John P. Cooke

Asymmetric Dimethylarginine (ADMA): A Novel Risk Factor for Endothelial Dysfunction Its Role in Hypercholesterolemia

BACKGROUND Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase. Because endothelial NO elaboration is impaired in hypercholesterolemia, we investigated whether plasma concentrations of ADMA are elevated in young, clinically asymptomatic hypercholesterolemic adults. We further studied whether such elevation of ADMA levels was correlated with impaired endothelium-dependent, NO-mediated vasodilation and urinary nitrate excretion. In a randomized, double-blind, placebo-controlled study, we investigated whether these changes could be reversed with exogenous L-arginine. METHODS AND RESULTS We measured plasma levels of L-arginine, ADMA, and symmetrical dimethylarginine (SDMA) by high-performance liquid chromatography in 49 hypercholesterolemic (HC) and 31 normocholesterolemic (NC) humans. In 8 HC subjects, endothelium-dependent forearm vasodilation was assessed before and after an intravenous infusion of L-arginine or placebo and compared with 8 NC control subjects. ADMA levels were significantly elevated by >100% (2.17+/-0.15 versus 1.03+/-0.09 micromol/L; P<0.05) in HC subjects compared with NC adults. L-Arginine levels were similar, resulting in a significantly decreased L-arginine/ADMA ratio in HC subjects (27.7+/-2.4 versus 55. 7+/-5.4; P<0.05). In 8 HC subjects, intravenous infusion of L-arginine significantly increased the L-arginine/ADMA ratio and normalized endothelium-dependent vasodilation and urinary nitrate excretion. ADMA levels were inversely correlated with endothelium-mediated vasodilation (R=0.762, P<0.01) and urinary nitrate excretion rates (R=0.534, P<0.01). CONCLUSIONS We find that ADMA is elevated in young HC individuals. Elevation of ADMA is associated with impaired endothelium-dependent vasodilation and reduced urinary nitrate excretion. This abnormality is reversed by administration of L-arginine. ADMA may be a novel risk factor for endothelial dysfunction in humans.

Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans.

The effect of hypercholesterolemia on vascular function was studied in humans. To eliminate the potential confounding effects of atherosclerosis, vascular reactivity was measured in the forearm resistance vessels of 11 normal subjects (serum LDL cholesterol = 111 +/- 7 mg/dl) and 13 patients with hypercholesterolemia (serum LDL cholesterol = 211 +/- 19 mg/dl, P less than 0.05). Each subject received intrabrachial artery infusions of methacholine, which releases endothelium-derived relaxant factor, and nitroprusside which directly stimulates guanylate cyclase in vascular smooth muscle. Maximal vasodilatory potential was determined during reactive hyperemia. Vasoconstrictive responsiveness was examined during intra-arterial phenylephrine infusion. Forearm blood flow was determined by venous occlusion plethysmography. Basal forearm blood flow in normal and hypercholesterolemic subjects was comparable. Similarly, reactive hyperemic blood flow did not differ between the two groups. In contrast, the maximal forearm blood flow response to methacholine in hypercholesterolemic subjects was less than that observed in normal subjects. In addition, the forearm blood flow response to nitroprusside was less in hypercholesterolemic subjects. There was no difference in the forearm vasoconstrictive response to phenylephrine in the two groups. Thus, the vasodilator responses to methacholine and nitroprusside were blunted in patients with hypercholesterolemia. We conclude that in humans with hypercholesterolemia, there is a decreased effect of nitrovasodilators, including endothelium-derived relaxing factor, on the vascular smooth muscle of resistance vessels.

L-arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans.

Endothelium-dependent vasodilation is impaired in hypercholesterolemia, even before the development of atherosclerosis. The purpose of this study was to determine whether infusion of L-arginine, the precursor of the endothelium-derived relaxing factor, nitric oxide, improves endothelium-dependent vasodilation in hypercholesterolemic humans. Vascular reactivity was measured in the forearm resistance vessels of 11 normal subjects (serum LDL cholesterol = 2.76 +/- 0.10 mmol/liter) and 14 age-matched patients with hypercholesterolemia (serum LDL cholesterol = 4.65 +/- 0.36 mmol/liter, P < 0.05). The vasodilative response to the endothelium-dependent vasodilator, methacholine chloride, was depressed in the hypercholesterolemic group, whereas endothelium-independent vasodilation, induced by nitroprusside, was similar in each group. Intravenous administration of L-arginine augmented the forearm blood flow response to methacholine in the hypercholesterolemic individuals, but not in the normal subjects. L-arginine did not alter the effect of nitroprusside in either group. D-arginine had no effect on forearm vascular reactivity in either group. It is concluded that endothelium-dependent vasodilation is impaired in hypercholesterolemic humans. This abnormality can be improved acutely by administration of L-arginine, possibly by increasing the synthesis of endothelium-derived relaxing factor.

Endogenous Nitric Oxide Synthase Inhibitor A Novel Marker of Atherosclerosis

BACKGROUND Exposure to risk factors such as hypertension or hypercholesterolemia decreases the bioavailability of endothelium-derived nitric oxide (NO) and impairs endothelium-dependent vasodilation. Recently, a circulating endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), has been detected in human plasma. The purpose of this study was to examine the relationship between plasma ADMA and atherosclerosis in humans. METHODS AND RESULTS Subjects (n=116; age, 52+/-1 years; male:female ratio, 100:16) underwent a complete history and physical examination, determination of serum chemistries and ADMA levels, and duplex scanning of the carotid arteries. These individuals had no symptoms of coronary or peripheral artery disease and were taking no medications. Univariate and multivariate analyses revealed that plasma levels of ADMA were positively correlated with age (P<0.0001), mean arterial pressure (P<0.0001), and Sigma glucose (an index of glucose tolerance) (P=0.0006). Most intriguingly, stepwise regression analysis revealed that plasma ADMA levels were significantly correlated to the intima-media thickness of the carotid artery (as measured by high-resolution ultrasonography). CONCLUSIONS This study reveals that plasma ADMA levels are positively correlated with risk factors for atherosclerosis. Furthermore, plasma ADMA level is significantly correlated with carotid intima-media thickness. Our results suggest that this endogenous antagonist of NO synthase may be a marker of atherosclerosis.

Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis.

We provide anatomic and functional evidence that nicotine induces angiogenesis. We also show that nicotine accelerates the growth of tumor and atheroma in association with increased neovascularization. Nicotine increased endothelial-cell growth and tube formation in vitro, and accelerated fibrovascular growth in vivo. In a mouse model of hind-limb ischemia, nicotine increased capillary and collateral growth, and enhanced tissue perfusion. In mouse models of lung cancer and atherosclerosis, we found that nicotine enhanced lesion growth in association with an increase in lesion vascularity. These effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant. The endothelial production of nitric oxide, prostacyclin and vascular endothelial growth factor might have a role in these effects.

Novel Mechanism for Endothelial Dysfunction Dysregulation of Dimethylarginine Dimethylaminohydrolase

BACKGROUND Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA. METHODS AND RESULTS ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity. CONCLUSIONS These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit.

The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.

Impaired nitric oxide synthase pathway in diabetes mellitus: Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase

Background—An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM. Methods and Results—Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (1.33±0.31 versus 0.48±0.08 &mgr;mol/L;P <0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P <0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-1) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol–conjugated superoxide dismutase (22 U/mL) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis. Conclusions—A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.

Does ADMA Cause Endothelial Dysfunction

Asymmetric dimethylarginine (ADMA) is an endogenous and competitive inhibitor of nitric oxide synthase. Plasma levels of this inhibitor are elevated in patients with atherosclerosis and in those with risk factors for atherosclerosis. In these patients, plasma ADMA levels are correlated with the severity of endothelial dysfunction and atherosclerosis. By inhibiting the production of nitric oxide, ADMA may impair blood flow, accelerate atherogenesis, and interfere with angiogenesis. ADMA may be a novel risk factor for vascular disease.

Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.

Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide.