Rainer H Böger
email: boeger@uke.uni-hamburg.de

Prof. Dr med H Rainer

Böger

Asymmetric dimethylarginine (ADMA) and cardiovascular disease: insights from prospective clinical trials

Keywords: atherosclerosis, coronary heart disease, endothelium, nitric oxide, oxidative stress, prognosis Vascular Medicine 2005, 10: S19-25

HAL is

Introduction

Evidence has accumulated in recent years that the endothelial production and release of nitric oxide (NO) plays a crucial role in the maintenance of physiological vascular tone and structure. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase. It inhibits vascular NO production at concentrations found in pathophysiological conditions, and also causes local vasoconstriction when infused intra-arterially. Thus, elevated ADMA levels may explain the 'L-arginine paradox', that is, the observation that supplementation with exogenous L-arginine improves NO-mediated vascular functions in vivo, although its baseline plasma concentration is about 25-fold higher than the K m of the isolated, purified endothelial NO synthase in vitro.

Dysfunction of the endothelial L-arginine/NO pathway is a common mechanism by which several cardiovascular risk factors mediate their deleterious effects on the vascular wall. Among them are hypercholesterolemia, [START_REF] Böger | ADMA: a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia[END_REF] hypertension, [START_REF] Panza | Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension[END_REF] smoking, [START_REF] Celermajer | Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilation in healthy young adults[END_REF] diabetes mellitus, [START_REF] Johnstone | Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus[END_REF] hyperhomocysteinemia, [START_REF] Tawakol | Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans[END_REF] and vascular inflammation. [START_REF] Hingorani | Acute systemic inflammation impairs endothelium-dependent dilatation in humans[END_REF] Vallance and coworkers first described ADMA as an endogenous inhibitor of NO synthase in 1992. [START_REF] Vallance | 339: 572-75. oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e)inemia. Effects of L-arginine and B vitamins[END_REF] Since their initial observation, the circulating concentration of ADMA has been shown to be increased in humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, congestive heart failure, diabetes mellitus, and other cardiovascular and metabolic diseases. Increased ADMA levels are associated with reduced NO synthesis and may therefore be a link between many other known and evolving cardiovascular risk factors and the progression of vascular disease. This review focuses on the evidence that has been gathered from clinical studies relating ADMA concentration to disease outcome.

Association of ADMA with cardiovascular disease: evidence from epidemiological studies

Numerous studies have been published that have demonstrated a relationship between high ADMA concentration and cardiovascular disease. Elevated ADMA has a high prevalence in hypercholesterolemia, 1 hyperhomocysteinemia, 8 diabetes mellitus, [START_REF] Abbasi | Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus[END_REF] peripheral arterial occlusive disease, [START_REF] Böger | Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease[END_REF] hypertension, [START_REF] Goonasekera | Nitric oxide synthase inhibitors and hypertension in children and adolescents[END_REF][START_REF] Surdacki | Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetrical dimethylarginine in men with essential hypertension[END_REF] chronic heart failure, [START_REF] Usui | Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure[END_REF] coronary artery disease, [START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF] and other diseases (Table 1). All of the studies cited above were designed as case-control studies, thereby not allowing a conclusion about the possible cause-effect relationship between ADMA and cardiovascular diseases. However, the observation that ADMA levels increased early during the development of atherosclerosis suggested that ADMA had the potential to be not only a marker but also a mediator of vascular lesions. Data have recently accumulated from a series of cross-sectional clinical studies that confirm this hypothesis.

Miyazaki and coworkers [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF] performed a study on 116 clinically healthy humans in whom intima-media thickness in the common carotid artery was determined. This had previously been shown to be a surrogate marker for atherosclerosis progression. In stepwise regression analysis including multiple cardiovascular risk markers they found that ADMA concentration was significantly associated with carotid intima-media thickness.

In another study a similar approach was used to assess intima-media thickness as determined by high-resolution ultrasound analysis in 90 patients with end-stage renal disease who were undergoing longterm hemodialysis. [START_REF] Zoccali | Asymmetric dimethylarginine (ADMA), C-reactive protein, and carotid intima media-thickness in end-stage renal disease[END_REF] Carotid intima-media thickness was related to various potential risk factors in a multiple regression analysis. Confirming the data of Miyazaki and coworkers, [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF] it was found that ADMA was highly significantly correlated with intima-media thickness in this high-risk population. Furthermore, in an extension of previous data, the progression of intimal thickening during 1 year of follow-up was best predicted by initial ADMA and C-reactive protein levels.

Valkonen and coworkers 14 performed a nested casecontrol study including 150 middle aged, nonsmoking men from eastern Finland. They found that those whose ADMA concentration was within the highest quartile of the distribution in this population (i.e. Ͼ0.62 mol/l) had a 3.9-fold elevation of the risk for acute coronary events compared with the other quartiles.

In a multicenter case-control study including a total of 816 patients with objectively proven coronary artery disease and matched controls, we recently found that ADMA was the marker with the best discriminative power to differentiate between cases and controls. [START_REF] Böger | Asymmetric dimethylarginine and the risk of coronary heart disease: relationship with traditional risk factors as assessed in the multicenter CARDIAC study [Abstract[END_REF] A close relationship has also been established between insulin resistance and ADMA by the work of Stühlinger et al. [START_REF] Stühlinger | Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor[END_REF] These investigators assessed insulin sensitivity in a group of 64 nondiabetic people using the insulin-glucose clamp technique. They found that a tight linear relationship existed between steady state plasma glucose (a measure of insulin resistance) and plasma ADMA. These data indicate that elevation of ADMA occurs before the manifestation of overt diabetes mellitus, which is also associated with elevated plasma ADMA. 9

ADMA is a novel cardiovascular risk factor: evidence from prospective clinical trials

In the first prospective trial studying ADMA as a potential new cardiovascular risk factor, at the University Hospital Hamburg-Eppendorf, we measured plasma ADMA as well as plasma levels of conventional and emerging cardiovascular risk factors in 225 patients on hemodialysis. After a median 33.4 months of follow-up, 120 major cardiovascular events and a total of 83 deaths (53 vascular deaths) had occurred. In a Cox's proportional hazards model ADMA and age were the strongest predictors of cardiovascular events and total mortality. [START_REF] Zoccali | Asymmetric dimethylarginine (ADMA): an endogenous inhibitor of nitric oxide synthase predicts mortality in end-stage renal disease (ESRD)[END_REF] Although the median plasma ADMA concentration in this population (2.52 mol/l) was higher than in healthy normal adults (usually below 1 mol/l), patients with an ADMA concentration above the 75th percentile within this group had a three-fold higher risk of death from any cause than those with ADMA levels below the median.

In another prospective study, Lu and coworkers 26 addressed the question of whether ADMA may predict outcome after percutaneous coronary intervention in patients with stable angina pectoris. A total of 153 consecutive patients were followed for a median of 16 months. There were 51 major cardiovascular events during follow-up. There was a clear increase in risk with rising ADMA levels, which was independent of other potential confounding factors in a multifactorial Cox's regression analysis (including age, smoking, hypercholesterolemia, the use of stents, and others). The remarkable outcome of this study is that ADMA levels were within a range that many would consider as 'normal'; patients in the lowest tertile of ADMA had a median circulating concentration of 0.42 mol/l compared with 0.75 mol/l in the highest tertile.

Nijveldt and coworkers analysed potential predictors of death in patients undergoing treatment on a multidisciplinary intensive care unit. They included patients with multiple organ failure and found that ADMA was the strongest predictor of death, with a 17-fold excess in mortality for patients in the highest ADMA quartile compared with those in the lowest quartile. [START_REF] Nijveldt | Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality[END_REF] ADMA was significantly associated with hepatic failure, and with lactic acid and bilirubin concentrations, suggesting that hepatic function is an important determinant of circulating ADMA concentration.

The currently available data on ADMA from prospective clinical studies are summarized in Table 2. Additional prospective clinical studies have been initiated in patients with congestive heart failure, acute coronary syndrome, stable coronary heart disease, heart transplantation, and pulmonary hypertension. These data will enable us more precisely to define the role of ADMA as an emerging cardiovascular risk factor.

The pathophysiological link between ADMA and cardiovascular disease: evidence from experimental studies in humans

Experimental studies performed in vitro or in animal models have recently been summarized in several reviews. [START_REF] Böger | The emerging role of ADMA as a novel cardiovascular risk factor[END_REF][START_REF] Cooke | Asymmetrical dimethylarginine: the Über marker?[END_REF][START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway[END_REF] This paper will therefore focus on data generated in humans.

In-vivo pathophysiological studies in humans have been performed by several groups of investigators. These data shed more light on the relationship between ADMA and vascular disease.

Intra-arterial infusion of ADMA into the brachial artery significantly reduced forearm blood flow. [START_REF] Calver | Effect of local intra-arterial asymmetric dimethylarginine (ADMA) on the forearm arteriolar bed of healthy volunteers[END_REF] However, ADMA concentrations reached in the local circulation after intra-arterial infusion by far outstrip those that can be measured in patients. Thus, two studies in which ADMA was administered intravenously added data to the dose-response relationship of ADMA in humans. In one study, ADMA was infused to reach a systemic plasma concentration of about 2.6 mol/l, [START_REF] Achan | Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolised by dimethylarginine dimethylaminohydolase[END_REF] which caused a significant elevation of arterial blood pressure and systemic vascular resistance, reduced cardiac output and heart rate, and decreased the vascular response to exercise. In another study it was shown that ADMA decreased plasma cyclic guanosine monophosphate concentrations at pathophysiologically relevant concentrations, whereas renal plasma flow and glomerular filtration rate were reduced only at very high concentrations. [START_REF] Kielstein | Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans[END_REF] Chan and coworkers studied the adhesion of peripheral blood monocytes to cultured human endothelial cells. They found that the adhesiveness index was related to the circulating ADMA level of the patient from whom the monocytes were isolated. Moreover, when they manipulated the ratio of L-arginine over ADMA by giving supplemental L-arginine, they found that monocyte adhesion normalized during 12 weeks of treatment. [START_REF] Chan | Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans[END_REF] Taken together, the above mentioned studies provide a plethora of evidence that ADMA modulates endothelial NO synthase activity within the concentration range found in patients with vascular and metabolic diseases. Moreover, this evidence also suggests that even small modifications of ADMA significantly change vascular NO production, vascular tone, and systemic vascular resistance.

One important question in interpreting and understanding these findings and their relationship to ADMA plasma or serum levels measured in prospective clinical trials is the following: why do such small differences in circulating ADMA concentration as reported from prospective trials indicate such dramatic increases in cardiovascular event rate if such high levels of ADMA need to be reached during its systemic administration? The current interpretation shared by many experienced investigators in the field is that circulating levels of endogenous ADMA are the result of a 'spillover' from ADMA released within the cytosol during protein degradation. As diemthylarginine dimethylaminohydrolase (DDAH) enzymatic activity is present in many cells, and as ADMA is rapidly cleared from plasma, small changes in circulating levels may indicate large changes in local and/or intracellular ADMA concentration that clearly affect NO production and function. However, when ADMA is administered exogenously, it enters the circulation first and has to be transported into cells by CAT (the cationic amino acid transporter system y ϩ ) before it can affect cellular function. Much higher increases in plasma ADMA must therefore be attained to induce pathophysiological changes when exogenous ADMA is administered.

Measurement of ADMA in serum and plasma: methodological considerations and clinical relevance as a diagnostic tool

Research into ADMA was an exotic, purely experimental topic during the 1990s. However, as the above data show, it is now coming of age as an emerging cardiovascular risk factor. Currently, effort is being made to define the normal range of ADMA concentration in healthy humans of different age groups and of both sexes. Moreover, an international research initiative has undertaken a correlation of analytical methods, in order to allow the correlation of data measured in different laboratories around the world. Finally, besides elaborate analytical methods such as highperformance liquid chromatography and mass spectrometry, more widely accessible and less time consuming (and therefore cheaper) ways of measuring ADMA have become available with the development and validation of a novel competitive enzyme immunoassay that allows the sensitive and specific assessment of ADMA levels. [START_REF] Schulze | Determination of ADMA using a novel ELISA assay[END_REF][START_REF] Böger | All about ADMA[END_REF] As the scene is set for a wider application of ADMA measurement in clinical routine, the question is: who should be tested? ADMA is elevated in association with almost all traditional cardiovascular risk factors and in patients with established cardiovascular or metabolic disorders. However, its concentration shows significant variability even within these patient populations. Moreover, multivariate regression analyses that have taken into consideration all of the traditional and some other novel risk factors have shown that ADMA remains independently associated with cardiovascular risk or total mortality even after adjustment for the other risk factors has been performed.

The steadily increasing number of prospective clinical trials show that ADMA not only helps to discriminate between health and cardiovascular disease (for which there are other established diagnostic criteria), but these data point out that, even within a group of patients who have previously been regarded as homogeneous for their similar risk factor profile, ADMA is a tool that can be used to identify individuals with a comparatively high probability of experiencing a major event. There is therefore the potential that cardiovascular risk assessment, which currently is achieved by risk factor scores such as the Framingham risk score, [START_REF]Estimating coronary heart disease (CHD) risk using Framingham Heart Study prediction score sheets[END_REF] the PROCAM risk score, [START_REF]PROCAM risk calculator[END_REF] or the risk charts developed by the European Society of Cardiology, [START_REF]CVD prevention score risk charts[END_REF] can be strengthened by adding ADMA as another risk indicator. This may hold especially true for patients who do not clearly fall into the high-risk (i.e. greater than 20% risk for a major event during the subsequent 10 years) or lowrisk (i.e. below 10% risk) categories, but who fall within the 'grey zone' of intermediate risk. Thus, ADMA levels will be helpful in estimating the risk for patients who deserve intensified treatment for the prevention of cardiac events. L-arginine may be one means of specifically antagonizing the deleterious effects of ADMA on endothelial function via nutritional supplementation in primary and secondary prevention.

When ADMA is elevated: therapeutic consequences?

Clinical and experimental evidence suggests that elevation of ADMA causes a relative L-arginine deficiency, even in the presence of 'normal' L-arginine plasma concentrations. As ADMA is a competitive inhibitor of NO synthase, its inhibitory action can be overcome by increasing the concentration of this enzyme's natural substrate, L-arginine. [START_REF] Tsikas | Hypothesis: endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox[END_REF][START_REF] Böger | Asymmetric dimethylarginine, and endogenous inhibitor of nitric oxide synthase, explains the 'L-arginine paradox' and acts as a novel cardiovascular risk factor[END_REF] Thus, it can be predicted that nutritional supplementation with L-arginine may help to restore the physiological balance by normalizing the L-arginine/ADMA ratio, whereas it will have little or no effect on humans with no disturbed L-arginine/ADMA balance. This view is supported by data from experimental studies with L-arginine supplementation.

Azuma and coworkers showed, in an experimental study, that ADMA accumulates in regenerated endothelial cells after balloon angioplasty. [START_REF] Azuma | Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L-arginine in regenerated endothelial cells[END_REF] It is well known that regenerated endothelial cells growing at the site of an arterial injury lack full functional capacity, with a major defect in the L-arginine-NO pathway. [START_REF] El-Tamimi | Response of human coronary arteries after injury by coronary angioplasty[END_REF] This finding may explain why the local or systemic administration of L-arginine improves local arterial function after injury. [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF] Few patients experience pathologically low L-arginine concentrations. However, clinical and experimental evidence suggests that elevation of ADMA may cause a relative L-arginine deficiency, even in the presence of 'normal' L-arginine levels. ADMA is a competitive inhibitor of nitric oxide synthase, so its inhibitory action can be overcome by increasing the concentration of the enzyme's substrate, L-arginine. The studies cited above have shown that ADMA levels may increase in situations associated with cardiovascular diseases, in which elevated ADMA concentration is one possible explanation for endothelial dysfunction and decreased NO production.

The beneficial vascular effects of cholesterollowering drugs -statins -have been shown to depend in part on their ability to upregulate endothelial NO synthase gene expression. [START_REF] Laufs | Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors[END_REF] However, in clinical studies, statins failed to improve endothelium-dependent vasodilation in about as many studies as there were in favour of this hypothesis. Once more, this discrepancy may be resolved with the help of ADMA. Janatuinen et al [START_REF] Janatuinen | Plasma asymmetric dimethylarginine modifies the effect of pravastatin on myocardial blood flow in young adults[END_REF] recently found that pravastatin was able to enhance myocardial blood flow (measured by positron electron resonance tomography) only in patients with a low ADMA level, whereas this drug was ineffective in those with an elevated ADMA. We speculated that ADMA may block NO synthase despite its upregulated gene expression after statin treatment, and that this blockade may be overcome by L-arginine supplementation. [START_REF] Böger | Asymmetric dimethylarginine (ADMA) modulates endothelial function -therapeutic implications[END_REF] Indeed, we found in a randomized, controlled trial that, in patients with an elevated ADMA concentration, simvastatin enhanced endothelium-dependent vasodilation only when it was combined with supplemental nutritional L-arginine. [START_REF] Böger | Improvement of endothelium-dependent vasodilation by simvastatin is potentiated by combination with L-arginine in patients with elevated asymmetric dimethylarginine levels [Abstract][END_REF] Thus, ADMA may explain the results of clinical trials in which L-arginine was shown either to improve endothelial function or fail to do so, a discrepancy that has so far remained unexplained and which has previously been called the 'L-arginine paradox'. [START_REF] Tsikas | Hypothesis: endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox[END_REF] Moreover, the ability of L-arginine to counteract the detrimental vascular effects of ADMA and thereby improve endothelial function as well as the symptoms of vascular disease demonstrates that the risk associated with elevated ADMA is modifiable. This is another critical aspect that needs to be addressed before one may call ADMA an 'emerging cardiovascular risk factor'.

The observation of an elevated ADMA concentration in a given patient may therefore justify L-arginine supplementation in order to improve the ability of the endothelium to generate NO, and thereby counteract the adhesion of circulating blood cells, reverse vasoconstriction, and attenuate the production of oxygen-derived free radicals.

  ADMA and cardiovascular disease S21Vascular Medicine 2005; 10: S19-25

Table 1

 1 Pathophysiological conditions in which ADMA has been reported to be elevated.

	Condition	x-fold increase	Reference
	Chronic renal failure	2-7-fold	Zoccali et al 2001 15
	Hypertension	2-fold	Surdacki et al 1999 12
	Childhood hypertension	2.3-fold	Goonasekera et al 1997 11
	Pregnancy-related hypertension	2-fold	Chen et al 1997 16
	Pre-eclampsia	2-fold	Pettersson et al 1998 17
	Pulmonary hypertension	2-3-fold	Gorenflo et al 2001 18
	Stable coronary artery disease	2-fold	Valkonen et al 2001 14
	Peripheral arterial occlusive disease	2-3-fold	Böger et al 1997 10
	Stroke	2-fold	Yoo et al 2001 19
	Hypercholesterolemia	2-fold	Böger et al 1998 1
	Hyperhomocysteinemia	2-fold	Sydow et al 2003 8
	Chronic heart failure	2-3-fold	Usui et al 1998 13
	Hyperthyroidism	2-fold	Hermenegildo et al 2002 20
	Diabetes mellitus type 2	2-3-fold	Abbasi et al 2001 9
	Insulin resistance	2-fold	Stühlinger et al 2002 21
	Liver failure	2-fold	Tsikas et al 2003 22

Table 2

 2 Results from published prospective studies of ADMA as a cardiovascular risk factor.

	Condition	x-fold risk excess	ADMA level	Reference
	Hemodialysis	3-fold	Highest quartile (Ͼ3.85 mol/l)	Zoccali et al 2001 15
	Stable coronary artery disease	3.9-fold	Highest quartile (Ͼ0.62 mol/l)	Valkonen et al 2001 14
	Intensive care unit treatment	17.2-fold	Highest quartile (Ͼ1.05 mol/l)	Nijveldt et al 2003 27
	Stable coronary artery disease	5.3-fold	Highest tertile (Ͼ0.62 mol/l)	Lu et al 2003 26

Vascular Medicine 2005; 10: S19-25

Acknowledgments

The work of the author was funded by the Deutsche Forschungsgemeinschaft (DFG Bo 1431/3-1) and by the Else-Kröner-Fresenius-Stiftung. The author owns a patent on an ADMA assay and receives royalties from the license.


Renke Maas
email: maas@uke.uni-hamburg.de

Keywords: asymmetric dimethylarginine (ADMA), cardiovascular disease, DDAH, drug therapy, nitric oxide synthase inhibitor

Pharmacotherapies and their influence on asymmetric dimethylargine (ADMA)

Introduction

The L-arginine-nitric oxide pathway is involved in the regulation of diverse physiological functions including endothelium-dependent vasodilation, platelet aggregation, neurotransmission, immune defense and apoptosis. [START_REF] Moncada | The L-arginine-nitric oxide pathway[END_REF] Accumulating evidence links asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), to human disease. [START_REF] Cooke | Asymmetrical dimethylarginine: the Über marker?[END_REF] Elevated plasma ADMA concentrations are found in various clinical settings ranging from renal failure and liver failure to atherosclerosis, hypertension, diabetes and pre-eclampsia. [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF][START_REF] Tsikas | Elevated plasma and urine levels of ADMA and 15(S)-8iso-PGF2alpha in end-stage liver disease[END_REF][START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF][START_REF] Kielstein | Marked increase of asymmetric dimethylarginine in patients with incipient primary chronic renal disease[END_REF][START_REF] Yoo | Elevated levels of plasma homocyst(e)ine and asymmetric dimethylarginine in elderly patients with stroke[END_REF][START_REF] Taddei | Aging and endothelial function in normotensive subjects and patients with essential hypertension[END_REF][START_REF] Tarnow | Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes[END_REF][START_REF] Stühlinger | Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor[END_REF][11] Moreover, in patients with cardiovascular disease, end-stage renal disease, or critical illness, elevated plasma ADMA concentrations independently predict progression of atherosclerosis, future vascular events and/or overall mortality. [START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF][START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Lu | Plasma levels of asymmetrical dimethylarginine and adverse cardiovascular events after percutaneous coronary intervention[END_REF][START_REF] Zoccali | Asymmetric dimethylarginine, C-reactive protein, and carotid intima-media thickness in end-stage renal disease[END_REF] Several lines of evidence suggest that ADMA, being a NOS inhibitor, may not only be a marker of disease but a player. In human volunteers, infusion of ADMA has been shown impair to endothelium-dependent vasodilation, increase systemic and renal vascular resistance, and decrease cardiac output. [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF][START_REF] Calver | Effect of local intra-arterial asymmetric dimethylarginine (ADMA) on the forearm arteriolar bed of healthy volunteers[END_REF][START_REF] Achan | Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Kielstein | Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans[END_REF] These data suggest that chronic elevation of ADMA may directly promote vascular disease, which makes ADMA a promising new drug target. Studying the effects of commonly used drugs on plasma ADMA levels may be a first step towards gaining some insights into the mechanisms leading to elevated plasma ADMA concentrations. As ADMA may be both a marker and a player in cardiovascular disease it may therefore be difficult to discern indirect drug effects on ADMA levels related to improvement of Pharmacotherapies and their influence on asymmetric dimethylargine (ADMA)

Renke Maas

Abstract: Elevated plasma concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) are found in various clinical settings, including renal failure, coronary heart disease, hypertension, diabetes and preeclampsia. In healthy people acute infusion of ADMA promotes vascular dysfunction, and in mice chronic infusion of ADMA promotes progression of atherosclerosis. Thus, ADMA may not only be a marker but also an active player in cardiovascular disease, which makes it a potential target for therapeutic interventions. This review provides a summary and critical discussion of the presently available data concerning the effects on plasma ADMA levels of cardiovascular drugs, hypoglycemic agents, hormone replacement therapy, antioxidants, and vitamin supplementation. We assess the evidence that the beneficial effects of drug therapies on vascular function can be attributed to modification of ADMA levels. To develop more specific ADMA-lowering therapies, mechanisms leading to elevation of plasma ADMA concentrations in cardiovascular disease need to be better understood. ADMA is formed endogenously by degradation of proteins containing arginine residues that have been methylated by S-adenosylmethionine-dependent methyltransferases (PRMTs). There are two major routes of elimination: renal excretion and enzymatic degradation by the dimethylarginine dimethylaminohydrolases (DDAH-1 and -2). Oxidative stress causing upregulation of PRMT expression and/or attenuation of DDAH activity has been suggested as a mechanism and possible drug target in clinical conditions associated with elevation of ADMA. As impairment of DDAH activity or capacity is associated with substantial increases in plasma ADMA concentrations, DDAH is likely to emerge as a prime target for specific therapeutic interventions.

the underlying condition from direct drug effects (such as drug-induced augmentation of dimethylarginine dimethylaminohydrolase (DDAH) activity).

It is the purpose of this review to evaluate possible mechanisms leading to the elevation of ADMA plasma concentration and critically to review first in vitro and in vivo data concerning potential ADMAlowering therapies.

Formation and elimination of ADMA: mechanisms and targets for therapeutic interventions

Asymmetric dimethylation of L-arginine residues in proteins

The initial step in the endogenous formation of ADMA (Figure 1) is the asymmetric dimethylation of L-arginine residues of various cellular proteins by specific protein L-arginine methyltransferases (PRMTs). [START_REF] Boisvert | A proteomic analysis of arginine-methylated protein complexes[END_REF][START_REF] Tang | PRMT1 is the predominant type 1 protein arginine methyltransferase in mammalian cells[END_REF] Type 1 PRMTs are considered the predominant enzymes responsible for asymmetrical dimethylation, [START_REF] Tang | PRMT1 is the predominant type 1 protein arginine methyltransferase in mammalian cells[END_REF] and S-adenosylmethionine (SAM) derived from L-methionine appears to be the principal methyl group donor for this reaction. [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases[END_REF] Moreover, elevation of SAM promotes PRMT activity and subsequent formation of ADMA. The product of this methylation reaction, S-adenosylhomocysteine (which inhibits PRMT activity) is hydrolysed to L-homocys- teine, which, in turn, can be remethylated by methionine synthase to L-methionine. [START_REF] Lentz | Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA[END_REF] This reaction, which closes the methylation cycle, depends on vitamin B 12 and 5-methyltetrahydrofolic acid as methyl group donors. In principle, each step of this methylationremethylation cycle constitutes a potential target for therapeutic approaches to lower SAM levels and thus reduce substrate availability for asymmetric dimethylation of L-arginine residues.

Practical and clinical considerations, however, make formation of SAM and PRMTs unlikely targets. First, post-translational methylation of L-arginine residues is considered a universal biological process affecting hundreds of proteins. [START_REF] Boisvert | A proteomic analysis of arginine-methylated protein complexes[END_REF] In most cases the effects of this methylation are still unknown. Thus, interference with this mechanism may have unforseen consequences. Secondly, based upon their role in the homocysteinemethionine cycle, one would expect folate and vitamin B 12 supplementation to increase synthesis of SAM and subsequent ADMA formation. However, it was shown in patients with increased plasma homocysteine levels that folate lowered elevated plasma ADMA concentrations. [START_REF] Holven | Folic acid treatment reduces elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects[END_REF] In this study, which unfortunately lacked a placebo group, the beneficial effect of folate on ADMA levels was attributed to the reduction of homocysteine by folate. In contrast, in another study, supplementation with folate and B vitamins had no impact on ADMA levels. [START_REF] Sydow | ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e)inemia: effects of L-arginine and B vitamins[END_REF] Proteolysis and formation of ADMA When proteins are degraded, asymmetrically dimethylated L-arginine residues are released as ADMA. Considering the wide variety of asymmetrically dimethylated proteins it is very unlikely that sufficiently specific inhibitors of the proteasome involved in degrading these proteins can be found. Moreover, specific or nonspecific inhibition of protein degradation could lead to accumulation of these proteins or peptide fragments, which potentially could cause a form of storage disease. Hence, proteolysis is unlikely to present a suitable drug target for reducing ADMA levels.

Specific metabolism of ADMA by DDAH

ADMA is selectively degraded by DDAH to citrulline and dimethylamine. [START_REF] Ogawa | Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney[END_REF] In humans there are two major isoforms of DDAH (DDAH-1 and DDAH-2) with partly overlapping tissue distributions [START_REF] Tran | Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins[END_REF] in kidney, liver and small intestine (Table 1). Presently, it is not known what percentage of the endogenously formed ADMA is directly metabolized in the cells where it is formed and how much is released from the cells into other tissues to be metabolized or excreted. It is also not known which DDAH isoform or which organ dominates ADMA degradation in humans in vivo. Rising plasma concentrations in renal [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] or liver failure 4 strongly suggest, however, that at least some tissues release a substantial fraction of the endogenously formed ADMA to be metabolized or excreted elsewhere. With an estimated 'organ weight' similar to that of the liver, the endothelium could be another major site of ADMA metabolism (Table 1). [START_REF] Mccarty | Vascular endothelium is the organ chiefly responsible for the catabolism of plasma asymmetric dimethylarginine -an explanation for the elevation of plasma ADMA in disorders characterized by endothelial dysfunction[END_REF] Current estimates of the relative expression of the two isoforms are mainly based on mRNA data. DDAH-1 expression tends to dominate in tissues with a high expression of the neuronal isoform of NOS (nNOS), whereas DDAH-2 expression seems to follow the expression pattern of endothelial NOS isoform (eNOS). Analysis of the DDAH-2 gene and its promotor regions revealed potential binding sites for regulators of transcription (STAT, PPAR/RXR, NF-B and IRF-1) as well as features consistent with the function of a housekeeping gene (absence of canonical TATA boxes and a CpG island around the transcription start sites). Specific activators of DDAH expression are not yet known, but activation of DDAH expression is of course an interesting drug target.

In cell culture all-trans-retinoic acid increased DDAH-2 expression. [START_REF] Achan | all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase[END_REF] Regulation of DDAH-1 expression is even less well understood. In smooth muscle cells interleukin-1 beta upregulates DDAH-1 expression at the protein level. [START_REF] Ueda | Regulation of cytokineinduced nitric oxide synthesis by asymmetric dimethylarginine: role of dimethylarginine dimethylaminohydrolase[END_REF] DDAH contains four highly redox sensitive cysteine groups, which appear to be crucial for its catalytic function. [START_REF] Ogawa | Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney[END_REF][START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF] DDAH activity may also be regulated by S-nitrosylation of a reactive cysteine residue (Cys-249), [START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF] providing some form of an indirect negative feedback, especially for inducible NOS (iNOS)-derived nitric oxide (NO).

Therapeutic augmentation of DDAH expression or activity: a word of caution

Although increased NO synthesis due to upregulation of DDAH expression or activity is usually desired in cardiovascular disease, it may have catastrophic effects when occurring in tumors (by promoting NOmediated angiogenesis). Indeed, recent studies suggest that DDAH activity is increased in human tumors. [START_REF] Kostourou | Effects of overexpression of dimethylarginine dimethylaminohydrolase on tumor angiogenesis assessed by susceptibility magnetic resonance imaging[END_REF] Moreover, overexpression of DDAH-1 or -2 enhances tumor growth and tumor angiogenesis. [START_REF] Kostourou | Dimethylarginine dimethylaminohydrolase 1 enhances tumour growth and angiogenesis[END_REF] Transport and exchange of ADMA When assessing drug effects on ADMA levels, a major drawback of all available clinical data has to be considered. We currently measure the plasma concentration, which may represent only some form of 'cellular spillover'. Little is known regarding the correlation of plasma ADMA and intracellular ADMA concentrations in vivo. Thus, even a normal or normalized plasma ADMA concentration may not completely exclude severe derangement of intracellular ADMA concentrations by disease or drugs.

Given its structural similarities to L-arginine, the cationic amino acid ADMA most likely shares carrier systems with L-arginine to leave or enter cells. [START_REF] Brosnan | Renal arginine metabolism[END_REF] Indeed, early in vitro data suggest that transport of ADMA may at least in part be mediated by y+ carriers. [START_REF] Closs | Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B[END_REF] Clinical data regarding the precise transport mechanisms for ADMA and their regulation in vivo are not currently available.

Renal filtration and excretion of ADMA

Elevated plasma levels of ADMA were first reported in patients with chronic renal failure by Vallance and co-workers. [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] We now know that the combination of renal filtration, tubular secretion, tubular reabsorption and degradation by renal DDAH result in a net renal extraction of about 16% of circulating ADMA. [START_REF] Nijveldt | Net renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine in fasting humans[END_REF] Presently it is difficult, however, to estimate the relative contribution of each mechanism. It is interesting that symmetric dimethylarginine, which is chemically very similar to ADMA but not metabolized by DDAH, has a renal extraction rate of only 10%. This difference may be interpreted as evidence for a substantial contribution of renal DDAH activity to the renal extraction of ADMA.

Exogenous sources of ADMA

Protein-L-arginine methylation is a universal biological principle found in bacteria as well as in plants and animals. [START_REF] Mcbride | State of the arg:protein methylation at arginine comes of age[END_REF] It is therefore very likely that free and protein-incorporated ADMA [START_REF] Banchaabouchi | NG-dimethylarginine and NG, NG-dimethylarginine in renal insufficiency[END_REF] is also taken in with the diet, modulated by dietary components, or formed by resident colonic bacteria. This offers several further targets for ADMA-lowering therapies that have not yet been investigated.

Major disease mechanisms leading to elevation of ADMA in human disease

Shear stress

In vitro, shear stress may have divergent effects on ADMA. [START_REF] Osanai | Effect of shear stress on asymmetric dimethylarginine release from vascular endothelial cells[END_REF] Moderate shear stress was found to upregulate PRMT1 expression while DDAH activity was unchanged, resulting in enhanced cellular ADMA release. In contrast, when cells were exposed to a higher shear stress, PRMT1 and DDAH activity both increased, resulting in normalization of ADMA levels.

High blood glucose and impaired glucose tolerance

Elevation of plasma ADMA levels have been observed in a rat model of diabetes, and DDAH activity was found to be significantly impaired in human cells exposed to high concentrations of glucose. [START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF] Since it was shown that impairment of DDAH activity could be alleviated by polyethylene glycol-conjugated superoxide dismutase, impairment of DDAH activity by glucose-induced oxidative stress was suggested as the major underlying mechanism. In humans the situation appears to be more complex. In volunteers without diabetes, plasma ADMA levels were found to be correlated with individual insulin sensitivity but not with blood glucose level. [START_REF] Stühlinger | Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor[END_REF] Clinical data concerning plasma ADMA levels in patients with diabetes are somewhat conflicting: some studies have shown an elevation of ADMA [START_REF] Abbasi | Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus[END_REF] while others demonstrated a reduction. 41

Oxidative stress

Several independent lines of evidence indicate that impairment of DDAH activity by oxidative stress is the final mechanism leading to elevation of ADMA in diverse clinical conditions. [START_REF] Sydow | ADMA and oxidative stress[END_REF] DDAH contains four highly redox sensitive cysteine groups, which appear to be crucial for its catalytic function. [START_REF] Ogawa | Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney[END_REF][START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF] Oxidized low-density lipoprotein (oxLDL) but not native LDL was found to decrease DDAH activity [START_REF] Ito | Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase[END_REF] and increase cellular ADMA liberation. In cell culture, oxLDL and high concentrations of native LDL lead to upregulation of PRMTs and cellular ADMA synthesis that could be prevented by antioxidants. [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases[END_REF] Unfortunately, impairment of DDAH activity by oxidative stress or upregulation of PRMTs may not be easy to overcome in humans. A Finnish study found no association between plasma ADMA levels and dietary vitamin E or C intake. [START_REF] Päivä | Dietary composition as a determinant of plasma asymmetric dimethylarginine in subjects with mild hypercholesterolemia[END_REF] In an open label study, 800 IU vitamin E over 6 weeks had no significant effect on plasma ADMA. [START_REF] Saran | Impact of vitamin E on plasma asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD): a pilot study[END_REF] Moreover, in a very recent clinical trial N-acetyl-cysteine had no effect on plasma ADMA levels. [START_REF] Doshi | Relationship between S-adenosylmethionine, S-adenosylhomocysteine, asymmetric dimethylarginine and endothelial function in healthy human subjects during experimental hyper-and hypohomocysteinemia[END_REF]

Hyperhomocysteinemia

In animals [START_REF] Böger | Plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is elevated in monkeys with hyperhomocyst(e)inemia or hypercholesterolemia[END_REF] and humans [START_REF] Stühlinger | Endothelial dysfunction induced by hyperhomocyst(e)inemia: role of asymmetric dimethylarginine[END_REF][START_REF] Böger | Elevation of asymmetrical dimethylarginine may mediate endothelial dysfunction during experimental hyperhomocyst(e)inaemia in humans[END_REF] experimental hyperhomocysteinemia is associated with elevation of plasma ADMA. Two (not necessarily exclusive) mechanisms have been proposed to explain this finding:

1. Homocysteine may be remethylated to form L- methionine, which, in turn, serves as a methyl group donator for the methylation of L-arginine. [START_REF] Lentz | Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA[END_REF] Increased ADMA release by endothelial cells under high concentrations of methionine or homocysteine was reduced by the methylation inhibitor S-adenosylmethionine. [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases[END_REF] In monkeys, dietary supplementation with B vitamins normalized homocysteine but did not alter ADMA levels or improve endothelial function. [START_REF] Böger | Plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is elevated in monkeys with hyperhomocyst(e)inemia or hypercholesterolemia[END_REF] 2. Homocysteine may also directly interact with the sulfhydryl groups of DDAH. In an in vitro binding assay, biotinylated homocysteine was found to bind directly to DDAH-1. [START_REF] Stühlinger | Endothelial dysfunction induced by hyperhomocyst(e)inemia: role of asymmetric dimethylarginine[END_REF] This binding could be prevented by an antioxidant.

Drug therapy and ADMA: early clinical data

Statin therapy

Statins are among the most effective drugs for lowering cardiovascular risk associated with elevation of LDL cholesterol. Several lines of evidence suggest that the beneficial effects of statins are at least in part mediated by LDL-independent (pleiotropic) mechanisms such as improvement of endothelial function by increasing the bioavailability of NO. In animal experiments as well as in patients a high LDL cholesterol level was found to be associated with elevated plasma ADMA concentrations. [START_REF] Böger | Plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is elevated in monkeys with hyperhomocyst(e)inemia or hypercholesterolemia[END_REF][START_REF] Böger | Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia[END_REF] Even though this finding does not seem to be found consistently in all patient populations, [START_REF] Päivä | Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors[END_REF] several investigators have tested the hypothesis that statin treatment may improve vascular function by reducing plasma ADMA (Table 2). In the majority of studies, statin treatment had no impact on plasma ADMA levels. [START_REF] Eid | Increased levels of asymmetric dimethylarginine in populations at risk for atherosclerotic disease. Effects of pravastatin[END_REF][START_REF] Janatuinen | Effect of lipidlowering therapy with pravastatin on myocardial blood flow in young mildly hypercholesterolemic adults[END_REF][START_REF] Pereira | Effects of simvastatin and L-arginine on vasodilation, nitric oxide metabolites and endogenous NOS inhibitors in hypercholesterolemic subjects[END_REF] This would clearly argue against a direct connection between ADMA and LDL cholesterol in humans. Only rosuvastatin was shown to lower ADMA 57 but, given the negative results for the other statins, this finding remains to be replicated in other populations. Irrespective of the potential effects of statins on ADMA, recent studies suggest that ADMA may modulate the effect of statins on vascular function. [START_REF] Janatuinen | Plasma asymmetric dimethylarginine modifies the effect of pravastatin on myocardial blood flow in young adults[END_REF]

Angiotensin converting enzyme inhibitors and angiotensin receptor blockers

In various conditions such as hypertension, diabetes and coronary artery disease, angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) were found to improve endothelial function and NO availability. [START_REF] Joannides | Chronic ACE inhibition enhances the endothelial control of arterial mechanics and flow-dependent vasodilatation in heart failure[END_REF][START_REF] Higashi | Effect of the angiotensin-converting enzyme inhibitor imidapril on reactive hyperemia in patients with essential hypertension: relationship between treatment periods and resistance artery endothelial function[END_REF][START_REF] Hornig | Comparative effect of ace inhibition and angiotensin II type 1 receptor antagonism on bioavailability of nitric oxide in patients with coronary artery disease: role of superoxide dismutase[END_REF] Several studies have assessed the effect of ACE inhibitors or ARBs on plasma ADMA levels in these patient groups (Table 3). [START_REF] Delles | Angiotensin converting enzyme inhibition and angiotensin II AT1-receptor blockade reduce the levels of asymmetrical N(G), N(G)-dimethylarginine in human essential hypertension[END_REF][START_REF] Chen | Long-term angiotensin-converting enzyme inhibition reduces plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X[END_REF][START_REF] Napoli | Sulfhydryl angiotensinconverting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension[END_REF][START_REF] Ito | Angiotensin-converting enzyme activity is involved in the mechanism of increased endogenous nitric oxide synthase inhibitor in patients with type 2 diabetes mellitus[END_REF][START_REF] Ito | Renin-angiotensin system is involved in the mechanism of increased serum asymmetric dimethylarginine in essential hypertension[END_REF] In patients with hypertension, diabetes or syndrome X, the reduction in plasma ADMA achieved by ACE inhibition was in the range of 12-20%, while plasma L-arginine levels did not seem to be affected. [START_REF] Chen | Long-term angiotensin-converting enzyme inhibition reduces plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X[END_REF] ARBs were found to reduce plasma ADMA by 10-16%. [START_REF] Delles | Angiotensin converting enzyme inhibition and angiotensin II AT1-receptor blockade reduce the levels of asymmetrical N(G), N(G)-dimethylarginine in human essential hypertension[END_REF][START_REF] Ito | Renin-angiotensin system is involved in the mechanism of increased serum asymmetric dimethylarginine in essential hypertension[END_REF] All of these studies were small and had rather short observation periods of 1-12 weeks. Thus, confirmation of these promising results by larger trials will be required.

The results of a recent study in eNOS knockout mice suggests that ADMA may also exert NO-independent effects via upregulation of ACE and augmentation of oxidative stress through AT 1 -dependent pathways. [START_REF] Suda | Asymmetric dimethylarginine produces vascular lesions in endothelial nitric oxide synthase-deficient mice: involvement of renin-angiotensin system and oxidative stress[END_REF] Thus, ACE inhibitors and ARBs may help to prevent ADMA-induced pathology independently of their effects on plasma ADMA levels.

Hypoglycemic agents

In patients with type 2 diabetes, 3 months of treatment with 0.5-1.0 g/d metformin alone (n = 16) or in combination with sulfonylurea (n = 15) reduced plasma ADMA concentrations by 28.5% and 32.0% respectively. [START_REF] Asagami | Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes[END_REF] Some caution is warranted, however, as no placebo data are available from that study. Furthermore, in contrast to what one would expect from the in vitro data, no relationship was observed between the decrease in ADMA concentration and glycemic control. Thus, it is possible that the reduction of ADMA levels by metformin is mediated via glucose-independent mechanisms. In this respect it may be of interest to note that metformin has structural similarities to ADMA. [START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway[END_REF] In an uncontrolled study the PPAR-gamma agonist rosiglitazone (12 weeks of treatment with a final dose of up to 8 mg/d) improved insulin sensitivity and reduced plasma ADMA concentrations by 30% in seven insulin-resistant patients. [START_REF] Stühlinger | Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor[END_REF] These data certainly await further confirmation by larger controlled trials because, in vitro, neither PPAR-gamma agonists (ciglitazone) nor PPAR-gamma antagonists (T007) significantly altered cellular ADMA liberation. [START_REF] Albsmeier | Bedeutang endogener Inhibitoren der NO-Synthase für kardiovaskuläre Erkrankungen und ihre pharmakologische Therapie (Relevance of endogenous NOS inhibitors for cardiovascular disease and its therapy)[END_REF] Apart from the effects on blood glucose there is, however, at least support from theoretical considerations because a PPAR/PXR binding site has been identified in the promotor region of DDAH-2. [START_REF] Achan | all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase[END_REF] Hormone replacement therapy Hormone replacement therapy (HRT) has frequently been shown to improve endothelial function and NO synthesis. [START_REF] Vehkavaara | Differential effects of oral and transdermal estrogen replacement therapy on endothelial function in postmenopausal women[END_REF][START_REF] Imthurn | Differential effects of hormone-replacement therapy on endogenous nitric oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 beta-estradiol valerate and cyproterone acetate or medroxyprogesterone acetate[END_REF] Moreover, ADMA levels were found to decrease in normal pregnancy. 11 The precise mechanism by which estrogen reduces ADMA has remained obscure so far. The effect of various forms of HRT on ADMA plasma concentration was explored in some small randomized controlled trials (Table 4). [73][START_REF] Post | Effect of hormone replacement therapy on plasma levels of the cardiovascular risk factor asymmetric dimethylarginine: a randomized, placebo-controlled 12-week study in healthy early postmenopausal women[END_REF][START_REF] Teerlink | Oestrogen replacement therapy lowers plasma levels of asymmetrical dimethylarginine in healthy postmenopausal women[END_REF] In all of these trials ADMA levels were modestly (5-21%) reduced by 17-beta-estradiol alone or when sequentially combined with cyclogesterone or trimegestone. Unfortunately, L-arginine levels also declined, resulting in no change or even a decrease in the L-arginine/ ADMA ratio. Thus, unless it can be proven that the decrease in L-arginine was the result of increased consumption in NO synthesis (and not upregulation of arginase activity), it remains questionable how much benefit can be conferred by HRT on patients with an elevated ADMA.

Conclusion and outlook

Specific ADMA-lowering therapies are not yet available. Promising results from first pilot studies with widely used drugs such as ACE-inhibitors, ARBs, hypoglycemic agents and HRT await confirmation in large placebo-controlled trials.

To develop more specific therapies, further insights into the mechanisms leading to an elevation of ADMA in vivo are needed. Tools such as high-throughput methods for the quantification of ADMA and isoformspecific DDAH inhibitors (and activators) will play a key role. Important insights may also come from the DDAH-transgenic mice [START_REF] Dayoub | Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence[END_REF] and DDAH1/2 knock out models currently under development. So far, most conditions associated with an elevation of ADMA are chronic, so ADMA-lowering therapies will have to be long term. Future studies will therefore have to address carefully the long-term effects of ADMAlowering therapies on angiogenesis and tumor growth.

Until specific ADMA-lowering therapies become available, treatment of modifiable risk factors leading to elevation of ADMA or depletion of L-arginine, and supplementation with L-arginine, may be the best available options for patients with high plasma ADMA concentrations because NO synthesis is correlated with the L-arginine/ADMA ratio. [START_REF] Tsikas | Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox[END_REF][START_REF] Böger | The clinical pharmacology of L-arginine[END_REF]

Figure 1

 1 Figure 1 Formation and elimination of ADMA: (1) Sadenosylmethionine is required as a source of methyl groups; (2) asymmetric methylation of L-arginine residues in proteins by type I protein-arginine methyltransferases (PRMTs); (3) proteolysis leads to liberation of ADMA; (4) degradation of ADMA by dimethylarginine dimethlyaminohydrolase (DDAH); (5) carrier-mediated excretion of ADMA; (6) carrier-mediated uptake of ADMA; (7) renal filtration of ADMA; (8) carrier-mediated secretion and re-uptake of ADMA.

  Influence of drugs on ADMA S51Vascular Medicine 2005; 10: S49-57

  Influence of drugs on ADMA S53Vascular Medicine 2005; 10: S49-57

Table 1

 1 Major sites of ADMA elimination.

		Kidney	Liver	Endothelium
	Predominant DDAH isoform	DDAH-1Ͼ2	DDAH-1ϾϾ2	DDAH-2ϾϾϾ1
	Organ weight (kg)	0.3 (0.15 ϫ 2)	1.5	~1.5
	Increase of ADMA in organ failure	Yes 1.5-10ϫ	Yes 6-12ϫ	Likely but causality not established
	Net organ extraction (%)	16 (arterial-venous)	~30 a	?

a Estimate based on rat data.

Table 2

 2 Effect of statin therapy on plasma ADMA levels.

	Drug (ref.)	Duration (weeks)	n	% change	p
	Pravastatin 40 mg/d (Eid et al 2003 54 )	8	3 2	Ϫ9	n s
	Pravastatin 40 mg/d (Janatuinen et al 2001 55 )	26	21	No change	ns
	Simvastatin 80 mg/d (Päivä et al 2003 53 )	8	24	No change	ns
	Atorvastatin 40 mg/d (Päivä et al 2003 53 )	8	24	No change	ns
	Simvastatin 20 mg/d (Pereira et al 2003 56 )	8	25	No change	ns
	Rosuvastatin 10 mg/d (Lu et al 2004 57 )	6	2 3	Ϫ18	Ͻ0.001
	ns, not significant.

Table 3

 3 Effect of ACE inhibitors and angiotensin receptor blockers on ADMA plasma concentration.

	Drug (ref.)

a Data extracted from graphs in publication.

Table 4

 4 Effect of hormone therapy on ADMA plasma concentration.

			ADMA		L-arginine		L-arginine/
	Drug and dose	Duration (% change;		(% change;		ADMA ratio
	(ref.)	(weeks) mol/l)	p	mol/l)	p	(% change)	p	Placebo
	17-beta-estradiol 2 mg/d	12	Ϫ5.2	0.03	Ϫ7.3	0.02	Ϫ1.6		0.75 Yes
	(Post et al 2003 74 )
	17-beta-estradiol 2 mg/d ϩ	12	Ϫ8.0	0.02	Ϫ7.9	0.14	0.3		0.83 Yes
	dydrogesterone 10 mg/d
	(Post et al 2003 74 )
	17-beta-estradiol 2 mg/d ϩ	12	Ϫ21	Ͻ0.01 Ϫ36	Ͻ0.001 Ϫ19	Ͻ0.01 Yes
	trimegestone 10 mg/d
	(Post et al 2003 74 )
	Conjugated equine	104	Ϫ9.7	Ͻ0.01 No change;	-	Not reported -	Yes
	estrogen 0.625 mg/d				data not
	(Teerlink et al 2003 75 )				reported
	17-beta-estradiol	2	Ϫ18	Ͻ0.05 Not reported -	Not reported -	No
	100 mg implant
	(Holden et al 2003 73 )
	-, data not cited.

Vascular Medicine 2005; 10: S49-57


Professor Stefanie M Bode-Böger
email: stefanie.bode-boeger@medizin.uni-magdeburg.de

Fortunato Scalera

Jens Martens-Lobenhoffer

Asymmetric dimethylarginine (ADMA) accelerates cell senescence

Keywords: asymmetric dimethylarginine, DDAH, oxidative stress, senescence, telomerase activity Vascular Medicine 2005, 10: S65-71

come

Introduction

The incidence of atherosclerosis and cardiovascular disease increases dramatically with age. The links between aging and atherosclerosis are not well established. Common pathways at the cellular level have been proposed for aging and atherosclerosis. [START_REF] Xu | Homocysteine accelerates endothelial cell senescence[END_REF][START_REF] Vasa | Nitric oxide activates telomerase and delays endothelial cell senescence[END_REF] There is accumulating evidence that telomeres and telomerase play a role in cellular senescence. Telomeres, the physical ends of the chromosomes, are involved in the control of chromosome stability, genetic integrity and cell viability. [START_REF] Blackburn | Telomere states and cells' fates[END_REF] Primary mammalian cells have a finite life span in tissue culture. Two sources of signals have been identified to cause the induction of senescence in cultured cells. Intrinsic signals can be induced by a critical telomere shortening, which is a consequence of telomere length reduction during each cell division ('mitotic clock'). [START_REF] Sherr | Cellular senescence: mitotic clock or culture shock?[END_REF] Cell culturing can induce stress signals that result in the expression of different cell cycle inhibitors, leading to a senescent phenotype. Furthermore, a disturbance of the telomere-associated proteins can cause induction of senescence. Telomere shortening forces human primary cells, including endothelial cells, to stop dividing when a critical minimum telomere length is reached. [START_REF] Colgin | Telomere maintenance mechanisms and cellular immortalization[END_REF] The enzyme telomerase, a ribonucleoprotein, counteracts the shortening of telomeres. Recently, it was reported that there is a higher rate of age-dependent telomere attrition in both the intima and the media of the distal compared with the proximal human abdominal aorta, accompanied by a negative correlation between telomere length and atherosclerotic grade. [START_REF] Okuda | Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis[END_REF] These links underscore the need for biological indicators of aging in evaluating the etiology of these age-related disorders. A novel risk factor for cardiovascular disease is asymmetric dimethylarginine (ADMA), [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF] an endogenous inhibitor of nitric oxide synthase (NOS). Elevations in plasma ADMA may contribute to the vascular pathophysiology observed in atherosclerosis, hypertension, hypercholesteremia and renal failure. Increased oxidative stress seems to play an important role in the pathogenesis of these clinical conditions. Moreover, ADMA appears to be an independent predictor of cardiovascular mortality. [START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF] Recent findings suggest a significant positive correlation between age and plasma ADMA levels. [START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Kielstein | Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects[END_REF] We have previously demonstrated that ADMA concentration dependently increased oxidative stress measured as superoxide production by cultured human endothelial cells. [START_REF] Böger | An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes[END_REF] It has been reported that cellular senescence, the limited ability of primary human cells to divide when cultured in vitro, is accelerated by increased oxidative stress [START_REF] Zglinicki | Oxidative stress shortens telomeres[END_REF] and is reduced by nitric oxide (NO) donor treatment. [START_REF] Vasa | Nitric oxide activates telomerase and delays endothelial cell senescence[END_REF] In the present study we therefore investigated if ADMA, a novel cardiovascular risk factor, influences cell senescence in cell culture and increases oxidative stress.

Materials and methods

Cell culture

Human umbilical vein endothelial cells (HUVECs, Clonetics, Germany) were cultured in endothelial basal medium until the tenth passage. ADMA, S-adenosylhomocysteine (SAH) and pyrrolidine dithiocarbamate (PDTC, Sigma, Germany) were replaced every 48 hours starting at the fourth passage. After reaching confluence (between 8 and 9 days) endothelial cells were trypsinized and seeded at a density of 2500 cells/cm 2 using 75 cm 2 flasks or six-well plates. Population doubling (PD) was calculated at each passage until growth arrest by using the following formula: PD ϭ (log 10 Y Ϫ log 10 X)/log 10 2 (Y indicates the number of cells counted at the end the passage; X is the number of cells seeded). Cumulative population doubling (CPD) was calculated as the sum of all the changes in PD. The endothelial cells were counted and the vitality was determined. Viability after trypsinization was usually Ͼ95%. HUVECs were characterized at passage 10 by specific staining for CD31 (PECAM-1) using the fluorescenceactivated flow cytometry assay (FACS). After detachment with trypsin telomerase activity, telomere length, FACS analyses or dimethylarginine dimethylaminohydrolase (DDAH) activity was performed.

Staining for senescence-associated ␤ ␤-galactosidase activity HUVECs were fixed and stained for senescenceassociated ␤-galactosidase (SA-␤-gal) activity according to the procedure developed by Dimri et al. [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF] The percentage of SA-␤-gal positive cells was determined by counting the number of blue cells in 1000 cells.

Telomerase assay and telomere length

Quantitative determination of telomerase activity was performed according to the manufacturer's protocol of the TeloTAGGG Telomerase PCR ELISA PLUS Kit (Roche Diagnostic GmbH, Mannheim, Germany), which is based on the telomeric repeat amplification protocol assay. Telomerase activity was measured by the polymerase chain reaction using 1.5 g protein.

Telomere length was analysed by the terminal restriction fragment length assay using a Telo TAGGG Telomere Length Assay Kit (Roche).

Measurement of nitrate and nitrite

The determination of nitrate and nitrite in cell culture supernatants was carried out in accordance with the method described by Tsikas et al. [START_REF] Tsikas | Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their 15N-labelled analogs[END_REF] DDAH activity assay DDAH activity in endothelial cells was estimated directly by measuring the amount of ADMA metabolized by the enzyme as described by Lin et al. [START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus. Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF] The ADMA level in each group was measured by highperformance liquid chromatography-mass spectrometry as previously described by our group. [START_REF] Martens-Lobenhoffer | Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation[END_REF]

Detection of oxidative stress

For the determination of allantoin (a marker of oxygen free radical generation) in cell culture supernatants we adopted the method for human plasma described by Doehner et al. [START_REF] Doehner | Effect of xantine oxidase inhibition with allopurinol on endothelial function and peripheral blood flow in hyperuricemic patients with chronic heart failure. Results from 2 placebo-controlled studies[END_REF] Dihydrorhodamine 123 (DHR) was used as a marker for intracellular reactive oxygen species (ROS). The cells were incubated with gentle agitation for 20 minutes at 37ЊC in the presence of 10 M DHR123. The reaction was stopped by cooling on dryice for 1 minute and the subsequent addition of 500 l phosphate buffered solution followed by two washing steps. After final fixation with 1% paraformaldehyde the cells were analyzed by flow cytometry (Epics XL-MCL, Coulter). The level of ROS was indicated by mean fluorescence intensities (mfi) of stained probes versus negative controls.

Measurement of monocyte chemotactic protein-1 and interleukin-8

For the quantitative determination of monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8 the cell supernatants were collected, centrifuged and stored at Ϫ20ЊC until assayed. The MCP-1 and IL-8 protein concentrations were assayed using the Quantikine human MCP-1 and IL-8 immunoassay (R&D Systems, Wiesbaden, Germany).

Statistical methods

All data were expressed as the mean Ϯ standard error of the mean (SEM) from at least three independent experiments. Statistical significance was tested with repeated analysis of variance (ANOVA) using a least significant difference post-hoc test or ANOVA for multiple comparisons (SPSS Software 11.0). Differences were considered significant at p Ͻ 0.05.

Results

ADMA accelerates endothelial senescence by inhibiting telomerase activity

To investigate the link between endothelial cell senescence and ADMA, human endothelial cells were cultured until CPD 40 and incubated in the presence of different concentrations of ADMA, which was replaced every 48 hours starting from CPD 20. All concentrations of ADMA (5, 50 and 100 mol/l) significantly increased SA-␤-gal activity compared with untreated cells in a dose-dependent manner (Figure 1A).

To examine the effect of ADMA on shortening of telomere length, genomic DNA was isolated and separated, and telomere restriction fragment length was measured. The telomere length was shortened with increasing passage in control cells (Figure 1B). The repeated addition of different concentrations of ADMA increased significantly the telomere shortening compared with control cells in a dose-dependent manner.

Because the synthesis of telomeres is associated with the enzymatic activity of an RNA-protein complex called telomerase, we measured the relative activity of telomerase in HUVECs. Telomerase activity decreased as a function of passage in control cells and was significantly further reduced in a dose-dependent manner in the ADMA treated cells (Figure 1C).

SAH-inhibited ADMA-accelerated senescence

To test whether SAH could attenuate the effect of ADMA (50 mol/l) on cellular senescence, HUVECs were incubated in the presence of SAH (100 mol/l) starting from CPD 20. It is interesting that a more significant decrease in SA-␤-gal activity was observed in endothelial cells treated with SAH compared with the control cells (Figure 2A), with a maximal effect at the tenth passage (CPD 40). Additionally, the shortening of telomere length was substantially delayed and the telomerase activity was significantly increased (Figures 2B and2C). The increased senescence in the presence of ADMA was completely reversed in the presence of SAH. Exposure of endothelial cells to ADMA resulted in a significant reduction of NO synthesis (CPD 40: ADMA, 2.59 Ϯ 0. ADMA-induced senescence was associated with an increase in oxidative stress and a decrease in DDAH enzyme activity Endogenous ROS formation was measured by detection of DHR during endothelial cell aging. The intracellular level of ROS was increased in control cells, starting at CPD 29 (Figure 3A). ADMA (5 mol/l) augmented ROS formation (p Ͻ 0.05). The antioxidant PDTC (10 mol/l) prevented an increase endogenous oxidative stress by ADMA.

To examine whether ADMA-induced oxidative stress is due to reduced degradation of ADMA by DDAH, DDAH activity was determined during continuous passaging of endothelial cells by assessing the rate of degradation of exogenous ADMA added to the control). Incubation with SAH significantly decreased allantoin formation. The stimulatory effects of ADMA on allantoin production were reversed by coincubating the cells with SAH. cell lysates. Concomitantly with the significant increase in ROS formation, DDAH activity significantly decreased at CPD 29 in control cells (Figure 3B). The treatment of endothelial cells with ADMA reduced DDAH enzyme activity in cell lysates compared with untreated cells ( p Ͻ 0.05). PDTC enhanced DDAH activity and prevented the impairment of DDAH activity by ADMA.

Furthermore, the effect of ADMA to inhibit NO synthesis was reversed by PDTC (data not shown). Moreover, PDTC abolished the effect of ADMA on allantoin production and endothelial senescence (data not shown).

MCP-1 and IL-8 secretion increased during ADMA-induced endothelial cell aging

The treatment of cultured endothelial cells with ADMA (50 mol/l) induced a significant secretion of MCP-1 and IL-8 compared with control (MCP-1: CPD 40 ADMA, 3073 Ϯ 67 pg/ml versus control, 2683 Ϯ 51 pg/ml; IL-8: ADMA, 2461 Ϯ 60 pg/ml versus control, 1968 Ϯ 30 pg/ml; p Ͻ 0.05 versus control, respectively). When cultured endothelial cells were incubated with SAH (100 mol/l), secretion of MCP-1 and IL-8 was significantly reduced compared with untreated cells (MCP-1: CPD 40 SAH, 2407 Ϯ 67 pg/ml; IL-8: SAH, 1753 Ϯ 58 pg/ml; p Ͻ 0.05 versus control, respectively). The stimulatory effects of ADMA on MCP-1 and IL-8 secretion were significantly diminished by SAH (MCP-1: SAH ϩ ADMA, 2663 Ϯ 52 pg/ml; IL-8: SAH ϩ ADMA, 1849 Ϯ 36 pg/ml; p Ͻ 0.05 versus ADMA, respectively).

Discussion

ADMA accelerates endothelial aging, as demonstrated by a more rapid reduction in telomere length and a decline in telomerase activity. The effect of ADMA on accelerating endothelial senescence seems to be due to increased oxidative stress because it can be reversed by the antioxidant PDTC. SAH significantly attenuates endothelial senescence, indicating that modest changes of intracellular ADMA levels are associated with substantial effects on endothelial cell senescence.

These data provide the first evidence for a novel mechanism of endothelial cell senescence mediated by ADMA.

What is the underlying mechanism by which ADMA accelerates cellular senescence? We speculate that ADMA increases oxidative stress, for example, by uncoupling NOS activity. Uncoupling of NOS catalytic activity is observed in the presence of N G- monomethyl-L-arginine (L-NMMA), [START_REF] Pritchard | Native lowdensity lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion[END_REF] an endogenous inhibitor of NOS. In this situation optimal electron flow within the two catalytic domains of NOS is impaired and molecular oxygen becomes the sole electron acceptor. This means that NOS generates superoxide radicals. We have shown that ADMA concentration dependently increases superoxide production by cultured human endothelial cells. [START_REF] Böger | An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes[END_REF] In the experiments presented here, the inhibition of NOS by ADMA decreases the elaboration of NO and increases endothelial oxidative stress as measured by intracellular ROS and allantoin production.

Telomere length is an important marker for cellular aging. Our data suggest that replicative senescence can be modulated by various intracellular processes. Endothelial cells are capable of synthesizing ADMA by the protein arginine methyltransferase type one. A major source of methyl groups used for various methylating reactions is S-adenosylmethionine, This methylation reaction is inhibited by SAH. [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells. Involvement of S-adenosylmethioninedependent methyltransferases[END_REF] We found that SAH attenuates endothelial senescence by increasing the activity of telomerase compared with control cells. The increased rate of endothelial senescence in the presence of ADMA was completely reversed in the presence of SAH.

The issue to be addressed is why the administration of exogenous SAH was seen to inhibit the effect of exogenous ADMA to produce its biological effects. However, it is hard to understand how an SAHinduced reduction of endogenous ADMA formation could fully counteract high concentrations of exogenous ADMA (50 M). A possible explanation would be the activation of DDAH, the enzyme that degrades ADMA. [START_REF] Macallister | Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase[END_REF] DDAH has a K m (Michaelis-Menten constant) for ADMA of about 180 M, so increased DDAH activity could overcome supraphysiological ADMA concentrations. This enzyme seems to be exquisitely sensitive to oxidative [START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus. Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Stühlinger | Homocysteine impairs the nitric oxide synthase pathway. Role of asymmetric dimethylarginine[END_REF] and nitrosative stress. [START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between S70 SM Bode-Böger et al Vascular Medicine 2005; 10: S65-71 nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF] We observed a decrease in the formation of allantoin, reflecting the reduced generation of superoxide anions. As recently shown by Cooke's group, even modest changes in ADMA levels induced by DDAH activity can have significant effects on NO synthesis. [START_REF] Dayoub | Dimethylarginine dimethyaminohydrolase regulates nitric oxide synthesis[END_REF] We were able to show in this study that incubation with SAH significantly increases the synthesis of NO.

This finding may indicate that intracellular levels of ADMA are more important than extracellular or systemic levels and/or that there may be compartmentalization of intracellular ADMA, with much higher levels in the vicinity of NOS.

Moreover, we observed in this investigation that ADMA-increased intracellular ROS formation was accompanied by a corresponding decrease in the activity of DDAH. This finding is supported by the observation that co-incubation with PDTC, a thiol antioxidant that protects sulfhydryl groups, reduced ROS formation and consequently the impairment of DDAH activity during ADMA-accelerated senescence. Recently, Haendler et al reported that the onset of replicative senescence of endothelial cells is regu-lated by ROS formation. [START_REF] Haendler | Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells[END_REF] Similarly, in the present study, we found that ADMA increased oxidative stress and accelerated the rate of cellular senescence. This effect of ADMA was reversed by the antioxidant PDTC. Thus, a reduction in ADMA synthesis induced by SAH and increased DDAH activity induced by PDTC both delayed the onset of cellular senescence.

We also investigated the effects of ADMA on the secretion of MCP-1 and IL-8 in endothelial cell supernatants. MCP-1 and IL-8 are major chemokines for leukocyte trafficking and have been found to be highly expressed in lesions of human atherosclerosis. [START_REF] Reape | Chemokines and atherosclerosis[END_REF] Upregulation of these proteins is induced by intracellular oxidative stress via the oxidant-responsive transcription factor NF-B. [START_REF] Böger | An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes[END_REF][START_REF] Zeng | Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes[END_REF] Indeed, we found that reduced NO production by ADMA was associated with increased oxidative stress and increased protein secretion of MCP-1 and IL-8. These effects were completely reversed by SAH.

Clinical relevance

Plasma ADMA levels are positively correlated with risk factors for atherosclerosis. Miyazaki et al reported a positive correlation for ADMA with age in individuals with no symptoms of coronary or peripheral artery disease. [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF] Celermajer et al reported that aging is associated with progressive endothelial dysfunction in normal humans. [START_REF] Celermajer | Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women[END_REF] Furthermore, endothelial senescence is associated with decreases in eNOS expression and activity, as well as with augmented endothelial adhesiveness for monocytes. These effects can be prevented by stably transfecting endothelial cells with human telomerase. [START_REF] Matsushita | ENOS activity is reduced in senescent human endothelial cells. Preservation by hTERT immortalization[END_REF] We speculate that high plasma levels of ADMA may accelerate the loss of telomerase activity, increase cellular senescence and induce endothelial dysfunction. Our data provide a new mechanism to explain the association of plasma ADMA or endothelial dysfunction with an increased risk of cardiovascular events.

Figure 1

 1 Figure 1 Effect of different concentrations of ADMA on senescence, telomere length and telomerase activity of endothelial cells, which were incubated with ADMA (5, 50 and 100 mol/L) starting from cumulative population doubling (CPD) 20 and replaced every 48 hours (A through C): (A) endothelial senescence was detected by senescence associated ␤-galactosidase (SA-␤-gal) positive cells; (B) terminal restriction fragment (TRF) lengths are plotted as a function of passage number; (C) relative telomerase activity (RTA). Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding CPD of control cells. (Figure 1A, B and C reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22.17)

  Figure 1 Effect of different concentrations of ADMA on senescence, telomere length and telomerase activity of endothelial cells, which were incubated with ADMA (5, 50 and 100 mol/L) starting from cumulative population doubling (CPD) 20 and replaced every 48 hours (A through C): (A) endothelial senescence was detected by senescence associated ␤-galactosidase (SA-␤-gal) positive cells; (B) terminal restriction fragment (TRF) lengths are plotted as a function of passage number; (C) relative telomerase activity (RTA). Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding CPD of control cells. (Figure 1A, B and C reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22.17)

S68Figure 2

 2 Figure 2 Effect of SAH on ADMA-accelerated senescence. Endothelial cells were incubated with ADMA (50 mol/L) and SAH (100 mol/L) starting from the fourth passage (young) and replaced every 48 hours until the tenth passage (old): (A) endothelial senescence was detected by senescence-associated ␤-galactosidase (SA-␤-gal) positive cells; (B) terminal restriction fragment (TRF) lengths are plotted as a function of passage number; (C) relative telomerase activity (RTA). Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding cumulative population doubling (CPD) of control cells.

Figure 3

 3 Figure 3 ADMA-inhibited NO synthesis was associated with an increase in endogenous oxidative stress and a decrease in endothelial DDAH enzyme activity. Endothelial cells were incubated with ADMA (5 mol/L) and pyrrolidine dithiocarbamate (PDTC) (10 mol/L) starting from the fourth passage (young) and added every 48 hours until the tenth passage (old): (A) endogenous reactive oxygen species formation was measured using the marker dihydrorhodamine (DHR) and the fluorescence-activated flow cytometry assay; (B) effect of ADMA and PDTC on endothelial DDAH activity. Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding cumulative population doubling (CPD) of control cells. (Figure 3B reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22.17)

17

 17 Figure 3 ADMA-inhibited NO synthesis was associated with an increase in endogenous oxidative stress and a decrease in endothelial DDAH enzyme activity. Endothelial cells were incubated with ADMA (5 mol/L) and pyrrolidine dithiocarbamate (PDTC) (10 mol/L) starting from the fourth passage (young) and added every 48 hours until the tenth passage (old): (A) endogenous reactive oxygen species formation was measured using the marker dihydrorhodamine (DHR) and the fluorescence-activated flow cytometry assay; (B) effect of ADMA and PDTC on endothelial DDAH activity. Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding cumulative population doubling (CPD) of control cells. (Figure 3B reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22.17)

  Figure 3 ADMA-inhibited NO synthesis was associated with an increase in endogenous oxidative stress and a decrease in endothelial DDAH enzyme activity. Endothelial cells were incubated with ADMA (5 mol/L) and pyrrolidine dithiocarbamate (PDTC) (10 mol/L) starting from the fourth passage (young) and added every 48 hours until the tenth passage (old): (A) endogenous reactive oxygen species formation was measured using the marker dihydrorhodamine (DHR) and the fluorescence-activated flow cytometry assay; (B) effect of ADMA and PDTC on endothelial DDAH activity. Each point represents the mean Ϯ SEM of results from three experiments. *p Ͻ 0.05 vs corresponding cumulative population doubling (CPD) of control cells. (Figure 3B reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22.17)

  4 nmol/mg protein versus control, 4.18 Ϯ 0.3 nmol/mg protein; p Ͻ 0.05 versus control). After SAH treatment, NO levels were increased significantly compared with control cells (CPD 40: SAH, 5.44 Ϯ 0.3 nmol/mg protein; p Ͻ 0.05 versus control). The inhibitory effects of ADMA on endothelial NO release were abolished by SAH (CPD 40: SAH ϩ ADMA, 4.03 Ϯ 0.7 nmol/mg protein; p Ͻ 0.05 versus ADMA). Incubation of endothelial cells with ADMA led to a significant increase in allantoin levels compared with control (CPD 40: ADMA, 41.2 Ϯ 1.8 nmol/mg protein versus control, 30.3 Ϯ 1.6 nmol/mg protein; p Ͻ 0.05 versus

Vascular Medicine 2005; 10: S65-71