Restoring vascular nitric oxide formation by l-arginine improves the symptoms of intermittent claudication in patients with peripheral arterial occlusive disease

AbstractBackground. Administration of l-arginine improves nitric oxide (NO) formation and endothelium-dependent vasodilation in atherosclerotic patients.Objectives. We investigated in this double-blind, controlled study whether prolonged intermittent infusion therapy with l-arginine improves the clinical symptoms of patients with intermittent claudication, as compared with the endothelium-independent vasodilator prostaglandin E1, and control patients.Methods. Thirty-nine patients with intermittent claudication were randomly assigned to receive 2 × 8 g l-arginine/day, or 2 × 40 μg prostaglandin E1(PGE1)/day or no hemodynamically active treatment, for 3 weeks. The pain-free and absolute walking distances were assessed on a walking treadmill at 3 km/h, 12% slope, and NO-mediated, flow-induced vasodilation of the femoral artery was assessed by ultrasonography at baseline, at 1, 2 and 3 weeks of therapy and 6 weeks after the end of treatment. Urinary nitrate and cyclic guanosine-3′, 5′-monophosphate (GMP) were assessed as indices of endogenous NO production.Results. l-Arginine improved the pain-free walking distance by 230 ± 63% and the absolute walking distance by 155 ± 48% (each p < 0.05). Prostaglandin E1improved both parameters by 209 ± 63% and 144 ± 28%, respectively (each p < 0.05), whereas control patients experienced no significant change. l-Arginine therapy also improved endothelium-dependent vasodilation in the femoral artery, whereas PGE1had no such effect. There was a significant linear correlation between the l-arginine/asymmetric dimethylarginine (ADMA) ratio and the pain-free walking distance at baseline (r = 0.359, p < 0.03). l-Arginine treatment elevated the plasma l-arginine/ADMA ratio and increased urinary nitrate and cyclic GMP excretion rates, indicating normalized endogenous NO formation. Prostaglandin E1therapy had no significant effect on any of these parameters. Symptom scores assessed on a visual analog scale increased from 3.51 ± 0.18 to 8.3 ± 0.4 (l-arginine) and 7.0 ± 0.5 (PGE1; each p < 0.05), but did not significantly change in the control group (4.3 ± 0.4).Conclusions. Restoring NO formation and endothelium-dependent vasodilation by l-arginine improves the clinical symptoms of intermittent claudication in patients with peripheral arterial occlusive disease

713-4 Inhibition of Vascular Superoxide Production in Hypercholesterolemic Rabbit Aorta by L-Arginine Contributes to Restored Endothelium-dependent Relaxation

Chronic oral administration of L-arginine (L-ARG) has been shown to enhance endothelial function in cholesterol (CHOL)-fed rabbits and to reduce atherogenesis. We investigated whether modulation of endogenous NO production (as assessed by urinary NO3-excretion) by L-ARG and the inhibitor of NO synthesis, L-NAME, affects vascular superoxide (O2-) production in hypercholesterolemic rabbits. Phorbol-myristate-acetate (PMA)-stimulated O2-production from isolated aortic rings was increased in rabbits given CHOL (+159±28%) or CHOL + L-NAME (+149±37%) as compared to controls (-22±7%), and endothelium-dependent relaxations by acetylcholine were diminished in both groups. In aortic rings from rabbits given CHOL + L-ARG, PMA-induced O2-production was restored to control levels (+14±17%; p&lt;0.05), and endothelium-dependent cholinergic relaxations were also partly restored. Urinary NO3-excretion decreased in all animals fed a CHOL-enriched diet (p&lt;0.01). As NO inactivated by O2-is also oxidized to NO3-, this indicates a decreased endothelial production of NO. NO3-excretion was further decreased by L-NAME (p&lt;0.05 vs. CHOL), and partly restored by L-ARG (p&lt;0.05). We conclude that both a decreased production of NO and an enhanced breakdown of NO by O2-contribute to the diminished biological activity of endothelial NO in hypercholesterolemia. L-ARG restores endothelial function by enhancing NO formation and by protecting NO from early breakdown by O2-

An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes

AbstractOBJECTIVESWe sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes.BACKGROUNDEndothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase.METHODSHuman umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2−) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-κB by electromobility gel shift assay.RESULTSIncubation of endothelial cells with ADMA (0.1 μM to 100 μM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 μM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2− to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-κB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody.CONCLUSIONSWe concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule

Indoleamine-2,3-dioxygenase activity in experimental human endotoxemia

Background: Excessive tryptophan metabolism to kynurenine by the rate-limiting enzyme endothelial indoleamine 2,3-dioxygenase 1 (IDO) controls arterial vessel relaxation and causes hypotension in murine endotoxemia. However, its relevance in human endotoxemia has not been investigated so far. We thus aimed to study changes in blood pressure in parallel with tryptophan and kynurenine levels during experimental endotoxemia in humans. Findings: Six healthy male volunteers were given E. coli lipopolysaccharide (LPS; 4 ng/kg) as a 1-min intravenous infusion. They had levels of soluble E-Selectin and soluble vascular cell adhesion molecule-1 as well as IDO activity assessed as the kynurenine-to-tryptophan plasma ratio by liquid chromatography-tandem mass spectrometry at various time points during a 24 h time course. During endotoxemia, IDO activity significantly increased, reaching peak levels at 8 h after LPS infusion (44.0 ± 15.2 vs. 29.4 ± 6.8 at baseline, P<0.0001). IDO activity correlated inversely with the development of hypotension as shown by random effects linear regression models. Finally, IDO activity exhibited a kinetic profile similar to that of soluble endothelial-specific adhesion molecules. Conclusions: LPS is a triggering factor for the induction of IDO in men. Our findings strongly support the concept that the induction of IDO in the vascular endothelium contributes to hypotension in human sepsis

A Novel Pathway for Metabolism of the Cardiovascular Risk Factor Homoarginine by alanine:glyoxylate aminotransferase 2

Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism - its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency

A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases

Supplementing essential amino acids with the nitric oxide precursor, l-arginine, enhances skeletal muscle perfusion without impacting anabolism in older men

Postprandial limb blood flow and skeletal muscle microvascular perfusion reduce with aging. Here we tested the impact of providing bolus essential amino acids (EAA) in the presence and absence of the nitric oxide precursor, l-Arginine (ARG), upon skeletal muscle blood flow and anabolism in older men. Healthy young (YOUNG: 19.7 ± 0.5 y, N = 8) and older men (OLD, 70 ± 0.8 y, N = 8) received 15 g EAA or (older only) 15 g EAA +3 g ARG (OLD-ARG, 69.2 ± 1.2 y, N = 8). We quantified responses in muscle protein synthesis (MPS; incorporation of 13C phenylalanine into myofibrillar proteins), leg and muscle microvascular blood flow (Doppler/contrast enhanced ultrasound (CEUS)) and insulin/EAA in response to EEA ± ARG. Plasma EAA increased similarly across groups but argininemia was evident solely in OLD-ARG (∼320 mmol, 65 min post feed); increases in plasma insulin (to ∼13 IU ml−1) were similar across groups. Increases in femoral flow were evident in YOUNG >2 h after feeding; these effects were blunted in OLD and OLD-ARG. Increases in microvascular blood volume (MBV) occurred only in YOUNG and these effects were isolated to the early postprandial phase (+45% at ∼45 min after feeding) coinciding with detectable arterio-venous differences in EAA reflecting net uptake by muscle. Increases in microvascular flow velocity (MFV) and tissue perfusion (MBV × MFV) occurred (∼2 h) in YOUNG and OLD-ARG, but not OLD. Postprandial protein accretion was greater in YOUNG than OLD or OLD-ARG; the latter two groups being indistinguishable. Therefore, ARG rescues aspects of muscle perfusion in OLD without impacting anabolic blunting, perhaps due to the “rescue” being beyond the period of active EAA-uptake