Possible role for K+ in endothelium-derived hyperpolarizing factor–linked dilatation in rat middle cerebral artery

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K+ are vasodilators in the cerebral circulation. Recently, K+ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K+ contributes as an EDHF. Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K+ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously. Results— SLIGRL (0.02 to 20 μmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of NG-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of KIR with Ba2+ (30μmol/L) and Na+/K+-ATPase with ouabain (1 μmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K+] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba2+ and ouabain. Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IKCa channels. Furthermore, similar inhibition of responses to EDHF and exogenous K+ with Ba2+ and ouabain suggests that K+ may contribute as an EDHF in the middle cerebral artery.

Thromboxane A2 inhibition of SKCa after NO synthase block in rat middle cerebral artery

NO/prostanoid independent, EDHF-mediated hyperpolarization and dilation in rat middle cerebral arteries is mediated solely by endothelial cell IK(Ca). However, when the NO-pathway is also active, both SK(Ca) and IK(Ca) contribute to EDHF responses. As the SK(Ca) component can be inhibited by stimulation of thromboxane A(2) (TxA(2)) TP receptors and NO has the potential ability to inhibit thromboxane synthesis, we investigated whether TxA(2) might explain loss of functional input from SK(Ca) during NOS inhibition in cerebral arteries. EXPERIMENTAL APPROACH: Rat middle cerebral arteries were mounted in a wire myograph. Endothelium-dependent responses to the PAR2 agonist, SLIGRL were assessed as simultaneous changes in smooth muscle membrane potential and tension. KEY RESULTS: Responses were obtained in the presence of L-NAME as appropriate. Inhibition of TP receptors with either ICI 192,605 or SQ 29,548, did not affect EDHF mediated hyperpolarization and relaxation, but in their presence neither TRAM-34 nor apamin (to block IK(Ca) and SK(Ca) respectively) individually affected the EDHF response. However, in combination they virtually abolished it. Similar effects were obtained in the presence of the thromboxane synthase inhibitor, furegrelate, which additionally revealed an iberiotoxin-sensitive residual EDHF hyperpolarization and relaxation in the combined presence of TRAM-34 and apamin. CONCLUSIONS AND IMPLICATIONS: In the rat middle cerebral artery, inhibition of NOS leads to a loss of the SK(Ca) component of EDHF responses. Either antagonism of TP receptors or block of thromboxane synthase restores an input through SK(Ca). These data indicate that NO normally enables SK(Ca) activity in rat middle cerebral arteries.

Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery

Endothelium-derived hyperpolarizing factor responses in the rat middle cerebral artery are blocked by inhibiting IKCa channels alone, contrasting with peripheral vessels where block of both IKCa and SKCa is required. As the contribution of IKCa and SKCa to endothelium-dependent hyperpolarization differs in peripheral arteries, depending on the level of arterial constriction, we investigated the possibility that SKCa might contribute to equivalent hyperpolarization in cerebral arteries under certain conditions. METHODS: Rat middle cerebral arteries (approximately 175 microm) were mounted in a wire myograph. The effect of KCa channel blockers on endothelium-dependent responses to the protease-activated receptor 2 agonist, SLIGRL (20 micromol/L), were then assessed as simultaneous changes in tension and membrane potential. These data were correlated with the distribution of arterial KCa channels revealed with immunohistochemistry. RESULTS: SLIGRL hyperpolarized and relaxed cerebral arteries undergoing variable levels of stretch-induced tone. The relaxation was unaffected by specific inhibitors of IKCa (TRAM-34, 1 micromol/L) or SKCa (apamin, 50 nmol/L) alone or in combination. In contrast, the associated smooth-muscle hyperpolarization was inhibited, but only with these blockers in combination. Blocking nitric oxide synthase (NOS) or guanylyl cyclase evoked smooth-muscle depolarization and constriction, with both hyperpolarization and relaxation to SLIGRL being abolished by TRAM-34 alone, whereas apamin had no effect. Immunolabeling showed SKCa and IKCa within the endothelium. CONCLUSIONS: In the absence of NO, IKCa underpins endothelium-dependent hyperpolarization and relaxation in cerebral arteries. However, when NOS is active SKCa contributes to hyperpolarization, whatever the extent of background contraction. These changes may have relevance in vascular disease states where NO release is compromised and when the levels of SKCa expression may be altered.

Regulation of KCa2.3 andendothelium-dependent hyperpolarization (EDH) in the rat middle cerebral artery: the role of lipoxygenase metabolites and isoprostanes

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium(KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries. Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes. Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized 14C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH. Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition,loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit.

Moderate Champagne consumption promotes an acute improvement in acute endothelial-independent vascular function in healthy human volunteers

Epidemiological studies have suggested an inverse correlation between red wine consumption and the incidence of CVD. However, Champagne wine has not been fully investigated for its cardioprotective potential. In order to assess whether acute and moderate Champagne wine consumption is capable of modulating vascular function, we performed a randomised, placebo-controlled, cross-over intervention trial. We show that consumption of Champagne wine, but not a control matched for alcohol, carbohydrate and fruit-derived acid content, induced an acute change in endothelium-independent vasodilatation at 4 and 8 h post-consumption. Although both Champagne wine and the control also induced an increase in endothelium-dependent vascular reactivity at 4 h, there was no significant difference between the vascular effects induced by Champagne or the control at any time point. These effects were accompanied by an acute decrease in the concentration of matrix metalloproteinase (MMP-9), a significant decrease in plasma levels of oxidising species and an increase in urinary excretion of a number of phenolic metabolites. In particular, the mean total excretion of hippuric acid, protocatechuic acid and isoferulic acid were all significantly greater following the Champagne wine intervention compared with the control intervention. Our data suggest that a daily moderate consumption of Champagne wine may improve vascular performance via the delivery of phenolic constituents capable of improving NO bioavailability and reducing matrix metalloproteinase activity.

Soya isoflavone-enriched cereal bars affect markers of endothelial function in postmenopausal women

Soya isoflavones are thought to be cardioprotective due to their structural similarity to oestrogen. In order to investigate the effect of soya isoflavones on markers of endothelial function we conducted a randomised, double-blind, placebo-controlled, cross-over study with thirty healthy postmenopausal women. The women consumed cereal bars, with or without soya isoflavones (50 mg/d), for 8 weeks, separated by an 8-week washout period. Systemic arterial complince (SAC), isobaric arterial compliance (IAC), flow-mediated endothelium-dependent vasodilation (FMD) and nitroglycerine-mediated endothelium-independent vasodilation (NMD) were measured at the beginning of the study and after each intervention period. Blood pressure (BP) and plasma concentrations of nitrite and nitrate (NOx) and endothelin-1 (ET-1) were measured at the beginning and end of each intervention period. NMD was 13.4 (sem 2.0) % at baseline and 15.5 (sem 1.1) % after isoflavone treatment compared with 12.4 (sem 1.0) % after placebo treatment (P=0.03). NOx increased from 27.7 (sem 2.7) to 31.1 (sem 3.2) mu m after isoflavones treatment compared with 25.4 (sem 1.5) to 20.4 (sem 1.1) mu m after placebo treatment (P=0.003) and a significant increase in the NOx:ET-1 ratio (P=0.005) was observed after the isoflavone treatment compared with placebo. A significant difference in SAC after the isoflavone and placebo treatment was observed (P=0.04). No significant difference was found in FMD, IAC, BP and ET-1. In conclusion, 8 weeks' consumption of cereals bars enriched with 50 mg soya isoflavones/d increased plasma NOx concentrations and improved endothelium-independent vasodilation in healthy postmenopausal women.

Effects of chronic and acute consumption of fruit- and vegetable-puree-based drinks on vasodilation, risk factors for CVD and the response as a result of the eNOS G298T polymorphism

The average UK adult consumes less than three portions of fruit and vegetables daily, despite evidence to suggest that consuming five portions daily could help prevent chronic diseases. It is recommended that fruit juice should only count as one of these portions, as juicing removes fibre and releases sugars. However, fruit juices contain beneficial compounds such as vitamin C and flavonoids and could be a useful source of dietary phytochemicals. Two randomised controlled cross-over intervention studies investigating the effects of chronic and acute consumption of commercially-available fruit- and vegetable-puree-based drinks (FVPD) on bioavailability, antioxidant status and CVD risk factors are described. Blood and urine samples were collected during both studies and vascular tone was measured using laser Doppler imaging. In the chronic intervention study FVPD consumption was found to significantly increase dietary carotenoids (P = 0.001) and vitamin C (P = 0.003). Plasma carotenoids were increased (P = 0.001), but the increase in plasma vitamin C was not significant. There were no significant effects on oxidative stress, antioxidant status and other CVD risk factors. In the acute intervention study FVPD were found to increase total plasma nitrate and nitrite (P = 0.001) and plasma vitamin C (P = 0.002). There was no effect on plasma lipids or uric acid, but there was a lower glucose and insulin peak concentration after consumption of the FVPD compared with the sugar-matched control. There was a trend towards increased vasodilation following both chronic and acute FVPD consumption. All volunteers were retrospectively genotyped for the eNOS G298T polymorphism and the effect of genotype on the measurements is discussed. Overall, there was a non-significant trend towards increased endothelium-dependent vasodilation following both acute and chronic FVPD consumption. However, there was a significant time x treatment effect (P < 0.05) of acute FVPD consumption in individuals with the GG variant of the eNOS gene.

Combined oral contraceptive pills containing desogestrel or drospirenone enhance large vessel and microvasculature vasodilation in healthy premenopausal women

OBJECTIVE: To determine the effects of different COCs on endothelial function. BACKGROUND: COCs all contain ethinylestradiol, but different progestins; three of the more common progestins are DSG, LN, and DR. Ethinylestradiol enhances some measures of vascular reactivity, but certain progestins may increase risk of vascular diseases and impair endothelial vasodilation. METHODS: Twenty-nine healthy women taking COCs containing 30 μg ethinylestradiol and 150 μg DSG (Marvelon, n = 10), 150 μg LN (Microgynon, n = 10), or 3 mg DR (Yasmin, n = 9) had their vascular reactivity measured using various techniques during their pill-free week (days 5-7) and the third week of active pills (days 26-28). A reference group (n = 10) underwent the same measurements on two consecutive cycles. RESULTS: FMD and LDI were significantly higher during active-pill visits than pill-free visits in women taking DSG and DR (p < 0.02), but not in women taking LN. There were no differences between the duplicate measures in the reference group. CONCLUSIONS: COCs containing 150 μg DSG or 3 mg DR significantly increase endothelium-dependent vasodilation in both large vessels and peripheral microvasculature. These effects may be due to the progestins exhibiting differential effects on eNOS expression.

The Glu298Asp single nucleotide polymorphism in the endothelial nitric oxide synthase gene differentially effects the vascular response to acute consumption of fruit and vegetable puree-based drinks

Scope Diets low in fruits and vegetables (FV) are responsible for 2.7 million deaths from cardiovascular diseases (CVD) and certain cancers annually. Many FV and their juices contain flavonoids, some of which increase endothelial nitric oxide synthase (eNOS) activity. A single nucleotide polymorphism in the eNOS gene, where thymine (T) replaces guanine (G) at position 894 predicting substitution of glutamate for aspartate at codon 298 (Glu298Asp), has been associated with increased CVD risk due to effects on nitric oxide synthesis and subsequently vascular reactivity. Individuals can be homozygous for guanine (GG), thymine (TT) or heterozygous (GT). Methods and results We investigated the effects of acute ingestion of a FV-puree-based-drink (FVPD) on vasodilation and antioxidant status in subjects retrospectively genotyped for this polymorphism. Healthy volunteers (n = 24; 11 GG, 11 GT, 2 TT) aged 30–70 were recruited to a randomized, controlled, crossover, acute study. We showed that acute consumption of 400 mL FVPD differentially affected individuals depending on their genotype. There was a significant genotype interaction for endothelium-dependent vasodilation measured by laser Doppler imaging with iontophoresis (P < 0.05) and ex vivo low-density lipoproteins (LDL) oxidation (P = 0.002). GG subjects had increased endothelium-dependent vasodilation 180 min (P = 0.028) and reduced ex vivo LDL oxidation (P = 0.013) after 60 min after FVPD compared with control, no differences were observed in GT subjects. Conclusion eNOS Glu298Asp genotype differentially affects vasodilation and ex vivo LDL oxidation after consumption of FV in the form of a puree-based drink.

Effects of chronic consumption of fruit and vegetable puree-based drinks on vasodilation, plasma oxidative stability and antioxidant status

Background: Fruit and vegetable-rich diets are associated with a reduced cardiovascular disease (CVD) risk. This protective effect may be a result of the phytochemicals present within fruits and vegetables (F&V). However, there can be considerable variation in the content of phytochemical composition of whole F&V depending on growing location, cultivar, season and agricultural practices, etc. Therefore, the present study investigated the effects of consuming fruits and vegetables as puree-based drinks (FVPD) daily on vasodilation, phytochemical bioavailability, antioxidant status and other CVD risk factors. FVPD was chosen to provide a standardised source of F&V material that could be delivered from the same batch to all subjects during each treatment arm of the study. Methods: Thirty-nine subjects completed the randomised, controlled, cross-over dietary intervention. Subjects were randomised to consume 200 mL of FVPD (or fruit-flavoured control), daily for 6 weeks with an 8-week washout period between treatments. Dietary intake was measured using two 5-day diet records during each cross-over arm of the study. Blood and urine samples were collected before and after each intervention and vasodilation assessed in 19 subjects using laser Doppler imaging with iontophoresis. Results: FVPD significantly increased dietary vitamin C and carotenoids (P < 0.001), and concomitantly increased plasma α- and β-carotene (P < 0.001) with a near-significant increase in endothelium-dependent vasodilation (P = 0.060). Conclusions: Overall, the findings obtained in the present study showed that FVPD were a useful vehicle to increase fruit and vegetable intake, significantly increasing dietary and plasma phytochemical concentrations with a trend towards increased endothelium-dependent vasodilation.

Effects of acute consumption of a fruit and vegetable puree-based drink on vasodilation and oxidative status

Epidemiological studies indicate that diets rich in fruits and vegetables (F&V) are protective against cardiovascular diseases (CVD). Pureed F&V products retain many beneficial components, including flavonoids, carotenoids, vitamin C and dietary fibres. This study aimed to establish the physiological effects of acute ingestion of F&V puree-based drink (FVPD) on vasodilation, antioxidant status, phytochemical bioavailability and other CVD risk factors. 24 Subjects, aged 30-70 years, completed the randomised, single-blind, controlled, crossover test meal study. Subjects consumed 400 ml FVPD, or fruit-flavoured sugar-matched control, after following a low-flavonoid diet for 5 days. Blood and urine samples were collected throughout the study day and vascular reactivity was assessed at 90-minute intervals using laser Doppler iontophoresis (LDI). FVPD significantly increased plasma vitamin C (P=0.002) and total nitrate/nitrite (P=0.001) concentrations. There was a near significant time by treatment effect on ex vivo LDL oxidation (P=0.068), with a longer lag phase after consuming FVPD. During the 6 hours after juice consumption the antioxidant capacity of plasma increased significantly (P=0.003) and there was a simultaneous increase in plasma and urinary phenolic metabolites (P<0.05). There were significantly lower glucose and insulin peaks after ingestion of FVPD compared with control (P=0.019 and P=0.003) and a trend towards increased endothelium-dependent vasodilation following FVPD consumption (P=0.061). Overall, FVPD consumption significantly increased plasma vitamin C and total nitrate/nitrite concentrations, with a trend towards increased endothelium-dependent vasodilation. Pureed F&V products are useful vehicles for increasing micronutrient status, plasma antioxidant capacity and in vivo NO generation, which may contribute to CVD risk reduction.

Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

Background and Purpose: Calcitonin gene‐related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor‐like receptor (CLR) and receptor activity‐modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin‐converting enzyme‐1 (ECE‐1). However, it is not known if ECE‐1 regulates the resensitization of CGRP‐induced responses in functional arterial tissue. Experimental Approach: CLR, ECE‐1a‐d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA‐SMCs) and mesenteric arteries was analyzed by RT‐PCR and by immunofluorescence and confocal microscopy. CGRP‐induced signaling in cells was examined by measuring cAMP production and ERK activation. CGRP‐induced relaxation of arteries was measured by isometric wire myography. ECE‐1 was inhibited using the specific inhibitor, SM‐19712. Key Results: RMA‐SMCs and arteries contained mRNA for CLR, ECE‐1a‐d and RAMP1. ECE‐1 was present in early endosomes of RMA‐SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium‐independent relaxation of arteries. ECE‐1 inhibition had no effect on initial CGRP‐induced responses but reduced cAMP generation in RMA‐SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions and Implications: ECE‐1 regulates the resensitization of responses to CGRP in RMA‐SMCs and mesenteric arteries. CGRP‐induced relaxation does not involve endothelium‐derived pathways. This is the first report of ECE‐1 regulating CGRP responses in SMCs and arteries. ECE‐1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (KCa2.3) in cerebral arteries

Background: In rat middle cerebral and mesenteric arteries the KCa2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.

Habitual energy expenditure modifies the association between NOS3 gene polymorphisms and blood pressue

BACKGROUND: The endothelial nitric-oxide synthase (NOS3) gene encodes the enzyme (eNOS) that synthesizes the molecule nitric oxide, which facilitates endothelium-dependent vasodilation in response to physical activity. Thus, energy expenditure may modify the association between the genetic variation at NOS3 and blood pressure. METHODS: To test this hypothesis, we genotyped 11 NOS3 polymorphisms, capturing all common variations, in 726 men and women from the Medical Research Council (MRC) Ely Study (age (mean +/- s.d.): 55 +/- 10 years, body mass index: 26.4 +/- 4.1 kg/m(2)). Habitual/non-resting energy expenditure (NREE) was assessed via individually calibrated heart rate monitoring over 4 days. RESULTS: The intronic variant, IVS25+15 [G-->A], was significantly associated with blood pressure; GG homozygotes had significantly lower levels of diastolic blood pressure (DBP) (-2.8 mm Hg; P = 0.016) and systolic blood pressure (SBP) (-1.9 mm Hg; P = 0.018) than A-allele carriers. The interaction between NREE and IVS25+15 was also significant for both DBP (P = 0.006) and SBP (P = 0.026), in such a way that the effect of the GG-genotype on blood pressure was stronger in individuals with higher NREE (DBP: -4.9 mm Hg, P = 0.02. SBP: -3.8 mm Hg, P= 0.03 for the third tertile). Similar results were observed when the outcome was dichotomously defined as hypertension. CONCLUSIONS: In summary, the NOS3 IVS25+15 is directly associated with blood pressure and hypertension in white Europeans. However, the associations are most evident in the individuals with the highest NREE. These results need further replication and have to be ideally tested in a trial before being informative for targeted disease prevention. Eventually, the selection of individuals for lifestyle intervention programs could be guided by knowledge of genotype.

Flavonoid-rich fruits and vegetables improve microvascular reactivity and inflammatory status in men at risk of cardiovascular disease – FLAVURS: a randomized controlled trial

BACKGROUND: Observed associations between increased fruit and vegetable (F&V) consumption, particularly those F&Vs that are rich in flavonoids, and vascular health improvements require confirmation in adequately powered randomized controlled trials. OBJECTIVE: This study was designed to measure the dose-response relation between high-flavonoid (HF), low-flavonoid (LF), and habitual F&V intakes and vascular function and other cardiovascular disease (CVD) risk indicators. DESIGN: A single-blind, dose-dependent, parallel randomized controlled dietary intervention study was conducted. Male and female low-F&V consumers who had a ≥1.5-fold increased risk of CVD (n = 174) were randomly assigned to receive an HF F&V, an LF F&V, or a habitual diet, with HF and LF F&V amounts sequentially increasing by 2, 4, and 6 (+2, +4, and +6) portions/d every 6 wk over habitual intakes. Microvascular reactivity (laser Doppler imaging with iontophoresis), arterial stiffness [pulse wave velocity, pulse wave analysis (PWA)], 24-h ambulatory blood pressure, and biomarkers of nitric oxide (NO), vascular function, and inflammation were determined at baseline and at 6, 12, and 18 wk. RESULTS: In men, the HF F&V diet increased endothelium-dependent microvascular reactivity (P = 0.017) with +2 portions/d (at 6 wk) and reduced C-reactive protein (P = 0.001), E-selectin (P = 0.0005), and vascular cell adhesion molecule (P = 0.0468) with +4 portions/d (at 12 wk). HF F&Vs increased plasma NO (P = 0.0243) with +4 portions/d (at 12 wk) in the group as a whole. An increase in F&Vs, regardless of flavonoid content in the groups as a whole, mitigated increases in vascular stiffness measured by PWA (P = 0.0065) and reductions in NO (P = 0.0299) in the control group. CONCLUSION: These data support recommendations to increase F&V intake to ≥6 portions daily, with additional benefit from F&Vs that are rich in flavonoids, particularly in men with an increased risk of CVD. This trial was registered at www.controlled-trials.com as ISRCTN47748735.