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.
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 micron 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.
anticoagulants; flow cytometry; microparticle; phosphatidylserine; pre-analytical and analytical variables
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.
Atherosclerosis; Carotid intima medial thickening; Coronary calcification; Endothelial function; Monocytes; Platelet secretion
Infection; inflammation; lipopolysaccharide; thrombosis; thrombotic 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.
Atherosclerosis; Carotid intima medial thickening; Coronary calcification; Endothelial function; Monocytes; Platelet secretion
Bacterial infection may increase risk for thrombosis and atherosclerosis. Human platelets express toll-like receptor 4 (TLR4), the receptor for gram-negative bacterial lipopolysaccharide (LPS). Experiments were designed to evaluate direct, acute effects of TLR4 activation on aggregation, secretion, and generation of pro-thrombogenic microparticles in vitro on platelets derived from healthy women at risk for development of cardiovascular disease because of their hormonal status. Platelet-rich plasma from recently menopausal women was incubated with ultrapure Escherichia coli LPS in the absence or presence of antibodies that neutralize the human TLR4. Incubating platelets with LPS (100 ng/mL) for 5 minutes decreased aggregation and dense granule adenosine triphosphate secretion induced by thrombin receptor agonist peptide (TRAP) but not by adenosine diphosphate or collagen. The antibody to TLR4 blocked this effect of LPS. TLR4 activation increased phosphorylation of p38 mitogen-activated protein kinase and decreased production of prothrombotic phosphatidylserine and P-selectin–positive microparticles in response to TRAP. Therefore, acute, direct activation of TLR4 reduces platelet reactivity to TRAP stimulation in vitro. Increased thrombotic and cardiovascular risk with bacterial infection most likely reflects the sum of TLR4 activation on other blood and vascular cells to release proinflammatory cytokines/chemokines, which indirectly affect platelet reactivity.
infection; innate immunity; thrombin receptor agonist peptide; ATP secretion; thrombotic risk
Collapse after prolonged endurance exercise is common and usually benign. This case study reports a triathlete who suffered a vaso‐vagal associated collapsed after exercise. Misdiagnosis of myocardial injury in the presence of elevated cardiac troponins and ECG anomalies led to inappropriate management and highlights the difficulty in treating the collapsed athlete following arduous exercise.
myocardial damage; Ironman triathlon; troponin
We recently reported that the antineoplastic thiodioxopiperazine natural product chaetocin potently induces cellular oxidative stress, thus selectively killing cancer cells. In pursuit of underlying molecular mechanisms, we now report that chaetocin is a competitive and selective substrate for the oxidative stress mitigation enzyme thioredoxin reductase-1 (TrxR1) with lower Km than the TrxR1 native substrate thioredoxin (Trx; chaetocin Km = 4.6 ± 0.6 μM, Trx Km = 104.7 ± 26 μM), thereby attenuating reduction of the critical downstream ROS remediation substrate Trx at achieved intracellular concentrations. Consistent with a role for TrxR1 targeting in the anticancer effects of chaetocin, overexpression of the TrxR1 downstream effector Trx in HeLa cells conferred resistance to chaetocin-induced, but not to doxorubicin-induced, cytotoxicity. As the TrxR/Trx pathway is of central importance in limiting cellular reactive oxygen species (ROS)—and as chaetocin exerts its selective anticancer effects via ROS imposition—the inhibition of TrxR1 by chaetocin has potential to explain its selective anticancer effects. These observations have important implications not just with regard to the mechanism of action and clinical development of chaetocin and related thiodioxopiperazines, but also with regard to the utility of molecular targets within the thioredoxin reductase/thioredoxin pathway in the development of novel candidate antineoplastic agents. Antioxid. Redox Signal. 11, 1097–1106.
Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females.
Aging; Estrogen receptors; Microparticles; Mitochondria; Platelet energy metabolism; Procoagulant activity
Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants (Kd) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the Kd values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.
doi:10.1021/bi702247d CCC: $40.75
The differentiation of physiological left ventricular hypertrophy (LVH) from hypertrophic cardiomyopathy (HCM) can prove challenging for even the most experienced cardiologists. The case is presented of a 17 year old elite swimmer who had electrocardiographic and echocardiographic features that were highly suggestive of HCM. However, indices of diastolic function were normal and cardiopulmonary exercise testing revealed high peak oxygen consumption in keeping with physiological LVH. To resolve the diagnostic dilemma, the patient underwent detraining for eight weeks, after which, there was complete resolution of the changes seen on electrocardiogram and echocardiogram, indicating physiological LVH rather than HCM.
hypertrophic cardiomyopathy; detraining; elite adolescent athlete; left ventricular hypertrophy
The case is reported of a male track and field athlete with breathing difficulties at rest and during exercise, which were exacerbated in the supine position and during water immersion. Right hemidiaphragmatic paralysis was diagnosed. The cause of this relatively benign disorder is not known and there are no serious clinical implications. There is no treatment, but a continuation of exercise together with interventions to strengthen the subsidiary inspiratory muscles is recommended.
diaphragm paralysis; electromagnetic stimulation; phrenic nerve
The reported prevalence of exercise induced asthma (EIA) in elite winter athletes ranges from 9% to 50%. Many elite winter athletes do not report symptoms of EIA. At present there is no gold standard test for EIA.
To establish the efficacy of screening for EIA and examine the role of the eucapnic voluntary hyperventilation (EVH) challenge and laboratory based and sport specific exercise challenges in the evaluation of elite winter athletes.
14 athletes (mean (SD) age 22.6 (5.7) years, height 177.2 (7.0) cm, body mass 68.9 (16.9) kg) from the Great Britain short‐track speed skating (n = 10) and biathlon teams (n = 4) were studied. Each athlete completed a laboratory based and sport specific exercise challenge as well as an EVH challenge, in randomised order.
All 14 athletes completed each challenge. Two had a previous history of asthma. Ten (including the two with a previous history) had a positive test to at least one of the challenges. Ten athletes had a positive response to EVH; of these, only three also had a positive response to the sport specific challenge. No athletes had a positive response to the laboratory based challenge.
Elite athletes should be screened for EIA. EVH is a more sensitive challenge in asymptomatic athletes than sport specific and laboratory based challenges. If sporting governing bodies were to implement screening programmes to test athletes for EIA, EVH is the challenge of choice.
exercise induced asthma; β2 agonist; eucapnic voluntary hyperventilation; exercise challenge
A fall in FEV1 of ⩾10% following bronchoprovocation (eucapnic voluntary hyperventilation (EVH) or exercise) is regarded as the gold standard criterion for diagnosing exercise induced asthma (EIA) in athletes. Previous studies have suggested that mid‐expiratory flow (FEF50) might be used to supplement FEV1 to improve the sensitivity and specificity of the diagnosis. A study was undertaken to investigate the response of FEF50 following EVH or exercise challenges in elite athletes as an adjunct to FEV1.
Sixty six male (36 asthmatic, 30 non‐asthmatic) and 50 female (24 asthmatic, 26 non‐asthmatic) elite athletes volunteered for the study. Maximal voluntary flow‐volume loops were measured before and 3, 5, 10, and 15 minutes after stopping EVH or exercise. A fall in FEV1 of ⩾10% and a fall in FEF50 of ⩾26% were used as the cut off criteria for identification of EIA.
There was a strong correlation between ΔFEV1 and ΔFEF50 following bronchoprovocation (r = 0.94, p = 0.000). Sixty athletes had a fall in FEV1 of ⩾10% leading to the diagnosis of EIA. Using the FEF50 criterion alone led to 21 (35%) of these asthmatic athletes receiving a false negative diagnosis. The lowest fall in FEF50 in an athlete with a ⩾10% fall in FEV1 was 14.3%. Reducing the FEF50 criteria to ⩾14% led to 13 athletes receiving a false positive diagnosis. Only one athlete had a fall in FEF50 of ⩾26% in the absence of a fall in FEV1 of ⩾10% (ΔFEV1 = 8.9%).
The inclusion of FEF50 in the diagnosis of EIA in elite athletes reduces the sensitivity and does not enhance the sensitivity or specificity of the diagnosis. The use of FEF50 alone is insufficiently sensitive to diagnose EIA reliably in elite athletes.
asthma; sensitivity; diagnosis; eucapnic voluntary hyperventilation; elite athletes
cardiac function; endurance exercise; marathon; troponin
Background: Since 2001 the International Olympic Committee-Medical Commission (IOC-MC) has required athletes using inhaled ß2 agonists to provide clinical evidence of their asthmatic condition. The aim of this study was to compare the reported prevalence of asthma at the 2000 and 2004 Olympic Games in the Great British Olympic team (Team GB).
Methods: Following local ethics committee approval, 271 athletes (165 men) from the 2004 Team GB volunteered and provided written informed consent. An athlete was confirmed asthmatic if he or she had a positive bronchoprovocation or bronchodilator test as defined by the IOC-MC. Pre-Olympic medical forms from the 2000 Team GB were also examined to establish the prevalence of asthma among the members of Team GB at the 2000 Olympic Games.
Results: The prevalence of asthma in the two teams at the 2000 and 2004 Olympic Games was similar (21.2% and 20.7%, respectively). In the 2004 Olympic Games 13 of 62 athletes (21.0%) with a previous diagnosis of asthma tested negative. A further seven with no previous diagnosis of asthma tested positive.
Conclusions: The prevalence of asthma within Team GB remained unchanged between 2000 and 2004. The IOC-MC requirement that asthmatic athletes must submit documented evidence of asthma has highlighted that 13 (21.0%) previously diagnosed as asthmatic failed to demonstrate evidence of asthma while seven athletes with no previous history or diagnosis of asthma tested positive. Screening for asthma within elite athletic populations using bronchoprovocation challenges appears warranted to assist athletes in preparing more effectively for major sporting events.
Objectives: To define physiological upper limits of left ventricular (LV) cavity size in trained adolescent athletes.
Design: Cross sectional echocardiographic study.
Setting: British national sports training grounds and Olympic Medical Institute.
Subjects: 900 elite adolescent athletes (77% boys) aged 15.7 (1.2) years participating in ball, racket, and endurance sports and 250 healthy controls matched for age, sex, and size.
Main outcome measures: LV end diastolic cavity size.
Results: Compared with controls, athletes had a larger LV cavity (50.8 (3.7) v 47.9 (3.5) mm), a difference of 6%. The LV cavity was > 54 mm in 18% athletes, whereas none of the controls had an LV cavity > 54 mm. The LV cavity exceeded predicted sizes in 117 (13%) athletes. Among the athletes with LV dilatation, 78% were boys, LV size ranged from 52–60 mm, and left atrial diameter and LV wall thickness were enlarged. Systolic and diastolic function were normal. None of the athletes in the study had an LV cavity size > 60 mm. LV cavity size correlated with age, sex, heart rate, and body surface area.
Conclusion: Highly trained junior athletes usually have only modest increases in LV cavity size. A proportion of trained adolescent athletes have LV cavity size exceeding predicted values but, in absolute terms, LV cavity rarely exceeds 60 mm as in patients with dilated cardiomyopathy. In highly trained adolescent athletes with an LV cavity size > 60 mm and any impairment of systolic or diastolic function, the diagnosis of dilated cardiomyopathy should be considered.
adolescent; elite athlete; athlete’s heart; cardiomyopathy; ventricular cavity dilatation
Objective: To determine whether capillary sampling could offer an alternative sampling method.
Methods: Capillary and venous blood samples were collected for determination of hGH at the end of each exercise stage during an incremental exercise test in 16 male club level competitive cyclists (mean (SD) age 30.8 (8.0) years, body mass 72.2 (7.1) kg, body fat 12.9 (3.5)%, peak oxygen consumption 4.18 (0.46) l•min–1). Linear regression, from a plot of venous v capillary blood hGH concentration, showed a correlation coefficient of r = 0.986 (p<0.001). When geometric means and log transformations were used, a coefficient of variation of 14.2% was demonstrated between venous and capillary flow for hGH concentration. The mean ratio limits of agreement were 0.62 (1.72)—that is, 95% of the ratios were contained between 0.36 and 1.07, with a mean of 0.62.
Conclusions: Capillary blood sampling is an acceptable alternative to venous sampling for determining hGH concentration during rest and exercise. Sample sites should not be used interchangeably: one site should be chosen and its use standardised.
Background: Prolonged steady state exercise can lead to a decrease in left ventricular (LV) function as well as promote the release of cardiac troponin T (cTnT). There is limited information on the effect of intermittent high intensity exercise of moderate duration.
Objectives: To determine the effect of intermittent high intensity exercise of moderate duration on LV function.
Methods: Nineteen male rugby and football players (mean (SD) age 21 (2) years) volunteered. Assessments, before, immediately after, and 24 hours after competitive games, included body mass, heart rate (HR), and systolic blood pressure (sBP) as well as echocardiography to assess stroke volume (SV), ejection fraction (EF), systolic blood pressure/end systolic volume ratio (sBP/ESV), and global diastolic filling (E:A) as well as to indirectly quantify preload (LV internal dimension at end diastole (LVIDd)). Serum cTnT was analysed using a 3rd generation assay. Changes in LV function were analysed by repeated measures analysis of variance. cTnT data are presented descriptively.
Results: SV (91 (26) v 91 (36) v 90 (35) ml before, after, and 24 hours after the game respectively), EF (71 (8) v 70 (9) v 71 (7)%), and sBP/ESV (4.2 (1.8) v 3.8 (1.9) v 4.1 (1.6) mm Hg/ml) were not significantly altered (p>0.05). Interestingly, whereas LVIDd was maintained after the game (50 (5) v 50 (6) mm), sBP was transiently but significantly reduced (131 (3) v 122 (3) mm Hg; p<0.05). E:A was moderately (p<0.05) reduced after the game (2.0 (0.4) v 1.5 (0.4)) but returned to baseline within 24 hours. No blood sample contained detectable levels of cTnT.
Conclusions: In this cohort, LV systolic function was not significantly altered after intermittent activity. A transient depression in global diastolic filling was partially attributable to a raised HR and could not be explained by myocyte disruption as represented by cTnT release.
Objective: To investigate if exercise induced cardiac fatigue and or cardiac damage occurs after prolonged exercise in a hypoxic environment.
Methods: Eight trained male triathletes volunteered for the study. Each completed two 50 mile cycle trials, randomly assigned from normobaric normoxia and normobaric hypoxia (15% FIO2). Echocardiographic assessment and whole blood collection was completed before, immediately after, and 24 hours after exercise. Left ventricular systolic and diastolic functional variables were calculated, and serum was analysed for cardiac troponin T. Results were analysed using a two way repeated measures analysis of variance, with α set at 0.05.
Results: No significant differences were observed in either systolic or diastolic function across time or between trials. Cardiac troponin T was detected in one subject immediately after exercise in the normobaric hypoxic trial.
Conclusions: A 50 mile cycle trial in either normobaric normoxia or normobaric hypoxia does not induce exercise induced cardiac fatigue. Some people, however, may exhibit minimal cardiac damage after exercise in normobaric hypoxia. The clinical significance of this is yet to be elucidated.
OBJECTIVES: To evaluate the spectrum of electrocardiographic (ECG) changes in 1000 junior (18 or under) elite athletes. METHODS: A total of 1000 (73% male) junior elite athletes (mean (SD) age 15.7 (1.4) years (range 14-18); mean (SD) body surface area 1.73 (0.17) m2 (range 1.09-2.25)) and 300 non-athletic controls matched for gender, age, and body surface area had a 12 lead ECG examination. RESULTS: Athletes had a significantly higher prevalence of sinus bradycardia (80% v 19%; p<0.0001) and sinus arrhythmia (52% v 9%; p<0.0001) than non-athletes. The PR interval, QRS, and QT duration were more prolonged in athletes than non-athletes (153 (20) v 140 (18) milliseconds (p<0.0001), 92 (12) v 89 (7) milliseconds (p<0.0001), and 391 (27) v 379 (29) milliseconds (p = 0.002) respectively). The Sokolow voltage criterion for left ventricular hypertrophy (LVH) and the Romhilt-Estes points score for LVH was more common in athletes (45% v 23% (p<0.0001) and 10% v 0% (p<0.0001) respectively), as were criteria for left and right atrial enlargement (14% v 1.2% and 16% v 2% respectively). None of the athletes with voltage criteria for LVH had left axis deviation, ST segment depression, deep T wave inversion, or pathological Q waves. ST segment elevation was more common in athletes than non-athletes (43% v 24%; p<0.0001). Minor T wave inversion (less than -0.2 mV) in V2 and V3 was present in 4% of athletes and non-athletes. Minor T wave inversion elsewhere was absent in non-athletes and present in 0.4% of athletes. CONCLUSIONS: ECG changes in junior elite athletes are not dissimilar to those in senior athletes. Isolated Sokolow voltage criterion for LVH is common; however, associated abnormalities that indicate pathological hypertrophy are absent. Minor T wave inversions in leads other than V2 and V3 may be present in athletes and non-athletes less than 16 but should be an indication for further investigation in older athletes.
OBJECTIVE: To assess cardiac structure and function in elite cross- trained male and female athletes (Alpine skiers). METHODS: Sixteen athletes (10 male, six female) and 19 healthy sedentary control subjects (12 male, seven female) volunteered to take part in the study. Basic anthropometry determined height, body mass, body surface area, and fat free mass. Cardiac dimensions and function were determined by two dimensional, M mode, and Doppler echocardiography. Absolute data and data corrected for body size (allometrically determined) were compared by two way analysis of variance and post hoc Scheffe tests. RESULTS: Absolute left ventricular internal dimension in diastole (LVIDd), septal and posterior wall thickness and left ventricular mass were larger in athletes than controls (p < 0.05) and also increased in the men (p < 0.05) compared with women (except for septal thickness in controls). An increased LVIDd, septal thickness, posterior wall thickness, and left ventricular mass in athletes persisted after correction for body size except when LVIDd was scaled by fat free mass. Cardiac dimensions did not differ between the sexes after correction for body size. All functional indices were similar between groups. CONCLUSION: There is evidence of both left ventricular chamber dilatation and wall enlargement in cross trained athletes compared with controls. Differences in absolute cardiac dimensions between the sexes were primarily due to greater body dimensions in the men.