Methods. Twenty young subjects were randomized to placebo (n = 10) or antioxidant supplementation (n = 10) for two weeks. Antioxidant status, vascular distensibility, and hemodynamics were obtained before, immediately, and 30 minutes after an acute bout of aerobic exercise both before and after supplementation. Results. Two weeks of antioxidant supplementation resulted in a greater systolic blood pressure (SBP) decrease during postexercise hypotension (PEH) and significant decreases in augmentation index versus placebo . Also ferric-reducing ability of plasma (FRAP) increased significantly (interaction P = 0.024) after supplementation. Conclusion. Supplementation showed an additive effect on PEH associated with increased FRAP values and decreases in systolic blood pressure and augmentation index.Antioxidants can decrease oxidative stress and combined with acute exercise they may lead to further decreases in blood pressure. The purpose of this study was to investigate the effects of 2 weeks of antioxidant supplementation on vascular distensibility and cardiovascular hemodynamics during postexercise hypotension.  Hypertension is defined as an elevation of blood pressure at or above a systolic blood pressure (SBP) of 140 mm Hg and/or a diastolic blood pressure (DBP) of 90 mm Hg. Pre-hypertension is designated as a systolic pressure from 120 to 139 mm Hg or a diastolic pressure from 80 to 89 mm Hg [Hypertension is widespread in the western world and it imposes a substantial burden on health care costs in the United States. According to the TROPHY study, it is possible to delay the progression from prehypertension to hypertension with medication , howeverIf it is possible to further decrease blood pressure during PEH due to these antioxidants and omega-3s, supplementation may be more favorable than pharmacological anti-hypertensives. The primary focus of this study was to investigate the potential additive effects of a novel omega-3 plus multiflavonoid supplement combined with aerobic exercise on PEH in young pre-hypertensives. We hypothesized that the supplement in combination with aerobic exercise will show an additive decrease in postexercise blood pressure (BP) and pulse wave velocity (PWV) changes while increasing arterial distensibility.P ⩽ 0.05.Prior to the start of this study, all methods were reviewed and approved by the University Institutional Review Board. Twenty participants , 18–26 years old, were recruited through posters/fliers located throughout the University Community. Participants on any medication, including aspirin or birth control, and/or with known cardiovascular disease, diabetes, or hypertension were excluded. Prior to the start of the study, each subject gave written informed consent and completed health history and lipid profile questionnaire to assess prior activity level . The sam2peak.Participants reported to a temperature controlled laboratory (22.5–23.5 degrees Celsius) on four separate occasions. Visit 1 consisted of informed consent, group randomization, body composition assessment (DEXA scan), and a laboratory equipment familiarization including laboratory equipment data collection procedures. This was followed by a graded exercise to test volitional exhaustion performed on a treadmill to determine VO The second visit consisted of a 12-hour fasted blood draw taken at 0700 hours by a certified phlebotomist in the laboratory. A 20 mL blood draw was obtained with the subject in the seated position using the antecubital vein. The blood was immediately centrifuged and the resulting plasma was aliquoted into cryovials and frozen in liquid nitrogen for later analyses of ferric-reducing ability of plasma and the oxygen radical absorbance capacity  and hematocrit.2peak HR for 30 minutes. Immediately following the termination of the exercise session, each participant underwent a seated postexercise blood draw and blood pressure measurement. Immediately following the blood draw and 30 minutes after exercise, each participant had the same measurements taken  immediately after and then 30 minutes following exercise.Upon entering the laboratory for Visit 3, the subject was seated and rested quietly for 15 minutes and had a resting blood pressure taken in their left arm using a manual sphygmomanometer and a stethoscope using the first and fourth Korotkoff sounds for SBP and DBP, followed by pulse wave analysis  measurements in the radial artery. Next, the subject was placed in a supine position and rested quietly for 10 minutes. Pulse wave velocity measurements were taken and then the subject was left in a quiet, darkened room while beat to beat blood pressure measurements were recorded for 10 minutes. Following these cardiovascular measures, the participants underwent a preexercise blood draw before running on the treadmill at a speed that elicited 65–70% of  VOAfter the third visit, the participants started a 14-day supplementation of an antioxidant mixture of quercetin, isoquercetin, omega 3 fatty acids, and EGCG, or a placebo. Both supplements were similar in composition and taste to a candied fruit chew, however the placebo lacked the active ingredients. The antioxidant daily dosage consisted of 1000 mg quercetin with 120 mg EGCG, 400 mg isoquercetin, and 400 mg omega 3 fatty acids from fish oil. The participants were instructed to take four chews a day of the supplement, with two taken immediately prior to lunch and two chews ingested immediately prior to dinner. All subjects were asked to maintain their daily eating habits and to return the empty packets of supplements at their next laboratory visit. Once the fourteen-day supplementation was completed, the participants reported back to the lab within 24 hours for the fourth visit and underwent the battery of tests performed in visit 3.Due to the confounding nature of estrogen on our cardiovascular parameters, the female participants had to arrange their supplementation and visits around their menstrual cycle and one female was counterbalanced for each treatment. Both visits two and three were performed on day two of their menstrual cycle, and supplementation commenced two weeks later; therefore when the fourteenth day of supplementation was finished, the female participants were on day two of their cycle for visit four.“Dual Energy X-ray Absorptiometry” or DEXA is a commonly used test for measuring bone mineral density and one of the most accurate ways to determine body composition. Subjects were placed in a Hologic Discovery QDR scanner in the supine position with their legs rotated inward to 25 degrees and a whole body scan was performed. Each scan was downloaded and analyzed by the same trained technician in one session.2 breath-by-breath metabolic system  for oxygen consumption, CO2 production, and respiratory exchange ratio. VO2peak was determined by averaging the VO2 values over the final 15 seconds of exercise. Maximal effort was considered to have been reached when participants met three of the following four criteria: (a) an RER of 1.15 or greater; (b) a plateau in HR despite an increase in workload; (c) a final RPE score of 17 or greater on the Borg scale ; and/or (d) a plateau in oxygen uptake despite an increase in workload.Aerobic exercise capacity was assessed using a modified Armstrong protocol, briefly, the subject begins the test at a comfortable running pace (between 5.0 and 6.0 miles per hour) at 0% grade with the incline increasing 2.5% every two minutes until volitional fatigue was reached. If the subject did not reach fatigue when the grade reached 10%, the speed was then increased by 2 mph every 2 minutes. Heart rate was measured using a Polar Heart Rate Monitor  and was recorded once during the final 15 seconds of every stage, along with a rating of perceived exertion RPE . Expired air was collected continuously throughout the test and analyzed using a Quark bDt) between proximal and distal foot waveforms as follows: PWV = D/Dt (m/s), where D is distance in meters and Dt is the time interval in seconds. Values attained from carotid to femoral artery were taken as an index of central compliance, while values attained from the carotid and radial artery along with the measurement from the femoral to dorsalis pedis were taken as an index of peripheral compliance. All data was stored and analyzed off line after completion of testing.All measurements were conducted in accordance with guidelines set forth by the Clinical Application of Arterial Stiffness, Task Force III . A SphygBeat-to-beat blood pressures were determined via the Finometer . With participants in a supine position in a quiet and darkened room, beat-to-beat blood pressures were recorded for 10 minutes via finger plethysmography. This noninvasive measurement of the change in blood pressure has been shown to be reliable when compared with measurements of intra-arterial blood pressure . The braFasted blood samples were drawn from an antecubital vein with participants in a seated position. The blood samples were centrifuged in EDTA tubes, and plasma was aliquoted and stored at 80°C prior to analysis for plasma antioxidant parameters Ferric Reducing Ability of Plasma (FRAP) and Oxygen Radical Absorptive Capacity (ORAC).μmol). Intra-assay and interassay coefficients of variation were less than 5% and 7%, respectively.Total plasma antioxidant potential was determined by the FRAP assay according to the methodology of Benzie and Strain . The basμM trolox solution was made with phosphate buffer solution to produce 25, 12.5, and 6.25 μM trolox standards. A fluorescein  working solution was made by pipetting 800 μL of stock solution into 50 mL phosphate buffer in a 50 mL conical tube. Before use, the solution was incubated in the water bath at 37°C until thoroughly heated. The 2,2′-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) solution was made by dissolving 0.108 g of AAPH  into 5 mL of incubated phosphate buffer immediately before the start of the assay. The microplate was prepared and loaded in a “forward-then-reverse” order to avoid possible positional errors. The edge wells were left empty or blank (phosphate buffer working solution) to reduce the impact of “edge effect” on samples and standards particularly from temperature effects on the outside wells. Twenty μL of sample, blank, and trolox standard solutions were pipetted into appropriate wells. Then, 200 μL fluorescein working solution were added to each well using an 8-channel micropipettor. A cover was placed on the microplate and the plate and contents incubated at 37°C for at least 20 minutes. Then, 20 μL AAPH working solution were added using an 8 channel micropipettor as quickly as possible. Final ORAC values (μM/L trolox) were made from the plate reader derived area under the curve (AUC). Then, the final ORACFL values were calculated by using a quadratic regression equation x = −b ± √b2 − 4ac + 4cy. Excitation wavelength was 485 nm and emission wavelength was 520 nm. Intra-assay and inter-assay coefficients of variation were less than 5% and 7%, respectively.The ORAC assay was a modification of the methodology of Ou et al. . Trolox t-test for unpaired values was used to evaluate the statistical differences in descriptive characteristics between groups. An ANOVA with repeated measures (group (treatment versus placebo) by time (baseline pre versus post) by exercise treatment (before versus after versus after 30)) was performed on all dependent variables. If a significant interaction was found, then the significant variables were compared using a Bonferroni post hoc test. SPSS version 17  statistical analysis software was used to perform all statistical calculations and all data is presented as means ± SEM.Student's n = 10) and the placebo group (n = 10) were found in age, height, weight, body mass, VO2peak, or resting heart rate which are presented in Table 1. There were also no significant differences between the treatment and placebo group in PWV , resting SBP, DBP, heart rate (HR), or ORAC between groups.No significant differences between the antioxidant treatment group (P < 0.05) decrease in blood pressure following an acute bout of exercise after antioxidant supplementation.Significant differences were found in 30-minute postexercise systolic blood pressure measurements in the treatment group following supplementation . The plaP = 0.01) decrease from before to after exercise, whereas there were no significant changes in the placebo group and no significant changes before supplementation.As shown in P = 0.001) increase was found in FRAP in the antioxidant supplementation group in the before exercise measurements. There were no significant changes in the placebo group following placebo supplementation. Following exercise, there were no significant differences in FRAP levels in supplement or placebo groups  or resting SBP, the magnitude of PEH was significantly decreased in the 30-minute postexercise period after supplementation for the treatment condition when compared to the placebo without showing a correlating decrease in HR or PWV. Although resting SBP did not change with supplementation, there was a significant decrease in the systolic component of PEH. Because there was no change in PWV, one can only postulate that arterial distensibility was not the cause of this reduction in blood pressure. One possible explanation is that supplementation caused a decrease in oxidative stress, potentially due to an increase in antioxidant power (an increase in FRAP). A reduction in ROS would increase the bioavailability of NO, stimulating vasodilation. A second theory would be a change in sympathovagal regulation in which the supplement may induce a sympatholytic effect on sympathetic tone, thereby increasing the vagal tone.The results of the present investigation support research done by Edwards et al.  who demoThe significant increase in FRAP in the treatment group demonstrates that the total plasma antioxidant capacity increased following supplementation which may be related to the further decrease in systolic blood pressure during the postexercise period. This increase in antioxidant capacity could lead to an increase of free radical scavenging by antioxidants, thereby decreasing oxidative stress and potentially increasing NO levels. An increase of NO in the vasculature would lead to vasodilation and a further decrease of the systolic blood pressure after exercise. Interestingly, resting blood pressure following two weeks of supplementation was unchanged, demonstrating that a decrease in blood pressure in the 30-minute postexercise period from antioxidant supplementation occurred in conjunction with postexercise vasodilation.The favorable effects of the supplement could be due to the multiflavonoid plus fish oil approach, unlike the single antioxidant approach that many previous research investigations have experimented with. The ingestion of many of the elements in the supplement has not shown consistent vascular benefits in humans. Chronic quercetin ingestion has not been proven to protect against exercise-induced oxidative stress or inflammation . EGCG waPeople with a high risk of ischemic heart disease and/or hypertension could benefit from eating fish as clinical trials have shown that undergoing supplementation of fatty acids like fish oil can improve arterial elasticity in subjects with diabetes or dyslipidemia and even improved large arterial elasticity in overweight hypertensive patients . A combiIn conclusion, we have found that a novel multiflavonoid plus fish oil supplement decreases augmentation index and systolic blood pressure with concomitant increases in FRAP, suggesting that cardiovascular benefits may be realized from this course of supplementation.


In multivariable analysis, E/I index was the strongest correlate of PWV . Cardiac parasympathetic function is a strong predictor of large arterial stiffness, in young T1D patients free of macrovascular and renal complications.Arterial stiffness is increased in type 1 diabetes (T1D), before any clinical complications of the disease are evident. The aim of the present paper was to investigate the association between cardiac autonomic function and arterial stiffness in a cohort of young T1D patients, without history of hypertension and any evidence of macrovascular and/or renal disease. Large artery stiffness was assessed by measurement of carotid-femoral pulse wave velocity (PWV). Cardiac autonomic function was assessed by the cardiovascular tests proposed by Ewing and Clarke. Patients with a high cardiac autonomic neuropathy score (≥4) had significantly higher PWV than those with a low score (0-1). A negative, heart rate-independent, correlation between PWV and heart rate variation during respiration was observed ( Arterial stiffness increases as a result of the aging process and as a consequence of many disease states, including diabetes mellitus (DM) –6. AortiDiabetic autonomic neuropathy (DAN) is a serious, often overlooked, complication of DM. Cardiovascular autonomic neuropathy (CAN) is the most studied form of DAN and its prevalence varies dramatically between cohorts of diabetic patients, due to differences in the methodology used for its diagnosis and the lack of method standardization –15. It hFew previous studies have explored the hypothesis that early impairment of the cardiovascular structure and function and early abnormalities of the autonomic nervous system in patients with T1D might be interrelated, and have shown conflicting results –22. AlthIn view of these considerations, the aim of the present study was to investigate whether there is an association between cardiac autonomic function and large artery compliance in a cohort of patients with T1D, without hypertension and/or any clinical evidence of macrovascular or renal disease.Patients with T1D were consecutively selected from the outpatient clinic of our institution. Inclusion criteria required that patients were under 40 years of age, with an HbA1c < 8%, and had no history of arterial hypertension, macrovascular disease, or renal disease, including microalbuminuria. Patients receiving statins were excluded from the study. Since the performance of reliable cardiovascular autonomic tests requires the exclusion of potential confounding factors, such us concomitant illnesses and drug use  those have been carefully ruled out . In addi2 = E*h/2rρ; where E is the slope of stress-strain relationship for a given vessel, Young's modulus; ρ is the density of fluid; h/2r is the wall thickness/diameter. PWV was calculated by measuring the time taken for the arterial pulse to propagate from the carotid to the femoral artery. The SphygmoCor system was used [Arterial stiffness was assessed noninvasively using the estimation of pulse wave velocity (PWV) which has emerged as the “gold-standard” for the measurement of regional arterial stiffness . The thewas used , which iE/I index). Heart rate response to standing (standing test) was calculated as the ratio of the longest R-R interval (found at about beat 30) to the shortest R-R interval (found at about beat 15) after standing up (30 : 15 ratio). Heart rate response to the Valsalva maneuver  was assessed by calculating the ratio of the longest R-R interval after the maneuver to the shortest R-R interval during or shortly after the maneuver (VM index). All calculations were undertaken by measuring ECG recordings of RR intervals automatically, using the computer-aided examination and evaluation system VariaCardio TF4  [Cardiac autonomic function was assessed using the battery of cardiovascular tests, proposed by Ewing et al.  and recoeds, UK) . The heart rate-based tests were evaluated according to published age-related tables . OrthostAll tests were carried out between 07:00 and 09:00 h, in a quiet environment with stable temperature (22–24°C). All PWV and CAN measurements were assessed by the same person.t-test or by ANOVA. Simple correlations were performed using Pearson's or Spearman's correlation coefficient, as appropriate. Multivariable stepwise linear regression analysis was used to assess the independent contribution of variables possibly associated with PWV. P values (two-tailed) < 0.05 were considered statistically significant.Statistical analysis was performed using the statistical package SPSS 15.0.1 . All data were assessed for normal distribution of the values. Categorical data were compared using a Chi-square test. Comparisons of normally distributed data, between groups, were performed by the independent samples Student's A total of 66 patients were included in the study, out of whom 31 (46.97%) were men. The mean age was 27.1 years while the mean duration of diabetes was 12.3 years. All patients were under intensive insulin treatment  and 11 (16.66%) received continuous subcutaneous insulin infusion via an insulin pump. The patients had decent glycemic control (mean HbA1c: 7.4%). The demographic and clinical characteristics of the study participants are shown in detail in P = 0.012) while borderline significance was observed regarding patients with high versus low total CAN score (P = 0.067).Patients with a high total CAN score (≥4) had a significantly higher PWV value than those with a low total score 0-1; . PWV wasr = 0.527, P < 0.001), diabetes duration , systolic blood pressure , diastolic blood pressure , waist circumference , and waist-to-hip ratio . No significant association was found between PWV and gender, BMI, smoking status, HbA1c, and plasma levels of HDL-C, LDL-C, and triglycerides.In univariate analysis, PWV correlated positively with age . A weaker negative correlation was observed between PWV and the 30 : 15 index , which was vanished after adjusting for heart rate . No significant correlation was observed between PWV and the Valsalva index as well as the change in blood pressure after standing up. After adjustment for age, PWV remained significantly correlated only to diastolic blood pressure , waist-to-hip ratio , and E/I index .A significant negative correlation was observed between PWV and the E/I index were independently associated with PWV. E/I index was the strongest predictor of PWV . E/I index, age and waist-to-hip ratio accounted for 43.7% of PWV variability , had no history of hypertension, and were free of macrovascular complications and nephropathy, including microalbuminuria. In addition, all patients were treated with intensive insulin therapy (either multiple daily injections or continuous subcutaneous insulin infusion), having a fairly decent glycemic control (mean HbA1c: 7.4%). It is known that renal disease, even in the microalbuminuric stage, is related to elevation of blood pressure, decreased blood pressure decline during sleep, increased arterial stiffness, and impaired autonomic nervous system function –31. HencE/I index (r = −0.533) and (to a lower extent) the 30 : 15 ratio (r = −0.275) but not to the VM index (r = −0.119). After adjustment for heart rate at rest, the association of E/I index and PWV remained significant , while the 30 : 15 ratio was not related significantly to PWV. After full adjustment for possible confounding factors, the association between E/I index and PWV not only remained significant  is considered as the most reproducible of the cardiac autonomic function tests [r = −0.288, P = 0.07) but neutral in smokers. It has been previously reported that smoking may attenuate the Valsalva heart rate response, due to dysfunction in some part of the autonomic reflex arch [The deep breathing test (expressed by the on tests . Intereslex arch .Both the deep breathing test and the standing-up test (as it concerns heart rate variation), reflect mainly parasympathetic activity of the heart innervation  , 35. On E/I index and aortic distensibility (measured noninvasively by ultrasound) was found in females  with type 1 diabetes [Previous cross-sectional studies have demonstrated an association between arterial stiffness and autonomic neuropathy in both types of diabetes (type 1 and type 2) –22, 36. diabetes . Patientdifference in duration of the RR interval of the ECG during deep inspiration and expiration while in the present study the same parameter was expressed as the ratio between the two respective values; second (2), Van Ittersum et al. performed adjustment for 24-hour heart rate while we only had one measurement of resting heart rate available; third (3), the demographic and clinical characteristics of patients included in the present study were suggestive of an even “earlier” T1D population than in the study by van Ittersum et al. Indeed, our cohort was younger (27.1 ± 6 versus 32.6 ± 11 years) and had a shorter duration of diabetes (12.3 ± 7.7 versus 14.6 ± 10 years). In addition, patients in our study had better glucose control than in the Dutch study (HbA1c: 7.4 ± 4.5 versus 8.1 ± 1.4). As a consequence, the mean PWV values of the two populations was different, with a  5.6 ± 0.9 m/s in our study, compared to a clearly higher value of 6.5 ± 1.8 m/s in the study by van Ittersum et al. It can be assumed that the presence of a less advanced disease in our population may have allowed the association between parasympathetic activity (expressed as the E/I ratio) and arterial stiffness to emerge. In a subsequent Dutch study by van Ittersum et al. , performThe association between PWV and cardiac autonomic function has been previously examined in a small study in healthy individuals as well . Similarr = 0.072, P = 0.59). It might be hypothesized, however, that a single measurement of HbA1c, undertaken some years after the initiation of diabetes, might not reflect the overall glycemic burden, acquired during the entire course of the disease.Both the decrease of large arteries elasticity and the dysfunction of autonomic nervous system appear early in the natural history of T1D –9. The qE/I index and PWV remained highly significant after adjustment for heart rate, a finding indicating that the association between parasympathetic dysfunction and arterial stiffness is not mediated by an increase in heart rate. Finally, alterations in autonomic tone may lead to artery stiffening through trophic influence, leading to changing the structure of vessels [A possible mechanism that might explain the first hypothesis is the impairment of baroreceptor function, induced by stiffening of the arterial wall . On the  vessels , 45. E/I ratio) is associated with increased arterial stiffness measures, 18 years later in life of patients with childhood-diagnosed T1D [In the Pittsburgh Epidemiology of Diabetes Complications (EDC) study, it has been shown prospectively that CAN  and arterial stiffness.


Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.28 patients  were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests .13 patients with mild asthma  and 11 healthy controls  had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients , but not of controls . The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection .Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma . Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.http://www.trialregister.nl): NTR1677.This trial was registered at the Dutch trial registry ( It is increasingly recognized that inflammation and haemostasis are interdependent and closely linked processes that can stimulate each other ,2. Many In addition to chronic inflammatory conditions of the lung, viral and bacterial infections have been shown to induce pulmonary coagulation as well . For exaAsthma exacerbations are most often caused by respiratory viruses ,18, in pWe hypothesized that rhinovirus infection enhances coagulation and reduces fibrinolysis in patients with asthma to a larger extent than in healthy control subjects. Therefore, the aim of the present study was to determine the effect of experimental rhinovirus infection on coagulation , endothelial activation (vWF) and fibrinolysis (PAPc and PAI-1) in peripheral blood and bronchoalveolar lavage (BAL) fluid of patients with mild allergic asthma and healthy control subjects. The second aim was to assess the relationship between coagulation and fibrinolytic parameters and markers of airway inflammation in BAL fluid.Patients with mild asthma had a doctor’s diagnosis of asthma and had to meet the following criteria: baseline FEV1 > 80% predicted, PC20 (methacholine) < 8.0 mg/ml, skin-prick test positive for at least one out of 12 common aeroallergens. Healthy control subjects had the following criteria: baseline FEV1 > 80% predicted, PC20 (methacholine) > 16.0 mg/ml, skin-prick test negative for 12 common aeroallergens. All volunteers were aged between 18 and 40 years, were non-smoking or had stopped smoking more than 12 months ago with ≤ 5 pack years (PY), were negative for neutralizing antibodies against rhinovirus type 16 and did not have concomitant disease or (chronic) inflammatory condition that would interfere with this study, according to the judgment of a pulmonary physician. Patients with asthma were not allowed to use asthma medication other than short-acting β2-agonists within 2 weeks prior to the start of this study until day 6 after rhinovirus infection. Informed consent was obtained from each individual before inclusion.In a prospective parallel design single center study all volunteers were experimentally infected with rhinovirus type 16 (RV16) as described previously , but nowThe study was approved by Medical Ethics Committee of the Academic Medical Centre in Amsterdam, The Netherlands. The study was registered at The Netherlands Trial Register (no. 1677). Written informed consent was obtained from all volunteers.The primary objective of the study was to evaluate tryptophan catabolism in patients with allergic asthma and healthy subjects and the change in this metabolism after experimental rhinovirus infection . CoagulaMeasurements of TATc , PAPc  and PAI-1 , were performed by ELISA. D-dimer levels were determined with a particle-enhanced immunoturbidimetric assay . vWF was determined by ELISA with a polyclonal rabbit anti-human vWF antibody (A0082) as catching antibody and a horse radish peroxidase-labeled rabbit anti-human vWF antibody (P0226) as detecting antibody .The presence of coagulant TF-exposing microparticles in BAL fluid was measured by a fibrin generation test (FGT) in autologous vesicle-depleted pool plasma as described before . In brieLeukocytes in BAL fluid were counted and differentiated on Quick Diff stained cytospin preparations. IL-8 was determined by Luminex according to the manufacturer's protocol . Myeloperoxidase (MPO) was measured by ELISA  using a rabbit anti-MPO (Dako A0398) as capture antibody and the biotinylated antibody as detection antibody with a well-characterised sample as MPO standard. Eosinophil cationic protein (ECP) was measured by ELISA  using a monoclonal mouse-anti-human ECP capture antibody , ECP standard  and a biotinylated polyclonal rabbit-anti-human ECP detection antibody . Both ELISAs were developed using streptavidin poly-HRP  and tetramethyl-benzidine .Coagulation was defined as a secondary endpoint. These endpoints were defined as the changes from baseline in TATc, vWF, D-dimer, PAPc, PAI-1, and ETP in plasma and changes from baseline in TATc, vWF, FGT, D-dimer, PAPc, and PAI-1 in BAL fluid. Changes from baseline were calculated by calculating the fold change, which is the ratio of pre- and post-interventional scores . Comparisons between both time points for each group were calculated by Wilcoxon signed rank test. Fold changes between both groups were analyzed by Mann–Whitney U test. Spearman rho correlations were used to determine associations between the fold changes of coagulation and inflammatory markers in BAL fluid. Linear correlations were calculated for the fold changes of coagulation in plasma and BAL fluid and the viral load detected in BAL-fluid. Differences were considered significant for all statistical tests at p-values less than 0.05. All reported p-values are two-sided. Analyses were performed with SPSS 18.0 .In total 28 volunteers  were included in the study. Out of the 28 patients infected with RV16, 13 patients with mild asthma and 11 healthy control subjects had a positive PCR at day 6  and/or a serologic conversion for RV16 at day 42. Two BAL fluid samples were excluded from the analysis as these samples contained >15% bronchial and squamous epithelial cells. Therefore disruption of the epithelial barrier and subsequent contamination of BAL fluid with plasma cannot be excluded for these samples. Clinical and virologic characteristics of patients and controls are shown in Table Measurements of haemostatic and fibrinolytic markers in venous plasma at baseline and after viral infection are shown in Table The fibrinolytic marker PAP and PAI-1 did not differ between both groups at baseline. Although the fold changes of the fibrinolytic parameters did not change significantly Figure E and F, Amongst the procoagulant markers , the endogenous capacity of the TF-exposing microvesicles (FGT) and the marker of endothelial activation (vWF) only TATc and FGT were detectable in BAL-fluid Table . TATc leOf the fibrinolytic markers, only PAPc was detectable in BAL-fluid. PAPc levels did not differ between both time points nor did the fold change from baseline differ between both groups.IL-8 = -0.455 for FGT (p = 0.03) and RIL-8 = 0.461 for TATc, both p = 0.02). No correlations were found for ECP nor for MPO. After viral infection, the fold changes of FGT and TATc in BAL-fluid showed a significant correlation with ECP and MPO, while TATc also correlated with IL-8 in BAL-fluid  and ECP  at both timepoints. Asthmatic individuals showed an increase in ECP  and IL-8  in BALF after rhinovirus exposure . However2 = 0.7409 p = 0.01). PAI-1 also showed a significant positive linear correlation with RV16-load , while an inverse linear correlation with PAPc was found . vWF did not show a significant correlation, because of a single outlier  When the outlier was omitted from the analyses, the relationship was very significant . The fold change in TATc and D-dimer did not correlate in these patients with the RV16-load in BAL fluid, nor did the haemostatic markers in BAL fluid correlate with the viral load in BAL fluid.RV-16 was detected in BAL fluid in 7 asthmatic patients and 3 healthy controls. Only the mild asthmatic patients were analysed as the group of healthy controls was too small. ETP showed a significant linear correlation with RV16-load in BAL fluid  in BAL fluid. Moreover, fold changes in eosinophil cationic protein and myeloperoxidase were both associated with the fold changes in FGT and TATc levels in BAL fluid after viral infection. This suggests that rhinovirus infection increases the local procoagulant activity in the airways through both eosinophilic and neutrophilic airway inflammation. In addition to local procoagulant activity, RV16-load showed linear correlations with markers of fibrinolysis and haemostatic activity in plasma.This is the first study investigating the effect of experimental virus infection on activation of coagulation in patients with asthma. We observed an increase in the intrinsic capability to coagulate in BAL fluid, which is associated with eosinophilic and neutrophilic inflammation. This confirms previous studies showing that inflammatory and allergic asthma exacerbations induce coagulation in the airways as reflected by increased levels of TATc, TF activity and factor Xa -34. BothThe increase in procoagulant activity in patients with asthma after rhinovirus infection can be explained by several mechanisms. Firstly, coagulant TF-exposing microparticles are likely to play a key role in the local activation of coagulation in the airways of asthmatic individuals, since these microparticles have been shown to be indispensable to initiate coagulation through the extrinsic pathway . BesidesTF-exposing microparticles derived from eosinophils and neutrophils have been described in related eosinophilic diseases, like Churg Strauss vasculitis and bullous pemphigoid -40. HoweThis study extends the role of increased procoagulant activity as described in previous studies to infectious aetiologies of asthma exacerbation ,32. For in vitro  [in vivo (elderly patients with acute respiratory tract infections) [Secondly, the linear correlations between RV16-load in BAL-fluid and systemic PAI-1 and ETP and inverse correlation with systemic PAPc observed for patients with mild asthma point towards endothelial activation by RV16. vWF and PAI-1 are stored in endothelial cells . Quiescenocytes) ,55 and iections) .The strength of our study is the experimental design, which provided standardized exposure and comparable time points of post-infection sampling. In addition, the patients were free of steroids, which may affect inflammatory and coagulant outcomes. However, we cannot exclude the influence of potential confounding factors in the present outcomes. First, the male/female ratio was different between the two groups, with more female subjects in the control group than in the group of patients with asthma. Since many women used oral contraceptives, this might have influenced coagulation activity ,58. HoweSecondly, we did not observe significant changes for some relevant coagulation parameters in BAL fluid and plasma. This might relate to the mild nature of the experimental rhinovirus infection (i.e. low infectious dose and mild rhinovirus strain). We used this method to infect volunteers because it most closely resembles the natural course of rhinovirus infection. In addition, the procedure has been proven to be safe in patients with mild asthma and resulted in increase in common cold symptoms in both asthmatic and healthy individuals. The effects on coagulation are likely more pronounced after a rhinovirus-induced exacerbation in patients with moderate to severe asthma or in more severe disease exacerbations (i.e. influenza).Another explanation for the non significant changes of coagulant parameters in BAL fluid and plasma could be the use of short-acting beta-2-agonists. Although it is suggested that beta-2 agonists may have a small anti-inflammatory effect ,60, we bThirdly, the study was limited by the fact that some haemostatic parameters were below the detection limit in BAL fluid which could be explained by the timing of the second bronchoscopy and/or the dilution involved with bronchoalveolar lavage. This is a common problem often encountered and difficult to circumvent in this type of studies due to the dilution involved with bronchoalveolar lavage. Still, our data provides evidence of increased procoagulant activity in the airways of patients with asthma after viral infection, which correlates with inflammation, and thus matches with our hypothesis.And finally, one could argue that a larger study population would have shown more procoagulant activity and systemic procoagulant changes. Based on our present findings, we calculated that we would need more than 1000 volunteers to reach statistical significance, which is neither ethical nor feasible. Evaluation of coagulation after rhinovirus infection in patients with more severe asthma would be more relevant as these patients are more at risk for venous thromboembolism .Although this study is observational in nature, the findings of the present study may have important clinical implications. Patients with asthma have an increased risk of pulmonary embolism ,13 whichIn conclusion we have provided evidence of local procoagulant activity after rhinovirus infection in the airways of patients with asthma, which is associated with the intensity of eosinophilic and neutrophilic airway inflammation. As rhinovirus infection is a very common trigger of asthma exacerbations the clinical consequences of this induction of procoagulant activity deserve further investigation, in particular in patients with severe asthma who have already an increased risk of myocardial infarction, stroke and venous thromboembolism ,61.BAL: Bronchoalveolar lavage; COPD: Chronic obstructive pulmonary disease; DD: Doubling doses of methacholine; ECP: Eosinophil cationic protein; ELISA: Enzyme-linkes immunosorbant assay; ETP: Endogenous thrombin potential; FENO: Fraction of exhaled Nitric oxide; FEV1: Forced expiratory volume in 1 second; FGT: Fibrin generation test; ICAM-1: Intercellular adhesion molecule 1; IFN-γ: Interferon gamma; IL-8: Interleukin 8; IQR: Interquartal range; mRNA: Messenger ribosomal nucleic acid; MPO: Myeloperoxidase; pb: Post bronchodilator; PAI-1: Plasminogen activator inhibitor-1; PAPc: Plasmin-α2-antiplasmin complexes; PC20: Provocative concentration of inhaled methacholine to cause a 20% falling in FEV1; PCR: Polymerase chain reaction; PE: Pulmonary embolism; PY: Packyears; RSV: Respiratory syncytial virus; RV16: Rhinovirus type 16; TATc: Thrombin-antithrombin complexes; TF: Tissue factor; vWF: von Willebrand factor.The authors declare that they have no competing interests.Conception and design: KS, RL, PS, CJM, PWK, and EHDB. Data collection: KS, MP CJM Analysis and interpretation: CJM, PWK, JCMM, RM, KCW, TP, RN, EHDB, SLJ, KS, Drafting and editing manuscript: all authors. All authors read and approved the final manuscript.Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study.Click here for fileAssociations between fold changes of inflammatory parameters and hemostatic proteins and fibrin generation test in BAL-fluid after RV16-infection.Click here for file


Men have higher blood pressure than women through much of life regardless of race and ethnicity. This is a robust and highly conserved sex difference that it is also observed across species including dogs, rats, mice and chickens and it is found in induced, genetic and transgenic animal models of hypertension. Not only do the differences between the ovarian and testicular hormonal milieu contribute to this sexual dimorphism in blood pressure, the sex chromosomes also play a role in and of themselves. This review primarily focuses on epidemiological studies of blood pressure in men and women and experimental models of hypertension in both sexes. Gaps in current knowledge regarding what underlie male-female differences in blood pressure control are discussed. Elucidating the mechanisms underlying sex differences in hypertension may lead to the development of anti-hypertensives tailored to one's sex and ultimately to improved therapeutic strategies for treating this disease and preventing its devastating consequences. Sry located on the Y chromosome that causes differentiation of testes and leading to a rise in the levels of testosterone (T) in utero. Since one's sex is defined phenotypically , a functioning Sry leads to the birth of a male while in its absence , ovaries develop and a female is born. Thus, Y dosage (0Y or 1Y) dictates the hormonal milieu in utero and throughout the life span. Physiological sex differences can also arise from differences in X dosage (2X or 1X) as a result of differences in parental imprinting, escape from X-inactivation and X-mosacism. This review will focus not only on the impact of the gonadal hormone milieu but also on the other effects that differences in the sex chromosome complement can exert on blood pressure control. The current state of knowledge regarding sex differences in the pathophysiology of human hypertension as well as the clinical implications of studies conducted in experimental animal models of hypertension will be addressed.Sex differences in biology arise from differences in sex chromosome dosage (2X vs. 1X or 0Y vs. 1Y)  than by telemetry  . A studyBlood pressure can also be measured under anesthesia using carotid arterial cannulation; however, blood pressure measured under anesthesia is typically 10-20 mm Hg lower than when measured in conscious freely moving animals ,89. ThisIn people, blood pressure is typically measured using a stethoscope and sphygmomanometer after an individual has been sitting calmly for a few minutes because blood pressure can be elevated by physical activity and emotional stress. In fact, there is a documented phenomenon called "white coat hypertension", which is defined as a normotensive individual who exhibits increased SBP and DBP of at least 20 mm Hg and 10 mm Hg, respectively, in the presence of a health care provider. Multivariate analysis of 5,716 patients in Ireland over a 22 year period demonstrated that females have a higher prevalence of white coat hypertension than males . A higheSS1a and SS1b), in the female only the SS1b was found to be a QTL [Sex differences in the genetic basis for salt-sensitivity was observed in the Sabra salt-sensitive (SS) rat by linkage analysis in segregating populations derived from the SS (a.k.a. SBH/y) and Sabra salt-resistant strain (SR a.k.a. SBN/y). Whereas two quantitative trait loci (QTL) for salt-sensitive blood pressure were found on chromosome 1 in the male  genetic background. MAP was measured in both male and female rats fed a high-salt 8.0% NaCl) diet for 3 weeks, and was found to be significantly greater in the male and female  SS/Mcwi rat than in the male and female  Brown Norway rats. Similarly, MAP was higher in males than females in certain consomic strains. Mattson et al.  found th.0% NaCl It has been difficult to disentangle the effects of ovarian function on arterial pressure in women from those of aging because ovarian hormone loss is an age-associated event. Cross-sectional studies Figures  &2 suggeComparing arterial pressure between young premenopausal women and women with premature ovarian failure provides another way to assess ovarian hormone loss on blood pressure since premature ovarian failure typically presents at 27 years of age. Women with premature ovarian failure have a higher incidence of hypertension than age-matched premenopausal women suggesting ovarian hormone loss raises arterial pressure independently of the effect of aging on blood pressure. These studies, however, are confounded by the possibility that premature ovarian failure represents an acceleration of the aging process .Turner syndrome provides a window into the blood pressure effects of ovarian function at an even earlier age since girls with Turner syndrome exhibit ovarian hormone deficiency during puberty. Compared to published population standards in girls, girls with Turner syndrome have higher blood pressure, a higher incidence of abnormal blood pressure circadian rhythms, and a higher incidence of idiopathic hypertension even in the absence of aortic coarctation, renal abnormalities or chronic urinary track infections ,107. TurLongitudinal studies investigating the relationship between arterial pressure and the age of menopause onset have not demonstrated an effect of menopause on arterial pressure that is distinct from the effect of aging. Hjortland et al.  conducteThe mRen2.Lewis rat exhibits salt-sensitive hypertension, which was not found to be magnified by ovariectomy ; howeverMuch can be learned by comparing models in which ovariectomy had and did not have an effect on arterial pressure. While ovariectomy increased arterial pressure in several animal models of hypertension including Ang II- ,39 and D2) treatment. E2 treatment prevented the blood pressure raising effects of ovariectomy in the Ang II- [2 to prevent the blood pressure raising effects of ovariectomy across these distinct experimental models of hypertension, suggests the mechanisms by which E2 loss raises blood pressure are robust and highly conserved and thus support the conclusion of cross-sectional studies showing that menopause increases arterial pressure in women.To address ovarian hormone regulation of blood pressure, most investigators have compared intact animals with ovariectomized animals treated with and without 17β-estradiol  receptor cause receptor down regulation [Most experimental studies of Ee et al.  addressee in MAP . At 1 ye DS rats . Therefogulation . Thus, i2. In 1997, Crofton et al. [2 treatment to attenuate the blood pressure raising effects of ovariectomy. Studies are clearly needed to investigate the role of progesterone in diverse experimental models of hypertension to fully understand the role of progestins in blood pressure control. This is particularly relevant to women on hormone replacement regimens. In fact, clinical studies have shown that some progestins impair endothelial function in women while others do not, suggesting that certain progestins like depot-medroxyprogesterone acetate can inhibit the beneficial effects of E2 while others like drospirenone are without effect [Far less is known regarding the effects of progesterone loss on blood pressure since few studies have investigated the role of ovarian hormones other than En et al.  found tht effect -120.2 alone or the ERα agonist [Ten years ago, studies of estrogen receptor alpha and beta (ERα & ERβ) null mice suggested that ERβ protects against the age-associated increase in arterial pressure since at 6-7 months of age compared to wild type (WT), SBP was shown to be elevated by 17 and 28 mm Hg in the female and male KO mice, respectively . In cont agonist ; however2 levels markedly increase by 5-10-fold [2 levels drop by greater than 10-fold compared to levels in normal cycling women [2 secretion from the ovarian follicles that occurs at this life transition [2 means that the T:E2 ratio markedly increases as a woman transitions into menopause.The gonadal hormone milieu changes markedly throughout the life of a woman. After puberty, E-10-fold ,124. Fivng women  has centansition ; however2 markedly reduces FSH and LH levels in postmenopausal women, although levels remain significantly higher than those found in premenopausal women [2 in the age-associated increases in arterial pressure are confounded by changes in these hormones of the hypothalamic-pituitary-ovarian axis and therefore, the effects of E2 deficiency can not be separated from the effects of rising FSH and LH levels.As women approach menopause, atresia of the ovarian follicles occurs, fertility declines and serum follicle stimulating hormone (FSH) and LH start to surge in an effort to produce more ovarian follicles . After mal women . Studies2 who were not receiving exogenous hormone or statin treatment. Total cholesterol and low density lipoprotein were significantly higher in women who had an FSH level ≥ 7 IU/I compared to those with an FSH ≤ 7 IU/I and these effects were independent of age. Patients with low FSH had E2 levels around 27 pg/ml whereas patients with high FSH had E2 levels around 52 pg/ml. If the increased prevalence of cardiovascular risk factors is solely due to lower E2 levels then the opposite findings would be expected, namely, that the premenopausal women with higher plasma E2 levels would have less cardiovascular risk factors than those women with lower plasma E2 levels. This study implicates a role for FSH and LH in and of themselves in cardiovascular risk.Some studies suggest that elevation of FSH contributes to hypertension-associated disease. Chu et al.  showed t2 levels than those without the A/A genotype (G/G or G/A). These studies suggest that this single nucleotide polymorphism is a susceptibility mutation for essential hypertension in women and underscores the value of future studies that focus on ovarian hormone changes in addition to E2 in blood pressure control. Surprisingly, the roles of T, LH and FSH and changes in their ratio to E2  throughout a woman's life span are generally neglected in hypertension research and consequently, much less is known regarding their specific effects on blood pressure control.Inactivating mutations in the FSH receptor gene have been reported to cause hereditary hypergonadotropic ovarian failure in women . More reThere are fewer studies investigating the influence of the testicular hormonal milieu on blood pressure compared to studies of the ovarian hormonal milieu. As in women, as men age, the prevalence of hypertension increases Figures  &2. Acco2, lower levels of T in men are also associated with lower levels of E2. LH receptor agonists not only reduced T from 14.5 to 1.2 nmol/liter, E2 was reduced by 3-fold [2 or changes in the T:E2 ratio.While it is difficult to isolate the effects of declining T levels on blood pressure from other effects of advancing age on hypertension-associated disease , studiesy 3-fold . Thus, iIn contrast to the clinical data suggesting that T deficiency exacerbates hypertension, T deficiency induced by orchiectomy lowers blood pressure in several experimental models of hypertension including Ang II infusion , DOCA-sa50 in the Ang II-blood pressure dose response curve between WT and ARKO mice.The absence of the androgen receptor (AR) did not affect arterial pressure under basal conditions as no differences in MAP were found between male WT and AR null mice (ARKO) nor was there any effect of AR deficiency on the magnitude of Ang II-induced hypertension . This st2 and mice with a targeted disruption of the aromatase gene are deficient in E2. No detectable differences in SBP were reported between the aromatase KO and WT female mice [2 induced by aromatase disruption had no detectable effect on SBP is consistent with studies in WT mice that showed E2 depletion by ovariectomy did not alter basal arterial pressure [2 deficiency or lowering of 10-nor-corticosteroids in these experiments.The cytochrome P450 enzyme aromatase produces Eale mice . The finpressure ,39 and epressure . The aropressure ,39. Thespressure  and the pressure ; howeverWhy then does this majority of the animal data directly conflict with the clinical data showing T deficiency is associated with blood pressure elevation? One explanation is the discrepancy in age. Animal castrations for the most part were performed on young animals while the clinical findings are primarily reported on men in their fifth to seventh decades of life. In fact, when orchiectomies in the SHR were conducted at 6 mo of age , there w2) may impact blood pressure. Clearly more research into of T action is needed to sort out the role of T, E2 and the ratio of T:E2 in blood pressure control in both the male and female.Studies of T and blood pressure have also been investigated in the female. In women with excess T production, T has a blood pressure elevating effect. Women with polycystic ovary syndrome have elevated T levels and there is a positive association between T levels and arterial pressure . This lam) while women express 50% of their X genes from their father (Xp) and 50% from their mother (Xm). Thus, men and women possess distinct differences in parental imprinting of the X chromosome which can have a profound effect on physiology and pathophysiology [Although gonadal sex is a major difference between men and women, it is not the only difference. The sex chromosome complement is also distinct. Men have one X and one Y chromosome whereas women have two X and no Y chromosomes. Thus, the sex chromosome gene dosage is different between the sexes. Men express Y chromosome genes that women do not. Moreover, the second X chromosome is inactivated in each cell in the woman's body, but this inactivation is not complete and 15-17% of genes on the inactivated X chromosome (approximately 200-300 genes) escape X-inactivation . Therefoysiology .mO versus XpO Turner women suggests maternal imprinting of the X chromosome adversely affects the lipid profile compared to paternal imprinting [mO compared to XpO. While blood pressure was not investigated in this study, the association between hypertension and adverse lipid profiles, suggests parental imprinting impacts blood pressure control as well.There are many men walking around with more than one Y chromosome, only they don't know it because it is rarely associated with a phenotype except perhaps that of being taller than usual. These XYY men have normal blood pressure, suggesting Y gene dosage does not adversely affect blood pressure . In contprinting . TriglycSry gene was deleted from the Y chromosome through a natural mutation (Y-) [- mouse does not develop testes, but instead, develops ovaries and expresses a female gonadal hormone phenotype. The Sry gene was also inserted onto an autosome, creating XY-Sry and XXSry transgenic mice that regardless of the sex chromosome complement (XY vs. XX) are gonadal males  . Thus, ty Arnold ).We have used the four core genotype mouse model to isolate sex chromosome effects from gonadal sex effects in the Ang II-induced model of hypertension. We found that the magnitude of the hypertension was greater in gonadectomized XX mice compared to gonadectomized XY mice regardless of whether the mice were born male or female  Figure . This fiCaeiro, X.E. et al.  recentlySry in the SHR genetic model of hypertension. Switching the Y chromosome between SHR and the normotensive Wistar-Kyoto (WKY) rat increases arterial pressure by 15-30 mm Hg in WKY and reduces the arterial pressure to a similar degree in SHR [Sry1 gene was delivered to the WKY kidney, the renal sympathetic nervous system [Sry gene on the Y chromosome that is contributing to the hypertension in the SHR. These Sry findings in the SHR do not, however, explain the observation of a sex chromosome effect in the four core genotype mice since the magnitude of the hypertension was higher in the gonadectomized XX mice compared to the XY mice independently of whether they were born with testes (+Sry) or ovaries (-Sry) [Studies implicate a role for e in SHR ,166. Furs system  was actis (-Sry) .While many studies have investigated the role of gonadal hormones on physiological parameters that are known to modulate arterial pressure , it is imperative to realize that demonstrating hormonal regulation of one of these parameters in either sex does not mean this mechanism is a major contributor to male-female differences in blood pressure control. Thousands of genes  are diffSex differences in blood pressure and the prevalence of hypertension are highly conserved across race, ethnicity and country of origin. This robust finding in humans is also observed among animal species including mammals and avian species. While differences in the gonadal steroid profile contribute to the sexual dimorphism in blood pressure control, the sex chromosomes independently of the gonadal hormone milieu also contribute. Gaps in our knowledge regarding sex differences in primary hypertension include the role of T, FSH and LH and the changes in the ratios of these steroids in impacting blood pressure in both men and women across the life span.in utero or during development. Alternatively, focusing on gonadal hormone regulation within one sex is a valuable strategy for gaining insights relevant to that specific sex. Moreover, focusing on genes on the Y chromosome that are not found on the X and the role of genes that escape X-inactivation or studying the influence of parental imprinting and mosacisim on arterial pressure is likely to uncover new insights into blood pressure control that may ultimately be used to develop novel targets for the treatment of hypertension and associated disease as well as provide the rationale for treating men and women with antihypertensive regimens that reach maximal optimization in both sexes.Studies comparing male and female differences in a physiological parameter like blood pressure have numerous confounds whereas studies conducted using the four core genotype mouse model are uniquely able to isolate sex chromosome from gonadal hormone effects that occur The authors declare that they have no competing interests.KS and HJ each contributed to the literature review and the writing of this manuscript. All authors read and approved the final manuscript.KS is the director of Center for the Study of Sex Differences in health, aging and disease, and the vice-chair for Research in Department of Medicine at Georgetown University Medical Center.


Excessive tryptophan metabolism to kynurenine by the rate-limiting enzyme endothelial indoleamine 2,3-dioxygenase 1 (IDO) controls arterial vessel relaxation and causes hypotension in murine endotoxemia. However, its relevance in human endotoxemia has not been investigated so far. We thus aimed to study changes in blood pressure in parallel with tryptophan and kynurenine levels during experimental endotoxemia in humans.Six healthy male volunteers were given E. coli lipopolysaccharide  as a 1-min intravenous infusion. They had levels of soluble E-Selectin and soluble vascular cell adhesion molecule-1 as well as IDO activity assessed as the kynurenine-to-tryptophan plasma ratio by liquid chromatography-tandem mass spectrometry at various time points during a 24 h time course. During endotoxemia, IDO activity significantly increased, reaching peak levels at 8 h after LPS infusion . IDO activity correlated inversely with the development of hypotension as shown by random effects linear regression models. Finally, IDO activity exhibited a kinetic profile similar to that of soluble endothelial-specific adhesion molecules.LPS is a triggering factor for the induction of IDO in men. Our findings strongly support the concept that the induction of IDO in the vascular endothelium contributes to hypotension in human sepsis. Loss of vascular tone is an important factor in the development of severe sepsis, septic shock and multiple organ failure. Recent work by Wang and colleagues provided experimental evidence that kynurenine, a metabolite of the amino acid L-tryptophan, is a mediator of vasodilation and subsequent hypotension during murine endotoxemia . In diffSix healthy male subjects, aged 32 ± 4 years, were admitted to the research unit of our intensive care department. The local medical ethics committee  approved the study and written informed consent was obtained from all subjects before enrolment. Subjects were admitted 15 h prior to endotoxin infusion. A radial artery catheter was placed for blood sampling and arterial blood pressure measurement. During the study, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and pulse rate were measured continuously. At time point zero (0 h) the volunteers received a 1-min infusion of endotoxin  at a dose of 4 ng/kg body weight  via an indwelling venous catheter. Blood samples for marker analysis were obtained pre-dose and at several time points up to 24 h after endotoxin infusion. All samples were immediately placed on ice, centrifuged  and stored at -70°C until analysis. Data from this study have been reported extensively elsewhere [c) as the kynurenine to tryptophan ratio x 1000. Soluble E-Selectin (sEsel) and soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were measured using Fluorokine® MultiAnalyte Profiling kits and a Luminex® Bioanalyzer  according to the manufacturers’ instructions and have been reported elsewhere [Levels of tryptophan and kynurenine were determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing deuterated internal standards for both analytes. The method was adapted from de Jong et al. . In shorlsewhere .c activity and hemodynamic parameters measured repeatedly at various time points in the same person was examined in random effects linear regression models treating hemodynamic parameters as dependent variables and using maximum likelihood estimators. The association between adhesion molecules and IDOc activity was assessed in a similar manner, treating IDOc activity as dependent variable. To fulfil the assumptions needed for the analysis, logarithmic (ln) transformation of sEsel was performed. Results are shown as beta-coefficients that correspond to a one SD higher value of each of the independent variables in each regression.Continuous variables are expressed as means ± one standard deviation (SD). A repeated-measures one-way analysis of variance with Dunnett’s test for multiple comparisons (two-sided) was used to demonstrate statistical changes in hemodynamic and laboratory variables after endotoxin infusion. Two-sided p-values < 0.05 were considered statistically significant. The association between IDOc activity significantly increased at 6.5 h and peaked at 8 h , p = 0.002; DBP: β = -3.907 (95% CI -7.448 to -0.336), p = 0.031; MAP: β = -5.725 (95% CI -9.922 to -1.529), p = 0.007).LPS infusion elicited a significant laboratory and clinical inflammatory response with chills, headache, muscle pain, increased heart rate, and overt hypotension Table . In briec in our human endotoxemia model ; p < 0.0001) and sEsel (β = 3.62 (95% CI 1.86 to 5.38); p < 0.0001) using random effects linear regression.Wang et al.  reportedl Figure . IDOc acc was associated with the extent of endothelial inflammation and correlated inversely with the development of hypotension. In support of our findings, Changsirivathanathamrong et al. [c activity correlates with vasopressor requirement in patients with septic shock. Darcy et al. could show that IDOc activity correlates with severity measures such as the Sequential Organ Failure Assessment (SOFA) score [Here we show that, as in mice , LPS is g et al.  convinciA) score . Zeden aA) score . AdditioA) score . Thus, oA possible limitation of the current study is that we cannot rule out loss of tryptophan and kynurenine due to deep-freeze storage for several years. However, values at baseline (i.e. before LPS infusion) match those of healthy controls from the literature. Furthermore, as the study is a re-analysis of blood samples from a placebo-controlled interventional trial on pharmacologic p38 MAP kinase inhibition in endotoxemia ,5, the iIn conclusion, our findings support the concept that IDO, the rate-limiting enzyme for tryptophan catabolism, is a novel and previously unrecognized mediator of hypotension that might be particularly significant in human sepsis .DBP: Diastolic blood pressure; IDO: Indoleamine 2,3-dioxygenase; LPS: Lipopolysaccharide; MAP: Mean arterial pressure; SBP: Systolic blood pressure; sEsel: Soluble E-selectin; sVCAM-1: Soluble vascular cell adhesion molecule-1.The authors declare that they have no competing interests.JP, JTK PK analysed the data and wrote the manuscript. CPK contributed to statistical analysis and critically revised the manuscript. JGZ collected the data and critically revised the manuscript; MM supervised the soluble ELISA measurements and critically revised the manuscript. JM and SMB carried out measurements of kynurenine and tryptophan and critically revised the manuscript. All authors read and approved the final manuscript.


Variability in arterial pressure and cerebral blood flow has traditionally been interpreted as a marker of cardiovascular decompensation, and has been associated with negative clinical outcomes across varying time scales, from impending orthostatic syncope to an increased risk of stroke. Emerging evidence, however, suggests that increased hemodynamic variability may, in fact, be protective in the face of acute challenges to perfusion, including significant central hypovolemia and hypotension (including hemorrhage), and during cardiac bypass surgery. This review presents the dichotomous views on the role of hemodynamic variability on clinical outcome, including the physiological mechanisms underlying these patterns, and the potential impact of increased and decreased variability on cerebral perfusion and oxygenation. We suggest that reconciliation of these two apparently discrepant views may lie in the time scale of hemodynamic variability; short time scale variability appears to be cerebroprotective, while mid to longer term fluctuations are associated with primary and secondary end-organ dysfunction. Traditionally, clinicians have assessed the cardiovascular status of their patients with static “snapshot” techniques, such as radial pulse for heart rate, brachial sphygmomanometry for arterial pressure, and chest excursions for respiration rate. Subsequently, clinical judgment about health status and identification of potential risk factors was based on average values, without consideration of the inherent dynamic nature of these variables. While the notion that arterial blood pressure is not constant, but fluctuates dynamically over time has been known since the 18th century, the clinical importance of this phenomenon is only now being recognized. There is growing recognition that assessment of hemodynamic variability  across multiple time scales may provide important insight into acute and long-term clinical outcomes, such as risk of stroke  ultrasound monitoring in the 1980s by Aaslid et al.  rapidly The evolving appreciation that measurement of hemodynamic variability provides important physiological insight is demonstrated by Newell et al. in the early 1990s who cautioned that the variability of cerebral blood velocity obtained from TCD may “interfere” with measurement of mean values   , BPV does not contribute to risk stratification over and beyond mean systolic pressure   as highlighted in the “foe” section of this review, the potentially protective effect of pulsatile arterial pressure and/or cerebral blood flow has also been demonstrated in a variety of experimental and clinical settings, focused primarily on what we define as “short-term” variability.While a wealth of studies have assessed the In 1972, Sanderson et al. , subsequ2. These investigators have demonstrated that periodic acceleration can increase vital organ blood flow in pigs, including the brain, heart, kidneys, and liver at rest  . Other investigations, however, have also assessed pulsatile flow at much lower frequencies, generally associated with patterns of respiration, sympathetic nerve activity, and myogenic activity, among other factors.and cerebral blood velocity in subjects with high tolerance (HT) to this stress compared with low tolerant (LT) subjects  exhibited reduced LF oscillations in arterial pressure   is associated with changes in vascular properties, such as arterial compliance (Schroeter et al., In this review we have attempted to highlight the complexities inherent in the characterization of hemodynamic variables such as blood pressure and cerebral blood flow. We have contrasted evidence that supports hemodynamic variability as a protective feature of physiology against evidence suggesting that hemodynamic variability heralds expansive damage to organ function. Our review suggests that reconciliation of these two apparently discrepant views may lie in the time scale of hemodynamic variability; short time scale variability appears to be cerebroprotective, while mid-to-longer term fluctuations are associated with primary and secondary end-organ dysfunction. The extent to which knowledge of the positive and deleterious influences of hemodynamic variability will lead to improve health outcomes are presently unknown, but the case is mounting against classical approaches to hemodynamic assessment that focuses narrowly on absolute blood pressure and/or cerebral blood flow.Caroline A. Rickards and Yu-Chieh Tzeng contributed equally to this manuscript in terms of conception of the work, drafting the work and revising it critically for important intellectual content, and final approval of the version to be published. Caroline A. Rickards and Yu-Chieh Tzeng agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vascular ultrasound was performed and the patients were divided into two groups according to the values of cIMT (< or ≥0.9 mm). Patients with greater cIMT presented significantly higher systolic blood pressure and pulse pressure. Serum HDL-cholesterol was significantly lower and CRP was significantly higher in the same group. There was a significant correlation between cIMT and age , systolic blood pressure , pulse pressure , and LDL-cholesterol . cIMT was correlated to CRP  and negatively correlated to HDL-cholesterol . In logistic regression, only HDL-cholesterol, CRP, and pulse pressure were shown to be independent variables associated to increased cIMT. In conclusion, pulse pressure, HDL-cholesterol, and CRP are variables correlated with cIMT in treated hypertensive women.It has been previously documented that carotid intima-media thickness (cIMT) is a predictor of cardiovascular disease. The aim of this study was to identify clinical parameters associated with an increased cIMT treated hypertensive women. Female patients ( Atherosclerosis and its complications are the main cause of morbidity and mortality in the Western world . TherefoIt has been previously evidenced that carotid intima-media thickness (IMT) is a noninvasive predictor of future cardiovascular disease (CVD) . The incThe identification of risk factors associated with early atherogenesis is important to stratify the individual risk and to predict cardiovascular events . It has The objective of this study was to identify clinical variables associated with subclinical atherosclerosis defined by an increased carotid IMT in treated hypertensive women.We studied hypertensive women referred to Outpatient Clinics of Hypertension and Associated Metabolic Diseases at State University of Rio de Janeiro, Brazil. Participants were 40 to 65 years old, with systolic blood pressure ≥140 mmHg, and/or diastolic blood pressure ≥90 mmHg at two different times and/or in antihypertensive treatment. Patients were excluded if they had a past history of cardiovascular disease, clinically evident infection or inflammatory disease, diabetes mellitus, renal dysfunction with estimated glomerular filtration rate <60 mL/min, evidence of secondary hypertension, or using lipid-lowering medication in the last year. Vascular ultrasound was performed and the patients were divided into two groups according to the values of carotid IMT (< or ≥0.9 mm) . The loc2). The patients were classified as eutrophic (18.5 to 24.9 Kg/m2), overweight (25.0 to 29.9 Kg/m2), or obese patients (30.0 Kg/m2 or more) [Height was measured on a clinic stadiometer. The body weight was assessed using a calibrated scale, with participants using light clothes and no shoes. The body mass index (BMI) was calculated as body weight (kg) divided by square height (mor more) .With a nonextensible tape measure, waist circumference was measured at the midway between the lower rib and the iliac crest. Waist circumference was considered increased for women when the value was higher than 88 cm . MetabolStandardized questionnaires were used to obtain information about physical activity, smoking history, medication use, family history of cardiovascular disease, and duration of hypertension. Systolic and diastolic blood pressure were measured three times after a 5-minute rest, in a seated position, using an electronic device .Venous blood samples were taken after the subject had fasted for 12 hours. The serum lipids  were analyzed by colorimetry (Bioclin). The LDL-cholesterol was calculated using the Friedewald formula  when triThe patient was supine with slight hyperextension and rotation of the neck in the direction opposite the probe. A linear array transducer with a multiple-frequency (7 to 12 MHz) attached to a high-resolution B-mode ultrasound system was used to acquire images by a single-sonographer blind to clinical data of subjects. Simultaneous ECG was recorded to assure the timing of end-diastolic images. Manual measurement of IMT was performed in the common carotid artery, at both sides, in a region free of plaque located approximately 20 mm from bulb. We analysed only patients without any atherosclerotic plaques. At least three values were obtained in different sites of this segment and the mean value of six measurements (three from each side) was used for analysis.t-test was used for comparison of continuous variables, with a confidence interval of 95% and P < 0.05 was considered statistically significant. Menopause and metabolic syndrome were considered as categorical variables and were compared by Chi-squared test. The Pearson coefficient was obtained in the correlation tests between continuous variables. Ordinal logistic regression was used to evaluate associations between increased carotid IMT and previously correlated variables. All statistical analyses were performed using SPSS statistical package .Data were expressed as mean ± standard deviation or proportions when appropriate. Unpaired Student's P < 0.001). Patients in the group with greater carotid IMT were older and presented significantly higher values of systolic blood pressure and pulse pressure. The two groups had mean BMI in the overweight range and increased values of waist circumference. Regarding the biochemical profile, serum HDL-cholesterol was significantly lower, and CRP was significantly higher in the group with greater carotid IMT. Plasma glucose, creatinine, and other parameters of lipid profile were not different between the two groups , physical activity  and menopause  were similar between the two groups. The most used antihypertensive medications were diuretics  and angiotensin converting enzyme inhibitors  and there was no significant difference between the two groups.The proportions of smoking , systolic blood pressure , pulse pressure , and LDL-cholesterol . Carotid IMT was also positively correlated to CRP  and negatively correlated to HDL-cholesterol  (Figures There was a weak but significant correlation between carotid IMT and age ( Figures , resp..  Figures .Carotid IMT has been related to cardiovascular events but the link between this finding and clinical parameters in specific populations is not clear yet. In this study involving only nondiabetic treated hypertensive women with no previous cardiovascular event, carotid IMT was correlated with some clinical factors, but only pulse pressure, HDL-cholesterol, and CRP were considered independent parameters related to carotid IMT. It is well established that age is an important determinant factor for carotid IMT value. In a population-based study, Pastorius et al. evaluated 1448 healthy adults with no known cardiovascular disease and demonstrated that age was an independent predictor factor for increased carotid IMT . In cont Blood pressure levels have also been associated with carotid IMT. In our study, all patients were in use of antihypertensive drugs and the mean diastolic blood pressure levels were in the normal range. Systolic blood pressure was higher in the group with subclinical atherosclerosis but only pulse pressure was independently correlated with carotid IMT. In agreement with this, some studies have already reported association between increased carotid IMT and isolated systolic hypertension , 17, 18. Several studies have already described the relationship between subclinical atherosclerosis and obesity indicated by elevated body mass index or waist circumference –24. HoweThe protective effect of HDL-cholesterol is already well debated and it is not clarified yet. A recent meta-analysis reported that the great majority of the included studies observed an inverse association between HDL-cholesterol and carotid atherosclerosis . HoweverElevated CRP has been considered a predictor of cardiovascular events. A clinical history carefully obtained and a complete physical examination are needed in order to rule out an obvious cause for increased CRP . In the  We concluded that pulse pressure, HDL-cholesterol, and high-sensitive CRP are variables correlated with carotid IMT in treated hypertensive women with no diabetes or previous cardiovascular events. Therefore, these parameters should be carefully followed in this population as potential markers of subclinical atherosclerosis. Noticeably, further studies are needed to establish the prognostic value of clinical and biochemical factors associated with increased carotid IMT in treated hypertensive patients.


To determine risk factors of superimposed preeclampsia in women with essential chronic hypertension receiving antihypertensive therapy prior to conception.A retrospective study of 211 patients that analyzed risk factors of superimposed preeclampsia at first prenatal visit. Variables with a p<.1 at univariate analysis were included in a logistic regression analysis. P<.05 was considered as significant.Superimposed preeclampsia occurred in 49 (23.2%) women. In logistic regression analysis, previous preeclampsia [OR: 4.05 (1.61–10.16)], and mean arterial blood pressure of 95 mmHg or higher [OR: 4.60 (1.94–10.93)] were associated with increased risk of superimposed preeclampsia. When both variables were present, sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio for superimposed preeclampsia were 43%, 94%, 70%, 85%, and 7.71 (95% CI: 3.20–18.57), respectively.In essential chronic hypertensive women, previous preeclampsia and mean arterial blood pressure of 95 mmHg or higher are associated with increased risks of superimposed preeclampsia. Chronic hypertension is a relatively common disorder occurring in approximately 1–5% of pregnant women; rates depending on the population studied and the criteria used for the diagnosis In women with chronic hypertension, the risk of superimposed preeclampsia is increased in black ethnic origin, raised body mass index (BMI), smoking, booking systolic blood pressure of 130 to 139 mm Hg, and diastolic blood pressure of 80 to 89 mm Hg The objective of this study was to identify risk factors for superimposed preeclampsia at first prenatal visit in women with essential chronic hypertension receiving antihypertensive medication prior to conception.This retrospective study included women with chronic hypertension delivered between 1 January 2004 and 31 December 2007 who were identified from the hospital computer databases of two university hospital centers . Every medical chart was reviewed to collect the data. The criteria used to select women with chronic hypertension was a diagnosis of hypertension that needed a treatment before the onset of the pregnancy. Exclusion criteria were: women with multiple pregnancies, women with secondary hypertension, women with proteinuria at less than 20 weeks’ gestation, women considered as having a chronic hypertension but without any treatment at first prenatal visit, women transferred from other maternities, pregnancies complicated by fetal malformations.The data collected from medical records included: age, pre-pregnancy BMI, parity, ethnic origin, tobacco use during pregnancy, duration of hypertension, past obstetric history, antihypertensive treatment, treatment with low dose aspirin, maternal systolic and diastolic blood pressure at booking, presence or absence of proteinuria at first prenatal visit, maternal and neonatal outcomes. The blood pressure was obtained with automated device, patient in sitting up position. The mean arterial pressure was calculated from brachial systolic and diastolic blood pressure (BP), according to the following formula: Mean arterial pressure = [systolic BP+(2*diastolic BP)]/3. Superimposed preeclampsia was defined as a new onset proteinuria (0.3 g of protein or more in a 24-hour specimen) after 20 weeks’ gestation and without proteinuria early in pregnancy (less than 20 weeks’ gestation). Fetal growth restriction (FGR) was defined as a birth weight <5th percentile 2X square or Fisher’s exact test and continuous variables with a two-tailed student t test. Variables with a p<.1 were included in a logistic regression analysis. Data are expressed as odds ratio (OR) with 95% confidence interval (CI). P<.05 was considered as significant. Positive likelihood ratio with 95% CI was calculated for the prediction analysis. The statistical software Statview 5.0 (SAS Institute) was used for statistical analysis, and REALbasic (2002) for Receiver Operative Characteristics (ROC) analysis.We analyzed the risk factors that may influence the rates of superimposed preeclampsia. Categorical variables are presented as percentage, and continuous variables as mean and SD. Categorical variables were compared with The evaluation of chronic hypertensive women did not need approval of our ethical committee because it was a retrospective analysis of data in women who received standard management at both hospitals. Therefore, our ethical committee has waved requirement for approval.The study population consisted of 362 women with chronic hypertension. Women transferred from other centers (n = 61), women with secondary hypertension or with chronic hypertension and pre-existing proteinuria before 20 weeks’ gestation (n = 20), women with fetal anomalies (n = 4), and women not receiving antihypertensive medication prior to pregnancy (n = 66) were excluded. The analysis concerned exclusively the 211 women with essential chronic hypertension who were receiving antihypertensive therapy prior to conception.The rate of superimposed preeclampsia was 23.2%. All women with HELLP syndrome had preeclampsia. We have chosen to include mean arterial blood pressure ≥95 mmHg at multivariate analysis and to analyze its predictive status, rather than systolic or diastolic blood pressure, because mean arterial blood pressure had the best prediction analysis for superimposed preeclampsia  and 95 mmHg was the best cut-off for sensitivity and specificity. Multivariate analysis showed that previous preeclampsia, and mean arterial blood pressure ≥95 mmHg were independently associated with the occurrence of superimposed preeclampsia .We analyzed the prediction for the occurrence of superimposed preeclampsia using the variables that were selected in logistic regression for superimposed preeclampsia at first prenatal visit: previous preeclampsia and mean arterial blood pressure. The ROC analysis of mean arterial blood pressure and previous preeclampsia showed an area under curve at of.77 (p<.0001). The prediction analysis of previous preeclampsia and MAP≥95 mmHg is shown in Of the 49 patients with superimposed preeclampsia, 19 occurred at less than 34 weeks’ gestation. Of all variables analyzed, only SBP at booking was associated with increased risk of early onset compared to late onset of preeclampsia .The main goal of this study was to analyze factors obtained in the first half of the pregnancy that may be associated with superimposed preeclampsia in women with essential chronic hypertension that needed a treatment before the onset of pregnancy. In contrast to other studies The main findings of the study were: 1- superimposed preeclampsia occurred in 23.2% of women, 2- previous preeclampsia, and mean arterial blood pressure ≥95 mmHg at booking was associated with increased risk of superimposed preeclampsia.The rate of superimposed preeclampsia of 23.2% observed in our study is in agreement with that reported in other studies, ranging from 17% to 34.9% In several of these studies, conflicting results concerning the relationship between a history of previous preeclampsia and the recurrence of preeclampsia in a subsequent pregnancy have been reported. Sibai et al. showed that the risk of superimposed preeclampsia was greater in chronic hypertensive women who had preeclampsia during a previous pregnancy  Maternal age and parity had no significant effect on the risk of superimposed preeclampsia in our study. A similar lack of effect of maternal age in women with chronic hypertension was found by Sibai and colleagues Duration of hypertension prior to pregnancy of more than 4 years has been associated with increased risk of superimposed preeclampsia The prevalence of black ethnicity (53.6%) in chronic hypertensive women is quite high and it does not reflect our general population. It is, however, important to note that ethnicity was not independently associated with superimposed preeclampsia in our study.In several studies, pregnant women with essential chronic hypertension had high rates of fetal growth restriction (<5th birth weight percentile) without superimposed preeclampsia Our study has some limitations. The number of patients is relatively small. It is to note, however, that our population concerned exclusively women with essential chronic hypertension who had a treatment at first prenatal visit, and this is not the case of the majority of published studies. Another limitation is related to the retrospective structure of the study, for instance, we did not make any confirmation on past obstetric history that were mentioned in the medical charts, and this may induce some errors in the analysis In essential chronic hypertensive women treated before the pregnancy, previous preeclampsia, and mean arterial blood pressure ≥95 mmHg are associated with increased risk of superimposed preeclampsia. Together, these two variables may select women at extreme high risk of superimposed preeclampsia.