Andrew J. Maxwell

Limb Blood Flow During Exercise Is Dependent on Nitric Oxide

BACKGROUND We have recently reported that hypercholesterolemia reduces aerobic exercise capacity in mice and that this is associated with a reduced endothelium-dependent vasodilator function, endothelium-derived nitric oxide (EDNO) production, and urinary nitrate excretion. These findings led us to test the hypothesis that EDNO production contributes significantly to limb blood flow during exercise and to determine whether loss of EDNO production is responsible for the decline in exercise capacity observed in hypercholesterolemia. METHODS AND RESULTS Twelve-week-old wild-type (E+; n=9) and apoE-deficient (E-; n=9) C57BL/6J mice were treadmill-tested to measure indices defining exercise capacity on a metabolic chamber-enclosed treadmill capable of measuring oxygen uptake and carbon dioxide excretion. Urine was collected before and after treadmill exercise for determination of vascular NO production assessed by urinary nitrate excretion. The wild-type mice were then given nitro-L-arginine (E+LNA) in the drinking water (6 mg/dL) for 4 days before undergoing a second treadmill testing and urinary nitrate measurement. An additional set of 12-week-old wild-type mice was divided into 2 groups: 1 receiving regular water (E+; n=8) and 1 administered LNA for 4 days (E+LNA; n=8). These mice, along with an additional set of E mice (n=8), underwent treadmill testing to determine maximal oxygen uptake (VO2max). The mice were then cannulated such that the tip of the tubing was positioned in the ascending aorta. Fluorescent microspheres (20000) were infused into the carotid cannula while the mice were sedentary and again while approaching VO2max. When the mice were euthanized, the running muscles were collected and fluorescence intensity was measured to determine the peak-exercise redistribution of blood flow to the running muscles (expressed as percentage of total cardiac output, %COrm) during both states. Both E+LNA and E- mice demonstrated a markedly reduced postexercise urinary nitrate excretion, aerobic capacity, and %COrm at VO2max compared with E+. CONCLUSIONS EDNO contributes significantly to limb blood flow during exercise. Conditions that reduce EDNO production disturb the hyperemic response to exercise, resulting in a reduced exercise capacity.

Nutritional therapy for peripheral arterial disease: a double-blind, placebo-controlled, randomized trial of HeartBar®

We investigated the clinical effects of a food bar enriched with l-arginine and a combination of other nutrients known to enhance the activity of endothelium-derived nitric oxide (EDNO) in individuals with claudication from atherosclerotic peripheral arterial disease. The study was a 2-week, double-blind, placebo-controlled trial of subjects randomized to three groups (two active bars, one active and one placebo bar, and two placebo bars per day) followed by an 8-week open-label period. Subjects (n = 41) were outpatient volunteers with intermittent claudication. Pain-free and total walking distances were measured by variable-grade, treadmill exercise testing. Quality of life was assessed using the Medical Outcome Survey (SF-36). After 2 weeks of treatment, the pain-free walking distance increased 66% while the total walking distance increased 23% in the group taking two active bars/day. The general and emotional/social functioning components of the SF-36 also improved. These effects were not observed in the one active bar/day and placebo groups. The effects were maintained after 10 weeks and, in addition, an improvement in walking distance was observed in the group taking one active bar. These findings reveal that use of a nutrient bar designed to enhance EDNO activity improves pain-free and total walking distance as well as quality of life in individuals with intermittent claudication.

Uric acid is closely linked to vascular nitric oxide activity: Evidence for mechanism of association with cardiovascular disease

OBJECTIVES The study was undertaken to determine whether the mechanism of association of elevated serum uric acid level (SUA) with cardiovascular disease (CVD) is secondary to a common link with vascular nitric oxide (NO) activity. BACKGROUND Epidemiologic studies demonstrate an association of elevated SUA with CVD. The mechanism of this association is unknown, but both may be linked via an impairment in vascular NO activity. To examine this, we determined the relationship of SUA to vascular NO activity and to CVD risk. We then determined the effect of enhancing vascular NO activity on SUA. METHODS In part 1, individuals with various degrees of CVD (n = 458) were surveyed and underwent measurement of flow-mediated brachial artery vasodilation (FMV), a measure of vascular NO activity. In part 2, we performed an analysis of data pooled from six separate clinical trials of a medical food designed to enhance vascular NO activity in individuals with CVD (n = 217 subjects representing 253 treatment periods) to determine the effect on SUA. RESULTS In part 1, of all risk factors tested, SUA was second only to age in correlation with FMV, accounting for 7% (p < 0.0001) of the variability in FMV. Both SUA and FMV were related to the degree of disease risk (p < 0.0001 and p = 0.00025 by analysis of variance, respectively). By multivariate analysis, SUA did not continue to contribute significantly to the determination of FMV. In part 2, enhancement of FMV (5.8 +/- 4 to 8.6 +/- 5, p < 0.0001) was associated with a decrease in SUA (5.5 +/- 1.5 to 5.0 +/- 1.5, p < 0.0001). There was no placebo effect on FMV or SUA. CONCLUSIONS These results suggest that the association of elevated SUA with CVD may be a consequence of an impairment of vascular NO activity. This may be owing to an ability of NO to modulate uric acid production through its influence on xanthine oxidase activity.

Impaired aerobic capacity in hypercholesterolemic mice: partial reversal by exercise training

The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised ([Formula: see text], n= 13; [Formula: see text], n = 7) and high-fat chow, sedentary ([Formula: see text], n = 6;[Formula: see text], n = 8) or exercised ([Formula: see text], n = 6;[Formula: see text], n = 7). Mice were trained on a treadmill 2 × 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake ( r = -0.35; P < 0.02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match[Formula: see text] (all P< 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold ( r = 0.36; P < 0.025) and maximal distance run ( r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

Cardiovascular effects of L-arginine.

Most of the known cardiovascular effects of L-arginine are exerted via its conversion to nitric oxide by nitric oxide synthase. Accumulating evidence indicates that supplemental administration of L-arginine is sufficient to restore endothelium-derived nitric oxide production in many disorders in which endothelium-derived nitric oxide production is altered. L-arginine may enhance nitric oxide production by competing as a substrate with an endogenous antagonist for nitric oxide synthase. In other cases, L-arginine may act by competing with molecular oxygen as a substrate so as to reduce the production of superoxide anion. It is likely that other mechanisms exist by which the nitric oxide synthase pathway can be perturbed. Regardless of the mechanism, a wide array of cardiovascular disorders characterized by endothelial dysfunction are reversible by L-arginine.

Clinical study: endothelial functionUric acid is closely linked to vascular nitric oxide activity: Evidence for mechanism of association with cardiovascular disease☆

OBJECTIVES The study was undertaken to determine whether the mechanism of association of elevated serum uric acid level (SUA) with cardiovascular disease (CVD) is secondary to a common link with vascular nitric oxide (NO) activity. BACKGROUND Epidemiologic studies demonstrate an association of elevated SUA with CVD. The mechanism of this association is unknown, but both may be linked via an impairment in vascular NO activity. To examine this, we determined the relationship of SUA to vascular NO activity and to CVD risk. We then determined the effect of enhancing vascular NO activity on SUA. METHODS In part 1, individuals with various degrees of CVD (n = 458) were surveyed and underwent measurement of flow-mediated brachial artery vasodilation (FMV), a measure of vascular NO activity. In part 2, we performed an analysis of data pooled from six separate clinical trials of a medical food designed to enhance vascular NO activity in individuals with CVD (n = 217 subjects representing 253 treatment periods) to determine the effect on SUA. RESULTS In part 1, of all risk factors tested, SUA was second only to age in correlation with FMV, accounting for 7% (p < 0.0001) of the variability in FMV. Both SUA and FMV were related to the degree of disease risk (p < 0.0001 and p = 0.00025 by analysis of variance, respectively). By multivariate analysis, SUA did not continue to contribute significantly to the determination of FMV. In part 2, enhancement of FMV (5.8 +/- 4 to 8.6 +/- 5, p < 0.0001) was associated with a decrease in SUA (5.5 +/- 1.5 to 5.0 +/- 1.5, p < 0.0001). There was no placebo effect on FMV or SUA. CONCLUSIONS These results suggest that the association of elevated SUA with CVD may be a consequence of an impairment of vascular NO activity. This may be owing to an ability of NO to modulate uric acid production through its influence on xanthine oxidase activity.

Endothelial dysfunction in hypercholesterolemia is reversed by a nutritional product designed to enhance nitric oxide activity.

The objective of this study was to investigate the vascular andbiochemical effects of a formulated product in the form of a bar enrichedwith a combination of nutrients known to enhance the synthesis oractivity of endothelium-derived nitric oxide (EDNO). Individuals withhypercholesterolemia manifest impaired flow-mediated vasodilation, which islargely due to a reduction in EDNO activity. Oral supplementation withlarge amounts (6–21 g/day) of L-arginine, the precursor of EDNO,have been shown to improve endothelium-mediated vasodilation inhypercholesterolemia. Such large doses are effective but may be impracticalto take in capsule form. Accordingly, we have developed a nutrient barenriched with L-arginine as well as other ingredients that additivelyenhance EDNO activity. A pilot study in 41 hypercholesterolemic individualsindicated that the bar was well tolerated, had no adverse effects on serumchemistries or lipid profile, and normalized endothelial vasodilatorfunction. To definitively determine if the nutrient bar normalizesendothelial function, a double-blind, placebo-controlled study wasperformed. Flow-mediated endothelium-dependent vasodilation was assessedby high-resolution ultrasonography before and after 1 week of bar use(2 bars/day) in an additional group of 43 volunteer subjects (57 ±10 years old; 22 men, 21 women) with hypercholesterolemia. Subjectsmanifested an impaired flow-mediated vasodilation before theintervention. Vasodilator function in the active bar group improved towithin a normal range (6.5 ± 3% before to10 ± 5% after, P = 0.02; normal, 12± 3%) and was significantly better (P < 0.01)than in the placebo bar group (7.1 ± 3% before to6.7 ± 4% after). These findings reveal that use of a nutrientbar designed to enhance EDNO activity improves flow-mediatedendothelium-dependent vasodilation in hypercholesterolemicindividuals.

MODULATION OF THE NITRIC OXIDE SYNTHASE PATHWAY IN ATHEROSCLEROSIS

Atherosclerosis is the leading cause of mortality in the developed world. In the United States alone there are approximately 700000 deaths per year attributable to coronary artery disease, stroke and peripheral vascular diseases (National Center for Health Statistics, 1994). Although national data demonstrate an increase in coronary artery disease prevalence since 1970 (Morano, 1996), there has been a significant decline in mortality from atherosclerosis over the past three decades. Much of this decline may be attributable to risk factor modification. Intriguingly, many of the risk factors for atherosclerosis (hypercholesterolaemia, hypertension, homocysteinaemia, diabetes mellitus and exposure to tobacco) are associated with a reduced elaboration of nitric oxide by the endothelium (endothelium-derived nitric oxide (EDNO)) (Luscher, Raij & Vanhoutte, 1987; Celermajer et al. 1993; Wu & Meininger, 1995). The result is a perturbation in the normal functions of the endothelium to maintain appropriate vessel calibre and resist the development of atherosclerosis. This endothelial impairment occurs well before any structural changes of atherogenesis are detected. Indeed when otherwise normal vessels are exposed in vitro to oxidized lipoproteins, an impairment in EDNO-dependent vasodilatation can be detected within minutes. It is well documented that risk factor modification has reduced the incidence of morbidity and mortality due to cardiovascular disease (Pasternak, Grundy, Levy & Thompson, 1996).

Hypercholesterolemia Impairs Exercise Capacity in Mice

Abstract We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study, we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction. Eight-week-old wild type (n = 123) and apoE knockout (n = 79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding with regular or high-fat diets. At various ages, the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NOx) activity, urinary excretion of NOx, running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured. At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the four cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity. In conclusion, aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.

Nitric Oxide and Vascular Disease

Publisher Summary Nitric oxide (NO) is a potent regulator of vessel tone, structure, and interaction with circulating blood elements. In healthy individuals, the vasodilation induced by NO enhances blood flow by increasing vascular compliance and reducing vascular resistance. NO inhibits the proliferation of vascular smooth muscle cells, and prevents adherence of platelets and leukocytes to the vessel wall. It also plays an important role in angiogenesis. In individuals with risk factors for atherosclerosis, NO activity and/or synthesis are impaired. The impairment is multifactorial and dependent upon associated conditions and the stage of vascular disease, but the end result is pathophysiological increases in vascular resistance and changes in vascular structure that result in atherosclerosis, medial hypertrophy, myointimal hyperplasia, and/or thrombus formation, as well as impaired angiogenic response to occlusive arterial disease. One of the derangements of the NO synthase pathway that contributes to these pathophysiologies is competitive inhibition of NO synthesis by the endogenous antagonist, asymmetric dimethylarginine. New insights into the mechanisms of this derangement may lead to a new therapeutic avenue in the treatment of vascular disease.