study, none was a current smoker, hypertensive, diabetic, or had a history of thrombotic disease. This study was approved by the parent KEEPS Ancillary Studies Committee and the Institutional Review Board at Mayo Clinic. All participants gave written informed consent. Because these tests were performed on individuals being screened for KEEPS, none was on active medication. However, some had endogenous estradiol levels that made them ineligible for randomization into KEEPS but eligible for this ancillary study.

Antibodies and other reagents. Recombinant annexin-V and mouse anti-human cell surface marker antibodies conjugated with fluorescein isothiocyanate or R-phycoerythrin and TruCOUNT beads were obtained from BD Biosciences (San Jose, CA). Amine-modified polystyrene fluorescent yellow-green latex beads (1 and 2 µm) were from Sigma-Aldrich (St. Louis, MO). Human factor Xa, factor Va, and prothrombin were obtained from Haematologic Technologies (Essex Junction, VT). All other reagents were analytical grade.

Coronary calcification. CAC images were obtained using a 64 detector computed tomography scanner (Siemens Sensation 64, Siemens Medical Solutions, Forcheim, Germany) with a scan configured to cover the heart. Scans were gated to the cardiac cycle and obtained in end inspiration using the following parameters: rotation time = 0.33 s and collimation = 24 x 1.2, with images reconstructed at 3-mm-thick slices; pitch = 0.2, peak kilovoltage = 120; and field of view = 30; milliamphere-second (mA-s) was varied according to width of the patient on the scout image as measured across the liver. Subjects who measured <32 cm had mA-s = 126. Subjects who measured 32–38 cm had mA-s = 385. Subjects who measured >38 cm had mA-s = 780. Images were reconstructed with a B35 kernel at 65% of the R-R interval.

The calcium score was calculated using commercially available semiautomated software (GE Smartscore, GE Healthcare, Milwaukee, WI) according to the Agatston method (1). Calcified plaque within each epicardial vessel was detected by four contiguous pixels and a peak density >130 Hounsfield units (HU). The product of the area and the respective density factor (1 = 130 to 199 HU; 2 = 200 to 299 HU; 3 = 300 to 399 HU, and 4 if ≥400) constituted the vessel-specific coronary artery calcium score. The calcium scores across each individual epicardial vessel were summed to determine the total coronary artery calcium score.

Blood sample collection. Venous blood samples were collected for measurements of lipids, hormones, and high-sensitivity C-reactive protein (hs-CRP), chemistries, and platelet functions. The anticoagulant used for each assay was dictated by the requirement of that assay. For microparticle analyses, blood (4 ml) was collected through a 19-gauge butterfly needle into a tube containing 0.5 ml of 0.11 M sodium citrate and 0.1 ml of 1 M benzamidinium chloride, which incapacitates platelets. The measurement of P-selectin and fibrinogen receptor on platelets under basal conditions and after stimulation with a thrombin receptor agonist peptide (10 µM) was evaluated using standard techniques (23).

Blood chemistries. Total cholesterol, low (LDL) and high (HDL) density cholesterol, triglycerides, blood glucose, follicle-stimulating hormone, 17β-estradiol, thyroid-stimulating hormone, hs-CRP, sodium, potassium, chloride, bicarbonate, creatinine, phosphorus, total protein, albumin, bilirubin, alkaline phosphatase, aspartate, alanine transaminases, and urea were measured by Kronos Science Laboratories (Phoenix, AZ) and the Mayo Clinic Clinical Laboratories (Rochester, MN). Total white blood cells, differential leukocytes, hemoglobin, and hematocrit were also determined by Mayo Clinic Hematopathology Laboratories. Platelet counts were measured with a Coulter counter T660.

Isolation of blood microparticles. Blood samples were centrifuged at 3,000 g for 15 min within 30 min of phlebotomy. The plasma was frozen at –40°C. Frozen samples were thawed in a 37°C water bath for 5 min, vortexed, and then centrifuged at 3,000 g for 15 min. In preliminary experiments, it was determined that the recovery of microparticles was the same from fresh compared with plasma that had undergone a single-freeze thaw cycle. After centrifugation, samples were evaluated with the Coulter counter for the presence of other cells. This step validated that platelet counts of ≤1 and other cells were absent from the sample. Thus it is improbable that microparticles could be generated from cells remaining in the plasma during the subsequent isolation process. After the validation step, the plasma sample (0.5 ml) was then centrifuged at 20,000 g for 30 min, which recovers >90% of microparticles (data not shown). Supernatants were removed, and the remaining pellets (microparticles) were reconstituted with 0.5 ml Hanks' solution with 0.05% glucose and buffered (pH 7.4) with 20 mM HEPES. Tubes containing reconstituted microparticles were vortexed and centrifuged again at 20,000 g for 30 min. After centrifugation, the pellets containing microparticles were reconstituted again (0.5 ml) and vortexed for 1 to 2 min before analysis. All buffers were filtered twice through a 0.2-µm membrane filter before use.

Microscopic observation of isolated microparticles. For scanning electron microscopy, a small drop of isolated microparticles was added to a parlodian carbon-coated grid and allowed to sit for 10 min. A small drop of 1% phosphotungstic acid (pH 7.2) was added to the grid, and, afterward, air drying was examined by scanning electron microscopy. For transmission electron microscopy, isolated microparticles were fixed and embedded in quetol resin by standard methods (31). Ultrathin (70–90 nm) sections were cut with a diamond knife, stained with lead citrate, and examined with a transmission electron microscope (FEI Technai-12 TEM, Hillsboro, Oregon). Freshly isolated microparticles were also observed using dual mode fluorescence and optical microscopy (CytoViva, Auburn, AL).

Identification of isolated microparticles. Flow cytometry (FACSCanto, BD Biosciences) was used to define microparticles by size and positive fluorescence using marker-specific antibodies. The gates to define size were set using an internal standard of 1- and 2-µm beads, with the lower limit set above the background noise level of the machine (Fig. 1). A calibrated quantity of TruCOUNT beads of 4.2 µm diameter were added to samples to enable the calculation of absolute particle counts (Fig. 1). All buffers and antibodies were filtered twice through a 0.2-µm filter to eliminate chemical particles and reduce instrument noise. Isolated microparticles (50 µl) were incubated with 4 µl of specified antibodies conjugated to fluorescein (FL) and phycoerythrin (PE) for 30 min. After incubation, the microparticles were fixed with 400 µl of 1% paraformaldehyde for 15 min. After fixation, 50 µl of the TruCOUNT beads were added immediately before analysis by flow cytometry.


Figure 1
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  		Fig. 1. Representative scatter plot obtained by FACSCanto flow cytometry. A: control gates of buffer with fluorescein-conjugated antibodies and calibration (size and TruCount beads) beads in the absence of sample. B: gates derived from adding a sample containing microparticles to the buffer with fluorescein-conjugated antibodies and calibration beads. Note that the gate to define the area of interest (P1) was set above the background noise of the machine. C and D: representative quadrants (Q) derived from the microparticle gates shown in A and B, respectively. Counts are separated by antibody binding with Q3 representing microparticles. FL, fluorescence. E and F: representative scanning (E) and transmission (F) electron microscopy of the isolated microparticles. Arrow heads, membranes. G: microparticles imaged by CytoViva dual fluorescence and optical microscopy at x150. H: fluorescent beads (1 µm in diameter) imaged by the CytoViva cell imaging system at x150.

 
Annexin-V binding as a measure of microparticle surface phosphatidylserine was measured with the use of annexin-V-FL. Stained microparticles were separated by quadrant (Q1–Q4, cf. Fig. 1). To validate specificity, the annexin-V assay was repeated with annexin-V-PE, which gave congruent counts (data not shown). Absolute numbers of the microparticles were calculated from the number of events in the region containing microparticles divided by the number of events in the calibration bead region times the number of beads per test volume (see Fig. 1). The same calculation applies to determine the absolute number of annexin-V-positive or other cell specific marker-positive (or negative) microparticles in each quadrant.

Nonspecific annexin-V labeling was evaluated by preparing microparticles in phosphate-buffered saline without calcium, which yielded no annexin-V-positive events.

To determine intra-assay variability and whether measurements of the total numbers of microparticles were affected by a specific antibody, the total numbers of microparticles were measured in aliquots from the same sample incubated with four different antibodies.

Source (cells of origin) of microparticles. Platelet-derived microparticles expressing phosphatidylserine were identified with mouse anti-human integrin-β3 (CD61) and glycoprotein IX (CD42a-PE). Granulocyte-, monocyte-, and endothelium-derived microparticles and phosphatidylserine expression were identified with, respectively, mouse anti-human CD11b-PE, CD14-PE, CD62-E-PE antibodies, and annexin-V-FL. Microparticles expressing the cellular adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were identified by ICAM-1-PE and VCAM-1-PE antibodies, respectively.

Procoagulant activity of microparticles. A prothrombinase activity assay was used to measure the phospholipid procoagulant activity of the microparticles. For this assay, defined numbers (10,000) of microparticles from each individual were incubated in Tyrode buffer containing (final concentrations in 100 µl) 5 nM Factor Xa and 10 nM Factor Va at 37°C for 3 min. Then, 2 µM prothrombin and 50 µM Asp-(Val-Pro-Arg)-6-amino-1-naphthalene-sulfonamide (final concentrations in 120 µl) were added, and changes in fluorescence (thrombin activity) were measured immediately with a Thermo Electron FluoroScan fluorometer (excitation = 355 nm; emission = 445 nm).

Statistics. Data are presented as means ± SE. One-way analysis of variance followed by Bonferroni's multiple comparison test was used to determine statistical differences in numbers of annexin-V-positive microparticles among groups of women with negative, low and high CAC scores. Spearman's rank correlation coefficient was used to determine the correlation of endothelium-derived microparticles (Q1 + Q2) with CAC scores. Bartlett's test was used to identify analytical variability in thrombin generating capacity of microparticles among groups. A repeated measures analysis of variance was performed on the natural log of the thrombin generating capacity. Statistically significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of the study population. Women were grouped according to their CAC scores as negative (no calcification), low (0 < 50; range, 0.3–32 AU) or high (>50, range, 93–315 AU). There were no significant differences among groups in age, months past menopause, body weight, body mass index (BMI), lipid profile, follicle stimulating hormone, 17β estradiol, and CRP concentrations (Table 1). The concentrations of thyroid-stimulating hormone were significantly higher in women with high calcium scores compared with the other groups (Table 1). Serum chemistries (data not shown), total or differential leukocytes, hemoglobin, hematocrits, and platelet counts all were within normal range and did not differ among the groups (Tables 2 and 3).


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  		Table 1. Characteristics of women participating in this study

 

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  		Table 2. Blood cell counts of study participants

 

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  		Table 3. Platelet characteristics of study participants

 
Validation of microparticle isolation and assay. Basal platelets were <5% P-selectin and fibrinogen receptor positive and showed expected positivity after the stimulation with thrombin receptor agonist peptide (Table 3). The number of events measured by flow cytometry from filtered buffers and antibodies ranged between 100 and 250 events for a 3-min run (Fig. 1, A and C). More than 25,000 events were counted in the same buffer containing microparticles (Fig. 1, B and D). Isolated microparticles were heterogeneous in size and <1 µm in diameter (Fig. 1, G and H).

Total numbers of microparticles (Q1 + Q2+ Q 3+ Q4) detected by flow cytometry varied among individuals (Fig. 2). However, the correlation between numbers of microparticles prepared in duplicated samples from single individuals and then analyzed with two different antibodies approached unity (Fig. 2). The coefficient of variation calculated from single samples measured with four different antibodies was 0.095.


Figure 2
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  		Fig. 2. Analytical variability in total numbers of microparticles between 2 aliquots of the same sample from individual women. Each point represents data from an individual woman.

 
Microparticles and CAC. The total numbers of microparticles increased with the CAC score (Fig. 3), and women with the highest calcium scores had significantly more microparticles positive for annexin-V than women in the other two groups. Annexin-V positive microparticles in women with high CAC scores were identified as being of platelet origin by positive binding to two different platelet-specific antibodies (Fig. 4), anti-CD61 (integrin-β3) and anti-CD42a (glycoprotein IX). Endothelium-derived (CD62-E positive) microparticles also were more abundant in women with high calcium scores (Fig. 5). In the 23 women with positive CAC scores, endothelium-derived microparticles were correlated to the Agatston score (Spearman's rank coefficient = 0.57, P = 0.005). Leukocyte (CD11b positive)- and monocyte (CD14 positive)-derived microparticles and those positive for ICAM-1 and VCAM-1 did not differ among groups (Table 4).


Figure 3
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  		Fig. 3. Representative quadrants derived from flow cytometric scatter plots (top) demonstrating annexin-V-negative (Q3) and -positive (Q4) microparticles. Bottom: cumulative data for numbers of total (annexin-V negative, Q3, white bars, plus annexin-V positive, Q4, black bars) microparticles from women with negative (0 Agatston units, n = 10 women) and positive coronary calcification scores (low, <50 Agatston units, n = 18 women; and high, >50 Agatston units, n = 5 women). Data are presented as means ± SE. *P < 0.05, statistical significance from those with zero calcium and low calcium scores. FL1, green fluorescence; FL2, red fluorescence.

 

Figure 4
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  		Fig. 4. Representative quadrants derived from flow cytometric scatter plots of microparticles labeled with platelet-specific antibodies CD61 (integrin-β3; top, left) and CD42a (glycoprotein IX; top, right) in combination with the antibody for annexin-V. Cumulative numbers of CD61 (bottom, left)- and CD42a (bottom, right)-positive microparticles from women with negative (0 Agatston units, n = 10 women) and positive coronary calcification scores (low, 0 < 50 Agatston units, n = 18 women; and high, >50 Agatston units, n = 5 women). Data are presented as means ± SE. *P < 0.05, statistical significance from those with zero calcium and low calcium scores.

 

Figure 5
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  		Fig. 5. Representative quadrants from flow cytometric scatter plot of microparticles labeled to identify those derived from the endothelium by CD62-E (E-selectin) antibody and annexin-V antibody (top). Cumulative data of CD62-E-phycoerythrin-positive microparticles with (black bars) and without (white bars) annexin-V-positive expression from women with negative (0 Agatston units, n = 10 women) and positive coronary calcification scores (low, 0 < 50 Agatston units, n = 17 women; and high, >50 Agatston units, n = 5 women). Inset: correlation of CD62-E-positive microparticles (MPs) with the coronary artery calcification (CAC) score. Data are presented as means ± SE. *P < 0.05, statistical significance from those with zero calcium and low calcium scores.

 

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  		Table 4. Leukocyte marker (CD11b and CD14)-, ICAM-1-, and VCAM-1-positive microparticles from women in whom CAC scores are known

 
There was large and significant variability in in vitro thrombin generation by microparticles containing large numbers of annexin-V-positive microparticles, i.e., those from women with high CAC (Fig. 6). Thrombin-generating capacity (compared by repeated-measures test of the log transformation of individual data) was statistically greater in the high CAC group compared with negative and low CAC groups (P < 0.03).


Figure 6
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  		Fig. 6. Prothrombinase assay of thrombin generation by microparticles (10,000) derived from women with negative (0 Agatston units, n = 10 women; {blacklozenge}) and positive coronary calcification scores (low, 0 < 50 Agatston units, n = 18 women; {square}, and high, >50 Agatston units; n = 5 women, bullet). Control for buffer with factors but no microparticles ({lozenge}; n = 2) is shown. Data are presented as means ± SE. *P < 0.03, significant variability by Bartlett's test and thrombin-generating capacity by log transformation in the high compared with low and zero CAC groups.

 
Microparticles and FRS. FRSs (10 yr) calculated using the National Cholesterol Education Program (16a) ranged ≤1–3% and so placed these women in a low risk group. The mean FRS did not differ among groups based on CAC scores (Table 1). Within this range of FRS, the total numbers of microparticles, those positive for annexin-V and percentages of platelet and endothelium-derived microparticles, were significantly higher in women with FRS of 2 to 3 compared with those with scores of ≤1. Granulocyte- and monocyte-derived microparticles and those positive for ICAM-1 and VCAM-1 did not correlate with FRS (Table 5).


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  		Table 5. Microparticle phenotype categorized by Framingham risk score

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study documents CAC in about 16% of women who are within three years of menopause, and on the basis of plasma lipids, the body mass index, CRP, triglycerides, or other blood parameters would be considered otherwise at low risk for cardiovascular disease. The percentage of women screened for KEEPS and positive for CAC is half that reported in women of comparable chronological age from the estrogen-only arm of the Women's Health Initiative. However, those women had undergone hysterectomies 10–20 yr before the CAC measurements, and thus were between 5–10 years beyond menopause (30).

An important novel finding of the present study is that both the total numbers of microparticles and those defined with a procoagulant marker (annexin-V binding to phosphatidylserine) were highest in women with CAC scores of >50 AU. We verified that the flow cytometric measure of annexin-V binding recapitulated procoagulant activity. Furthermore, procoagulant microparticles were both of platelet and endothelial origin. Endothelium-derived microparticles identified by E-selectin antibody (CD62-E) (35) and a marker of possible endothelial dysfunction (21) were about tenfold higher in plasma of women with CAC. While this marker may not account for all endothelium-derived microparticles, it reflects those released during perturbation of the endothelium by inflammatory processes (13), perhaps related to the calcification process, rather than those released because of endothelial cell apoptosis (24). Microparticles of endothelial origin have been identified also by CD31+/CD41– and CD31+/CD42– staining (2, 3, 11, 19, 21, 24, 37). However, those methods identify endothelial microparticles by elimination and are not specific, since CD31 may be present on microparticles derived from leukocytes, and do not differentiate microparticles derived from apoptotic compared with activated cells (36, 44). Therefore, in the present study, microparticles expressing CD62-E unambiguously reflect those generated from possibly dysfunctional rather than apoptotic vascular endothelial cells.

The correlation of CAC canonical risk markers have been both supported and refuted (6, 14, 25, 26, 28, 39). Differences among studies reflect the multifactorial nature of the coronary calcification process, which, in some individuals, may have an etiology independent of abnormalities in lipid metabolism and may include polymorphisms associated with bone matrix proteins (16, 17), alterations in calcium and phosphate metabolism (22, 42), or infection (8, 32). The intensities of calcification observed in these women were low, and it is possible that microparticles of other origins would be more prominent if calcification scores were >500 AU or in individuals with advanced disease (12, 27). It is important to emphasize that tests used in this study identified differences in circulating populations of microparticles in asymptomatic women lacking the usual group of risk factors for cardiovascular disease, except for menopause. This consideration is important because other studies have identified differences in populations of microparticles only in individuals with usual risk factors or symptomatic disease, such as hypercholesterolemia, myocardial infarction, coronary artery disease, renal disease, and pulmonary hypertension (2, 3, 10, 37). Although one study reported an elevation of leukocyte-derived microparticles in some asymptomatic subjects (12), the study represented only 25% women. Furthermore, 23% of the entire cohort was smokers and 17% was using lipid-lowering or antihypertensive medications. These percentages are in contrast to the population of the present study who were all women, nonsmokers and of whom none was using lipid lowering or antihypertensive medication.

It is not possible from this study to establish a cause-and-effect relationship between platelet-endothelium-derived microparticles and coronary calcification. However, the origin and activation state of populations of microparticles in the blood may be of physiological significance marking a cell-cell interaction resulting from early endothelial dysfunction or other cellular processing in the initial stages of vascular disease. Using these particles as a window into the intravascular cellular interactions may allow for the identification of a subset of women with early calcification and in whom follow-up screening or additional testing might be warranted. The association of coronary calcification with elevation in thyroid-stimulating hormone warrants further evaluation in larger groups of women. The procoagulant nature of microparticles derived from women with the highest levels of calcification may identify women with increased propensity for thrombosis at sites of vascular lesions and may increase the specificity for risk stratification in women, especially those who are not smokers (see Table 5).

This study has some limitations. The number of individuals screened was 146, and as expected in this age group of women, the incidence of CAC was low. However, despite this low incidence, statistical significance prevailed. Clearly, these measurements need to be extended to larger and broader populations, including middle-aged men without elevated lipids and in older populations in whom coronary calcification is expected.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Kronos Longevity Research Institute; a National Heart, Lung, and Blood Institute Grant HL-78638; and the Mayo Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Jayachandran, Medical Science Bldg., Mayo Clinic College of Medicine, 200 1st St. SW, Rochester, MN 55905 (e-mail: jaya.m{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M Jr, Detrano R. Quantification of coronary artery calcium using ultrafast computed tomography. J Am Coll Cardiol 15: 827–832, 1990.[Abstract]
  2. Amabile N, Guerin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, London GM, Tedgui A, Boulanger CM. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 16: 3381–3388, 2005.[Abstract/Free Full Text]
  3. Amabile N, Heiss C, Real WM, Minasi P, McGlothlin D, Rame EJ, Grossman W, De Marco T, Yeghiazarians Y. Circulating endothelial microparticle levels predict hemodynamic severity of pulmonary hypertension. Am J Respir Crit Care Med 177: 1268–1275, 2008.[Abstract/Free Full Text]
  4. Andersson T, Flockhart DA, Goldstein DB, Huang SM, Kroetz DL, Milos PM, Ratain MJ, Thummel K. Drug-metabolizing enzymes: evidence for clinical utility of pharmacogenomic tests. Clin Pharmacol Ther 78: 559–581, 2005.[CrossRef][Web of Science][Medline]
  5. Bairey Merz CN, Shaw LJ, Reis SE, Bittner V, Kelsey SF, Olson M, Johnson BD, Pepine CJ, Mankad S, Sharaf BL, Rogers WJ, Pohost GM, Lerman A, Quyyumi AA, Sopko G. Insights from the NHLBI-Sponsored Women's Ischemia Syndrome Evaluation (WISE) Study: Part II: gender differences in presentation, diagnosis, and outcome with regard to gender-based pathophysiology of atherosclerosis and macrovascular and microvascular coronary disease. J Am Coll Cardiol 47: S21–S29, 2006.[Abstract/Free Full Text]
  6. Berckmans RJ, Neiuwland R, Boing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 85: 639–646, 2001.[Web of Science][Medline]
  7. Bobryshev YV, Lord RS, Tran D. Chlamydia pneumoniae in foci of "early" calcification of the tunica media in arteriosclerotic arteries: an incidental presence? Am J Physiol Heart Circ Physiol 290: H1510–H1519, 2006.[Abstract/Free Full Text]
  8. Boulanger CM, Amabile N, Tedgui A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension 48: 180–186, 2006.[Free Full Text]
  9. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, Mallat Z. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 104: 2649–2652, 2001.[Abstract/Free Full Text]
  10. 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. J Card Fail 14: 336–340, 2008.[CrossRef][Web of Science][Medline]
  11. Chironi G, Simon A, Hugel B, Del Pino M, Gariepy J, Freyssinet JM, Tedgui A. Circulating leukocyte-derived microparticles predict subclinical atherosclerosis burden in asymptomatic subjects. Arterioscler Thromb Vasc Biol 26: 2775–2780, 2006.[Abstract/Free Full Text]
  12. Corti R, Hutter R, Badimon JJ, Fuster V. Evolving concepts in the triad of atherosclerosis, inflammation and thrombosis. J Thromb Thrombolysis 17: 35–44, 2004.[CrossRef][Web of Science][Medline]
  13. Desai MY, Nasir K, Braunstein JB, Rumberger JA, Post WS, Budoff MJ, Blumenthal RS. Underlying risk factors incrementally add to the standard risk estimate in detecting subclinical atherosclerosis in low- and intermediate-risk middle-aged asymptomatic individuals. Am Heart J 148: 871–877, 2004.[CrossRef][Web of Science][Medline]
  14. Distler JH, Huber LC, Gay S, Distler O, Pisetsky DS. Microparticles as mediators of cellular cross-talk in inflammatory disease. Autoimmunity 39: 683–690, 2006.[CrossRef][Medline]
  15. Doherty TM, Fitzpatrick LA, Shaheen A, Rajavashisth TB, Detrano RC. Genetic determinants of arterial calcification associated with atherosclerosis. Mayo Clin Proc 79: 197–210, 2004.[Abstract/Free Full Text]
  16. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 285: 2486–2497, 2001.[Free Full Text]
  17. Fitzpatrick LA, Turner RT, Ritman ER. Endochondral bone formation in the heart: a possible mechanism of coronary calcification. Endocrinology 144: 2214–2219, 2003.[Abstract/Free Full Text]
  18. Harman SM, Brinton EA, Cedars M, Lobo R, Manson JE, Merriam GR, Miller VM, Naftolin F, Santoro N. KEEPS: The Kronos Early Estrogen Prevention Study. Climacteric 8: 3–12, 2005.[CrossRef][Web of Science][Medline]
  19. Hartman A, Saeed M, Bing RJ. Release of endothelium-derived relaxing factor from freshly harvested porcine endothelial cells. Circ Res 61: 548–554, 1987.[Abstract/Free Full Text]
  20. Healy AM, Pickard MD, Pradhan AD, Wang Y, Chen Z, Croce K, Sakuma M, Shi C, Zago AC, Garasic J, Damokosh AI, Dowie TL, Poisson L, Lillie J, Libby P, Ridker P, Simon DI. Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events. Circulation 113: 2278–2284, 2006.[Abstract/Free Full Text]
  21. Horstman LL, Jy W, Jimenez JJ, Ahn YS. Endothelial microparticles as markers of endothelial dysfunction. Front Biosci 9: 1118–1135, 2004.[Web of Science][Medline]
  22. Hsu HH, Tawfik O, Sun F. Mechanisms of calcification by vesicles isolated from atherosclerotic rabbit aortas. Biochim Biophys Acta 1563: 18–22, 2002.[Medline]
  23. Jayachandran M, Brunn GJ, Karnicki K, Miller RS, Owen WG, Miller VM. In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk. J Appl Physiol 102: 429–433, 2007.[Abstract/Free Full Text]
  24. Jimenez JJ, Jy W, Mauro LM, Soderland C, Horstman LL, Ahn YS. Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis. Thromb Res 109: 175–180, 2003.[CrossRef][Web of Science][Medline]
  25. Lakoski SG, Cushman M, Blumenthal RS, Kronmal R, Arnett D, D'Agostino RB Jr, Detrano RC, Herrington DM. Implications of C-reactive protein or coronary artery calcium score as an adjunct to global risk assessment for primary prevention of CHD. Atherosclerosis 193: 401–407, 2007.[Medline]
  26. Lakoski SG, Greenland P, Wong ND, Schreiner PJ, Herrington DM, Kronmal RA, Liu K, Blumenthal RS. Coronary artery calcium scores and risk for cardiovascular events in women classified as "low risk" based on Framingham risk score: The Multi-Ethnic Study of Atherosclerosis (MESA). Arch Intern Med 167: 2437–2442, 2007.[Abstract/Free Full Text]
  27. Leroyer AS, Isobe H, Leseche G, Castier Y, Wassef M, Mallat Z, Binder BR, Tedgui A, Boulanger CM. Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 49: 772–777, 2007.[Abstract/Free Full Text]
  28. Libby P. Inflammation in atherosclerosis. Nature 420: 868–874, 2002.[CrossRef][Web of Science][Medline]
  29. Lynch SF, Ludlam CA. Plasma microparticles and vascular disorders. Br J Haematol 137: 36–48, 2007.[Web of Science][Medline]
  30. Manson J, Allison M, Rossouw JE, Carr J, Langer R, Hsia J, Kuller L, Cochrane B, Hunt J, Ludlam S, Pettinger M, Gass M, Margolis K, Nathan L, Ockene J, Prentice R, Robbins JR, Stefanick M. Estrogen therapy and coronary-artery calcification. N Engl J Med 356: 2591–2602, 2007.[Abstract/Free Full Text]
  31. McDowell EM, Trump BF. Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100: 405–414, 1976.[Web of Science][Medline]
  32. Miller VM, Rodgers G, Charlesworth JA, Kirkland B, Severson SR, Rasmussen TE, Yagubyan M, Rodgers JC, Cockerill FR 3rd, Folk RL, Kumar V, Farell-Baril G, Lieske JC. Evidence of nanobacterial-like structures in human calcified arteries and cardiac valves. Am J Physiol Heart Circ Physiol 287: H1115–H1124, 2004.[Abstract/Free Full Text]
  33. Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L, Dignat-George F, Freyssinet JM. Procoagulant microparticles: disrupting the vascular homeostasis equation? Arterioscler Thromb Vasc Biol 26: 2594–2604, 2006.[Abstract/Free Full Text]
  34. Morel O, Toti F, Hugel B, Freyssinet JM. Cellular microparticles: a disseminated storage pool of bioactive vascular effectors. Curr Opin Hematol 11: 156–164, 2004.[CrossRef][Web of Science][Medline]
  35. Mutin M, Dignat-George F, Sampol J. Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules. Tissue Antigens 50: 449–458, 1997.[Web of Science][Medline]
  36. Newman PJ. The biology of PECAM-1. J Clin Invest 99: 3–8, 1997.[Web of Science][Medline]
  37. Pirro M, Schillaci G, Paltriccia R, Bagaglia F, Menecali C, Mannarino MR, Capanni M, Velardi A, Mannarino E. Increased ratio of CD31+/CD42– microparticles to endothelial progenitors as a novel marker of atherosclerosis in hypercholesterolemia. Arterioscler Thromb Vasc Biol 26: 2530–2535, 2006.[Abstract/Free Full Text]
  38. Pradhan AD, Manson JE, Rossouw JE, Siscovick DS, Mouton CP, Rifai N, Wallace RB, Jackson RD, Pettinger MB, Ridker PM. Inflammatory biomarkers, hormone replacement therapy, and incident coronary heart disease. Prospective analysis from the Women's Health Initiative Observational Study. JAMA 288: 980–987, 2002.[Abstract/Free Full Text]
  39. Ridker PM. C-reactive protein, inflammation, and cardiovascular disease: clinical update. Tex Heart Inst J 32: 384–386, 2005.[Web of Science][Medline]
  40. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol 57: 791–804, 1995.[CrossRef][Web of Science][Medline]
  41. Shaw LJ, Bairey Merz CN, Pepine CJ, Reis SE, Vittner V, Kelsey SF, Olson M, Delia Johnson B, Mankad S, Sharaf BL, Rogers WJ, Wessel TR, Arant CB, Pohost GM, Lerman A, Quyyumi AA, Sopko G. Insights from the NHLBI-sponsored Women's Ischemia Syndrome Evaluation (WISE) Study. J Am Coll Cardiol 47: 4S–20S, 2006.[Abstract/Free Full Text]
  42. Tintut Y, Demer LL. Recent advances in multifactorial regulation of vascular calcification. Curr Opin Lipidol 12: 555–560, 2001.[CrossRef][Web of Science][Medline]
  43. VanWijk MJ, VanBavel E, Sturk A, Nieuwland R. Microparticles in cardiovascular diseases. Cardiovasc Res 59: 277–287, 2003.[Abstract/Free Full Text]
  44. 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. Arterioscler Thromb Vasc Biol 26: 112–116, 2006.[Abstract/Free Full Text]
  45. Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89: 1121–1132, 1997.[Free Full Text]



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