Muthuvel Jayachandran

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Methodology for Isolation, Identification and Characterization of Microvesicles in Peripheral Blood

RATIONALE Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated. OBJECTIVE To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine. METHODS AND RESULTS Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 μm latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics. CONCLUSION This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.

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

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.

Atherosclerosis and sex hormones: current concepts

CVD (cardiovascular disease) is the leading cause of death for women. Considerable progress has been made in both our understanding of the complexities governing menopausal hormone therapy and our understanding of the cellular and molecular mechanisms underlying hormone and hormone receptor function. Understanding the interplay of atherosclerosis and sex steroid hormones and their cognate receptors at the level of the vessel wall has important ramifications for clinical practice. In the present review, we discuss the epidemiology of CVD in men and women, the clinical impact of sex hormones on CVD, and summarize our current understanding of the pathogenesis of atherosclerosis with a focus on gender differences in CVD, its clinical presentation and course, and pathobiology. The critical animal and human data that pertain to the role of oestrogens, androgens and progestins on the vessel wall is also reviewed, with particular attention to the actions of sex hormones on each of the three key cell types involved in atherogenesis: the endothelium, smooth muscle cells and macrophages. Where relevant, the systemic (metabolic) effects of sex hormones that influence atherogenesis, such as those involving vascular reactivity, inflammation and lipoprotein metabolism, are discussed. In addition, four key current concepts in the field are explored: (i) total hormone exposure time and coronary heart disease risk; (ii) the importance of tissue specificity of sex steroid hormones, critical timing and the stage of atherosclerosis in hormone action; (iii) biomarkers for atherosclerosis with regard to hormone therapy; and (iv) the complex role of sex steroids in inflammation. Future studies in this field will contribute to guiding clinical treatment recommendations for women and help define research priorities.

Human platelets contain estrogen receptor α, caveolin-1 and estrogen receptor associated proteins

Venous thrombosis is a risk factor for oral estrogen treatment in men and women. Platelet responses mediated through estrogen receptors may be involved in these processes. However, expression of estrogen receptors and estrogen receptor-associated protein have not been defined clearly in human platelets. Therefore, the present study was designed to characterize estrogen receptors and estrogen receptor-associated proteins in human platelets. Platelets were obtained from normal female and male blood donors between the ages of 25 and 65 years. Estrogen receptors (ER f and ER g ), heat shock proteins (hsp70 and hsp90), endothelial nitric oxide synthases (eNOS), caveolin-1, -2 and -3 and calmodulin were analyzed in total platelet lysate by immunoblotting. Expression of ER f and ER g , hsp70, hsp90, eNOS, calmodulin, and caveolin-1 were observed in both sexes. Expression of all proteins was similar in platelets from males and females. Results of the present study suggest for the first time that both estrogen receptors f and g , caveolin-1 and other proteins associated with estrogen receptors and production of nitric oxide are present in platelets of both males and females.

Alterations in Platelet Function and Cell-Derived Microvesicles in Recently Menopausal Women: Relationship to Metabolic Syndrome and Atherogenic Risk

A woman’s risk for metabolic syndrome (MS) increases at menopause, with an associated increase in risk for cardiovascular disease. We hypothesized that early menopause-related changes in platelet activity and concentrations of microvesicles derived from activated blood and vascular cells provide a mechanistic link to the early atherothrombotic process. Thus, platelet functions and cellular origin of blood-borne microvesicles in recently menopausal women (n = 118) enrolled in the Kronos Early Estrogen Prevention Study were correlated with components of MS and noninvasive measures of cardiovascular disease [carotid artery intima medial thickness (CIMT), coronary artery calcium (CAC) score, and endothelial reactive hyperemic index (RHI)]. Specific to individual components of the MS pentad, platelet number increased with increasing waist circumference, and platelet secretion of ATP and expression of P-selectin decreased with increasing blood glucose (p = 0.005) and blood pressure (p < 0.05), respectively. Waist circumference and systolic blood pressure were independently associated with monocyte- and endothelium-derived microvesicles (p < 0.05). Platelet-derived and total procoagulant phosphatidylserine-positive microvesicles, and systolic blood pressure correlated with CIMT (p < 0.05), but not with CAC or RHI. In summary, among recently menopausal women, specific platelet functions and concentrations of circulating activated cell membrane-derived procoagulant microvesicles change with individual components of MS. These cellular changes may explain in part how menopause contributes to MS and, eventually, to cardiovascular disease.

Thrombogenic microvesicles and white matter hyperintensities in postmenopausal women

Objective: To determine the association of conventional cardiovascular risk factors, markers of platelet activation, and thrombogenic blood-borne microvesicles with white matter hyperintensity (WMH) load and progression in recently menopausal women. Methods: Women (n = 95) enrolled in the Mayo Clinic Kronos Early Estrogen Prevention Study underwent MRI at baseline and at 18, 36, and 48 months after randomization to hormone treatments. Conventional cardiovascular risk factors, carotid intima-medial thickness, coronary arterial calcification, plasma lipids, markers of platelet activation, and thrombogenic microvesicles were measured at baseline. WMH volumes were calculated using a semiautomated segmentation algorithm based on fluid-attenuated inversion recovery MRI. Correlations of those parameters with baseline WMH and longitudinal change in WMH were adjusted for age, months past menopause, and APOE ε4 status in linear regression analysis. Results: At baseline, WMH were present in all women. The WMH to white matter volume fraction at baseline was 0.88% (0.69%, 1.16%). WMH volume increased by 122.1 mm3 (95% confidence interval: −164.3, 539.5) at 36 months (p = 0.003) and 155.4 mm3 (95% confidence interval: −92.13, 599.4) at 48 months (p < 0.001). These increases correlated with numbers of platelet-derived and total thrombogenic microvesicles at baseline (p = 0.03). Conclusion: Associations of platelet-derived, thrombogenic microvesicles at baseline and increases in WMH suggest that in vivo platelet activation may contribute to a cascade of events leading to development of WMH in recently menopausal women.

Circulating microparticles and endogenous estrogen in newly menopausal women

Background Estrogen modulates antithrombotic characteristics of the vascular endothelium and the interaction of blood elements with the vascular surface. A marker of these modulatory activities is formation of cell-specific microparticles. This study examined the relationship between blood-borne microparticles and endogenous estrogen at menopause. Methods Platelet activation and plasma microparticles were characterized from women being screened (n = 146) for the Kronos Early Estrogen Prevention Study. Women were grouped according to serum estrogen (< 20 pg/ml; low estrogen, n = 21 or > 40 pg/ml; high estrogen, n = 11). Results Age, body mass index, blood pressure and blood chemistries were the same in both groups. No woman was hypertensive, diabetic or a current smoker. Platelet counts, basal and activated expression of P-selectin on platelet membranes were the same, but activated expression of glycoprotein IIb/IIIa was greater in the high-estrogen group. Numbers of endothelium-, platelet-, monocyte- and granulocyte-derived microparticles were greater in the low-estrogen group. Of the total numbers of microparticles, those positive for phosphatidylserine and tissue factor were also greater in the low-estrogen group. Conclusion These results suggest that, with declines in endogenous estrogen at menopause, numbers of procoagulant microparticles increase and thus may provide a means to explore mechanisms for cardiovascular risk development in newly menopausal women.

A history of preeclampsia is associated with a risk for coronary artery calcification 3 decades later.

BACKGROUND A history of preeclampsia is an independent risk factor for cardiac events and stroke. Changes in vasculature structure that contribute to these associations are not well understood. OBJECTIVE The aim of this study was to quantify coronary artery calcification (CAC), a known risk factor for cardiac events, in a prospective cohort of women with and without histories of preeclampsia. STUDY DESIGN Women without prior cardiovascular events (40 with and 40 without histories of preeclampsia, matched for parity and age at index birth) were recruited from a large population-based cohort of women who were residents of Olmsted County, Minnesota, and who delivered from 1976 through 1982. Computed tomography was performed to measure CAC in Agatston units. All pregnancy histories and covariates were confirmed by review of the medical records. Current clinical variables were assessed at the time of imaging. Differences between women with and without histories of preeclampsia were examined using χ(2) tests and tests; CAC, in particular, was compared as a categorical and ordinal variable, with a χ(2) test and with Wilcoxon 2-sample tests and ordinal logistic regression, as appropriate. RESULTS Mean age (SD) at imaging was 59.5 (±4.6) years. Systolic and diastolic blood pressures, hyperlipidemia, and current diabetes status did not differ between women with and without histories of preeclampsia. However, the frequencies of having a current clinical diagnosis of hypertension (60% vs 20%, P < .001) and higher body mass index in kg/m(2) (expressed as median [25th-75th percentile], 29.8 [25.9-33.7] vs 25.3 [23.1-32.0], P = .023) were both greater in the women with histories of preeclampsia compared to those without. The frequency of a CAC score >50 Agatston units was also greater in the preeclampsia group (23% vs 0%, P = .001). Compared to women without preeclampsia, the odds of having a higher CAC score was 3.54 (confidence interval [CI], 1.39-9.02) times greater in women with prior preeclampsia without adjustment, and 2.61 (CI, 0.95-7.14) times greater after adjustment for current hypertension. After adjustment for body mass index alone, the odds of having a higher CAC based on a history of preeclampsia remained significant at 3.20 (CI, 1.21-8.49). CONCLUSION In this first prospective cohort study with confirmation of preeclampsia by medical record review, a history of preeclampsia is associated with an increased risk of CAC >30 years after affected pregnancies, even after controlling individually for traditional risk factors. A history of preeclampsia should be considered in risk assessment when initiating primary prevention strategies to reduce cardiovascular disease in women. Among women with histories of preeclampsia, the presence of CAC may be able to identify those at a particularly high cardiovascular risk, and should be the subject of future studies.

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

BackgroundSex differences in incidence of cardiovascular disease may reflect age-associated intravascular cellular activation resulting in shedding of cell membrane-derived bioactive microvesicles (MV or microparticles) into the blood. Concentrations of cell-specific MV in blood have the potential to be a diagnostic/prognostic marker of pathology, but ranges of MV must first be established in healthy individuals. This study identified cellular origin of blood-borne MV >0.2 μm in blood of apparently healthy women and men aged from 20–70 years.MethodsVenous blood from apparently healthy participants in the Mayo Clinic Biobank was collected into tubes containing protease inhibitors as the anticoagulant. MV were isolated by standardized differential centrifugation and characterized by digital flow cytometer. Each cellular origin of MV was verified by two different antibodies with strong correlation between the two distinct antibodies (e.g., for platelet-derived MV, r2 = 0.97).ResultsMV derived from platelets were the most abundant type of MV in blood from women and men in all age groups. Total numbers of phosphatidylserine, P-selectin, and platelet- and endothelium-derived MV were significantly (P < 0.05) greater in women than men. Numbers of MV from erythrocytes and stem/progenitor cells were significantly lower in premenopausal women than age-matched men. Number of tissue factor pathway inhibitor positive MV were significantly (P < 0.05) lower whereas erythrocyte-derived MV were significantly higher in postmenopausal women compared to premenopausal women. In women, there was a positive relationship between age and erythrocyte-derived MV (ρ = 0.28; P = 0.009), while in men adipocyte-derived MV increased with age (ρ = 0.33; P = 0.01).ConclusionsThis study provides ranges for cellular origin of blood-borne MV in age-matched, apparently healthy women and men from which to compare diagnostic and prognostic uses of blood-borne MV in larger studies and patient population. In addition, sex- and age-specific differences in phosphatidylserine, platelet-, endothelium-, erythrocyte-, and adipocyte-derived blood-borne MV may contribute to differential progression of cardiovascular disease in women compared to men.