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. 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. 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. This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.

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.

Sex 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. Venous 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, r 2 = 0.97). MV 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). This 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.

Non‐invasive tests to identify age and early disease‐associated pathology within the kidney are needed. Specific populations of urinary extracellular vesicles (EVs) could potentially be used for such a diagnostic test. Random urine samples were obtained from age‐ and sex‐stratified living kidney donors before kidney donation. A biopsy of the donor kidney was obtained at the time of transplantation to identify nephron hypertrophy (larger glomerular volume, cortex per glomerulus and mean profile tubular area) and nephrosclerosis (% fibrosis, % glomerulosclerosis and arteriosclerosis). Renal parenchymal‐derived EVs in cell‐free urine were quantified by digital flow cytometry. The relationship between these EV populations and structural pathology on the kidney biopsy was assessed. Clinical characteristics of the kidney donors (n=138, age range: 20–70 years, 50% women) were within the normative range. Overall, urine from women contained more EVs than that from men. The number of exosomes, juxtaglomerular cells and podocyte marker–positive EVs decreased (p<0.05) with increasing age. There were fewer total EVs as well as EVs positive for mesangial cell, parietal cell, descending limb of Henle's loop (simple squamous epithelium), collecting tubule‐intercalated cell and monocyte chemoattractant protein‐1 markers (p<0.05) in persons with nephron hypertrophy. The number of EVs positive for intercellular adhesion molecule‐1, juxtaglomerular cell, podocyte, parietal cell, proximal tubular epithelial cell, distal tubular epithelial cell and collecting duct cells were fewer (p<0.05) in persons with nephrosclerosis. EVs carrying markers of cells from the renal pelvis epithelium did not associate with any indices of nephron hypertrophy or nephrosclerosis. Therefore, specific populations of EVs derived from cells of the glomerulus and nephron associate with underlying kidney structural changes. Further validation of these findings in other cohorts is needed to determine their clinical utility.

Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

Understanding the biology of sex differences is integral to personalized medicine. Cardiovascular disease and cognitive decline are two related conditions, with distinct sex differences in morbidity and clinical manifestations, response to treatments, and mortality. Although mortality from all-cause cardiovascular diseases has declined in women over the past five years, due in part to increased educational campaigns regarding the recognition of symptoms and application of treatment guidelines, the mortality in women still exceeds that of men. The physiological basis for these differences requires further research, with particular attention to two physiological conditions which are unique to women and associated with hormonal changes: pregnancy and menopause. Both conditions have the potential to impact life-long cardiovascular risk, including cerebrovascular function and cognition in women. This review draws on epidemiological, translational, clinical, and basic science studies to assess the impact of hypertensive pregnancy disorders on cardiovascular disease and cognitive function later in life, and examines the effects of post-menopausal hormone treatments on cardiovascular risk and cognition in midlife women. We suggest that hypertensive pregnancy disorders and menopause activate vascular components, i.e., vascular endothelium and blood elements, including platelets and leukocytes, to release cell-membrane derived microvesicles that are potential mediators of changes in cerebral blood flow, and may ultimately affect cognition in women as they age. Research into specific sex differences for these disease processes with attention to an individual’s sex chromosomal complement and hormonal status is important and timely.