Effects of polycythemia on systemic endothelial function in chronic hypoxic lung disease

Chronic obstructive pulmonary disease (COPD) is a major risk factor for cardiovascular disease. Polycythemia, a common complication of hypoxic COPD, may affect systemic vascular function by altering blood viscosity, vessel wall shear stress (WSS), and endothelium-derived nitric oxide (NO) release. Here, we evaluated the effects of hypoxia-related polycythemia on systemic endothelial function in patients with COPD. We investigated blood viscosity, WSS, and endothelial function in 15 polycythemic and 13 normocythemic patients with COPD of equal severity, by recording brachial artery diameter variations in response to hyperemia and by using venous occlusion plethysmography (VOP) to measure forearm blood flow (FBF) responses to a brachial artery infusion of acetylcholine (ACh), bradykinin (BK), sodium nitroprusside (SNP), substance P (SP), isoptin, and N-monomethyl-L-arginine (L-NMMA). At baseline, polycythemic patients had higher blood viscosity and larger brachial artery diameter than normocythemic patients but similar calculated WSS. Flow-mediated brachial artery vasodilation was increased in the polycythemic patients, in proportion to the hemoglobin levels. ACh-induced vasodilation was markedly impaired in the polycythemic patients and negatively correlated with hemoglobin levels. FBF responses to endothelium- (BK, SP) and non-endothelium-dependent (SNP, isoptin) vasodilators were not significantly different between the two groups. L-NMMA infusion induced a similar vasoconstrictor response in both groups, in accordance with their similar baseline WSS. In conclusion, systemic arteries in polycythemic patients adjust appropriately to chronic or acute WSS elevations by appropriate basal and stimulated NO release. Overall, our results suggest that moderate polycythemia has no adverse effect on vascular function in COPD.     

Effects of chronic sympathectomy on vascular function in the human forearm.

To determine whether endothelial function is altered by chronic surgical sympathectomy, we infused ACh, isoproterenol, nitroprusside (NTP), and the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA) into the brachial arteries of nine patients 5-64 mo after thoracic sympathectomy for hyperhidrosis. Age- and gender-matched controls were also studied. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. Lower body negative pressure was used to assess reflex vasoconstrictor responses. Tyramine, which acts locally and causes norepinephrine release from sympathetic nerves, was also administered via the brachial artery. FBF at rest was 2.5 +/- 0.4 ml x dl-1 x min-1 in the patients and 2.5 +/- 0.3 ml x dl-1 x min-1 in the controls (P = 0.95). The normal vasoconstrictor responses to lower body negative pressure were abolished in the patients. By contrast, tyramine produced dose-dependent vasoconstriction in the patients that was identical to that of controls. The dose-response curves to ACh were similar in patients and controls, with maximum values of 19.3 +/- 4.4 vs. 25.5 +/- 2.8 ml x dl-1 x min-1, respectively. L-NMMA reduced baseline FBF similarly and reduced the maximal FBF response to ACh in both groups (patients 8.9 +/- 3.5 vs. controls 9.7 +/- 2.5 ml x dl-1 x min-1). The vasodilation to isoproterenol was similar and blunted to the same extent in both groups by L-NMMA. The responses to NTP in patients and controls were similar and not affected by L-NMMA. We conclude that, in humans, chronic surgical sympathectomy does not cause major disruptions in vascular function in the forearm. The normal vasoconstrictor responses to tyramine indicate that there were viable sympathetic nerves in the forearm that were not engaged by LBNP.     

Comparison of resistance and conduit vessel nitric oxide-mediated vascular function in vivo: effects of exercise training.

Exercise training improves vascular function in subjects with cardiovascular disease and risk factors, but there is mounting evidence these vascular adaptations may be vessel bed specific. We have therefore examined the hypothesis that exercise-induced improvements in conduit vessel function are related to changes in resistance vessel function. Endothelium-dependent and -independent conduit vessel function were assessed by using wall-tracking of high-resolution brachial artery ultrasound images of the response to flow-mediated dilation (FMD) and nitroglycerine [glyceryl trinitrate (GTN)] administration. Resistance vessel endothelium-dependent and -independent function were assessed using intrabrachial administration of acetylcholine (ACh) and nitroprusside (SNP). Randomized crossover studies of 8-wk exercise training were undertaken in untreated hypercholesterolemic (n = 10), treated hypercholesterolemic (n = 10), coronary artery disease (n = 8), and Type 2 diabetic subjects (n = 15). Exercise training significantly enhanced responses to ACh (P < 0.05) and FMD (P < 0.0001). There were no significant changes in either SNP or GTN responses. The correlation between ACh and FMD responses at entry was not significant (r = 0.186; P = 0.231), and training-induced changes in the ACh did not correlate with those in FMD (r = -0.022; P = 0.890). Similarly, no correlation was evident between the SNP and GTN responses at entry (r = -0.010; P = 0.951) or between changes in these variables with training (r = -0.211; P = 0.191). We conclude that, although short-term exercise training improves endothelium-dependent nitric oxide-mediated vascular function in both conduit and resistance vessels, the magnitude of these improvements are unrelated.     

Exercise training improves conduit vessel function in patients with coronary artery disease.

It is well established that endothelial dysfunction is present in coronary artery disease (CAD), although few studies have determined the effect of training on peripheral conduit vessel function in patients with CAD. A randomized, crossover design determined the effect of 8 wk of predominantly lower limb, combined aerobic and resistance training, in 10 patients with treated CAD. Endothelium-dependent dilation of the brachial artery was determined, by using high-resolution vascular ultrasonography, from flow-mediated vasodilation (FMD) after ischemia. Endothelium-independent vasodilation was measured after administration of glyceryl trinitrate (GTN). Baseline function was compared with that of 10 control subjects. Compared with matched healthy control subjects, FMD and GTN responses were significantly impaired in the untrained CAD patients [3.0 +/- 0.8 (SE) vs. 5.8 +/- 0.8% and 14.5 +/- 1.9 vs. 20.4 +/- 1.5%, respectively; both P < 0.05]. Training significantly improved FMD in the CAD patients (from 3.0 +/- 0.8 to 5.7 +/- 1.1%; P < 0.05) but not responsiveness to GTN (14.5 +/- 1.9 vs. 12.1 +/- 1.4%; P = not significant). Exercise training improves endothelium-dependent conduit vessel dilation in subjects with CAD, and the effect, evident in the brachial artery, appears to be generalized rather than limited to vessels of exercising muscle beds. These results provide evidence for the benefit of exercise training, as an adjunct to routine therapy, in patients with a history of CAD.     

Sex differences in salt sensitivity to nitric oxide dependent vasodilation in healthy young adults

Dietary sodium and blood pressure regulation differs between normotensive men and women, an effect which may involve endothelial production of nitric oxide (NO). Therefore, we tested the hypothesis that differences in the NO component of endothelium-dependent vasodilation between low and high dietary sodium intake depend on sex. For 5 days prior to study, healthy adults consumed a controlled low-sodium diet (10 mmol/day, n = 30, mean age ± SE: 30 ± 1 yr, 16 men) or high-sodium diet (400 mmol/day, n = 36, age 23 ± 1 yr, 13 men). Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of acetylcholine (ACh, 4 μg·100 ml tissue(-1)·min(-1)) were measured before and after endothelial NO synthase inhibition with N(G)-monomethyl-l-arginine (l-NMMA, 50 mg bolus + 1 mg/min infusion). The NO component of endothelium-dependent dilation was calculated as the response to ACh before and after l-NMMA accounting for changes in baseline FBF: [(FBF ACh - FBF baseline) - (FBF ACh(L-NMMA) - FBF baseline(L-NMMA))]. This value was 5.7 ± 1.3 and 2.5 ± 0.8 ml·100 ml forearm tissue(-1)·min(-1) for the low- and high-sodium diets, respectively (main effect of sodium, P = 0.019). The sodium effect was larger for the men, with values of 7.9 ± 2.0 and 2.2 ± 1.4 for men vs. 3.1 ± 1.3 and 2.7 ± 1.0 ml·100 ml forearm tissue(-1)·min(-1) for the women (P = 0.034, sex-by-sodium interaction). We conclude that the NO component of endothelium-dependent vasodilation is altered by dietary sodium intake based on sex, suggesting that endothelial NO production is sensitive to dietary sodium in healthy young men but not women.     

Plasma nitrite reserve and endothelial function in the human forearm circulation

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.     

Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans

Whether a cytochrome P-450 (CYP)-related endothelium-derived hyperpolarizing factor (EDHF), acting through calcium-activated potassium (K(Ca)) channels, interacts with nitric oxide (NO) to regulate the basal diameter of human peripheral conduit arteries is unexplored in vivo. Radial artery diameter (echo tracking) and blood flow (Doppler) were measured, after oral aspirin (500 mg), in eight healthy volunteers during local infusion for 8 min of tetraethylammonium chloride (TEA; 9 micromol/min), as K(Ca) channel inhibitor, and fluconazole (0.4 micromol/min), as CYP inhibitor, alone and in combination with N(G)-monomethyl-L-arginine (L-NMMA; 8 micromol/min), as endothelial NO synthase inhibitor. Endothelium-independent dilatation was assessed by using sodium nitroprusside (SNP). Radial diameter was unaffected by L-NMMA (0.4 +/- 0.9%) and fluconazole (-1.6 +/- 0.8%) but was decreased by TEA (-5.0 +/- 1.0%), L-NMMA + fluconazole (-5.3 +/- 0.5%), and L-NMMA + TEA (-9.9 +/- 1.3%). These effects are still significant even when the concomitant decreases in blood flow induced by L-NMMA (-24 +/- 4%), TEA (-21 +/- 3%), L-NMMA + fluconazole (-26 +/- 5%), and L-NMMA + TEA (-35 +/- 4%) were taken as covariate into analysis. Conversely, fluconazole alone slightly but not significantly increased radial flow (13 +/- 6%). L-NMMA alone or with TEA and with fluconazole enhanced radial artery dilatation to SNP, whereas TEA and fluconazole alone did not modify this response. These results confirm in humans the involvement of NO and K(Ca) channels in the regulation of basal conduit artery diameter. Moreover, the synergistic effect of combined inhibition of NO synthesis and CYP on the decrease in radial diameter in the absence of such effect after L-NMMA and fluconazole alone unmasks the role of CYP in this regulation and shows the presence of an interaction between NO and a CYP-related EDHF to maintain peripheral conduit artery diameter in vivo. Furthermore, the higher vasoconstrictor effect of TEA compared with fluconazole suggests that different K(Ca) channel-dependent hyperpolarizing mechanisms could exist in conduit arteries.     

Agonist-dependent variablity of contributions of nitric oxide and prostaglandins in human skeletal muscle.

The relative contributions of endothelium-dependent dilators [nitric oxide (NO), prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF)] in human limbs are poorly understood. We tested the hypothesis that relative contributions of NO and PGs differ between endothelial agonists acetylcholine (ACh; 1, 2, and 4 microg.dl(-1).min(-1)) and bradykinin (BK; 6.25, 25, and 50 ng.dl(-1).min(-1)). We measured forearm blood flow (FBF) using venous occlusion plethysmography in 50 healthy volunteers (27 +/- 1 yr) in response to brachial artery infusion of ACh or BK in the absence and presence of inhibitors of NO synthase [NOS; with NG-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (COX; with ketorolac). Furthermore, we tested the idea that the NOS + COX-independent dilation (in the presence of L-NMMA + ketorolac, presumably EDHF) could be inhibited by exogenous NO administration, as reported in animal studies. FBF increased approximately 10-fold in the ACh control; L-NMMA reduced baseline FBF and ACh dilation, whereas addition of ketorolac had no further effect. Ketorolac alone did not alter ACh dilation, but addition of L-NMMA reduced ACh dilation significantly. For BK infusion, FBF increased approximately 10-fold in the control condition; L-NMMA tended to reduce BK dilation (P < 0.1), and addition of ketorolac significantly reduced BK dilation. Similar to ACh, ketorolac alone did not alter BK dilation, but addition of L-NMMA reduced BK dilation. To test the idea that NO can inhibit the NOS + COX-independent portion of dilation, we infused a dose of sodium nitroprusside (NO-clamp technique) during ACh or BK that restored the reduction in baseline blood flow due to L-NMMA. Regardless of treatment order, the NO clamp restored baseline FBF but did not reduce the NOS + COX-independent dilation to ACh or BK. We conclude that the contribution of NO and PGs differs between ACh and BK, with ACh being more dependent on NO and BK being mostly dependent on a NOS + COX-independent mechanism (EDHF) in healthy young adults. The NOS + COX-independent dilation does not appear sensitive to feedback inhibition from NO in the human forearm.     

Insulin impairs endothelium-dependent vasodilation independent of insulin sensitivity or lipid profile

Insulin resistance is a risk factor for atherosclerosis and is associated with hyperinsulinemia, abnormal lipid profile, and hypertension. Whether hyperinsulinemia affects vascular function independent of insulin resistance or other metabolic risk factors is unknown. This investigation aimed to assess the effects of hyperinsulinemia on endothelial function in subjects with a spectrum of insulin sensitivity and lipid profile. Endothelium-dependent (flow-mediated dilation, FMD) and -independent (nitroglycerin) responses of the brachial artery were studied by high-resolution ultrasound before and during hyperinsulinemia (euglycemic clamp) in 25 normoglycemic, normotensive subjects. Participants were divided into an insulin-sensitive and an insulin-resistant subgroup based on their sensitivity index values, with a cutoff of 8, and into a normal-cholesterol and a high-cholesterol subgroup based on their total cholesterol levels, with a cutoff of 5.2 mmol/l (200 mg/dl). In the whole population, FMD was lower during hyperinsulinemia compared with baseline (2.3 +/- 0.6% vs. 6 +/- 0.6%; P < 0.001). Resting FMD was lower in the insulin-resistant subgroup compared with the insulin-sensitive subgroup (4.2 +/- 0.9% vs. 7.4 +/- 0.8%; P = 0.014) and in the high-cholesterol subjects compared with the normal-cholesterol subjects (4.4 +/- 0.7% vs. 8 +/- 0.7%; P = 0.002). Hyperinsulinemia decreased FMD in both the insulin-sensitive (from 7.4 +/- 0.8% to 3.6 +/- 0.4%; P < 0.001) and insulin-resistant (from 4.2% to 1.22%; P = 0.012) subgroups and in both the normal-cholesterol (from 8 +/- 0.7% to 3.9 +/- 0.4%; P < 0.001) and high-cholesterol (from 4.4 +/- 0.7% to 1.1 +/- 0.8%; P = 0.01) participants. Acute hyperinsulinemia impairs conduit vessel endothelial function independent of insulin sensitivity and lipid profile. Insulin may trigger endothelial dysfunction and promote atherosclerosis.     

Exercise-induced improvement in endothelial dysfunction is not mediated by changes in CV risk factors: pooled analysis of diverse patient populations

We have pooled data from a series of our exercise training studies undertaken in groups with a broad range of vascular (dys) function to the examine the hypothesis that exercise-induced improvements in the conduit and/or resistance vessel function are related to improvements in risk factors for cardiovascular (CV) disease. Endothelium-dependent and -independent conduit vessel function were assessed by using wall tracking of high-resolution ultrasound images of the brachial artery response to flow-mediated dilation (FMD) and glyceryl trinitrate. Resistance vessel function was assessed using intrabrachial administration of acetylcholine (ACh), sodium nitroprusside, and NG-monomethyl-l-arginine. Randomized cross-over studies of 8-wk exercise training were undertaken in untreated hypercholesterolemic (n = 11), treated hypercholesterolemic (n = 11), coronary artery disease (n = 10), chronic heart failure (n = 12), Type 2 diabetic (n = 15), and healthy control subjects (n = 16). Exercise training did not significantly alter plasma lipids, blood pressure, blood glucose, waist-to-hip ratio, or body mass index values, despite significant improvement in both FMD and ACh responses. There were no correlations between changes in any risk factor variables and indexes of either resistance or conduit vessel function. We conclude that, in these subjects with antecedent vascular dysfunction, the beneficial effects of relatively short-term exercise training on vascular function are not solely mediated by the effects of exercise on CV risk factors.     

Heterogenous vasodilator pathways underlie flow-mediated dilation in men and women

This study investigated the sex differences in the contribution of nitric oxide (NO) and prostaglandins (PGs) to flow-mediated dilation (FMD). Radial artery (RA) FMD, assessed as the dilatory response to 5-min distal cuff occlusion, was repeated after three separate brachial artery infusions of saline (SAL), N(G)-monomethyl-L-arginine (L-NMMA), and ketorolac (KETO) + L-NMMA in healthy younger men (M; n = 8) and women (W; n = 8). In eight subjects (4 M, 4W) RA FMD was reassessed on a separate day with drug order reversed (SAL, KETO, and L-NMMA + KETO). RA FMD was calculated as the peak dilatory response observed relative to baseline (%FMD) and expressed relative to the corresponding area under the curve shear stress (%FMD/AUC SS). L-NMMA reduced %FMD similarly and modestly (P = 0.68 for sex * trial interaction) in M and W (all subjects: 10.0 ± 3.8 to 7.6 ± 4.7%; P = 0.03) with no further effect of KETO (P = 0.68). However, all sex * trial and trial effects on %FMD/AUC SS for l-NMMA and KETO + l-NMMA were insignificant (all P > 0.20). There was also substantial heterogeneity of the magnitude and direction of dilator responses to blockade. After l-NMMA infusion, subjects exhibited both reduced (n = 14; range: 11 to 78% decrease) and augmented (n = 2; range: 1 to 96% increase) %FMD. Following KETO + l-NMMA, seven subjects exhibited reduced dilation (range: 10 to 115% decrease) and nine subjects exhibited augmented dilation (range: 1 to 212% increase). Reversing drug order did not change the nature of the findings. These findings suggest that RA FMD is not fully or uniformly NO dependent in either men or women, and that there is heterogeneity in the pathways underlying the conduit dilatory response to ischemia.     

Heterogeneity in conduit artery function in humans: impact of arterial size

To determine whether conduit artery size affects functional responses, we compared the magnitude, time course, and eliciting shear rate stimulus for flow-mediated dilation (FMD) in healthy men (n = 20; 31 +/- 7 yr). Upper limb (brachial and radial) and lower limb (common and superficial femoral) FMD responses were simultaneously assessed, whereas popliteal responses were measured in the same subjects during a separate visit. Glyceryl trinitrate (GTN)-mediated responses were similarly examined. Edge detection and wall tracking of high-resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate conduit artery diameter, blood flow, and shear rate continuously across the cardiac cycle. Baseline artery size correlated inversely with the FMD response (r = -0.57, P < 0.001). Within-artery comparisons revealed a significant inverse correlation between artery size and FMD% for the radial (r = -0.66, P = 0.001), brachial (r = -0.55, P = 0.01), and popliteal artery (r = -0.48, P = 0.03), but not for the superficial and common femoral artery. Normalization of FMD responses for differences in eliciting shear rate did not abolish the between-artery relationship for artery function and size (r = -0.48, P < 0.001), suggesting that differences between artery function responses were not entirely due to size-related differences in shear rate. This was reinforced by a significant between-artery correlation for GTN responses and baseline artery size (r = -0.74, P < 0.001). In summary, systematic differences exist in vascular function responses of conduit arteries that differ in size. This raises the possibility that differences in artery size within or between individuals may influence functional responses.     

Forearm vascular control during acute hyperglycemia in healthy humans

The vascular endothelium is a site of pathological changes in patients with diabetes mellitus that may be related to severe chronic hyperglycemia. However, it is unclear whether transient hyperglycemia alters vascular function in an otherwise healthy human forearm. To test the hypothesis that acute, moderate hyperglycemia impairs endothelium-dependent forearm vasodilation, we measured vasodilator responses in 25 healthy volunteers (11 F, 14 M) assigned to one of three protocols. In protocol 1, glucose was varied to mimic a postprandial pattern (i.e., peak glucose approximately 11.1 mmol/l) commonly observed in individuals with impaired glucose tolerance. Protocol 2 involved 6 h of mild hyperglycemia (approximately 7 mmol/l). Protocol 3 involved 6 h of euglycemia. Glucose concentration was maintained with a variable systemic glucose infusion. Insulin concentrations were maintained at approximately 65 pmol/l by means of a somatostatin and "basal" insulin infusion. Glucagon and growth hormone were replaced at basal concentrations. Forearm blood flow (FBF) was calculated from Doppler ultrasound measurements at the brachial artery. In each protocol, FBF dose responses to intrabrachial acetylcholine (ACh) and sodium nitroprusside (NTP) were assessed at baseline and at 60, 180, and 360 min of glucose infusion. Peak endothelium-dependent vasodilator responses to ACh were not diminished by hyperglycemia in any trial. For example, peak responses to ACh during protocol 2 were 307 +/- 47 ml/min at euglycemic baseline and 325 +/- 52, 353 +/- 65, and 370 +/- 70 ml/min during three subsequent hyperglycemic trials (P = 0.46). Peak endothelium-independent responses to NTP infusion were also unaffected. We conclude that acute, moderate hyperglycemia does not cause short-term impairment of endothelial function in the healthy human forearm.     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.     

High-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance

Endothelial dysfunction is a hallmark of Type 2 diabetes related to hyperglycemia and oxidative stress. Nitric oxide-dependent vasodilator actions of insulin may augment glucose disposal. Thus endothelial dysfunction may worsen insulin resistance. Intra-arterial administration of vitamin C improves endothelial dysfunction in diabetes. In the present study, we investigated effects of high-dose oral vitamin C to alter endothelial dysfunction and insulin resistance in Type 2 diabetes. Plasma vitamin C levels in 109 diabetic subjects were lower than healthy (36 +/- 2 microM) levels. Thirty-two diabetic subjects with low plasma vitamin C (<40 microM) were subsequently enrolled in a randomized, double-blind, placebo-controlled study of vitamin C (800 mg/day for 4 wk). Insulin sensitivity (determined by glucose clamp) and forearm blood flow in response to ACh, sodium nitroprusside (SNP), or insulin (determined by plethysmography) were assessed before and after 4 wk of treatment. In the placebo group (n = 17 subjects), plasma vitamin C (22 +/- 3 microM), fasting glucose (159 +/- 12 mg/dl), insulin (19 +/- 7 microU/ml), and SI(Clamp) [2.06 +/- 0.29 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)] did not change significantly after placebo treatment. In the vitamin C group (n = 15 subjects), basal plasma vitamin C (23 +/- 2 microM) increased to 48 +/- 6 microM (P < 0.01) after treatment, but this was significantly less than that expected for healthy subjects (>80 microM). No significant changes in fasting glucose (156 +/- 11 mg/dl), insulin (14 +/- 2 microU/ml), SI(Clamp) [2.71 +/- 0.46 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)], or forearm blood flow in response to ACh, SNP, or insulin were observed after vitamin C treatment. We conclude that high-dose oral vitamin C therapy, resulting in incomplete replenishment of vitamin C levels, is ineffective at improving endothelial dysfunction and insulin resistance in Type 2 diabetes.     

Evidence against oxidative stress as mechanism of endothelial dysfunction in methionine loading model

Endothelial dysfunction reflects reduced nitric oxide (NO) bioavailability due to either reduced production, inactivation of NO, or reduced smooth muscle responsiveness. Oral methionine loading causes acute endothelial dysfunction in healthy subjects and provides a model in which to study mechanisms. Endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery in humans. Three markers of oxidative stress were measured ex vivo in venous blood. NO responsiveness was assessed in vascular smooth muscle and platelets. Oral methionine loading induced endothelial dysfunction (FMD decreased from 2.8 +/- 0.8 to 0.3 +/- 0.3% with methionine and from 2.8 +/- 0.8 to 1.3 +/- 0.3% with placebo; P < 0.05). No significant changes in measures of plasma oxidative stress or in vascular or platelet sensitivity to submaximal doses of NO donors were detected. These data suggest that oxidative stress is not the mechanism of endothelial dysfunction after oral methionine loading. Furthermore, the preservation of vascular and platelet NO sensitivity makes a signal transduction abnormality unlikely.     

Effects of combined inhibition of ATP-sensitive potassium channels, nitric oxide, and prostaglandins on hyperemia during moderate exercise.

ATP-sensitive potassium (KATP) channels have been suggested to contribute to coronary and skeletal muscle vasodilation during exercise, either alone or interacting in a parallel or redundant process with nitric oxide (NO), prostaglandins (PGs), and adenosine. We tested the hypothesis that KATP channels, alone or in combination with NO and PGs, regulate exercise hyperemia in forearm muscle. Eighteen healthy young adults performed 20 min of moderate dynamic forearm exercise, with forearm blood flow (FBF) measured via Doppler ultrasound. After steady-state FBF was achieved for 5 min (saline control), the KATP inhibitor glibenclamide (Glib) was infused into the brachial artery for 5 min (10 microg.dl(-1).min(-1)), followed by saline infusion during the final 10 min of exercise (n = 9). Exercise increased FBF from 71 +/- 11 to 239 +/- 24 ml/min, and FBF was not altered by 5 min of Glib. Systemic plasma Glib levels were above the therapeutic range, and Glib increased insulin levels by approximately 50%, whereas blood glucose was unchanged (88 +/- 2 vs. 90 +/- 2 mg/dl). In nine additional subjects, Glib was followed by combined infusion of NG-nitro-L-arginine methyl ester (L-NAME) plus ketorolac (to inhibit NO and PGs, respectively). As above, Glib had no effect on FBF but addition of L-NAME + ketorolac (i.e., triple blockade) reduced FBF by approximately 15% below steady-state exercise levels in seven of nine subjects. Interestingly, triple blockade in two subjects caused FBF to transiently and dramatically decrease. This was followed by an acute recovery of flow above steady-state exercise values. We conclude 1) opening of KATP channels is not obligatory for forearm exercise hyperemia, and 2) triple blockade of NO, PGs, and KATP channels does not reduce hyperemia more than the inhibition of NO and PGs in most subjects. However, some subjects are sensitive to triple blockade, but they are able to restore FBF acutely during exercise. Future studies are required to determine the nature of these compensatory mechanisms in the affected individuals.     

Endothelium-dependent and -independent vasodilation of the superficial femoral artery in spinal cord-injured subjects

Extreme inactivity of the legs in spinal cord-injured (SCI) individuals does not result in an impairment of the superficial femoral artery flow-mediated dilation (FMD). To gain insight into the underlying mechanism, the present study examined nitric oxide (NO) responsiveness of vascular smooth muscles in controls and SCI subjects. In eight healthy men (34 +/- 13 yr) and six SCI subjects (37 +/- 10 yr), superficial femoral artery FMD response was assessed by echo Doppler. Subsequently, infusion of incremental dosages of sodium nitroprusside (SNP) was used to assess NO responsiveness. Peak diameter was examined on a second day after 13 min of arterial occlusion in combination with sublingual administration of nitroglycerine. Resting and peak superficial femoral artery diameter in SCI subjects were smaller than in controls (P < 0.001). The FMD response in controls (4.2 +/- 0.9%) was lower than in SCI subjects (8.2 +/- 0.9%, P < 0.001), but not after correcting for area under the curve for shear rate (P = 0.35). When expressed as relative change from baseline, SCI subjects demonstrate a significantly larger diameter increase compared with controls at each dose of SNP. However, when expressed as a relative increase within the range of diameter changes [baseline (0%) - peak diameter (100%)], both groups demonstrate similar changes in response to SNP. Changes in diameter during SNP infusion and FMD response are larger in SCI subjects compared with controls. When these results are corrected, superficial femoral artery FMD and NO sensitivity in SCI subjects are not different from those in controls. This illustrates the importance of appropriate data presentation and suggests that, subsequent to structural inward remodeling of conduit arteries as a consequence of extreme physical inactivity, arterial function is normalized.     

Effects of glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable coronary artery disease

GLP-1 stimulates insulin secretion, suppresses glucagon secretion, delays gastric emptying, and inhibits small bowel motility, all actions contributing to the anti-diabetogenic peptide effect. Endothelial dysfunction is strongly associated with insulin resistance and type 2 diabetes mellitus and may cause the angiopathy typifying this debilitating disease. Therefore, interventions affecting both endothelial dysfunction and insulin resistance may prove useful in improving survival in type 2 diabetes patients. We investigated GLP-1's effect on endothelial function and insulin sensitivity (S(I)) in two groups: 1) 12 type 2 diabetes patients with stable coronary artery disease and 2) 10 healthy subjects with normal endothelial function and S(I). Subjects underwent infusion of recombinant GLP-1 or saline in a random crossover study. Endothelial function was measured by postischemic FMD of brachial artery, using ultrasonography. S(I) [in (10(-4) dl.kg(-1).min(-1))/(muU/ml)] was measured by hyperinsulinemic isoglycemic clamp technique. In type 2 diabetic subjects, GLP-1 infusion significantly increased relative changes in brachial artery diameter from baseline FMD(%) (3.1 +/- 0.6 vs. 6.6 +/- 1.0%, P < 0.05), with no significant effects on S(I) (4.5 +/- 0.8 vs. 5.2 +/- 0.9, P = NS). In healthy subjects, GLP-1 infusion affected neither FMD(%) (11.9 +/- 0.9 vs. 10.3 +/- 1.0%, P = NS) nor S(I) (14.8 +/- 1.8 vs. 11.6 +/- 2.0, P = NS). We conclude that GLP-1 improves endothelial dysfunction but not insulin resistance in type 2 diabetic patients with coronary heart disease. This beneficial vascular effect of GLP-1 adds yet another salutary property of the peptide useful in diabetes treatment.