The endogenous methylarginines N G, N G -dimethyl-larginine

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1 Brief Review Endogenous Nitric Oxide Synthase Inhibitors in the Biology of Disease Markers, Mediators, and Regulators? Ben Caplin, James Leiper Abstract The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring, and modulating circulating asymmetric dimethylarginine alone is inadequate. This review outlines the basic biochemistry and physiology of endogenous methylarginines, examines both the experimental and observational evidence for a role in disease pathogenesis, and examines the potential for therapeutic regulation of these molecules. (Arterioscler Thromb Vasc Biol. 2012;32: ) Key Words: kidney nitric oxide nitric oxide synthase vascular biology asymmetric dimethylarginine The endogenous methylarginines N G, N G -dimethyl-larginine (asymmetric dimethylarginine; ADMA); N G, N G - dimethyl-l-arginine (symmetric dimethylarginine; SDMA); and N G -monomethyl-l-arginine (monomethylarginine; L-NMMA) are formed by the liberation of constituent methylated arginine residues from intracellular proteins. 2 These molecules are of interest as the enzymatic synthesis of the ubiquitous biological messenger nitric oxide (NO) is inhibited by a number of arginine analogues. It is now established, in vitro 3 and in vivo, 4 that micromolar concentrations of ADMA and L-NMMA can compete with the substrate L-arginine, reducing NO formation, whereas the isomer of ADMA and SDMA does not. The NO pathway has been of particular interest in cardiovascular disease once its identity with endothelium derived relaxing factor was demonstrated. 5 Specifically, there is strong evidence that NO plays a critical role in preatherogenic endothelial dysfunction. 6 Although now undergoing a degree of reappraisal, direct targeting of the NO pathway has proved to be of limited use to date. 7 NO donors can have substantial adverse effects such as headache along with the potential for production of free radicals and the development of nitrate tolerance, the precise mechanisms of which remain unclear. 8 Conversely, direct inhibition of NO synthesis in vasodilatory septic shock, a condition of pathological NO excess, has shown no survival benefit in randomized trials. 9 Targeting regulators of the NO pathway rather than increasing NO directly has been shown to be a successful approach as illustrated by the use of phosphodiesterase-5 inhibitors in pulmonary hypertension and erectile dysfunction. 10 This strategy might allow both tissue specific targeting of pharmacological actions as well as the potential to enhance or inhibit endogenous homeostatic mechanisms, eg, acting only where NO synthesis is dysregulated while preserving constitutive NO production. Furthermore, there is evidence that a number of vascular conditions are characterized by aberrant regulation of NO synthesis suggesting that some of these endogenous control mechanisms might not only provide potential therapeutic targets but also underlie pathogenesis of chronic cardiovascular diseases. The endogenous methylarginines are candidates as NO pathway regulators in the above respects. There is not only evidence that these molecules modulate NO synthesis in vivo, but also that this pathway has a primary role in the pathogenesis of disease. In addition, a degree of tissue/cell specificity of the enzymes controlling methylarginine levels along with potential for a key homeostatic role in NO synthesis makes this pathway an attractive target for therapeutic intervention. There have been a series of in vitro, in vivo, and clinical investigations with the aim of characterizing the pathological role of the endogenous methylarginines in human health and disease. Specifically, the role of the ADMA has been the focus of interest in cardiovascular conditions. However, the accumulating evidence implies that a simplistic strategy targeting circulating ADMA in chronic disease may not be successful. This article reviews the biology, summarizes the state of the field, details some of the controversies, and proposes avenues for further study. Received on: February 14, 2012; final version accepted on: March 14, From the Centre for Nephrology, UCL Medical School, London (B.C.); and Nitric Oxide Signalling Group, MRC Clinical Sciences Centre, London, UK (J.L.). The online-only Data Supplement is available with this article at Correspondence to Ben Caplin, Centre for Nephrology, UCL Medical School, Royal Free Campus 2nd Floor, Rowland Hill St, London NW3 2PF. b.caplin@ucl.ac.uk 2012 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at doi: /ATVBAHA

2 1344 Arterioscler Thromb Vasc Biol June 2012 Figure 1. The structure and metabolism of the endogenous methylarginines. A, The structure of L-arginine and the free endogenous methylarginines; asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethylarginine (L-NMMA), which are formed following the liberation of methylated arginines from proteins; B, Dimethylarginine dimethylaminohydrolase (DDAH) mediated hydrolysis of ADMA to form dimethylamine (DMA) and L-citrulline. L-NMMA also undergoes hydrolysis (not shown); C, Alanine-glyoxylate aminotransferase-2 (AGXT2) mediated deamination of ADMA to form asymmetric -keto- -dimethylguanidino valeric acid (ADGV). Both pyruvate and glyoxylate can act as the amino-acceptor in this reaction. L-NMMA and SDMA are also thought to undergo deamination (not shown). Identification and Association of Endogenous Methylarginines With Disease In 1992, Vallance et al 4 described a marked increase in the plasma concentration of ADMA in a cohort of patients with end-stage renal failure, reporting an 8-fold higher ADMA level in patients on dialysis compared with healthy controls. In addition, these authors described an increase in blood pressure in guinea pigs as well as a reduction in endothelium-dependent forearm blood flow, consistent with reversible inhibition of nitric oxide synthase (NOS), in healthy volunteers following administration of high-dose intravenous ADMA. The monomethylated arginine, L-NMMA in common with ADMA inhibits all 3 isoforms of NOS 3 but is present in the human plasma at a 5- to10-fold lower concentration than ADMA. 11 The endogenous production of ADMA has been proposed as an explanation for the L-arginine paradox, 12 the observation that exogenous L-arginine in vivo or in vitro increases NO production despite baseline concentrations of L-arginine that should saturate NOS. A competitive antagonist, such as ADMA or L-NMMA, present at the active site of the enzyme is consistent with these observations. The techniques used to quantify methylarginines have improved substantially over the past 2 decades such that the threshold of what constitutes normal levels has fallen. Recent epidemiological studies suggest ADMA and SDMA concentrations in normal (50 75 years old) human plasma lie in the range (2 standard deviations) of 0.43 μmol/l to 0.56 μmol/l and 0.38 μmol/l to 0.73 μmol/l, respectively, using high performance liquid chromatography. 13 The highest circulating levels of methylarginines in humans are observed in chronic renal disease with median ADMA and SDMA concentrations of 1.08 μmol/l (interquartile range ) and 2.42 μmol/l (interquartile range ), respectively, reported in a recent large study of patients on dialysis, again using high-performance liquid chromatography. 14 Evidence for increased levels of ADMA in disease is wide ranging. In general, higher ADMA levels have been found in case-control studies involving subjects of cardiovascular, endocrine, and immune mediated disease, although there are a

3 Caplin and Leiper Endogenous NOS Inhibitors 1345 Table 1. Examples of Disease Associations With (Plasma Unless Stated) ADMA Levels Renal failure 14 Peripheral vascular disease 92 Pulmonary hypertension 93 Septic shock 94 Atherosclerotic coronary disease 95 Diabetes mellitus 96,97 Diabetes mellitus (aqueous humor) 98 Preeclampsia 99 Alzheimer disease 100 Connective tissue disease 101 Liver disease 102 Hyperthyroidism 103 Hypothyroidism 104 Stroke 105 Sickle cell disease 106 Alzheimer disease (plasma) 107,108 Diabetes mellitus 109,110 Alzheimer disease (CSF) 108,111 Higher ADMA levels in human disease Lower ADMA levels in disease ADMA indicates asymmetric dimethylarginine; CSF; cerebral spinal fluid. few notable exceptions where lower levels have been reported (Table 1). More convincing evidence for a pathological role comes from longitudinal cohort studies where higher methylarginine levels have been associated with cardiovascular and other adverse outcomes many years later (Table 2). However, Table 2. Clinical Outcomes Independently Associated With Raised ADMA in Longitudinal Studies causal associations remain difficult to establish given the number of significant relationships between ADMA and both traditional and nontraditional cardiovascular risk factors (Table 3) as well as the interaction with impaired kidney function, also an independent risk factor for vascular disease. So, despite attempts at adjustment, the possibility of confounding in these studies remains. Similarly, SDMA has been reported to be as good a surrogate for kidney function as routinely used clinical measures 15 making inferences as to any role in pathophysiology using observational studies even more difficult than with ADMA. Therefore, an understanding of the biology of the endogenous methylarginines is required, both to describe the potential mechanistic pathways and to develop useful models to address any causal role in disease. Distribution of ADMA and SDMA In healthy subjects by extrapolation from studies of methylarginine metabolism, total ADMA production can be estimated at 100 to 250 μmol per day. 16,17 Early investigations into the fate of infused radio-labeled methylarginines in rodents suggest that it is taken up rapidly from the extracellular compartment and found at high concentrations within tissues, specifically kidney and liver. 18 In these studies, approximately half of the radioactivity from infused ADMA (but not SDMA) was found reincorporated into the protein fraction within an hour. Given that this is thought to only occur after enzymatic degradation (see below) this again suggests the intra-cellular compartment is the key site of interest. Other investigations in rats have suggested that under basal conditions the kidney and liver contain the highest concentrations of free methylarginines whereas other tissues such as the lung contain larger amounts of incorporated methylated protein arginine Group n Outcomes Investigated Follow-Up Reference Healthy subjects 3320 Death (No association with CV events) 10.9 y 112 Healthy women 880 CV events (No association with death) 24 y 113 Combined healthy men and men 150 Coronary events 5 y 114 with coronary artery disease 342 Coronary events (nonsmokers only) 6.2 y 115 Patients with coronary artery disease 193 Death; MI 2 y Death or nonfatal MI 2.6 y Death or nonfatal MI 16 mo 118 Patients with diabetes mellitus 170 Death; death or CV event 2 y Cardiovascular event 21 mo 120 Patients with diabetic nephropathy 397 Fatal and nonfatal CV event 11 y 121 (Borderline association with renal decline) Patients with peripheral vascular 496 CV events 19 mo 122 disease 125 CV events 35 mo 123 Patients with chronic kidney disease 131 Death or kidney failure 27 mo Progression of kidney dysfunction 82 mo Death; CV mortality 10 y 126 (No association with kidney failure) Patients with pulmonary hypertension 57 Death 26 mo 127 ADMA indicates asymmetric dimethylarginine; CV, cardiovascular; MI, myocardial infarction.

4 1346 Arterioscler Thromb Vasc Biol June 2012 Table 3. Relationship Between Circulating ADMA and Potential CV Risk Factors Age Association with age 113,128,129 No association with age 130 Sex Higher in women 13 No association with sex 130 Lipids Association with higher total cholesterol 113,130,131 Association with lower HDL 130 Association with lower triglycerides 130 Tobacco Association with smoking status 113,130 No association with smoking status 128 Blood pressure Association with higher BP 128,129 Association with lower SBP 130 Inflammation Association with lower CRP 130 Homocysteine Association with higher plasma homocysteine 130 Obesity Association with higher BMI 113,130 Diabetes mellitus Association with impaired glucose tolerance 128 Association with lower plasma insulin 130 Association with diabetes mellitus 96 Kidney function No association with creatinine clearance in subjects without kidney disease 131 No association with egfr in subjects without kidney disease 130 ADMA indicates asymmetric dimethylarginine; CV, cardiovascular; HDL, high density lipoprotein; BP, blood pressure; SBP, systolic blood pressure; CRP, c-reactive protein; BMI, body mass index; egfr, estimated glomerular filtration rate. residues. 19 Further evidence for the importance of the kidney in total methylarginine metabolism is provided by the reports of significant arterio-venous gradients for ADMA across this organ in both rodents 20 and humans. 21 Mechanisms by which the endogenous methylarginines move between the extracellular and intracellular compartments are not well characterized but there is evidence the y+ cation transporter may be involved. 22 Although this transporter may have a higher affinity for ADMA than L-arginine, 23 the orders of magnitude excess of the unmodified plasma amino acid means that unless L-arginine concentrations fall severalfold the endogenous methylarginines are unlikely to provide physiologically relevant mechanism to control L-arginine uptake into cells from the extracellular fluid. Actions of Endogenous Methylarginines Although circulating L-arginine levels may be >100 times those of ADMA, recent systematic investigations have shown that (1) intracellular ADMA:L-Arginine ratios are significantly higher than those observed in plasma and (2) using electron paramagnetic resonance spectroscopy and spin trapping to quantify NO, significant NOS inhibition is achieved in endothelial cells at physiological levels of endogenous methylarginines (both ADMA and L-NMMA) in the presence of L-arginine concentrations close to those observed in healthy humans. 23 Indeed Cardounel et al 23 estimated that at physiological levels of ADMA and L-arginine endothelial NOS activity is subject to 10% tonic inhibition. As the K i of ADMA for NOS is only 0.9 μmol/l 23 the asymmetric methylarginines would be expected to inhibit NO production over a wide range of L-arginine concentrations, ie, in conditions (sepsis and exercise) where substrate availability varies markedly. Although the endogenous methylarginines have also been reported to modulate the production of superoxide by constitutive NOS the direction of effect appears to be dependent on the NOS isoform and prevailing substrate and cofactor concentrations. 24,25 There is also evidence that ADMA can lead to reactive oxygen species mediated apoptosis in endothelial cells through both NOS-dependent and -independent pathways. 26 Exogenous ADMA has been shown to alter transcription of a number of genes, including the protein-methyl transferases (see below) through both NO-dependent and -independent mechanisms. 27 There is also increasing evidence from in vitro studies that the endogenous methylarginines will modulate cell processes that are thought to be critical in the pathogenesis of atherosclerotic vascular disease. Published reports suggest ADMA can increase cell adhesion, 28,29 inhibit endothelial-cell motility, 30 and inhibit Rac-1 mediated vascular endothelial growth factor induced angiogenesis 31 through NO-dependent mechanisms, all of which might be thought to promote atherogenesis. Conversely, ADMA has also been shown to inhibit cytokine induced inos mediated-no production in vascular smooth muscle cells, 32 which in turn would be predicted to downregulate the proliferation of smooth muscle cells and free radical formation in atherosclerosis. Therefore there is strong in vitro evidence to suggest that the endogenous methylarginines can inhibit NO production through the competitive inhibition of NOS within cells, potentially both inhibiting and upregulating critical downstream functions. In experimental systems it has been demonstrated that this NOS inhibition occurs at ADMA concentrations that are observed in humans after accounting for the differences between circulating and intracellular concentrations of both substrate and inhibitor. Consistent with the evidence for NOS inhibition, reductions in downstream markers of NO signaling have been found in vivo, at least with the acute administration of ADMA at pharmacological doses in normal subjects. 33 However, although never tested in humans for obvious reasons, the long-term response to administration of exogenous ADMA in mice has not always produced reductions in systemic measures of NO production consistent with the acute effects 34 suggesting other counterregulatory systems are also active. In fact in these same studies, ADMA infusion led to vascular lesions in coronary microvessels in both wild-type mice and enos knockout mice and these findings were not reversed by L-arginine supplementation providing further evidence for NO-independent actions. Administration of exogenous L-arginine to increase NOS substrate in the face of increased circulating ADMA concentrations would be predicted to overcome methylarginine mediated inhibition of NOS, and in small, short-term studies using biochemical and surrogate clinical vascular end points findings compatible with improvements in NO bioavailability were reported. 35,36 However, no benefits and potential harm was observed in a 6-month controlled trial of oral L-arginine supplementation in patients with peripheral vascular disease. 37 Over the follow-up period, no increase in NO production, or

5 Caplin and Leiper Endogenous NOS Inhibitors 1347 outcome benefit, was found in the treatment group despite higher levels of circulating L-arginine. Given the demonstrated increase in the plasma L-arginine:ADMA ratio, these data suggest, at least in patients with peripheral vascular disease, that either: (1) the relative concentrations of the endogenous methylarginines and arginine in the plasma compartment are not reflected at the active site of NOS, or (2) other counterregulatory mechanisms prevent an increase in NO production in situations where chronic inhibition of NOS by ADMA is overcome by increases in substrate availability. Recognition of the time- and tissue compartment-dependent variation in methylarginine handling as well as potential NO-independent effects is essential to the interpretation of many of the in vivo studies. If the major site of action and handling of the endogenous methylarginines is within cells, relatively small changes in the plasma ADMA concentrations observed in chronic illness may be the consequence of substantially altered local metabolism and tissue-no dysregulation. However, infusion of methylarginines that achieve similar circulating levels but which do not impact (fold-higher) prevailing intracellular concentrations will not necessarily simulate disease-associated pathology. Figure 2. Formation, distribution, metabolism and actions of endogenous methylarginines. L-arginine (L-Arg) is present in the circulation at >100 times the concentrations of the free endogenous methylarginines: ADMA and symmetric dimethylarginine (SDMA). ADMA but not SDMA inhibits all 3 isoforms of nitric oxide synthase (NOS), decreasing the production of nitric oxide. L-arginine and the free methylarginines are thought to enter the cell (shown on the left) through the y+ transporter. ADMA and SDMA are generated intracellularly following the methylation, by protein-arginine methyltransferases (PRMT), and subsequent proteolysis, of constituent protein arginine residues. ADMA can regulate protein expression through both NO-dependent and -independent pathways. ADMA but not SDMA is hydrolyzed by DDAH to form dimethylamine (DMA) and L-citrulline (L-Cit), which can be reincorporated into proteins. The majority of ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAHs) with the product DMA excreted in the urine whereas SDMA is excreted intact. Both SDMA and ADMA are substrates for alanine-glyoxylate aminotransferase-2 (AGXT2), which is expressed only in kidney (right) and liver (not shown), leading to the formation of symmetrical and asymmetrical -keto- dimethylguanidino valeric acid (DGV) that is also excreted in the urine. Dotted arrows show metabolic pathways for which limited in vivo evidence is available. Acetylation of ADMA has also been described but is not shown. Monomethylarginine (not shown for clarity) is thought to have similar actions, distribution, and degradation pathway to ADMA. Although ADMA and L-NMMA have been relatively well- studied, little is known about the actions, if any, of the symmetrically methylated arginine. SDMA does not appear to inhibit NO synthesis directly. 4,38 Millimolar concentrations of SDMA have been reported to competitively inhibit arginine uptake in vitro, 38 but similar to ADMA, it is difficult to see how this mechanism would be physiologically important given the relative circulating concentrations of these amino acids observed in vivo. Inhibition of renal tubular L-arginine uptake by SDMA has also been reported, 39 but again the millimolar concentrations used in these experiments make it difficult to infer any physiological role. Other authors have reported effects of SDMA on leucocytes in vitro at concentrations seen in vivo 40 but further work in this area is clearly needed. Regulation of ADMA The Dimethylarginine Dimethylaminohydrolases The formation, actions and breakdown of the endogenous methylarginines are outlined in Figure 2. Although the metabolic fate of ADMA has been studied extensively in rodents and humans, 17,18,41 the factors controlling the production of endogenous methylarginines are poorly understood. The upstream methylation of protein arginine residues, but not free L-arginine, is catalyzed by the protein-methyl transferase family of enzymes of which at least 8 mammalian isoforms have been described. 42 The functional consequences of protein arginine methylation are diverse and the impact on cellular processes is far wider than the NO pathway. These include: localization of RNA through modification of RNAbinding proteins; 43 transcriptional regulation by methylation of histones; 44 and signal transduction through protein-protein interactions. 2 Given that it is only following subsequent proteolysis that free endogenous methylarginines are formed, and that protein-methyl transferase activity regulates a number of vital cellular functions, it would seem unlikely that these enzymes act as the rate-limiting factor in controlling ADMA or L-NMMA levels. Although the rate of proteolyisis following ubiquitination or similar would also seem unlikely to represent a regulatory step in methylarginine production, there have been reports that inhibition of alternative pathways of protein degradation (proteasomic versus macroautophagy) lead to differential production of ADMA and SDMA. 45 It is now clear that unlike SDMA, which is mainly renally excreted, the main mechanism for ADMA clearance is through hydrolysis, with the formation of dimethylamine and citrulline. 46 Methylated arginine residues within proteins are not thought to be substrates for the dimethylarginine dimethylaminohydrolases (DDAHs), although recent reports suggest other enzymes may perform this role. 47 L-NMMA is thought to undergo a similar metabolic fate as ADMA whereas dimethylamine along with SDMA is excreted intact in the urine. 17,48 In healthy humans, it has been estimated that 10% to 20% of ADMA is excreted unchanged but the remaining 80% to 90% is actively metabolized, predominantly by the DDAH enzymes 17 of which there are 2 isoforms (DDAH1 and DDAH2). 49 DDAHs colocalize with NOS isoforms 50 providing further indirect evidence that these enzymes might provide

6 1348 Arterioscler Thromb Vasc Biol June 2012 a mechanism for controlling the local availability of NO and downstream vascular endothelium-dependent responses. Studies in rodent-tissue homogenates have demonstrated that the (nonisoform specific) DDAH K m for ADMA is 180 µmol/ L, 48 although recent experiments using recombinant human enzyme have reported values closer to a third of this concentration for DDAH1, 51 which would be consistent with physiologically significant enzyme activity at prevailing intracellular methylarginine concentrations. Unfortunately published data on the enzyme kinetics of DDAH2 are not available as recombinant expression of this enzyme is technically challenging. Although there have been some recent insights from in vivo studies (see below) the relative contribution of the 2 DDAH isoforms to overall methylarginine metabolism as well as any substrate specificity remains far from clear. The DDAHs are 285 residue cytosolic proteins highly conserved across species with the 2 isoforms of DDAH present in all mammals and a similar enzyme present in prokaryotes. 50 Although the 2 DDAHs are structurally similar at the protein level (60% amino acid identity), the genes encoding the enzymes differ significantly. The DDAH1 gene has been mapped to chromosome 1p22 and is 167-kb in length, whereas DDAH2 has been mapped to chromosome 6p21 and is <4-kb. Functional mutations in the DDAH genes appear to be very rare (see below). The active site of both enzymes contains a cysteine-histidine-glutamate catalytic triad, 52 but the 2 isoforms of DDAH appear to have different tissue distributions. DDAH2 is the primary isoform found in the heart, lung, placenta, and fetal tissues whereas DDAH1, although widespread, is the only isoform found in neuronal tissues. 49,50 Further detailed gene expression and electron immunohistochemical analysis of the distribution of these enzymes suggests that DDAH2 is the primary isoform present in rat mesenteric resistance vessels and is found in the apical membrane of endothelial cells and smooth muscle fibrils. 53 Whereas data from rat 54 and human (Caplin and Leiper, unpublished observation, 2009) kidney suggests that DDAH1 is primarily an enzyme of epithelial cells whereas DDAH2 is present in blood vessels. Table 4. Summary of Rodent Models of Genetic/Pharmacological Modulation in DDAH Manipulation of ADMA by Modulating DDAH Over recent years, the impact of genetic manipulation of the DDAH axis using viral transfection, 55 knockout, 56 and overexpressing mice, 57,58 as well as administration of small interfering RNAs 59 has been examined (Table 4). This results in intracellular manipulation of methylarginine levels, which can in turn can result in modulation of circulating endogenous ADMA levels consistent with those observed in human disease. 56 Results from these genetic studies have demonstrated that knockout or RNA interference of DDAH1 is associated with an increase in ADMA and leads to a cardiovascular phenotype, ie, hypertension, consistent with NOS inhibition. DDAH1 deletion has been achieved by constitutive replacement of exon 1 (encoding the initiating methionine and first 100 amino acids) 56 or using LoxP/Cre strategy to target exon 4 (which includes the enzymatic active site). 60,61 The constitutive homozygous knockout initially developed by our group died in utero failing to develop beyond blastocyst stage, 62 but the LoxP/Cre null animal appears to develop normally. 60 This anomaly appears to be related to the difference in targeting strategy used to generate DDAH1 null alleles as LoxP/Cre-targeted exon 1 homozygous animals are viable (Dowsett and Leiper, unpublished observation, 2012). The LoxP/Cre system has also been used to generate endothelium-specific DDAH1 exon 4 deletion and has been reported to lead to the absence of enzyme expression in all tissues including kidney and liver and with a consequent rise in plasma and tissue ADMA. 61 These data appear at odds with the demonstration of DDAH1 in other cell types, 63,64 so further studies are required to resolve this apparent contradiction. In general agreement with the data on DDAH1 insufficiency over expression of the same enzyme in transgenic mice or through adenoviral transfection reduces circulating ADMA and leads to a phenotype suggesting an increase in NO bioavailability. 55,57 The data with respect to DDAH2 are less clear. Small interfering RNAs targeted to DDAH2 have been reported to not alter plasma ADMA or induce hypertension but do reduce endothelium-dependent responses in vessels ex vivo. 59 DDAH2 overexpression using a transgenic mouse has been reported to reduce circulating ADMA levels but only reduce Change in DDAH Technique Species ADMA EDV BP DDAH1 Constitutive exon 1 haploinsuffiency 56 Mouse Cre/LoxP exon 4 global null 60 Mouse ND Endothelium targeted exon 4 null 61 Mouse sirna 59 Rat DDAH1 Transgenic over expression 57 Mouse ND Adenoviral transfection 55 Rat (CKD model) ND DDAH2 sirna 59 Rat DDAH2 Transgenic over expression 58 Mouse ND Mouse (ADMA infusion) ND DDAH1/2 Pharmacological 56 Rat (sepsis model) DDAH indicates dimethylarginine dimethylaminohydrolase; sirna, small interfering RNA; CKD, complete knock down; ADMA, asymmetric dimethylarginine; EDV, endothelium dependent vasodilation; BP, blood pressure; ND, not done/not reported ;, increase;, decrease;, unchanged.

7 Caplin and Leiper Endogenous NOS Inhibitors 1349 blood pressure in the face of additional exogenous ADMA administration. 58 Furthermore, the globally deleted Cre/LoxP exon 4 DDAH1 knockout mouse shows little detectable DDAH activity in several tissues, 60 further calling into question the degree to which DDAH2 contributes to ADMA metabolism at least under basal conditions. Although the catalytic triad of residues responsible for the breakdown of ADMA in DDAH1 are preserved in DDAH2, the in vivo findings along with the technical difficulties in expression of active recombinant protein mean that definitive evidence for a role in methylarginine degradation is lacking. However, given the tissues where DDAH2 appears to be the only potential route for ADMA catabolism (leucocytes) and the chromosomal position of the gene (proximity to the major histocompatibility complex locus), it is tempting to speculate this enzyme might have an important role in regulating inos mediated inflammatory responses perhaps following immune activation. As decreases in endothelial NO bioavailability are thought to be proatherogenic in humans, 65 it would be predicted that manipulating DDAH1 in rodents would alter the course of models of atherosclerosis. Crossing of proatherogenic apolipoprotein E mice onto a DDAH1 overexpressing transgenic background not only reduces plaque formation but substantially altered the distribution of plaque within the aorta. 66 These data raise the intriguing possibility that modulating methylarginine metabolism may impact atherogenesis through altering systemic hemodynamics as well as through local endothelial injury. However, overexpressing DDAH1 does not appear to ubiquitously improve outcomes in murine disease models with a recent report suggesting it exacerbates bleomycin-induced lung fibrosis. 67 Genetic variation in the DDAHs has not only been shown to alter circulating methylarginine levels in rodents. Taking a candidate gene approach, both our group 68 and others 69 have recently shown that variation in a region of intron-1 of the human DDAH1 gene is associated with differences in circulating ADMA concentrations in both healthy subjects and patient groups. Naturally occurring promoter variation, demonstrated to alter transcription in vitro, has also been reported for both DDAH1 (a 396 4N insertion disrupting MRTF-1 induced transcription) 70 and DDAH2 ( 871 6G/7G repeat adjacent to a putative NGFIC/Egr-2 binding domain). 71 Together, these data suggest that the effect of organism-wide alterations in methylarginine metabolizing enzyme gene expression can be detected in plasma ADMA levels. In contrast, the evidence for DDAH gene variation leading to human cardiovascular disease is less clear. Genome-wide association studies in cases of coronary artery disease, 72 stroke, 73 or chronic kidney disease 74 have consistently failed to implicate either of the DDAH genes in these conditions. Such case-control studies have been restricted to certain ethnic groups, only examine cross-sectional outcome data, and require very large numbers of subjects to maintain adequate power after correction for the many thousands of comparisons, so clinical studies of other designs have been employed. A rare coding mutation in DDAH1 has been described in a Finnish population 75 with a prevalence of the heterozygous genotype reported as <1%. Carriers of this mutation were reported to have higher plasma- ADMA concentrations and an increased risk of hypertension compared with noncarrier family members. The DDAH N promoter insertion mutation described above, which was shown to decrease DDAH1 transcription in vitro, is present at an increased prevalence in patients with stroke and coronary artery disease in a Han Chinese population. 70 Assuming a dominant effect of the mutation, odds ratios were reported to be close to 1.5 and were not substantially altered by adjustment for differences in known cardiovascular risk factors. Although replication in other ethnic groups is required, these findings suggest naturally occurring DDAH1 promoter variation can have an important role in the development of cardiovascular disease. We have also shown that DDAH1 genetic variation is associated with human pathology. Intron-1 DDAH1 polymorphisms were associated with rate of decline in estimated glomerular filtration rate in patients with chronic kidney disease >2 years follow-up. 68 Interestingly however, it was the genotypes associated with lower ADMA levels that were associated with a steeper decline in kidney function, suggesting that the increases in the endogenous methylarginines may have protective effects in addition to pathological roles dependent on the organ system studied. Regulation of DDAHs Given the potential pathophysiological significance of these enzymes, there has been increasing interest in the regulation of the DDAH enzymes with evidence that this occurs both at the transcriptional and posttranslational level. DDAH levels have been shown to be altered by a number of endocrine and cytokine factors in cell culture, 76 although the mechanisms through which this occurs have not been fully elucidated. There is good evidence that nuclear receptor ligands regulate transcription of both DDAH1 77 and DDAH2, 78 although the contribution of these pathways to DDAH gene expression in vivo remains to be determined. If minute-to-minute control of NOS inhibition was a function of these enzymes, then posttranslational modification would be expected to be a key regulatory mechanism. Millimolar concentrations of NO donors have been shown to nitrosylate cysteine residues and reduce the activity of pseudomonal DDAH, 79 bovine DDAH1, 80 and total renal DDAH, 81 potentially providing a negative-feedback mechanism for the control of NO synthesis if this were to occur at physiological concentrations of NO. Conversely NO donors have been reported to increase DDAH2-mediated metabolism of ADMA via transcriptional and posttranslational mechanisms resulting in a positive-feedback loop. 82 These data imply that the effects of NO on the DDAHs may be concentration-and isoform-specific. Homocysteine has been shown to bind recombinant DDAH1 and inhibit DDAH activity both in cell culture and cell-free systems in a dose-dependent mechanism. 83 This effect was reversed by antioxidants 83 and investigators have proposed that the DDAHs are redox-sensitive enzymes. 76,81 Pharmacological Manipulation of DDAH-ADMA Numerous drugs have been shown to alter circulating ADMA levels in humans (Table I in the online-only Data Supplement) with drugs that target the renin-angiotensin system showing

8 1350 Arterioscler Thromb Vasc Biol June 2012 the most consistent effects. Whether the changes in methylarginine levels are mediated by an impact on synthesis, distribution, or degradation has generally not been explored. Given the limited knowledge regarding posttranslational regulation of DDAH, there is clearly a need for these mechanisms to be investigated in greater detail if targeting of the enzyme for pharmacotherapy is to be considered. However, recent reports suggest an indirect approach using combined phosphodiesterase-3/4 inhibition with the drug tolafentrine (leading to an increase in cyclic AMP) increases DDAH2 transcription and ADMA metabolism both in vitro and in vivo along with amelioration of pulmonary and vascular pathology in a rat model of pulmonary hypertension. 84 It is also important to recognize that any therapy based on the chronic widespread upregulation of the DDAHs would raise concern over potential enhanced tumor growth, 85 although whether this is entirely mediated by increases in ADMA metabolism has recently been questioned. 86 In contrast, small-molecule (isoform-specific) inhibition of the DDAHs might be achieved using synthetic arginine analogues assuming off-target inhibition of NOS can be avoided and the potential for this approach has recently been demonstrated in models of vasodilatory septic shock. 56 Mitochondrial Methylarginine Metabolism by Alanine-Glyoxylate Aminotransferase-2 Two other ADMA breakdown products have also been identified. 18 α-keto-δ-dimethylguanidino valeric acid (DGV), has been isolated from rodent urine and is the product of the metabolism of SDMA and ADMA by alanine-glyoxylate aminotransferase-2 (AGXT2). 18 Dimethylguanidino valeric acid has not been studied in detail but previous investigation of short-chain guanidino compounds suggests that inhibition of NOS occurs only at very high (>100 µm) concentrations. 3 AGXT2 is a 514-amino-acid pyridoxal-phosphate-dependent mitochondrial aminotransferase and the 50-kb gene is located on chromosome 5p13. The enzyme has attracted little interest as early studies suggested that it contributed to <5% of overall ADMA metabolism 18 and it had a K m far higher than endogenous methylarginine concentrations 87 raising doubt for its physiological significance. Expression of AGXT2 has been reported only in kidney and liver. 88 Recently, viral transfection of AGXT2 has been shown to decrease ADMA in vivo and increase NO production in vitro, 89 providing experimental evidence that this enzyme not only can metabolize methylarginines at physiological concentrations, but that the products of this reaction do not inhibit NOS. However, the role of methylarginine metabolism in the mitochondria is unclear. Whether mitochondria are a source of NO remains controversial, 90,91 and there is currently no published evidence for NO-independent actions of ADMA in this organelle. Therefore, the contribution of AGXT2 to both the metabolism of ADMA/ L-NMMA overall and the bioavailability of mitochondrial NO in either human health or disease remains unknown. Lastly ADMA and SDMA can both be acetylated, 18 although the precise pathways and physiological relevance are not yet characterized. Acetyl-methylarginines are excreted in the urine. Summary and Implications There is increasing evidence that the endogenous methylarginines play a significant role in the disease. The observations that ADMA levels predict future outcomes in cohort studies demonstrates the potential for methylarginines to act as markers for pathophysiology, but are only circumstantial evidence for a role as either a regulator or mediator of disease. It is the data from the studies on DDAH that provide evidence for causal associations between ADMA and pathophysiology. Both transgenic animals and analysis of human DDAH1 variation demonstrate that alterations in DDAH expression will change outcomes in animal models or the course/occurrence of clinical disease. However, it is important to recognize that depending on the different disease state studied, the evidence suggests reducing ADMA may either ameliorate (eg, hypertension or vascular disease) or exacerbate (eg, lung fibrosis or progressive chronic kidney disease) pathophysiology. No therapies in current use specifically target endogenous methylarginines. Although certain classes of drugs such as angiotensin-converting-enzyme inhibitors do consistently reduce ADMA, whether the beneficial effects of these drugs are in any way related to the effects on the DDAH-ADMA-NO axis remains to be established. It might be predicted that reducing ADMA in patients with vascular disease or hypertension would be beneficial given the evidence for a causal relationship between reduced ADMA metabolism and stroke and myocardial infarction, although whether any disruption of the DDAH-ADMA-NO occurs as an early or late event in pathogenesis of these diseases needs to be addressed. Even if pharmacotherapy specifically targeting endogenous methylarginines can be developed, a deeper understanding of the tissue specific roles of these amino acids in different disease states will be needed. Despite the recognition of circulating endogenous methylarginines almost 20 years ago, it is only in recent years that we have clear evidence for a causal role for the DDAH- ADMA pathway in human diseases. It appears that ADMA (and potentially L-NMMA) may have important regulatory, as well as pathological, roles in health and disease so a targeted approach to intervention in this pathway will be required. There is still inadequate understanding of the production of endogenous methylarginines, but with characterization of the different enzymes that degrade these competitive NOS inhibitors the possibility of developing specific and effective therapies targeting this pathway becomes closer. Sources of Funding Ben Caplin receives support from the Academy of Medical Sciences (in collaboration with the Welcome Trust and British Heart Foundation). James Leiper receives support from the Medical Research Council and British Heart Foundation. None. Disclosures References 1. Kakimoto Y, Akazawa S. Isolation and identification of N-G,N-G- and N-G,N -G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine,

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