Cardiovascular complications are the major cause of

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1 Impaired Nitric Oxide Synthase Pathway in Diabetes Mellitus Role of Asymmetric Dimethylarginine and Dimethylarginine Dimethylaminohydrolase Ken Y. Lin; Akira Ito, MD, PhD; Tomoko Asagami, MD, PhD; Philip S. Tsao, PhD; Shanthi Adimoolam, PhD; Masumi Kimoto, PhD; Hideaki Tsuji, PhD; Gerald M. Reaven, MD; John P. Cooke, MD, PhD 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 ( versus 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. (Circulation. 2002;106: ) Key Words: nitric oxide endothelium oxidative stress Cardiovascular complications are the major cause of mortality and morbidity for the 135 million individuals worldwide afflicted by type 2 diabetes mellitus (DM). 1 3 Endothelial dysfunction is a common feature in diabetic patients 4 6 and may contribute to cardiovascular morbidity. 7 9 Mechanisms of diabetes-induced endothelial dysfunction include the production of prostanoid vasoconstrictors and the increased oxidative degradation of NO. 10,11 Deficiency of NO increases vascular resistance and promotes atherogenesis. 12 In addition to its increased oxidative degradation, another possible mechanism for NO deficiency and cardiovascular morbidity is reduced NO synthesis caused by asymmetric dimethylarginine (ADMA). 13 ADMA is an endogenous competitive inhibitor of NO synthase (NOS). 14 This modified amino acid is derived from proteins that have been posttranslationally methylated and subsequently hydrolyzed. 15 ADMA is in part cleared by renal excretion. 16 Reduced clearance of ADMA in renal failure is associated with endothelial vasodilator dysfunction, reversible by administration of L-arginine 14,17 or by dialysis, which removes plasma ADMA. 18 However, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) accounts for most of the clearance of ADMA. 19 DDAH metabolizes ADMA to L-citrulline and dimethylamine. 20 ADMA is elevated to a level that can significantly inhibit NOS activity in individuals with hypercholesterolemia, hypertension, hyperhomocyst(e)inemia, tobacco exposure, and hyperglycemia We have proposed that in each of these conditions, the elevation in ADMA is a result of oxidative stress, which impairs the ability of DDAH to metabolize ADMA. 21,24 Hyperglycemia can elevate intracellular oxidative stress through multiple mechanisms. 25,26 Accordingly, we tested the hypothesis that hyperglycemia-induced oxidative stress impairs DDAH activity, leading to an elevation of ADMA and inhibition of endothelium-derived synthesis of NO. Methods Metabolic Studies Ten male Sprague-Dawley rats (Harlan Sprague Dawley Inc, Indianapolis, Ind) were assigned to either normal chow (control, n 5) or Received March 7, 2002; revision received May 23, 2002; accepted May 24, From the Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, Calif (K.Y.L., A.I., T.A., P.S.T., S.A., G.M.R., J.P.C.), and the Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama, Japan (M.K., H.T.). Correspondence to John P. Cooke, MD, PhD, Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA john.cooke@stanford.edu 2002 American Heart Association, Inc. Circulation is available at DOI: /01.CIR

2 988 Circulation August 20, 2002 high-fat diet (60% fat; Harlan Teklad) and streptozotocin (STZ; 35 mg/kg) injection (non insulin-dependent diabetes mellitus, NIDDM, n 5). The latter diet, combined with a low dose of STZ (to induce a moderate pancreatic injury), is an established model of NIDDM. 27 After 4 weeks, rats were killed after 12 hours of fasting, and blood was collected for determination of plasma glucose (in mg/dl), 28 insulin (in U/mL), 29 triglyceride (in mg/dl), 30 and ADMA (in mol/l) concentrations. These protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. ADMA Measurement Concentrations of plasma L-arginine, symmetric DMA (SDMA), and ADMA were measured by high-performance liquid chromatography (HPLC) as previously described, 21 with the following modification. A gradient mobile phase, consisting of potassium phosphate buffer (ph 6.5) and HPLC graded methanol, was used. The HPLC process is initiated with a mobile phase containing 15% methanol, which is increased linearly to 25% over the first 20 minutes, and then to 27% over the subsequent 15 minutes. This modification reduces peak broadening, enhances separation, and decreases procedure time. Western Blotting After removal, abdominal aortas were rapidly rinsed and placed on ice in lysis buffer containing 10 mmol/l sodium phosphate buffer, 1% Igepal CA-630 (Sigma), trypsin inhibitor, leupeptin, pepstatin, and antipain (each 10 mg/l). Tissues were homogenized and centrifuged. Protein concentration of supernatants was measured by Lowry assay. Hybond enhanced chemiluminescence nitrocellulose membranes with proteins transferred from SDS-PAGE gels were incubated with a mouse monoclonal antibody (1:500) against purified human DDAH-1 15 in Tris-buffered saline containing 4% nonfat milk and probed with a peroxidase-conjugated anti-mouse antibody (1:1000), detected by enhanced chemiluminescence. Cell Culture HMEC-1, a human dermal microvascular endothelial cell line, 31 was grown in DMEM (Gibco BRL) containing 5.5 mmol/l glucose, 20% FBS, 84 mg/l L-arginine, 110 mg/l sodium pyruvate, 1% glutamine, and 1% gentamicin and incubated at 37 C in a humidified atmosphere of 5% CO 2. Cells were seeded at a density of 5000 cells/cm 2 and grown to 80% confluence (3 to 4 days). Subconfluent (80%) HMEC-1s were washed twice with PBS (ph 7.0) and cultured in control (5.5 mmol/l D-glucose), osmotic-control (control with 20 mmol/l mannose), or high-glucose (25.5 mmol/l) medium containing 5% FBS, with or without polyethylene glycol conjugated superoxide dismutase (PEG-SOD; 22 U/mL). Cells and media were harvested after 48 hours for DMA concentrations. DDAH activity was determined in confluent cells. Rat vascular smooth muscle cells (RVSMCs) were isolated from abdominal aortas after 1 hour of incubation in RPMI containing collagenase (2 mg/ml, type IV) and elastase (0.4 mg/ml pancreatic) at 37 C. Cells were then grown in control, osmotic control, or high-glucose medium for 3 passages. DDAH activity and expression were determined on confluence of the third passage. All measurements were performed in triplicate, and cells were counted using a hemocytometer. Rat Tissue and Cell DDAH Activity Assay DDAH activity was assayed by determining L-citrulline formation in tissue homogenates as previously described. 32 As negative controls, tissue homogenates were boiled for 10 minutes to inactivate the enzyme. Background values obtained were subtracted from the experimental data to provide the genuine DDAH activity. One unit of the enzyme was defined as the amount that catalyzed the formation of 1 mol L-citrulline from ADMA per minute at 37 C. Figure 1. Effect of hyperglycemia on plasma ADMA levels in rats. High-fat diet (administered for 4 weeks) and STZ (35 mg/kg) were given to create NIDDM rats. Plasma ADMA ( mol/l) was measured by HPLC. *P 0.05 vs control rats; n 5. Endothelial DDAH Activity Assay Hitherto, DDAH activity assay has depended on measuring the formation of L-citrulline. However, DDAH is not the sole source of citrulline. NOS, for instance, also produces citrulline. Accordingly, we circumvented this problem by directly measuring the amount of ADMA metabolized by the enzyme. In an ice bath, cell lysates were divided into 2 groups, and ADMA was added (final concentration 500 mol/l). To inactivate DDAH, 30% sulfurosalicylic acid was immediately added to 1 experimental group. This group provided a baseline of 0% DDAH activity. The other lysate was incubated at 37 C for 2 hours before the addition of 30% sulfurosalicylic acid. The ADMA level in each group was measured by HPLC as described above. The difference in ADMA concentration between the 2 groups reflected the DDAH activity. For every experiment, DDAH activity of HMEC-1s exposed to normal glucose levels is defined as 100%, and DDAH activities in other conditions were expressed as percentages of the ADMA metabolized compared with the control. Determination of cgmp Levels in Endothelial Cells Subconfluent HMEC-1s cultured in medium containing normal glucose level (5.5 mmol/l, with 20% FBS) were exposed to the experimental conditions for 48 hours. To assess synthesis of bioactive NO, HMEC-1s were stimulated with calcium ionophore (A23187, 10 mol/l) after 15 minutes of preincubation with zaprinast (30 mol/l; a type V phosphodiesterase inhibitor). HMEC-1s were incubated with A23187 and zaprinast for 2 hours, after which the media were aspirated. Intracellular cgmp was assayed by electroimmunoassay according to the protocol provided by the manufacturer (Amersham). Statistical Analysis Data are expressed as mean SEM. Comparisons of multiple means were made by ANOVA followed by a Fisher s protected least significant difference test. Pearson correlation coefficients were calculated when indicated. Comparisons between 2 groups were made by Students t test for unpaired observations. Probability values of P 0.05 denote statistical significance. Results Metabolic Studies Significant increases in both plasma glucose and triglyceride concentrations were observed in the NIDDM rats (glucose: versus mg/dl in the control; triglyceride: versus mg/dl; P 0.05 in both cases). Insulin levels were not significantly different between the NIDDM and the control rats ( versus U/dL). Plasma ADMA concentrations were significantly elevated in NIDDM rats (Figure 1).

3 Lin et al Hyperglycemia Elevates ADMA 989 Figure 2. A, Abdominal aortic DDAH expression in NIDDM and control rats. Abdominal aortas from control (n 5) and NIDDM (n 5) rats were removed for Western analysis. Amount of proteins in each lane was 5 g. Protein size markers are shown on left. Molecular mass of DDAH protein is 34 kda. Aortic DDAH expression was unaffected by NIDDM. B, Abdominal aortic DDAH activity in NIDDM and control rats. One unit of enzyme activity was defined as amount catalyzing formation of 1 mol L-citrulline from ADMA per minute at 37 C. *P 0.05 vs control rats; n 5. Aortic DDAH activity was reduced in NIDDM. Aortic DDAH Expression and Activity No difference in aortic DDAH expression was observed between control and NIDDM rats (Figure 2A). In contrast, aortic DDAH activity was significantly reduced in NIDDM rats ( versus U/g protein in control, P 0.05; Figure 2B). In addition, aortic DDAH activity negatively correlated with the levels of plasma ADMA (Figure 3). DDAH Activity and Expression in Cultured Vascular Cells Protein expression and activity were assessed in RVSMCs incubated with a normal or a high level of glucose. The high-glucose condition did not change DDAH expression (Figure 4A). However, DDAH activity was significantly reduced in RVSMCs exposed to high glucose (Figure 4B). Like VSMCs, HMEC-1s exposed to high-glucose conditions manifested a reduced DDAH activity (Figure 5). Mannose had no effect on endothelial DDAH activity (osmotic control, Figure 5). Reduction of endothelial DDAH activity in cells exposed to high glucose was reversed by addition of PEG-SOD. Figure 4. A, Vascular DDAH expression in cultured RVSMCs. Isolated from aortas, RVSMCs were grown in control (5.5 mmol/l D-glucose) and high-glucose (25.5 mmol/l glucose) medium for 3 passages. Expression of DDAH was not altered by high-glucose conditions. B, Effects of high-glucose condition on vascular DDAH activity. DDAH activity in RVSMCs was determined on confluence of third passage grown in medium with normal (Control) or high level of glucose (High Glucose) medium. One unit of enzyme was defined as amount catalyzing formation of 1 mol L-citrulline from ADMA per minute at 37 C. DDAH activity was impaired by high-glucose condition. Endothelial ADMA and SDMA Elaboration The reduction in endothelial DDAH activity was associated with an elevation of ADMA concentration in the medium. Glucose dose-dependently elevated ADMA accumulation over 48 hours. Coincubation with the antioxidant PEG-SOD blocked the effect of glucose on ADMA accumulation (Table). Mannose had no effect on ADMA ( versus mol/l, mannose condition versus control, data not shown). SDMA accumulation in the medium was unaf- Figure 3. Significant negative correlation between aortic DDAH activity and plasma ADMA in control and NIDDM rats. Figure 5. Effects of high-glucose condition on endothelial DDAH activity. Cultured HMEC-1s were exposed to control (5.5 mmol/l glucose), osmotic control (5.5 mmol/l glucose and 20 mmol/l mannose), high glucose (25.5 mmol/l glucose), or antioxidant (25.5 mmol/l glucose with 22 U/mL of PEG-SOD). DDAH activity in cell lysates from each condition is expressed as percentage of amount of ADMA metabolized by control, which is defined as 100% for every experiment. *P 0.05 vs control (normal glucose). DDAH activity was impaired by highglucose condition, an effect that was prevented by coincubation with antioxidant PEG-SOD.

4 990 Circulation August 20, 2002 Effect of Glucose and/or PEG-SOD on Human Endothelial ADMA Elaboration Glucose Concentration, ADMA SDMA mmol/l ( PEG-SOD) ( PEG-SOD) ( PEG-SOD) ( PEG-SOD) * * * Values are expressed as mol/l ADMA or SDMA per 10 7 cells. *P 0.05 from the control (5 mmol/l glucose); P 0.05 from corresponding control values (without PEG-SOD). fected by glucose concentration and was not altered by the addition of PEG-SOD (Table). Endothelial cgmp Levels To confirm the pathological relevance of elevated ADMA to the NOS pathway, endothelial cgmp levels were assayed and found to be significantly reduced in cells exposed to high glucose (Figure 6). Mannose did not alter endothelial cgmp level. Addition of PEG-SOD to the high-glucose medium restored cgmp production to a level comparable to that observed during the normal-glucose condition. Discussion The salient findings of the present investigation are as follows: (1) plasma ADMA concentrations are elevated in NIDDM rats and are associated with reduced DDAH activity; (2) prolonged exposure to high glucose decreases DDAH activity in cultured endothelial cells or VSMCs; (3) elevated glucose impairs DDAH activity; through this impairment, glucose dose-dependently elevates endothelial ADMA elaboration to reduce nitric oxide synthesis; and (4) these effects of elevated glucose are reversed by the intracellular antioxidant PEG-SOD. Vascular NO activity is reduced in diabetes, leading to impaired endothelium-dependent vasodilation 33 and elevated platelet aggregation. 34,35 It is possible that reduced NO Figure 6. Effects of high-glucose condition on endothelial cgmp elaboration. Subconfluent HMEC-1s were exposed to control (5.5 mmol/l glucose), osmotic control (5.5 mmol/l glucose and 20 mmol/l mannose), high glucose (25.5 mmol/l glucose), or antioxidant (25.5 mmol/l glucose with 22 U/mL of PEG-SOD) for 48 hours. Cell extracts were assayed for cgmp after 2 hours of incubation with zaprinast and calcium ionophore. *P 0.05 vs control (normal glucose). Stimulated accumulation of cgmp was reduced by high-glucose condition, an effect that was prevented by coincubation with antioxidant PEG-SOD. activity contributes to the increased cardiovascular morbidity observed in patients with diabetes. Of note, elevated levels of ADMA are predictive of carotid artery disease. 21 Furthermore, 2 recent studies indicate that plasma ADMA is an independent predictor of cardiovascular events and total mortality. 21,36 Previous studies have shown that exogenous ADMA in concentrations between 1 and 10 mol/l dose-dependently reduces endothelium-dependent NO-mediated vasodilation in isolated rat mesenteric vessels 37 and cerebral vessels, 38 inhibits NO production by cultured macrophages, 39 and increases endothelial adhesiveness to monocytes. 40 ADMA elevations in diabetic individuals have been reported previously. 21,23 Results from the present investigation are consistent with these observations. We found that plasma ADMA concentrations are elevated 3-fold in NIDDM rats. The increase of ADMA may have pathophysiological significance, because its concentration falls into the range shown to inhibit the activity of NOS. ADMA is generated from the hydrolysis of ubiquitous proteins containing methylated arginine residues. The nuclear protein arginine N-methyltransferase (protein methylase I) has been shown to methylate internal arginine residues in a variety of proteins. The methyl groups may be distributed symmetrically or asymmetrically to the guanidinium nitrogens of arginine, resulting in SDMA, N-monomethylarginine, and ADMA, with ADMA being the predominant isomer. 41 These methylated arginines are excreted in the urine. In addition, the metabolism of ADMA and N-monomethylarginine, but not SDMA, occurs via hydrolytic degradation to L-citrulline and dimethylamine by DDAH. We recently showed that reduced DDAH activity, but not expression, is responsible for plasma ADMA elevation in hypercholesterolemia and hyperhomocyst(e)inemia. 21,22 Similarly, in the present study, we found that NIDDM rats manifest reduced DDAH activity in the absence of any change in DDAH expression. DDAH activity of the aorta was significantly decreased in NIDDM rats (Figure 4, A and B). One caveat to note is that there are 2 isoforms of DDAH. Whereas our antibody revealed no change in DDAH-1 expression, it is possible that there was an undetected change in DDAH-2 expression that could explain in part the reduction in DDAH activity. Diabetes is a constellation of physiological abnormalities. A number of metabolic syndromes probably contribute to endothelial dysfunction. We decided to focus on the specific effect of high glucose on ADMA. The decline in DDAH activity was strongly associated with elevated ADMA levels in the plasma in vivo and in the conditioned medium in vitro. This effect occurred without any change in SDMA, which is not metabolized by DDAH. Hence, elevated glucose raises endothelial elaboration of ADMA by reducing its breakdown (by DDAH), not by increasing protein methylation. DDAH is an oxidant-sensitive enzyme. The sulfhydryl group of its cysteine 249 residue participates in the reactionintermediate formation by its nucleophilic attack of guanidinium carbon of ADMA. 20 Sulfhydryl-blocking agents such as p-chloromercuribenzoate and HgCl 2 are known to inhibit the activity of the enzyme. 41 DDAH dysfunction hence seems

5 Lin et al Hyperglycemia Elevates ADMA 991 plausible, especially in the setting of DM, in which hyperglycemia has been known to elevate oxidative stress. 25,26 Several pathways have been characterized to account for the increased production of free radicals in hyperglycemia. For instance, elevated glucose may activate the polyol pathway, leading to the oxidation of sorbitol to fructose, coupled by the reduction of NAD to NADH. 42,43 The increased ratio of NADH/NAD may in turn promote free-radical production by activating xanthine oxidase and inactivating intracellular and extracellular SOD. It is possible that these processes contribute to reduced DDAH activity. We hypothesized that glucose-induced oxidative stress would impair DDAH activity. This hypothesis was supported by the observation that the antioxidant PEG-SOD restored DDAH activity in the high-glucose condition, with a corresponding reduction in endothelial ADMA accumulation. We assessed the effect of high glucose on the NOS pathway by detecting endothelial cgmp production rather than by measuring the levels of NO x using chemiluminescence, because the former better reflects bioactive NO. Measurement of nitrate and nitrite levels by chemiluminescence or Griess reaction reflects NO metabolites as well as bioactive NO. Although NO metabolites may be increased in diabetes, 44 bioactive NO is reduced, 7,8 as confirmed in this study. Furthermore, we observed that the antioxidant PEG- SOD reversed the effect of high glucose levels on cgmp production. This is not surprising, because the effect of PEG-SOD to scavenge O 2 radicals may enhance cgmp production at 2 levels: (1) by restoring the activity of DDAH and thus lowering ADMA and (2) by reducing the oxidative degradation of NO. ADMA and DDAH are widely distributed in tissues 16 and appear to play a seminal role in regulating NO synthesis in physiological and/or pathological states. In this study, we found that hyperglycemia elevates ADMA by impairing DDAH activity in vascular smooth muscle and the endothelium. The effect of hyperglycemia to impair DDAH activity is probably mediated by oxidative stress. This is a novel mechanism for diabetes-induced endothelial vasodilator dysfunction, which may contribute to the cardiovascular morbidity observed in patients with DM. Acknowledgments This study was supported in part by grants (to Dr Cooke) from the National Institutes of Health (R01-HL and PO1-AI-50153), from the Tobacco-Related Diseases Research Program of the University of California (7RT-0128), and from the Phillip Morris External Research program. Dr Cooke is an Established Investigator of the American Heart Association. Dr Ito is a recipient of a Japan Heart Foundation and a Bayer Yakuhin Research Grant Abroad. Dr Tsao is a recipient of a Scientist Development Grant from the American Heart Association. References 1. Nathan DM. Treating type 2 diabetes with respect. Ann Intern Med. 1999;130: Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 1999;22:S5 S Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications, I: diagnosis and classification of diabetes mellitus: provisional report of a WHO consultation. Diabet Med. 1998;15: Weis U, Turner B, Gibney J, et al. Long-term predictors of coronary artery disease and mortality in type 1 diabetes. QJM. 2001;94: Van de Ree MA, Huisman MV, de Man FH, et al. Impaired endothelium-dependent vasodilation in type 2 diabetes mellitus and the lack of effect of simvastatin. Cardiovasc Res. 2001;52: Dogra G, Rich L, Stanton K, et al. Endothelium-dependent and independent vasodilation studies at normoglycemia in type I diabetes mellitus with and without microalbuminuria. Diabetologia. 2001;44: Miyazaki H, Matsuoka H, Cooke JP, et al. Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation. 1999; 99: Schächinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000;101: Suwaidi JA, Hamasaki S, Higano ST, et al. Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. Circulation. 2000;101: Tesfamariam B, Cohen RA. Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol. 1992;263: H321 H Tesfamariam B, Brown ML, Cohen RA. 15-Hydroxyeicosatetraenoic acid and diabetic endothelial dysfunction in rabbit aorta. J Cardiovasc Pharmacol. 1995;25: Cooke JP, Dzau VJ. Nitric oxide synthase: role in the genesis of vascular disease. Annu Rev Med. 1997;48: Valkonen V-P, Päivä H, Salonen JT, et al. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet. 2001; 358: Vallance P, Leone A, Calver A, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet. 1992; 339: MacAllister RJ, Parry H, Kimoto M, et al. Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase. Endothelium. 1993;1: Kimoto M, Whitley GS, Tsuji H, et al. Detection of N G,N G - dimethylarginine dimethylaminohydrolase in human tissues using a monoclonal antibody. J Biochem. 1995;117: Kakimoto Y, Akazawa S. Isolation and identification of N G,N G - and N G,N G -dimethylarginine, N -mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl- -hydroxylysine from human urine. J Biol Chem. 1970;245: Kielstein J, Boeger R, Bode-Boeger S, et al. Asymmetric dimethylarginine plasma concentrations differ in patients with end-stage renal disease: relationship to treatment method and atherosclerotic disease. J Am Soc Nephrol. 1999;10: McDermott. Studies on the catabolism of N G -methylarginine, N G,N G - dimethylarginine and N G,N G -dimethylarginine in the rabbit. Biochem J. 1976;154: Murray-Rust J, Leiper J, McAlister M, et al. Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase. Nat Struct Biol. 2001;8: Boger RH, Bode-Boger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation. 1998;98: Stühlinger MC, Tsao PS, Her J-H, et al. Homocysteine impairs the NO synthase pathway: role of ADMA. Circulation. 2001;104: Fard A, Tuck CH, Donis JA, et al. Acute elevations of plasma asymmetric dimethylarginine and impaired endothelial function in response to a high-fat meal in patients with type 2 diabetes. Arterioscler Thromb Vasc Biol. 2000;20: Ito A, Tsao PS, Adimoolam S, et al. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation. 1999;24: Powell LA, Nally SM, McMaster D, et al. Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions. Free Radic Biol Med. 2001;31: Duckworth WC. Hyperglycemia and cardiovascular disease. Curr Atheroscler Rep. 2001;3: Luo J, Quan J, Tsai J, et al. Nongenetic mouse models of non-insulin dependent diabetes mellitus. Metabolism. 1998;47:

6 992 Circulation August 20, Kadish AH, Little RL, Sternberg JC. A new and rapid method for determination of glucose by measurement of rate of oxygen consumption. Clin Chem. 1968;14: Hales CN, Randle PJ. Immunoassay of insulin with insulin antibody precipitate. Biochem J. 1963;88: Washfield AW. Triglyceride determination after enzymatic hydrolysis. In: Bergmayer HW, ed. Methods in Enzymatic Analysis. San Diego, Calif: Academic Press; Ades EW, Candal FJ, Swerlick RA, et al. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 1992;99: Ogowa T, Kimoto M, Sasaoka K. Purification and properties of a new enzyme, N G,N G -dimethylarginine dimethylaminohydrolase, from rat kidney. J Biol Chem. 1989;264: Giugliano D, Ceriello A. Oxidative stress and diabetic vascular complications. Diabet Care. 1996;19: Tsao PS, Theilmeier G, Singer AH, et al. L-Arginine attenuates platelet reactivity in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14: Shukla SD, Paul A, Klachko DM. Hypersensitivity of diabetic human platelets to platelet activating factor. Thromb Res. 1992;66: Zoccali C, Bode-Boger S, Mallamaci F, et al. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet. 2001;358: Kurose I, Wolf R, Grisham MB, et al. Effects of an endogenous inhibitor of nitric oxide synthesis on postcapillary venules. Am J Physiol. 1995; 268:H2224 H Faraci FM, Brian JE, Heistad DD. Response of cerebral blood vessels to an endogenous inhibitor of nitric oxide synthase. Am J Physiol. 1995; 269:H1522 H Vallance P, Leone AM, Calver A, et al. Endogenous dimethylarginine as an inhibitor of nitric oxide synthase. J Cardiovasc Pharmacol. 1992;20: Boger RH, Bode-Boger SM, Tsao PS, et al. An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes. J Am Coll Cardiol. 2000;36: Ogawa T, Kimoto M, Sasaoka K. Occurrence of a new enzyme catalyzing the direct conversion of N G,N G -dimethyl-l-arginine to L-citrulline in rats. Biochem Biophys Res Commun. 1987;148: Williamson JR, Chang K, Frangos M, et al. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes. 1993;42: Tilton RG, Chang K, Nyengaard JR, et al. Inhibition of sorbitol dehydrogenase: effects on vascular and neural dysfunction in streptozotocininduced diabetic rats. Diabetes. 1995;44: Cosentino F, Hishikawa K, Katusic ZS, et al. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation. 1997;96:25 28.