Differences in vascular nitric oxide and endothelium-derived hyperpolarizing factor bioavailability in blacks and whites.

OBJECTIVE: Abnormalities in nitric oxide (NO) bioavailability have been reported in blacks. Whether there are differences in endothelium-derived hyperpolarizing factor (EDHF) in addition to NO between blacks and whites and how these affect physiological vasodilation remain unknown. We hypothesized that the bioavailability of vascular NO and EDHF, at rest and with pharmacological and physiological vasodilation, varies between whites and blacks. APPROACH AND RESULTS: In 74 white and 86 black subjects without known cardiovascular disease risk factors, forearm blood flow was measured using plethysmography at rest and during inhibition of NO with N(G)-monomethyl-L-arginine and of K(+) Ca channels (EDHF) with tetraethylammonium. The reduction in resting forearm blood flow was greater with N(G)-monomethyl-L-arginine (P=0.019) and similar with tetraethylammonium in whites compared with blacks. Vasodilation with bradykinin, acetylcholine, and sodium nitroprusside was lower in blacks compared with whites (all P<0.0001). Inhibition with N(G)-monomethyl-L-arginine was greater in whites compared with blacks with bradykinin, acetylcholine, and exercise. Inhibition with tetraethylammonium was lower in blacks with bradykinin, but greater during exercise and with acetylcholine. CONCLUSIONS: The contribution to both resting and stimulus-mediated vasodilator tone of NO is greater in whites compared with blacks. EDHF partly compensates for the reduced NO release in exercise and acetylcholine-mediated vasodilation in blacks. Preserved EDHF but reduced NO bioavailability and sensitivity characterizes the vasculature in healthy blacks. CLINICAL TRIAL REGISTRATION URL: http://clinicaltrials.gov/. Unique identifier: NCT00166166.

Contribution of endothelium-derived hyperpolarizing factor to exercise-induced vasodilation in health and hypercholesterolemia.

The role of endothelium-derived hyperpolarizing factor (EDHF) in either the healthy circulation or in those with hypercholesterolemia is unknown. In healthy and hypercholesterolemic subjects, we measured forearm blood flow (FBF) using strain-gauge plethysmography at rest, during graded handgrip exercise, and after sodium nitroprusside infusion. Measurements were repeated after l-NMMA, tetraethylammonium (TEA), and combined infusions. At rest, l-NMMA infusion reduced FBF in healthy but not hypercholesterolemic subjects. At peak exercise, vasodilation was lower in hypercholesterolemic compared to healthy subjects (274% vs 438% increase in FBF, p=0.017). TEA infusion reduced exercise-induced vasodilation in both healthy and hypercholesterolemic subjects (27%, p<0.0001 and -20%, p<0.0001, respectively). The addition of l-NMMA to TEA further reduced FBF in healthy (-14%, p=0.012) but not in hypercholesterolemic subjects, indicating a reduced nitric oxide and greater EDHF-mediated contribution to exercise-induced vasodilation in hypercholesterolemia. In conclusion, exercise-induced vasodilation is impaired and predominantly mediated by EDHF in hypercholesterolemic subjects. CLINICAL TRIAL REGISTRATION IDENTIFIER NCT00166166:

Endothelium-derived hyperpolarizing factor mediates bradykinin-stimulated tissue plasminogen activator release in humans.

AIMS: Bradykinin (BK) stimulates tissue plasminogen activator (t-PA) release from human endothelium. Although BK stimulates both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) release, the role of EDHF in t-PA release remains unexplored. This study sought to determine the mechanisms of BK-stimulated t-PA release in the forearm vasculature of healthy human subjects. METHODS: In 33 healthy subjects (age 40.3 ± 1.9 years), forearm blood flow (FBF) and t-PA release were measured at rest and after intra-arterial infusions of BK (400 ng/min) and sodium nitroprusside (3.2 mg/min). Measurements were repeated after intra-arterial infusion of tetraethylammonium chloride (TEA; 1 µmol/min), fluconazole (0.4 µmol·min(-1)·l(-1)), and N(G)-monomethyl-L-arginine (L-NMMA, 8 µmol/min) to block nitric oxide, and their combination in separate studies. RESULTS: BK significantly increased net t-PA release across the forearm (p < 0.0001). Fluconazole attenuated both BK-mediated vasodilation (-23.3 ± 2.7% FBF, p < 0.0001) and t-PA release (from 50.9 ± 9.0 to 21.3 ± 8.9 ng/min/100 ml, p = 0.02). TEA attenuated FBF (-14.7 ± 3.2%, p = 0.002) and abolished BK-stimulated t-PA release (from 22.9 ± 5.7 to -0.8 ± 3.6 ng/min/100 ml, p = 0.0002). L-NMMA attenuated FBF (p < 0.0001), but did not inhibit BK-induced t-PA release (nonsignificant). CONCLUSION: BK-stimulated t-PA release is partly due to cytochrome P450-derived epoxides and is inhibited by K(+)Ca channel blockade. Thus, BK stimulates both EDHF-dependent vasodilation and t-PA release.

Endothelium-derived hyperpolarizing factor determines resting and stimulated forearm vasodilator tone in health and in disease.

BACKGROUND: We assessed the contribution of endothelium-derived hyperpolarizing factors to resting and agonist-stimulated vasodilator tone in health and disease. Tetraethylammonium chloride (TEA) was used to inhibit K(+)(Ca) channel activation and fluconazole was used to inhibit cytochrome P450 2C9-mediated epoxyeicosatrienoic acid synthesis. We hypothesized that endothelium-derived hyperpolarizing factors contribute to resting vascular tone by K(+)(Ca) channel activation and epoxyeicosatrienoic acid release and that endothelium-derived hyperpolarizing factors compensate for reduced nitric oxide bioavailability at rest and with endothelium-dependent vasodilators. METHODS AND RESULTS: In 103 healthy subjects and 71 nonhypertensive subjects with multiple risk factors, we measured resting forearm blood flow (FBF) using venous occlusion plethysmography before and after intra-arterial infusions of N(G)-monomethyl-l-arginine (L-NMMA), TEA, fluconazole, and their combination. The effects of these antagonists on resting FBF and on bradykinin- and acetylcholine-mediated vasodilation were studied. Resting FBF decreased with TEA and L-NMMA in all subjects (P<0.001); however, the vasoconstrictor response to L-NMMA was greater (P=0.04) and to TEA was lower (P=0.04) in healthy subjects compared with those with risk factors. Fluconazole decreased resting FBF in all subjects, and the addition of TEA further reduced FBF after fluconazole, suggesting that cytochrome P450 metabolites and other hyperpolarizing factor(s) activate K(+)(Ca) channels. Both L-NMMA and TEA attenuated bradykinin-mediated vasodilation in healthy and hypercholesterolemic subjects (P<0.001). In contrast, acetylcholine-mediated vasodilation remained unchanged with TEA in healthy subjects but was significantly attenuated in hypercholesterolemia (P<0.04). CONCLUSIONS: First, by activating TEA-inhibitable K(+)(Ca) channels, endothelium-derived hyperpolarizing factors, together with nitric oxide, contribute to resting microvascular dilator tone. The contribution of K(+)(Ca) channel activation compared with nitric oxide is greater in those with multiple risk factors compared with healthy subjects. Second, activation of K(+)(Ca) channels is only partly through epoxyeicosatrienoic acid release, indicating the presence of other hyperpolarizing mechanisms. Third, bradykinin, but not acetylcholine, stimulates K(+)(Ca) channel-mediated vasodilation in healthy subjects, whereas in hypercholesterolemia, K(+)(Ca) channel-mediated vasodilation compensates for the reduced nitric oxide activity. Thus, enhanced endothelium-derived hyperpolarizing factor activity in conditions of nitric oxide deficiency contributes to maintenance of resting and agonist-stimulated vasodilation. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00166166.

Nitric Oxide Contributes to Vasomotor Tone in Hypertensive African Americans Treated With Nebivolol and Metoprolol.

Endothelial dysfunction is more prevalent in African Americans (AAs) compared with whites. The authors hypothesized that nebivolol, a selective ß1 -antagonist that stimulates nitric oxide (NO), will improve endothelial function in AAs with hypertension when compared with metoprolol. In a double-blind, randomized, crossover study, 19 AA hypertensive patients were randomized to a 12-week treatment period with either nebivolol 10 mg or metoprolol succinate 100 mg daily. Forearm blood flow (FBF) was measured using plethysmography at rest and after intra-arterial infusion of acetylcholine and sodium nitroprusside to estimate endothelium-dependent and independent vasodilation, respectively. Physiologic vasodilation was assessed during hand-grip exercise. Measurements were repeated after NO blockade with L-N(G) -monomethylarginine (L-NMMA) and after inhibition of endothelium-derived hyperpolarizing factor (EDHF) with tetraethylammonium chloride (TEA). NO blockade with L-NMMA produced a trend toward greater vasoconstriction during nebivolol compared with metoprolol treatment (21% vs 12% reduction in FBF, P=.06, respectively). This difference was more significant after combined administration of L-NMMA and TEA (P<.001). Similarly, there was a contribution of NO to exercise-induced vasodilation during nebivolol but not during metoprolol treatment. There were significantly greater contributions of NO and EDHF to resting vasodilator tone and of NO to exercise-induced vasodilation with nebivolol compared with metoprolol in AAs with hypertension.

Differential effects of nebivolol and metoprolol on arterial stiffness, circulating progenitor cells, and oxidative stress.

Unlike traditional beta receptor antagonists, nebivolol activates nitric oxide. We hypothesized that therapy with nebivolol compared with metoprolol would improve arterial stiffness, increase levels of circulating progenitor cells (PC), and decrease oxidative stress (OS). In a randomized, double-blind, cross-over study, 30 hypertensive subjects received either once daily nebivolol or metoprolol succinate for 3 months each. Pulse wave velocity and augmentation index were measured using tonometry. Flow cytometry was used to measure circulating PC. OS was measured as plasma aminothiols. Measurements were performed at baseline, and repeated at 3 and 6 months. No significant differences were present between the levels of OS, arterial stiffness, and PC numbers during treatment with metoprolol compared with nebivolol. In subgroup analyses of beta-blocker naïve subjects (n = 19), nebivolol reduced pulse wave velocity significantly compared with metoprolol (-1.4 ± 1.9 vs. -0.1 ± 2.2; P = .005). Both nebivolol and metoprolol increased circulating levels of CD34+/CD133 + PC similarly (P = .05), suggesting improved regenerative capacity.

Presence of endometrial epithelial cells in the peritoneal cavity and the mesothelial inflammatory response.

OBJECTIVE: To determine the contribution of endometrial cells in the development of endometriosis. Specifically the response of the mesothelium to endometrial cells in the production of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and IL-8 was studied. DESIGN: In vitro study. SETTING: University Research Laboratory. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cellular MCP-1, IL-6 secretion and MCP-1, and IL-6 and IL-8 messenger RNA expression were evaluated by ELISA and reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULT(S): The mesothelial cells produced more MCP-1 and IL-6 than endometrial epithelial and stromal cells. Mesothelial cells cultured in the presence of endometrial epithelial cells produced even greater levels of MCP-1 and IL-6 than those cultured in the presence of stromal cells or cultured alone. The MCP-1, IL-6, and IL-8 mRNA expression also increased when mesothelial cells were co-cultured with endometrial epithelial cells. CONCLUSION(S): The results suggest that endometrial epithelial cells may be important in evoking the inflammatory reaction in the peritoneal cavity during retrograde menstruation and that mesothelial cells may play an important role in the chemotaxis of monocytes and in the inflammatory process during the development of endometriosis.

Antioxidant supplementation reduces endometriosis-related pelvic pain in humans.

We previously suggested that women with endometriosis have increased oxidative stress in the peritoneal cavity. To assess whether antioxidant supplementation would ameliorate endometriosis-associated symptoms, we performed a randomized, placebo-controlled trial of antioxidant vitamins (vitamins E and C) in women with pelvic pain and endometriosis. Fifty-nine women, ages 19 to 41 years, with pelvic pain and history of endometriosis or infertility were recruited for this study. Patients were randomly assigned to 2 groups: vitamin E (1200 IU) and vitamin C (1000 mg) combination or placebo daily for 8 weeks before surgery. Pain scales were administered at baseline and biweekly. Inflammatory markers were measured in the peritoneal fluid obtained from both groups of patients at the end of therapy. Our results indicated that after treatment with antioxidants, chronic pain ("everyday pain") improved in 43% of patients in the antioxidant treatment group (P = 0.0055) compared with the placebo group. In the same group, dysmenorrhea ("pain associated with menstruation") and dyspareunia ("pain with sex") decreased in 37% and 24% patients, respectively. In the placebo group, dysmenorrhea-associated pain decreased in 4 patients and no change was seen in chronic pain or dyspareunia. There was a significant decrease in peritoneal fluid inflammatory markers, regulated upon activation, normal T-cell expressed and secreted (P ≤ 0.002), interleukin-6 (P ≤ 0.056), and monocyte chemotactic protein-1 (P ≤ 0.016) after antioxidant therapy compared with patients not taking antioxidants. The results of this clinical trial show that administration of antioxidants reduces chronic pelvic pain in women with endometriosis and inflammatory markers in the peritoneal fluid.

A genetic risk variant for myocardial infarction on chromosome 6p24 is associated with impaired central hemodynamic indexes.

BACKGROUND: Genome-wide association studies (GWAS) have identified novel variants associated with myocardial infarction (MI) in Caucasians. We hypothesized that those variants whose mechanism of risk is currently unknown, confer risk via pathways mediating arterial wave reflections which is an increasingly recognized risk factor for cardiovascular disease. METHODS: Single-nucleotide polymorphisms (SNPs) at eight MI risk loci were genotyped and correlated with noninvasively determined pulse wave analysis (PWA)-derived central hemodynamic indexes (augmentation index (AIx); augmented pressure (AP); time to reflected wave (TrW) and central systolic blood pressure (SBP) and diastolic BP (DBP)) in two independent Caucasian populations including (i) those free of measured cardiovascular risk factors (n = 133) and (ii) a community-based population (n = 270). RESULTS: Of the eight SNPs examined in the healthy group, the variants at loci 6p24 (AIx and AP both P < 0.001, TrW P = 0.02) and 21q22 (AIx P = 0.002, TrW P = 0.037) were significantly associated with PWA indexes. In the replication group, only the 6p24 variant correlated with these phenotypes (AIx P = 0.005, AP P = 0.049, TrW P = 0.013). In the pooled population (n = 403), no new associations were identified but the association with 6p24 and AIx remained significant even after Bonferroni correction and adjustment for covariates including age, mean arterial pressure, height, gender, glucose, cholesterol, body mass index (BMI), and smoking (AIx (P = 0.03)). Each copy of the risk allele C increased the AIx by 3.5%. CONCLUSIONS: The GWAS discovered MI risk variant at 6p24 in the protein phosphatase 1 regulator gene (PHACTR1) is associated with adverse arterial wave reflection indexes and may mediate MI risk through this pathway.

Vitamin D status is associated with arterial stiffness and vascular dysfunction in healthy humans.

OBJECTIVES: The primary objective of this study was to elucidate mechanisms underlying the link between vitamin D status and cardiovascular disease by exploring the relationship between 25-hydroxyvitamin D (25-OH D), an established marker of vitamin D status, and vascular function in healthy adults. BACKGROUND: Mechanisms underlying vitamin D deficiency-mediated increased risk of cardiovascular disease remain unknown. Vitamin D influences endothelial and smooth muscle cell function, mediates inflammation, and modulates the renin-angiotensin-aldosterone axis. We investigated the relationship between vitamin D status and vascular function in humans, with the hypothesis that vitamin D insufficiency will be associated with increased arterial stiffness and abnormal vascular function. METHODS: We measured serum 25-OH D in 554 subjects. Endothelial function was assessed as brachial artery flow-mediated dilation, and microvascular function was assessed as digital reactive hyperemia index. Carotid-femoral pulse wave velocity and radial tonometry-derived central augmentation index and subendocardial viability ratio were measured to assess arterial stiffness. RESULTS: Mean 25-OH D was 31.8 ± 14 ng/ml. After adjustment for age, sex, race, body mass index, total cholesterol, low-density lipoprotein, triglycerides, C-reactive protein, and medication use, 25-OH D remained independently associated with flow-mediated vasodilation (ß = 0.1, p = 0.03), reactive hyperemia index (ß = 0.23, p < 0.001), pulse wave velocity (ß = -0.09, p = 0.04), augmentation index (ß = -0.11, p = 0.03), and subendocardial viability ratio (ß = 0.18, p = 0.001). In 42 subjects with vitamin D insufficiency, normalization of 25-OH D at 6 months was associated with increases in reactive hyperemia index (0.38 ± 0.14, p = 0.009) and subendocardial viability ratio (7.7 ± 3.1, p = 0.04), and a decrease in mean arterial pressure (4.6 ± 2.3 mm Hg, p = 0.02). CONCLUSIONS: Vitamin D insufficiency is associated with increased arterial stiffness and endothelial dysfunction in the conductance and resistance blood vessels in humans, irrespective of traditional risk burden. Our findings provide impetus for larger trials to assess the effects of vitamin D therapy in cardiovascular disease.

Oxidative stress is associated with impaired arterial elasticity.

AIMS: Arterial stiffening may lead to hypertension, greater left ventricular after-load and adverse clinical outcomes. The underlying mechanisms influencing arterial elasticity may involve oxidative injury to the vessel wall. We sought to examine the relationship between novel markers of oxidative stress and arterial elastic properties in healthy humans. METHODS AND RESULTS: We studied 169 subjects (mean age 42.6 ± 14 years, 51.6% male) free of traditional cardiovascular risk factors. Indices of arterial stiffness and wave reflections measured included carotid-femoral Pulse Wave Velocity (PWV), Augmentation Index (Aix) and Pulse Pressure Amplification (PPA). Non-free radical oxidative stress was assessed as plasma oxidized and reduced amino-thiol levels (cysteine/cystine, glutathione/GSSG) and their ratios (redox potentials), and free radical oxidative stress as derivatives of reactive oxygen metabolites (dROMs). Inflammation was assessed as hsCRP and interleukin-6 levels. The non-free radical marker of oxidative stress, cystine was significantly correlated with all arterial indices; PWV (r=0.38, p<0.001), Aix (r=0.35, p<0.001) and PPA (r=-0.30, p<0.001). Its redox potential, was also associated with PWV (r=0.22, p=0.01), while the free radical marker of oxidative stress dROMS was associated with Aix (r=0.25, p<0.01). After multivariate adjustment for age, gender, arterial pressure, height, weight, heart rate and CRP, of these oxidative stress markers, only cystine remained independently associated with PWV (p=0.03), Aix (p=0.01) and PPA (p=0.05). CONCLUSIONS: In healthy subjects without confounding risk factors or significant systemic inflammation, a high cystine level, reflecting extracellular oxidant burden, is associated with increased arterial stiffness and wave reflections. This has implications for understanding the role of oxidant burden in pre-clinical vascular dysfunction.