Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans

Antibodies and other reagents

A detailed summary of proteins and antibodies used in this study for identification
and quantification of blood-borne MV 0.2 ?m by digital flow cytometer is presented
in Table 1. Fluorescein isothiocyanate (FITC) or R-phycoerythrin (PE)-conjugated rat anti-mouse
IgG and mouse anti-rabbit IgG isotype control antibodies were purchased from BD Biosciences,
San Jose, CA, USA and Santa Cruz Biotechnology, Inc., Dallas, TX, USA, respectively.
FITC and/or PE-conjugated recombinant annexin-V, mouse anti-human cluster differentiation
2 (CD2), CD3, CD11c, CD14, CD19, CD34, CD41, CD42a, CD45, CD54 (ICAM-1), CD62P, CD62E,
CD68, CD70, CD86, CD106 (VCAM-1), CD144, CD146, CD235a, and p16 set antibodies and
TruCOUNT™ (4.2 ?m) beads were purchased from BD Biosciences, San Jose, CA, USA. Fluorescent
latex beads (1 and 2 ?m) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Fluoresbrite®
microparticles (0.2, 0.5, 1, and 2 ?m) were purchased from Polysciences, Inc., Warrington,
PA, USA. PE-conjugated anti-human glycophorin C (CD236) antibody was purchased from
Novus Biologicals, LLC, Littleton, CO, USA. FITC-conjugated mouse anti-human tissue
factor antibody was purchased from American Sekisui Diagnostics, LLC, Stamford, CT,
USA. FITC-conjugated rabbit anti-human tissue factor pathway inhibitor and rabbit
anti-FUCA1 (alpha-L-fucosidase) antibodies were purchased from Bioss Inc., Woburn,
MA, USA. PE- or FITC-conjugated rabbit anti-human fatty acid binding protein 4 (FABP4),
anti-preadipocyte factor-1 (Pref-1), and mouse anti-human C-kit/CD117 antibodies were
purchased from LifeSpan BioSciences, Inc., Seattle, WA, USA. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) and Hanks’ balanced salts were purchased from Sigma Chemicals Co., St.
Louis, MO, USA. All other reagents and solvents used in this study were of analytical/reagent
grade.

Table 1. Fluorophore conjugated proteins/antibodies used to characterize blood-borne microvesicles
by flow cytometry

Study participants

This study was approved by the Institutional Review Board at Mayo Clinic, Rochester,
MN. Participants in this study were women (n?=?82) and men (n?=?62) aged 20–70 years who volunteered to be part of the Mayo Clinic Individualized
Medicine Biobank. The inclusion and exclusion criteria used to obtain samples from
the Individualized Medicine Biobank were based on those used for the Kronos Early
Estrogen Prevention Study and for what might be considered ‘apparently healthy’ persons
who volunteered for the Biobank Study 30],31]. Women were divided into two groups based on self-reported menstrual status (premenopausal
and postmenopausal). No woman less than 50 years old reported being postmenopausal.
Men were matched by age with women in each group. Exclusion criteria included self-reported
(for women) known BRCA mutation positive genotype, complex endometrial disease, endometrial
cancer, hysterectomy, use of oral contraceptives, and use of selective estrogen receptor
modulators (SERMs) such as Raloxifene, Tamoxifen. Exclusion for both women and men
were in utero exposure to diethylstilbestrol (DES; maternal treatment); current smoking more than
ten cigarettes/day; body mass index 35 (kg/m²); history of clinical cardiovascular disease including myocardial infarction, angina,
or congestive heart failure; history of cerebrovascular disease including stroke or
transient ischemic attack; history of thromboembolic disease (deep vein thrombosis
or pulmonary embolus); history of untreated (no cholecystectomy) gallbladder disease;
dyslipidemia (LDL cholesterol 190 mg/dL); current or recent (3 months) use of lipid-lowering
medications or supplements (e.g., statin, fibrate, 500 mg/day of niacin, red rice
yeast); nut allergy; uncontrolled hypertension (systolic BP 150 and/or diastolic
BP?95); and history of, or prevalent, chronic diseases including any cancer (other
than basal cell skin cancers), renal failure, cirrhosis, diabetes mellitus, and endocrinopathies
other than adequately treated thyroid disease, known HIV infection and/or medications
for HIV infection, active severe clinical depression, and dementia.

Blood sample collection

Venous blood was collected into protease inhibitors (1 ?M hirudin to inhibit thrombin
plus 10 ?M soybean trypsin to inhibit factor Xa) to prepare platelet-free plasma by
double centrifugation at 3,000 g for 15 min within 30 min of blood collection 29]; aliquots of platelet-free plasma were frozen at ?70°C until MV analysis. Freeze
and thaw of plasma do not affect the concentration of microvesicles 29]. Serum was not collected in this study, and sex hormones were not measured.

Blood-borne MV isolation, identification, and characterization by flow cytometry

The detailed method for the isolation, identification, separation, and quantification
of blood-borne MV is published by our group 10],22],29],32]. Briefly, plasma was separated from whole blood by double centrifugation at 3,000
g for 15 min. Contamination of the plasma by platelets and other cells was monitored
by Coulter counter and flow cytometry. After validation, this plasma sample was centrifuged
at 20,000 g for 30 min for MV isolation 29]. The pellets of MV were washed and reconstituted with twice filtered (0.2 ?m 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, digital flow cytometer (FACSCantoâ„¢,
BD Biosciences, San Jose, CA, USA) was used to define MV by size calibration beads
and positive annexin-V-fluorescence 29]. Gates to define size are set using an internal standard of 0.2, 0.5, 1, and 2 ?m
latex or silicon beads 29]. The lowest detection limit for the digital flow cytometer based on size calibration
beads is 0.2 ?m 10],29]; therefore, MV detection was set at this limit. For quantification, samples included
a known quantity of beads (TruCOUNT™, BD Biosciences, San Jose, CA, USA) of 4.2 ?m
diameter.

All antibodies were directly conjugated with either fluorescein (FITC) or PE. Cellular
origins of blood-borne MV were verified using two different fluorophores (FITC and
PE) conjugated to two distinct cell surface marker antibodies considered to be specific
for each cell type (Table 1). 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 29],33]. MV were separated by fluorescence scatter or dot plot quadrants (Q) derived MV gate
of light scatter plot in the presence PE (Q1+Q2) and FITC (Q4+Q2) or absence of both
(Q3) of staining (Figure 1). 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 TruCOUNTâ„¢ bead
region × number of beads per test (spiked known count)/test volume 29]. The same calculation applied to quantitation of MV positive or negative for annexin-V
and each cell membrane-specific antibody. Numbers of heterogeneous size of isolated
blood-borne MV from 0.2–1 ?m are reported in this study.

Figure 1. Typical fluorescence dot plot and correlation of two distinct antibodies for platelet-specific
antigens. Left, typical fluorescence dot plot (Q; quadrant) from the MV gate of the
light scatter by FACSCantoâ„¢ flow cytometer showing the spectra for CD41and CD42a positive
MV of activated platelets from apparently healthy women and men. All (100%) platelet-derived
MV did not carry both CD41and CD42a antigens (Q2). Right, correlation of two distinct
antibodies for platelet-specific antigens [CD41 (Q4+Q2) and CD42a (Q1+Q2)] binding
to MV isolated from the same blood sample of apparently healthy women and men. A similar
pattern was observed using two distinct antibodies for other cell-specific identification
of the MV (data not shown).

Statistical analysis

Analyses of MV data from the flow cytometry were performed using the software JMP
from the Statistical Analysis System (S.A.S.), Cary, NC, USA. The distribution of
blood-borne MV (normal or skewed) depends on the cell of origin. Data from each marker
positive blood-borne MV are presented as median with 25th and 75th percentiles. Differences
in concentration of MV between women and men were analyzed by Wilcoxon/Kruskal-Wallis
test (rank sum test) or Student’s t-test using JMP or Sigma plot software with significance accepted at P??0.05. Nonparametric Spearman’s rank correlation coefficient test was performed
to determine the association between MV identified by two different cell surface-specific
markers and to determine age-associated changes in blood-borne MV numbers.