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Cocoa flavanols (CFs) can improve flow-mediated dilation (FMD), blood pressure, and vascular stiffness in healthy subjects. Endothelial microparticles (EMPs) are markers of endothelial functional integrity, reflecting activation and injury. In plasma samples, we investigated whether age-dependent changes in circulating EMPs exist and whether CFs decrease EMPs in healthy humans. The concentrations of CD31 + /41 - , CD144 + , and CD62e + EMPs (flow cytometry) were increased in healthy elderly ( n = 19) compared to young ( n = 20) non-smokers. EMPs correlated with age, systolic blood pressure, and pulse wave velocity. CD31 + /41 - and CD62e + EMPs inversely correlated with FMD. Following 2 weeks twice-daily CF consumption (450 mg), CD31 + /41 - and CD144 + EMPs decreased in both young and elderly subjects compared to the CF-free control. The EMP decrease inversely correlated with FMD improvements. Cardiovascular aging is associated with increased EMPs that can be modulated by dietary flavanols along with improvements in vascular function. This indicates that flavanol consumption can improve endothelial functional integrity in healthy humans.

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether β1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of β1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for β1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that β1 integrin-blocking antibody or endothelial cell-specific depletion of β1 integrin attenuated FMD of the femoral artery, and blocking of β1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial β1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

Potential health benefits of blueberries may be due to vascular effects of anthocyanins that predominantly circulate in blood as phenolic acid metabolites. We investigated which role blueberry anthocyanins and circulating metabolites play in mediating improvements in vascular function and explore potential mechanisms using metabolomics and nutrigenomics. Purified anthocyanins exerted a dose-dependent improvement of endothelial function in healthy humans, as measured by flow-mediated dilation. The effects were similar to those of wild blueberries containing similar amounts of anthocyanins, whereas control drinks containing fiber, minerals, or vitamins had no significant effect. Daily 1-month wild blueberry consumption increased flow-mediated dilation and lowered 24-hour ambulatory systolic blood pressure. Of the 63 anthocyanin plasma metabolites quantified, 14 and 21 correlated with acute and chronic flow-mediated dilation improvements, respectively. Injection of these metabolites improved flow-mediated dilation in mice. Daily wild blueberry consumption led to differential expression (>1.2-fold) of 608 genes and 3 microRNAs, with Mir-181c showing a 13-fold increase in peripheral blood mononuclear cells. Patterns of 13 metabolites were independent predictors of gene expression changes and pathway enrichment analysis revealed significantly modulated biological processes involved in cell adhesion, migration, immune response, and cell differentiation. Our results identify anthocyanin metabolites as major mediators of vascular bioactivities of blueberries and changes of cellular gene programs. Trial registration: NCT025208., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Previous studies have shown that ultraviolet light can lead to the release of nitric oxide from the skin and decrease blood pressure. In contrast to visible light the local application of ultraviolet light bears a cancerogenic risk. Here, we investigated whether whole body exposure to visible blue light can also decrease blood pressure and increase endothelial function in healthy subjects., Methods: In a randomised crossover study, 14 healthy male subjects were exposed on 2 days to monochromatic blue light or blue light with a filter foil (control light) over 30 minutes. We measured blood pressure (primary endpoint), heart rate, forearm vascular resistance, forearm blood flow, endothelial function (flow-mediated dilation), pulse wave velocity and plasma nitric oxide species, nitrite and nitroso compounds (secondary endpoints) during and up to 2 hours after exposure., Results: Blue light exposure significantly decreased systolic blood pressure and increased heart rate as compared to control. In parallel, blue light significantly increased forearm blood flow, flow-mediated dilation, circulating nitric oxide species and nitroso compounds while it decreased forearm vascular resistance and pulse wave velocity., Conclusion: Whole body irradiation with visible blue light at real world doses improves blood pressure, endothelial function and arterial stiffness by nitric oxide released from photolabile intracutanous nitric oxide metabolites into circulating blood.

Raspberries are a rich source of ellagitannins and anthocyanins. The aim of this work was to investigate whether raspberry consumption can improve vascular function and to understand which phenolic metabolites may be responsible for the effects. A 3 arm double-blind randomized controlled crossover human intervention trial was conducted in 10 healthy males. Flow-mediated dilation (FMD) was measured at baseline, 2 h, and 24 h post-consumption of 200 g and 400 g of red raspberries containing 201 or 403 mg of total (poly)phenols, or a matched control drink. Raspberry (poly)phenol metabolites were analyzed in plasma and urine by UPLC-QTOF mass spectrometry using authentic standards. Significant improvements in FMD were observed at 2 h (1.6% (95%CI 1.2, 1.9) and 1.2% (95% CI 0.8, 1.5)) and 24 h (1.0% (95% CI 0.6, 1.2) and 0.7% (95%CI 0.2, 0.9)) post-consumption of the 200 and 400 g raspberry drinks as compared to control, respectively. Plasma ellagic acid, urolithin A-3-glucuronide and urolithin A-sulfate correlated with the improvements in FMD at 2 and 24 h post consumption, respectively. Consumption of dietary achievable amounts of red raspberries acutely improves endothelial function up to 24 h and ellagitannins may be responsible for the observed effect., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Scope: Cranberries are rich in potentially bioactive (poly)phenols. The aim of this paper was to investigate whether cranberry juice intake can improve vascular function in healthy men in a dose- and time-dependent manner, and to understand which of the circulating (poly)phenol metabolites correlate with vascular effects., Methods and Results: A double-blind randomized controlled crossover trial was conducted in ten healthy males. Flow-mediated dilation (FMD), blood pressure, pulse wave velocity and augmentation index were investigated at baseline, 1, 2, 4, 6, and 8 h post-consumption of cranberry juices containing 409, 787, 1238, 1534, and 1910 mg of total cranberry (poly)phenols (TP), and a control drink. Plasma (poly)phenol metabolites were analyzed by UPLC-Q-TOF MS using authentic standards. We observed dose-dependent increases in FMD at 1, 2, 4, 6, and 8 h with a peak at 4 h and maximal effects with juice containing 1238 mg TP. A total of 60 metabolites were quantified in plasma after cranberry consumption. Twelve (poly)phenol metabolites significantly correlated with the increases in FMD, including ferulic and caffeic acid sulfates, quercetin-3-O-ß-D-glucuronide and a γ-valerolactone sulfate., Conclusion: (Poly)phenols in cranberry juice can improve vascular function in healthy males and this is linked to the presence of specific newly identified plasma metabolites., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cocoa flavanol (CF) intake improves endothelial function in patients with cardiovascular risk factors and disease. We investigated the effects of CF on surrogate markers of cardiovascular health in low risk, healthy, middle-aged individuals without history, signs or symptoms of CVD. In a 1-month, open-label, one-armed pilot study, bi-daily ingestion of 450 mg of CF led to a time-dependent increase in endothelial function (measured as flow-mediated vasodilation (FMD)) that plateaued after 2 weeks. Subsequently, in a randomised, controlled, double-masked, parallel-group dietary intervention trial (Clinicaltrials.gov: NCT01799005), 100 healthy, middle-aged (35-60 years) men and women consumed either the CF-containing drink (450 mg) or a nutrient-matched CF-free control bi-daily for 1 month. The primary end point was FMD. Secondary end points included plasma lipids and blood pressure, thus enabling the calculation of Framingham Risk Scores and pulse wave velocity. At 1 month, CF increased FMD over control by 1·2 % (95 % CI 1·0, 1·4 %). CF decreased systolic and diastolic blood pressure by 4·4 mmHg (95 % CI 7·9, 0·9 mmHg) and 3·9 mmHg (95 % CI 6·7, 0·9 mmHg), pulse wave velocity by 0·4 m/s (95 % CI 0·8, 0·04 m/s), total cholesterol by 0·20 mmol/l (95 % CI 0·39, 0·01 mmol/l) and LDL-cholesterol by 0·17 mmol/l (95 % CI 0·32, 0·02 mmol/l), whereas HDL-cholesterol increased by 0·10 mmol/l (95 % CI 0·04, 0·17 mmol/l). By applying the Framingham Risk Score, CF predicted a significant lowering of 10-year risk for CHD, myocardial infarction, CVD, death from CHD and CVD. In healthy individuals, regular CF intake improved accredited cardiovascular surrogates of cardiovascular risk, demonstrating that dietary flavanols have the potential to maintain cardiovascular health even in low-risk subjects.

Aims: Degenerative aortic valve stenosis (AVS) is independently associated with endothelial dysfunction and increased levels of circulating endothelium-derived microparticles (EMPs) as a marker of compromised endothelial integrity. The aim of this study was to investigate whether therapy for severe AVS by transcatheter aortic valve implantation (TAVI) improves endothelial function and decreases EMPs., Methods and Results: Fifty-six patients with indication for TAVI due to symptomatic severe AVS were prospectively enrolled. Brachial wall shear stress (WSS), endothelial function and circulating microparticles (MPs) were measured before and three months following TAVI. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. MP subpopulations were discriminated by flow cytometry according to the expression of established surface antigens: CD31+/CD41-, CD144+ and CD62E+ as EMPs and CD41+ as platelet-derived MPs (PMPs). In patients with severe AVS, decreased brachial WSS was an independent predictor of low FMD. At three-month follow-up after TAVI, WSS and FMD increased along with decreased levels of EMPs as compared to pre TAVI. Decrease of CD31+/CD41-, CD144+ and CD62E+ EMP levels correlated with the increase of FMD., Conclusions: Therapy for AVS by TAVI was associated with improved endothelial function and integrity indicating beneficial effects of TAVI on systemic arterial function.

Background: Endothelial dysfunction is a key factor in the development of atherosclerosis. Commonly, endothelial function is determined in the brachial artery, whereas patients with peripheral artery disease (PAD) present with lower limb atherosclerosis. We hypothesized that in PAD, a segmental or local association exists between endothelial dysfunction and atherosclerotic structural changes., Methods and Results: We used ultrasound to study endothelial function as flow-mediated vasodilation, intima media thickness, and local stiffness of the superficial femoral artery (SFA) and brachial artery (BA). PAD patients with symptomatic SFA or below-the-knee disease were compared with age-matched patients without PAD and young healthy controls. PAD patients with SFA or below-the-knee disease exhibited endothelial dysfunction of the proximal SFA (flow-mediated vasodilation: 3.9±0.6%, 3.7±0.6%) compared with healthy controls (7.4±1.0%) and patients without PAD (5.4±0.6%). Brachial artery flow-mediated vasodilation values were not different in PAD patients with SFA or below-the-knee disease compared with patients without PAD, but they were significantly lower than those of healthy controls. Endothelial dysfunction correlated with increased intima media thickness or plaque thickness at the site of flow-mediated vasodilation measurement across vascular sites. In PAD patients with SFA disease, SFA flow-mediated vasodilation was further impaired within and distal to stenosis (prestenosis 3.9±0.6%, intrastenosis 2.3±0.7%, poststenosis 2.5±0.6%) and recovered within 24 hours after SFA balloon angioplasty to prestenotic values but not to the brachial artery or SFA values in patients without PAD or controls., Conclusion: A close association exists between local endothelial function and atherosclerotic structural remodeling, suggesting that in PAD, local and segmental factors-in addition to systemic factors-influence local endothelial function. Our data point toward a pathophysiological role for lower extremity endothelial dysfunction in PAD., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Scope: Blueberries are a rich source of flavonoids and phenolic acids. Currently, little information is available regarding the impact of processing on the bioavailability and the bioactivity of blueberry (poly)phenols., Methods and Results: In a randomized, controlled crossover trial, ten healthy volunteers consumed (a) blueberry-containing baked products, (b) an unprocessed blueberry drink containing the same amount of freeze-dried blueberry powder as used in the baked products, and (c) matched control baked products. Endothelial function was measured as flow-mediated dilation (FMD) and plasma samples taken at baseline and at 1, 2, 4, and 6 h postconsumption. Although processing did not significantly change the total (poly)phenolic amount, the processed products contained significantly less anthocyanins (-42%), more chlorogenic acid (23%), no flavanol nonamers or decamers, and significantly more flavanol dimers and trimers (36% and 28%, respectively). FMD increased after 1, 2, and 6 h consumption of the baked products to a similar degree as the unprocessed blueberries, despite significant differences in the levels of individual plasma metabolites. No changes were observed after the consumption of the control product., Conclusion: Careful processing can preserve important biological activities of blueberries despite changing the blueberry (poly)phenol composition and plasma metabolite profile., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: We sought to determine the effects of brief exposures to low concentrations of tobacco secondhand smoke (SHS) on arterial flow-mediated dilation (FMD, a nitric oxide-dependent measure of vascular endothelial function), in a controlled animal model never before exposed to smoke. In humans, SHS exposure for 30 min impairs FMD. It is important to gain a better understanding of the acute effects of exposure to SHS at low concentrations and for brief periods of time., Methods: We measured changes in FMD in rats exposed to a range of real-world levels of SHS for durations of 30 min, 10 min, 1 min, and 4 breaths (roughly 15 s)., Results: We observed a dose-response relationship between SHS particle concentration over 30 min and post-exposure impairment of FMD, which was linear through the range typically encountered in smoky restaurants and then saturated at higher concentrations. One min of exposure to SHS at moderate concentrations was sufficient to impair FMD., Conclusions: Brief SHS exposure at real-world levels reversibly impairs FMD. Even 1 min of SHS exposure can cause reduction of endothelial function.

Current evidence suggests that regenerative v. degenerative endothelial responses can be integrated in a clinical endothelial phenotype, reflecting the net result between damage from risk factors and endogenous repair capacity. We have previously shown that a cocoa flavanol (CF) intervention can improve endothelial function and increase the regenerative capacity of the endothelium by mobilising circulating angiogenic cells in patients with coronary artery disease (CAD). The aim of the present study was to investigate whether CF can lower the levels of circulating endothelial microparticles (EMP), markers of endothelial integrity, along with improvements in endothelial function. The levels of EMP in the frozen plasma samples of CAD patients were measured along with endothelial function (flow-mediated vasodilation, FMD); n 16, FMD data published previously), and these data were compared with those of young (n 12) and age-matched (n 12) healthy control subjects. The CAD patients exhibited significantly increased levels of EMP along with impaired FMD when compared with the healthy control subjects. The levels of CD144⁺ and CD31⁺/41⁻ EMP were inversely correlated with FMD (r -0.67, P=0.01 and r -0.59, P=0.01, respectively). In these CAD patients, the levels of EMP were measured after they had consumed a drink containing 375 mg of CF (high-CF intervention, HiFI) or 9 mg of CF (macro- and micronutrient-matched low-CF control, LoFl) twice daily over a 30-d period in a randomised, double-blind, cross-over study. After 1 month of HiFI, the levels of CD31⁺/41⁻ and CD144⁺ EMP decreased (-25 and -23%, respectively), but not after LoFl. Our data show that flavanols lower the levels of EMP along with higher endothelial function, lending evidence to the novel concept that flavanols may improve endothelial integrity.

Background: Microparticles (MPs) are circulating membrane particles of less than a micrometer in diameter shed from endothelial and blood cells. Recent literature suggests that MPs are not just functionally inert cell debris but may possess biological functions and mediate the communication between vascular cells. As a significant proportion of MPs originate from platelets and endothelial cells, we hypothesized that MPs may harbor functional enzymes including an endothelial NO synthase (eNOS)., Methods and Results: Using immunoprecipitation and Western blot analysis, we found that human circulating MPs carry an eNOS. Ca(2+) and l-arginine-dependent NOS activity of crude enzyme extract from MPs was determined by measuring the conversion of [(3)H]-L-arginine to [(3)H]-citrulline and NOS-dependent nitrite production. NOS-dependent NO production in intact MPs was assessed by the NO-specific fluorescent probe MNIP-Cu. In patients with cardiovascular disease, endothelial dysfunction was associated with an increase in the total number of circulating MPs as well as a significant decrease in the expression and activity of eNOS in MPs. No difference in reactive oxygen species was noted in MPs isolated from either group., Conclusions: Our data further support the concept that circulating MPs may not only retain phenotypic markers but also preserve the functionality of enzymes of the cells they originate from, including eNOS.

Objectives: In patients with coronary artery disease (CAD) medically managed according to currently accepted guidelines, we tested whether a 1-month dietary intervention with flavanol-containing cocoa leads to an improvement of endothelial dysfunction and whether this is associated with an enhanced number and function of circulating angiogenic cells (CACs)., Background: Dietary flavanols can improve endothelial dysfunction. The CACs, also termed endothelial progenitor cells, are critical for vascular repair and maintenance of endothelial function., Methods: In a randomized, controlled, double-masked, cross-over trial, 16 CAD patients (64+/-3 years of age) received a dietary high-flavanol intervention (HiFI [375 mg]) and a macronutrient- and micronutrient-matched low-flavanol intervention (LoFI [9 mg]) twice daily in random order over 30 days., Results: Endothelium-dependent vasomotor function, as measured by flow-mediated vasodilation of the brachial artery, improved by 47% in the HiFI period compared with the LoFI period. After HiFI, the number of CD34+/KDR+-CACs, as measured by flow cytometry, increased 2.2-fold as compared with after LoFI. The CAC functions, as measured by the capacity to survive, differentiate, proliferate, and to migrate were not different between the groups. The HiFI led to a decrease in systolic blood pressure (mean change over LoFI: -4.2+/-2.7 mm Hg), and increase in plasma nitrite level (mean change over LoFI: 74+/-32 nM). Applying a mixed-effects linear regression model, the results demonstrated a significant increase in flow-mediated vasodilation and a decrease in systolic blood pressure with increasing levels of CD34+/KDR+-CACs., Conclusions: Sustained improvements in endothelial dysfunction by regular dietary intake of flavanols are associated with mobilization of functional CACs. (Effect of Cocoa Flavanols on Vascular Function in Optimally Treated Coronary Artery Disease Patients: Interaction Between Endothelial Progenitor Cells, Reactivity of Micro- and Macrocirculation; NCT00553774)., (Copyright (c) 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: This study sought to characterize the impact of hemodialysis (HD)-induced release of hemoglobin on the bioavailability of nitric oxide (NO) and endothelial function., Background: Patients on chronic HD suffer from endothelial dysfunction and a massively increased risk for cardiovascular events. Although dialysis-dependent and -independent factors are discussed, the exact mechanisms are not fully understood., Methods: In 14 HD patients (56+/-15 years of age), endothelial function was determined by measuring flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound before and after treatment. The NO consumption activity of plasma isolated from patients before and after hemodialysis was studied with an NO-sensitive electrode., Results: HD impaired FMD (3.5+/-2.6% to 1.7+/-1.4%, p=0.04) without affecting brachial artery diameter (4.7+/-0.6 mm vs. 4.4+/-0.9 mm, p=0.27). This was accompanied by an increase in cell-free plasma hemoglobin (196+/-43 mg/l to 285+/-109 mg/l, p=0.01), which led to a decrease in the bioavailability of free NO by more than 70%. Oxidation of the released plasma ferrous hemoglobin prevented the consumption of NO. The amount of decompartmentalized hemoglobin after HD correlated inversely with the change in FMD (r=-0.65, p=0.041)., Conclusions: Our data support a role of HD-induced release of hemoglobin in the pathogenesis of endothelial dysfunction in patients with end-stage renal disease. Approaches that oxidize free plasma hemoglobin may restore NO bioavailability and may have potential beneficial effects on vascular function. (Influence of Hemodialysis on Endothel-Depending Dilatation of Peripheral Arteries; NCT00764192)., (Copyright (c) 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Endothelial and leukocytes-derived microparticles (EMPs and LMPs, respectively) are increased in patients with pulmonary hypertension (PH). We hypothesized that the levels of circulating EMPs and LMPs could predict outcome in these patients., Methods: Patients undergoing right heart catheterization for untreated pre-capillary PH were eligible for the study. Baseline hemodynamics and biologic and clinical parameters were measured at the time of enrollment. Measurements of CD62e(+), CD144(+) and CD31(+)/CD41(-) EMPs and CD45(+) LMPs were performed using flow cytometry in venous platelet-free plasma samples. After inclusion, patients were treated at the discretion of the physician and prospectively followed for 12 months. The primary end-point was the combined occurrence of death and re-admission for right heart failure (RHF) or worsening of RHF symptoms., Results: Seven of 21 patients (mean age 54.1 +/- 3.5 years, 62% female) experienced the primary end-point during the study period. These patients had higher baseline levels of CD62e(+) EMPs, LMPs and hsCRP (high sensitivity C-reactive protein) compared to patients without events (p < 0.05), whereas no difference was observed for other microparticles and functional and hemodynamics parameters. Receiver operating curve analysis showed that baseline CD62e(+) EMPs levels of >353 events/microl predicted clinical complications. Kaplan-Meier analysis revealed that patients with baseline CD62e(+) EMPs above this cut-off value had a significantly worse prognosis compared with those subjects who had levels below this cut-off (p = 0.02, log-rank statistics)., Conclusions: Elevated levels of circulating CD62e(+) EMPs but not LMPs in PH patients prior to treatment are associated with adverse clinical events. Assessment of CD62e(+) EMPs levels may represent a new tool for stratification of PH patients.

Background: Conduit arteries respond to increases in flow by dilating, which is mediated by endothelium-derived nitric oxide. The significance of the interaction between microcirculation and macrocirculation in the flow-mediated dilation (FMD) is poorly understood., Hypothesis: We hypothesize that baseline conduit artery vasomotor tone (CAVT) and resting microvascular resistance (MVR) predict FMD., Methods: We investigated resting diameter and FMD of the brachial artery using high-resolution ultrasound and forearm blood flow with plethysmography in 60 healthy individuals. CAVT was calculated as change of the arterial diameter from baseline to maximal dilation expressed as percentage of the maximum dilation. Resting MVR was determined as quotient of mean arterial blood pressure and forearm blood flow., Results: Mean FMD was 10.7+/-2.0%, indicating normal endothelial function. The extent of brachial artery FMD was not only related to baseline CAVT (r=0.70, P<0.01), but inversely to resting MVR (r=-0.69, P<0.01). Moreover, in a simple regression analysis, baseline CAVT was inversely related to resting MVR (r=-0.82, P<0.01). In a multivariate linear regression analysis, baseline CAVT and MVR were identified as independent predictors of brachial artery FMD., Conclusion: Our data imply a close interaction of resting microvascular resistance and baseline CAVT modulating flow-mediated conduit artery dilation.

Objectives: We investigated whether qualitative or quantitative alterations of the endothelial progenitor cell (EPC) pool predict age-related structural vessel wall changes., Background: We have previously shown that age-related endothelial dysfunction is accompanied by qualitative rather than quantitative changes of EPCs. Animal studies suggest that impaired EPC functions lead to accelerated arterial intimal thickening., Methods: Intima-media thickness (IMT) was measured in the common carotid artery in our previously published groups of younger (25 +/- 1 years, n = 20) and older (61 +/- 2 years, n = 20) healthy non-smoking volunteers without arterial hypertension, hypercholesterolemia, and diabetes mellitus. Endothelial progenitor cells (EPCs, KDR(+)/CD34(+) and KDR(+)/CD133(+)) were counted in peripheral blood using flow cytometry. In ex vivo expanded EPCs, the function was determined as chemotaxis to VEGF, proliferation, and survival., Results: We observed thicker IMT in older as compared to younger subjects (0.68 +/- 0.03 mm Vs. 0.48 +/- 0.02 mm, P < 0.001). Importantly, there were significant inverse univariate correlations between IMT, EPC chemotaxis, and survival (r = -0.466 P < 0.05; r = -0.463, P < 0.01). No correlation was observed with numbers of circulating EPCs. Multivariate regression analysis revealed that age, mean arterial pressure and migration of EPCs were independent predictors of IMT (R (2 )= 0.58)., Conclusion: Impaired EPC function may lead to accelerated vascular remodeling due to chronic impairment of endothelial maintenance.

Objectives: Our goal was to test feasibility and efficacy of a dietary intervention based on daily intake of flavanol-containing cocoa for improving vascular function of medicated diabetic patients., Background: Even in fully medicated diabetic patients, overall prognosis is unfavorable due to deteriorated cardiovascular function. Based on epidemiological data, diets rich in flavanols are associated with a reduced cardiovascular risk., Methods: In a feasibility study with 10 diabetic patients, we assessed vascular function as flow-mediated dilation (FMD) of the brachial artery, plasma levels of flavanol metabolites, and tolerability after an acute, single-dose ingestion of cocoa, containing increasing concentrations of flavanols (75, 371, and 963 mg). In a subsequent efficacy study, changes in vascular function in 41 medicated diabetic patients were assessed after a 30-day, thrice-daily dietary intervention with either flavanol-rich cocoa (321 mg flavanols per dose) or a nutrient-matched control (25 mg flavanols per dose). Both studies were undertaken in a randomized, double-masked fashion. Primary and secondary outcome measures included changes in FMD and plasma flavanol metabolites, respectively., Results: A single ingestion of flavanol-containing cocoa was dose-dependently associated with significant acute increases in circulating flavanols and FMD (at 2 h: from 3.7 +/- 0.2% to 5.5 +/- 0.4%, p < 0.001). A 30-day, thrice-daily consumption of flavanol-containing cocoa increased baseline FMD by 30% (p < 0.0001), while acute increases of FMD upon ingestion of flavanol-containing cocoa continued to be manifest throughout the study. Treatment was well tolerated without evidence of tachyphylaxia. Endothelium-independent responses, blood pressure, heart rate, and glycemic control were unaffected., Conclusions: Diets rich in flavanols reverse vascular dysfunction in diabetes, highlighting therapeutic potentials in cardiovascular disease.

Objectives: This study sought to analyze the effects of acute secondhand smoke (SHS) exposure on the number and function of endothelial progenitor cells (EPCs) over 24 h., Background: Secondhand smoke increases the risk of vascular disease and is a major public health concern, but the mechanism(s) of action are not fully understood., Methods: Healthy nonsmokers (age SEM 30.3 +/- 1.3 years, n = 10) were exposed to 30 min of SHS yielding cotinine levels commonly observed in passive smokers and to smokefree air on 2 separate days. Measurements were taken before exposure (baseline), immediately after (0 h), and at 1 h, 2.5 h, and 24 h after. The EPCs (CD133(+)/KDR(+), CD34(+)/KDR(+)) and endothelial microparticles (EMPs: CD31(+)/CD41(-), CD144(+), CD62e(+)) were determined in blood using flow cytometry. The EPC chemotaxis toward vascular endothelial growth factor was measured. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound., Results: Secondhand smoke exposure increased EPCs and plasma vascular endothelial growth factor and completely abolished EPC chemotaxis during 24 h after exposure. Secondhand smoke increased EMPs and decreased FMD. Although FMD returned to baseline at 2.5 h, EMPs and vascular endothelial growth factor levels remained elevated at 24 h, suggesting endothelial activation and injury with functional impairment of the vascular endothelium. Exposure to smokefree air had no effect. Incubation of EPCs from nonexposed subjects with plasma isolated from SHS-exposed subjects in vitro decreased chemotaxis by blockade of vascular endothelial growth factor-stimulated nitric oxide production., Conclusions: Brief exposure to real-world levels of SHS leads to sustained vascular injury characterized by mobilization of dysfunctional EPCs with blocked nitric oxide production. Our results suggest that SHS not only affects the vascular endothelium, but also the function of EPCs.

Age-dependent alterations of the vessel wall may predispose older individuals to increased cardiovascular pathology. Aging is associated with an impaired bioactivity of nitric oxide (NO). Plasma nitrite reflects NO-synthase activity under fasting conditions and is an important storage pool of NO. To test the hypothesis that aging is associated with an impaired capacity of the vasculature to increase plasma nitrite during exercise, 29 young and 28 old healthy individuals (25 +/- 1 years and 58 +/- 2 years; P < 0.001) without major cardiovascular risk factors were enrolled. Exercise stress was similar in both groups. Baseline nitrite did not differ (107 +/- 8 vs. 82 +/- 10 nmol/l, young vs. old; n.s.) although a trend toward higher nitrite levels in young individuals was seen. In young subjects, exercise increased plasma nitrite by 38 +/- 7% (P < 0.001) compared to only 13 +/- 8% (P = n.s.) in older subjects. L-NMMA blocked increases of nitrite. Endothelial function, as defined by flow-mediated-dilation (FMD) of the brachial artery via ultrasound, was impaired in older subjects (5.4 +/- 0.4% vs. 6.7 +/- 0.3%; P < 0.01). Multivariate analysis showed that age (P = 0.007), BMI (P = 0.010), and LDL (P = 0.021) were independent predictors of nitrite increase. The fact that aging is associated with an impaired capacity of the vasculature to adequately increase nitrite to physiological stimuli may contribute to attenuated maintenance and further deterioration of vascular homeostasis with aging.

A single-dose ingestion of flavanol-rich cocoa acutely reverses endothelial dysfunction. To investigate the time course of endothelial function during daily consumption of high-flavanol cocoa, we determined flow-mediated dilation (FMD) acutely (for up to 6 hours after single-dose ingestion) and chronically (administration for 7 days). The study population represented individuals with smoking-related endothelial dysfunction; in addition to FMD, plasma nitrite and nitrate were measured. The daily consumption of a flavanol-rich cocoa drink (3 x 306 mg flavanols/d) over 7 days (n=6) resulted in continual FMD increases at baseline (after overnight fast and before flavanol ingestion) and in sustained FMD augmentation at 2 hours after ingestion. Fasted FMD responses increased from 3.7 +/- 0.4% on day 1 to 5.2 +/- 0.6%, 6.1 +/- 0.6%, and 6.6 +/- 0.5% (each P < 0.05) on days 3, 5, and 8, respectively. FMD returned to 3.3 +/- 0.3% after a washout week of cocoa-free diet (day 15). Increases observed in circulating nitrite, but not in circulating nitrate, paralleled the observed FMD augmentations. The acute, single-dose consumption of cocoa drinks with 28 to 918 mg of flavanols led to dose-dependent increases in FMD and nitrite, with a maximal FMD at 2 hours after consumption. The dose to achieve a half-maximal FMD response was 616 mg (n=6). Generally applied biomarkers for oxidative stress (plasma, MDA, TEAC) and antioxidant status (plasma ascorbate, urate) remained unaffected by cocoa flavanol ingestion. The daily consumption of flavanol-rich cocoa has the potential to reverse endothelial dysfunction in a sustained and dose-dependent manner.

Attenuation of endothelium-derived nitric oxide (NO) synthesis is a hallmark of endothelial dysfunction. Early detection of this disorder may have therapeutic and prognostic implications. Plasma nitrite mirrors acute and chronic changes in endothelial NO-synthase activity. We hypothesized that local plasma nitrite concentration increases during reactive hyperemia of the forearm, reflecting endothelial function. In healthy subjects (n = 11) plasma nitrite and nitrate were determined at baseline and during reactive hyperemia of the forearm using reductive gas-phase chemiluminescence and flow-injection analysis, respectively. Endothelium-dependent dilation of the brachial artery was measured as flow-mediated dilation (FMD) using high-resolution ultrasound. Results were compared to patients with endothelial dysfunction as defined by reduced FMD (n = 11). Reactive hyperemia of the forearm increased local plasma nitrite concentration from 68 +/- 5 to 126 +/- 13 nmol/L (p < 0.01), whereas in endothelial dysfunction nitrite remained unaffected (116 +/- 12 to 104 +/- 10 nmol/L; n.s.), corresponding to nitrite reserves of 94 +/- 21 and -8 +/- 4%. This was accompanied by a significantly greater increase in brachial artery diameter (FMD: 8.5 +/- 0.4% vs 2.9 +/- 0.5%, for healthy subjects and endothelial dysfunction, respectively; p < 0.001). This observation suggests that nitrite changes reflect endothelial function. Assessment of local plasma nitrite during reactive hyperemia may open new avenues in the diagnosis of vascular function.

Objectives: We investigated whether plasma nitros(yl)ated species (RXNOs) that mediate systemic nitric oxide (NO) bioactivity are depleted in individuals with cardiovascular risk factors and endothelial dysfunction., Background: Endothelium-derived NO acts not only as a regional messenger but exerts significant systemic effects via formation of circulating RXNOs delivering NO to sites of impaired production., Methods: Endothelial function was assessed in 68 patients with one to four major cardiovascular risk factors (RF) and 39 healthy control subjects (C) by measurement of flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound. In parallel, plasma RXNOs were determined by reductive gas phase chemiluminescence., Results: Increasing numbers of risk factors were accompanied by a progressive decrease in FMD: 6.5 +/- 0.4% (C); 4.7 +/- 0.5% (one RF); 2.8 +/- 0.4% (two RF); 2.2 +/- 0.4% (three RF); and 1.0 +/- 0.3% (four RF). Progressively impaired vascular function was associated with a concomitant decrease in plasma RXNOs (p < 0.01): 39 +/- 2 nmol/l (C); 30 +/- 2 nmol/l (one RF); 24 +/- 3 nmol/l (two RF); 22 +/- 3 nmol/l (three RF); and 15 +/- 2 nmol/l (four RF), with univariate correlation between FMD and RXNO (r = 0.41, p < 0.001). In a multivariate regression model, RXNO was an independent predictor of endothelial function., Conclusions: Endothelial dysfunction in patients with cardiovascular risk factors is associated with decreased levels of circulating RXNOs. Plasma RXNOs may be diagnostically useful markers of NO bioavailability and a surrogate index of endothelial function. Whether the observed decrease in concentration reflects impaired NO formation, accelerated decomposition, and/or consumption of RXNOs and whether these processes play a causal role in the pathophysiology of arteriosclerosis remain to be investigated.

Atherosclerosis is the major cause for chronic vascular diseases. The key event in the pathogenesis of atherosclerosis is believed to be dysfunction of the endothelium and disruption of endothelial homeostasis, leading to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and proliferation of vascular smooth muscle cells. Endothelium-derived nitric oxide (NO) plays a major role in vascular homeostasis and a decrease in NO-bioavailability accelerates the development of atherosclerosis. Given that endothelial dysfunction is at least in part reversible, the characterization of endothelial function and therapeutical approaches have gained much attention over the past years. Recent studies demonstrated that especially the consumption of plant-derived foods rich in certain flavonoids can improve endothelial function in both compromised and healthy humans. Furthermore, various physiologic and biochemical measures have been used previously as biomarkers for the assessment of the proposed beneficial effects of flavonoids in this context. More recently, the analysis of plasma nitros(yl)ated species (RXNOs), referred to as the circulating NO pool, has gained recognition, especially as a marker for endothelial function. This review is aimed at evaluating the suitability of quantifying this NO pool as a biomarker for cardiovascular function in humans, in particular during dietary interventions with flavonoid-rich foods.

Endothelial dysfunction is the pathophysiologic principle involved in the initiation and progression of arteriosclerosis, thus endothelial function serves as a "barometer" for cardiovascular health that can be used for the evaluation of new therapeutic strategies. This review provides an introduction to the concept of endothelial dysfunction, and it explores the importance of this prognostic marker in the context of clinical, dietary interventions in humans. Moreover, we summarize and evaluate the findings of various clinical trials that demonstrated an improvement of endothelial dysfunction in subjects with cardiovascular risk factors after the acute and chronic consumption of flavanol-rich foods, including cocoa products, red wine, and tea.

Objectives: This study was designed to assess the effect of flavanol-rich food on the circulating pool of bioactive nitric oxide (NO) and endothelial dysfunction in smokers., Background: Studies suggest that smoking-related vascular disease is caused by impaired NO synthesis and that diets rich in flavanols can increase bioactive NO in plasma., Methods: In smokers (n = 11), the effects of flavanol-rich cocoa on circulating NO species in plasma (RXNO) measured by reductive gas-phase chemiluminescence and endothelial function as assessed by flow-mediated dilation (FMD) were characterized in a dose-finding study orally administering cocoa containing 88 to 370 mg flavanols and in a randomized double-blind crossover study using 100 ml cocoa drink with high (176 to 185 mg) or low (<11 mg) flavanol content on two separate days. In addition to cocoa drink, ascorbic acid and NO-synthase inhibitor L-NMMA (n = 4) were applied., Results: There were significant increases in RXNO (21 +/- 3 nmol/l to 29 +/- 5 nmol/l) and FMD (4.5 +/- 0.8% to 6.9 +/- 0.9%, each p < 0.05) at 2 h after ingestion of 176 to 185 mg flavanols, a dose potentially exerting maximal effects. These changes correlated with increases in flavanol metabolites. Cocoa-associated increases in RXNO and FMD were reversed by L-NMMA. Ascorbic acid had no effect., Conclusions: The circulating pool of bioactive NO and endothelium-dependent vasodilation is acutely increased in smokers following the oral ingestion of a flavanol-rich cocoa drink. The increase in circulating NO pool may contribute to beneficial vascular health effects of flavanol-rich food.

Objectives: We investigated whether human age-related endothelial dysfunction is accompanied by quantitative and qualitative alterations of the endothelial progenitor cell (EPC) pool., Background: Circulating progenitor cells with an endothelial phenotype contribute to the regeneration and repair of the vessel wall. An association between the loss of endothelial integrity and EPC modification may provide a background to study the mechanistic nature of such age-related vascular changes., Methods: In 20 old and young healthy individuals (61 +/- 2 years and 25 +/- 1 year, respectively) without major cardiovascular risk factors, endothelial function, defined by flow-mediated dilation of the brachial artery via ultrasound, as well as the number and function of EPCs isolated from peripheral blood, were determined., Results: Older subjects had significantly impaired endothelium-dependent dilation of brachial artery (flow-mediated dilation [FMD] 5.2 +/- 0.5% vs. 7.1 +/- 0.6%; p < 0.05). Endothelium-independent dilation after glycerol trinitrate (GTN) was not different, but the FMD/GTN ratio was significantly lower in old subjects (49 +/- 4% vs. 37 +/- 3%; p < 0.05), suggesting endothelial dysfunction. There were no differences in the numbers of circulating EPCs, defined as CD34/KDR or CD133/KDR double-positive cells in peripheral blood. In contrast, lower survival (39 +/- 6 cells/mm(2) vs. 65 +/- 11 cells/mm(2); p < 0.05), migration (80 +/- 12 vs. 157 +/- 16 cells/mm(2); p < 0.01), and proliferation (0.20 +/- 0.04 cpm vs. 0.44 +/- 0.07 cpm; p < 0.05) implicate functional impairment of EPCs from old subjects. The FMD correlated univariately with EPC migration (r = 0.52, p < 0.05) and EPC proliferation (r = 0.49, p < 0.05). Multivariate analysis showed that both functional features represent independent predictors of endothelial function., Conclusions: Maintenance of vascular homeostasis by EPCs may be attenuated with age based on functional deficits rather than depletion of CD34/KDR or CD133/KDR cells.

Background: A diminished flow reserve in resistance vessels is a hallmark of hypertensive microvascular disease. Hypertension is associated with structural alterations in the microcirculation and a reduced endothelium-dependent dilation in conduit arteries. Both have been demonstrated to predict future cardiovascular events., Objective: We hypothesized that a reduced peripheral flow reserve impairs endothelial function in upstream conduit arteries in patients with arterial hypertension., Design: In 43 hypertensive patients (HT) and 38 normotensive controls (NT) endothelial function of the brachial artery was assessed by measurement of flow-mediated dilatation (FMD), using high-resolution ultrasound. Peripheral flow reserve (FR) was determined via measurements of forearm blood flow at rest and during increments of reactive hyperaemia, using venous occlusion plethysmography., Results: FMD was markedly impaired in HT (3.6 +/- 0.3%) as compared with NT (10.2 +/- 0.3%), whereas maximum brachial artery diameter following endothelium-independent dilatation was similar in both groups. In hypertensive patients FR was significantly reduced (HT, 3.2 versus NT, 6.0) during reactive hyperaemia after 5 min of ischaemia. FR was associated with FMD (r = 0.68, P < 0.01). Multiple stepwise regression analysis identified FR as a strong independent variable determining the extent of FMD (r2 = 0.46, P < 0.01). In HT the dose-response curve of FMD upon stepwise increases of FR was shifted significantly to the right. Normalization of FR improved FMD in HT by more than 60%., Conclusions: In essential hypertension a reduced FR contributes to the endothelial dysfunction of upstream conduit arteries. These findings may have therapeutic and prognostic implications in patients with arterial hypertension.