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Keywords:

  • Asymmetric dimethylarginine;
  • atherosclerosis;
  • cardiovascular risk;
  • dimethylarginine dimethylaminohydrolase;
  • nitric oxide;
  • renal dysfunction

Abstract

  1. Top of page
  2. Abstract
  3. The ADMA–NO connection
  4. Conclusion
  5. References

Endothelial dysfunction as a result of reduced bioavailability of nitric oxide (NO) plays a central role in the process of atherosclerotic vascular disease. In endothelial cells NO is synthesized from the amino acid l-arginine by the action of the NO synthase (NOS), which can be blocked by endogenous inhibitors such as asymmetric dimethylarginine (ADMA). Acute systemic administration of ADMA to healthy subjects significantly reduces NO generation, and causes an increase in systemic vascular resistance and blood pressure. Increased plasma ADMA levels as a result of reduced renal excretion have been associated with atherosclerotic complications in patients with terminal renal failure. However, a significant relationship between ADMA and traditional cardiovascular risk factors such as advanced age, high blood pressure and serum LDL-cholesterol, has been documented even in individuals without manifest renal dysfunction. As a consequence, the metabolism of ADMA by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) has come into the focus of cardiovascular research. It has been proposed that dysregulation of DDAH with consecutive increase in plasma ADMA concentration and chronic NOS inhibition is a common pathophysiological pathway in numerous clinical conditions. Thus, ADMA has emerged as a potential mediator of atherosclerotic complications in patients with coronary heart disease, peripheral vascular disease, stroke, etc., being the culprit and not only an innocent biochemical marker of the atherosclerotic disease process.


The ADMA–NO connection

  1. Top of page
  2. Abstract
  3. The ADMA–NO connection
  4. Conclusion
  5. References

It is now widely accepted that endothelial dysfunction as a result of reduced bioavailability of nitric oxide (NO) plays a central role in the process of atherosclerotic vascular disease [1]. NO is a very active but short-living molecule that is released in the circulation from endothelial cells. It is a potent vasodilator that regulates vascular tone and tissue blood flow, and inhibits platelet aggregation and leukocyte adhesion on the endothelial surface. It is synthesized by stereospecific oxidation of the terminal guanidino nitrogen of the amino acid l-arginine by the action of a family of NO synthases (NOS) [2]. In endothelial cells, NO is synthesized by the endothelial NOS (eNOS), which converts the amino acid l-arginine into l-citrulline and NO. In clinical studies, impairment of the l-arginine/NO pathway independently predicted cardiovascular complications related to atherosclerosis [3–5]. Consequently, guanidino-substituted analogues of l-arginine which competitively block the NOS active site, i.e. endogenous NOS inhibitors such as asymmetric dimethylarginine (ADMA) and N-monomethylarginine (MMA), have gained much interest in cardiovascular medicine over the past decade [6,7]. Experimental and clinical research has focused on ADMA, however, because it is the predominant NOS inhibitor in humans with plasma levels 10-fold greater than those of MMA [8]. ADMA is released in endothelial cells (and other cell lines) after post-translational methylation from proteins involved in RNA processing and transcriptional control [9,10] (Fig. 1). The enzyme protein arginine methyltransferase type I (PRMT I) produces ADMA, whereas PRMT II produces symmetric dimethylarginine (SDMA), i.e. the stereoisomer of ADMA which has no proven direct inhibitory effect on NOS [11]. Meanwhile several PRMTs that produce methylarginines have been described [12].

image

Figure 1. Biochemical pathways for generation and degradation of asymmetric dimethylarginine (ADMA). PRMT I = protein arginine methyltransferase type I; NOS = nitric oxide synthase; DDAH = dimethylarginine dimethylaminohydrolase; DPT = dimethylarginine pyruvate aminotransferase. For detailed explanation please see text.

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Data from experimental studies document that biologically relevant ADMA blood levels significantly inhibit NOS and reduce NO generation in cultured endothelial cells and in isolated human blood vessels [13–15]. Further, administration of ADMA to laboratory animals caused an increase in renal, mesenteric and hindquarters vascular resistance and an increase in blood pressure [16,17]. In humans, indirect evidence for a biologically relevant action of ADMA comes from a study in renal patients, in whom plasma ADMA concentrations were markedly increased as compared with healthy controls [18]. Their blood significantly inhibited NO production in cultured endothelial cells ex vivo. In addition, Chan et al. have found that mononuclear cell adhesiveness ex vivo significantly correlates with plasma ADMA levels [19]. The authors could also enhance leukocyte adhesiveness by coculturing them with ADMA-stimulated endothelial cells. Moreover, local infusion of ADMA into the brachial artery significantly attenuated endothelial-dependent vasodilatation in healthy volunteers [8,20]. Finally, we and others have demonstrated that systemic administration of ADMA to healthy subjects causes a dose-dependent and sustained reduction of NO production and cardiac output, and an increase in peripheral vascular resistance accompanied by a rise in mean arterial blood pressure (Fig. 2) [21,22]. In addition, infusion of ADMA significantly reduced renal perfusion and increased sodium reabsorption. These renal effects can be induced even with low dose ADMA administration, i.e. a dose which does not cause an (acute) increase in blood pressure [23]. Importantly, these adverse cardiovascular effects were documented at plasma ADMA concentrations that are encountered in patients with different cardiovascular diseases [7,21]. Taken together, in vitro and in vivo findings confirm that ADMA is a potent and long-lasting (endogenous) NOS inhibitor.

image

Figure 2. Effect of systemic intravenous administration of asymmetric dimethylarginine (ADMA) to healthy subjects on cardiac output (a) and systemic vascular resistance (b) measured invasively using a right heart catheter. The infusion of ADMA caused an immediate significant increase in systemic vascular resistance and a decrease in cardiac output. The cardiovascular effects of ADMA persisted for at least 2 h after discontinuation of the infusion (with permission from reference [21]).

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The role of renal dysfunction in the metabolism of ADMA

In their seminal paper, Vallance et al. reported increased plasma concentrations of methylarginines (i.e. ADMA and SDMA) in a small group of patients on maintenance haemodialysis [8]. They hypothesized that the high incidence of hypertension and atherosclerosis encountered in patients with terminal renal failure might be caused, at least in part, by dysfunction of the l-arginine/NO pathway secondary to accumulation of ADMA because of declining renal excretion. Indeed, in several subsequent studies, markedly increased plasma ADMA levels have been documented in patients with terminal renal failure [24–28]. As pointed out previously, plasma ADMA concentrations in these patients are certainly high enough to significantly reduce NO production. Indirect evidence for a pathophysiological role of ADMA comes from the observation that in patients on maintenance haemodialysis with vascular complications, plasma ADMA levels are significantly higher than in patients without manifest atherosclerotic disease [26]. Moreover, in a prospective study in 225 patients with terminal renal failure, increased plasma ADMA concentrations were not only significantly related to the severity of carotid atherosclerosis and left ventricular dysfunction but, in addition, were the second strongest predictor (after age) of all-cause and cardiovascular mortality (Fig. 3) among several traditional and nontraditional risk factors assessed [27,29,30]. A subsequent analysis of the same study population revealed that ADMA also significantly correlates with plasma noradrenaline levels in these patients [31]. Because of the compelling data obtained in patients with renal failure, ADMA has been regarded by many rather as a uraemic toxin than a substance with more general significance in cardiovascular medicine.

image

Figure 3. ADMA and cardiovascular morbidity and mortality in 225 patients with terminal renal failure. In the Cox's regression model plasma ADMA concentration ranked as the second factor (after age) predicting cardiovascular outcome. The overall risk of fatal and nonfatal cardiovascular events (adjusted for age and sex) was progressively higher from the 50th percentile of plasma ADMA levels onwards (Adapted from data in reference [27]).

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DDAH and ADMA – a less nephrocentric view of the problem?

The assumption that renal excretion of ADMA is the main route of elimination has been recently questioned. Particularly our observation of significantly increased plasma ADMA concentrations in patients with incipient renal disease, i.e. in patients with normal glomerular filtration rate as documented by invasive clearance measurements, support the notion that other degradation pathways must also be involved [32]. It is now well established that the key elimination route for ADMA is enzyme degradation by dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyses ADMA to dimethylamine and l-citrulline (Fig. 1) [33,34]. So far, two isoforms of DDAH have been characterized; DDAH2 is the predominant form in tissues expressing eNOS. It has been estimated that in humans, approximately 300 µmol of ADMA is generated per day, of which approximately 250 µmol is metabolized by DDAHs, whereas only a minor amount is excreted by the kidneys [18]. Moreover, the finding that DDAH and NOS are colocalized in cells supports the hypothesis that intracellular ADMA concentration is actively regulated in NO-generating cells [35]. Because NO has tremendous biological activity, its tissue and/or blood concentration must be kept within narrow limits in order to prevent harmful effects on cell activity. This could be accomplished by cell-specific competitive NOS inhibitors such as ADMA. Indeed, recent experimental results even point to the possibility that DDAH activity is directly regulated by S-nitrosylation of its active site by NO, thereby creating a regulatory feedback loop between NO, DDAH, ADMA and NOS (Fig. 1) [36]. The implication of this finding is that under conditions of increased NO production, such as in inflammation where the inducible NOS (iNOS) generates abundant NO, S-nitrosylation diminishes DDAH activity and this in turn would lead to accumulation of ADMA and to NOS inhibition. This putative feedback loop would also help explain, at least in part, the link between infectious diseases, inflammation and atherosclerosis [37,38].

The finding that DDAH is present in abundance in endothelial cells within the glomerulus and renal vessels, and particularly in renal tubular cells supports the concept that impaired ADMA degradation by renal DDAH rather than by reduced renal filtration is the major cause of increased plasma ADMA concentrations in patients with renal disease [7,32]. DDAH in renal cells regulates (intra)cellular methylarginine levels thereby governing cell-specific l-arginine uptake and NO generation [35]. Indirect proof for the assumption that destruction of DDAH-rich renal tissue could impair ADMA degradation comes from metabolic balance studies in laboratory animals and in healthy subjects with normal renal function, which have revealed that the kidney is a major extraction site for ADMA from the circulation [39,40]. Collectively, these data may explain why plasma ADMA concentrations can increase in patients with even minor renal dysfunction, potentially contributing to their significantly increased cardiovascular morbidity and mortality [41,42]. Another important aspect of NOS inhibition by increased ADMA levels in patients with renal dysfunction is the progression of renal disease. Experimental studies have revealed that reduced NO bioavailability plays a critical role in progression, and increased ADMA blood levels may contribute to this process [43]. In this respect, it is of interest that plasma ADMA levels significantly correlate with the age-related decrease in renal perfusion in elderly subjects [44]. Prospective clinical studies are warranted to clarify the role of ADMA in the progression of renal disease.

NOS inhibition by ADMA – a common pathway of endothelial dysfunction and atherosclerosis

A growing number of published studies in individuals without manifested renal disease documented a strong relationship between several traditional cardiovascular risk factors and increased plasma ADMA levels (Table 1). For example, results from a cross-sectional study in 116 otherwise healthy Japanese subjects revealed that plasma ADMA concentrations were positively correlated with age, mean arterial blood pressure and glucose intolerance [45]. Most strikingly, plasma ADMA levels were also significantly correlated with the intima-media thickness of the carotid arteries, an established surrogate parameter of atherosclerosis. Moreover, in a large cohort of Finish men with normal renal function in whom several (traditional) cardiovascular risk factors were present, increased plasma ADMA levels were predictive for future coronary events [69].

Table 1.  Clinical conditions, diseases and experimental settings in which increased plasma asymmetric dimethylarginine (ADMA) concentrations have been reported. ADMA may serve as a cardiovascular risk marker in many of these conditions, whereas in some it is thought to play a definite pathophysiological role
Condition/diseaseClinical evidence [reference]Experimental evidence [reference]
Age/senescence[44,45][46]
Salt intake/high blood pressure[44,45,47,48][49]
Hypercholesterolemia[50,51][52–54]
Hypertriglyceridemia[55,56] 
Hyperhomocystinaemia[57,58][53,59]
Insulin resistance/hyperglycaemia[45,60][61]
Hypertension (essential)[45,47,51,62–64][65]
Hypertension (pulmonary)[66,67][68]
Coronary heart disease[27,69–72] 
Vascular disease/stroke[26,73–76][77]
Heart failure[78,79][80]
Pre-eclampsia[81] 
Diabetes[82][83,84]
Hyperthyrosis[85] 
Infection[86][52,87]

The key mechanism for an increase of plasma ADMA concentrations in populations without renal dysfunction is thought to be dysregulation of DDAH, i.e. inhibition of DDAH activity by hypercholesterolemia (LDL-cholesterol), hyperglycemia, inflammation, etc. This concept is also supported by data obtained in various experimental settings (Table 1). It is therefore conceivable that an increase in plasma ADMA concentrations as a result of DDAH inhibition may be a common pathway through which traditional as well as nontraditional cardiovascular risk factors may cause chronic endothelial dysfunction leading to atherosclerotic vascular disease (Fig. 1) [6]. Further evidence for this idea comes from a recent experiment with transgenic mice harbouring the human DDAH gene [88]. In these animals, additional DDAH activity lowers plasma ADMA concentration and increases NO bioavailability. As a result, vascular resistance and blood pressure are significantly lower than in wild type animals.

Based on the clinical and experimental data reviewed in this article, the hypothesis has been put forward that chronically elevated plasma ADMA concentrations may be of definite relevance in human cardiovascular biology, i.e. ADMA being the culprit and not only an innocent biochemical bystander of the atherosclerotic disease process [6,7,89]. Several recently published experimental studies revealing direct adverse effects of ADMA at the cellular and tissue level have provided further evidence in favour of this hypothesis [90–92]. In this respect, the discovery of a DDAH2 promoter polymorphism in human tissue is of particular interest, as it may be responsible for individual differences in the ability to metabolize ADMA [93]. This could theoretically contribute to the individual susceptibility for atherosclerosis and related vascular disorders.

Therapeutic perspectives

Because ADMA is thought to be a competitive NOS inhibitor, the logical therapeutic intervention would be administration of l-arginine in order to overcome NOS inhibition. To date, a large body of evidence has accumulated on the beneficial role of l-arginine administration on several aspects of endothelial (dys)function, but surprisingly few studies have specifically addressed the potential role of ADMA [6]. Böger et al. showed that administration of l-arginine to laboratory animals and to patients with peripheral vascular disease increased the l-arginine/ADMA ratio and thereby NO production and, in addition, also ameliorated clinical symptoms in patients [94,95]. Moreover, several pharmaceutical interventions have been tested in clinical trials, e.g. oral oestrogen, lipid-lowering drugs and ACE inhibitors (Table 2). There is evidence that hormone replacement therapy in postmenopausal women can reduce plasma ADMA concentrations via stimulation of DDAH activity [98–100]. In contrast, most studies examining the effect of HMG-CoA-reductase inhibitors (statins) on plasma ADMA levels were disappointing in this respect (Table 2). Results from two smaller studies in patients with essential hypertension and type 2 diabetes mellitus have suggested that pharmacological treatment with ACE inhibitors and/or AT1-receptor antagonists may reduce plasma ADMA concentrations [108,109], but we were not able to confirm this finding in a recent double-blind placebo-controlled trial with olmesartan [110]. Finally, improvement of insulin sensitivity was accompanied by a decrease in plasma ADMA concentrations in nondiabetic as well as in diabetic subjects [60,111].

Table 2.  The effect of various therapeutic interventions on plasma asymmetric dimethylarginine (ADMA) concentrations. Studies that have reported reduction of plasma ADMA levels are indicated as positive evidence, whereas reports in which no significant effect on ADMA has been observed are listed as negative evidence
Therapeutic interventionPositive evidence [reference]Negative evidence [reference]
Intravenous l-arginine[94–96][97]
Oral estrogen[98–100] 
Lipid lowering agents (statins/fibrates)[101,102][103–107]
ACE inhibitors/AT1-receptor antagonists[108,109][110]
Glucose lowering agents[60,111] 

Despite the theoretical possibility of therapeutic DDAH modulation and hence, plasma ADMA concentration, it has yet to be proven in controlled clinical studies that lowering ADMA also improves cardiovascular outcome in populations at risk. A major obstacle for the conduction of large-scale intervention trials as well as of epidemiological surveys is the measurement of ADMA, however. The available analytical methods of choice – high performance liquid chromatography (HPLC) and tandem liquid chromatography-mass spectrometry (LC-MS) – are rather sophisticated, impractical for routine use and quite expensive. In addition, most laboratories using these methods report different values for the normal range. In face of the growing importance of ADMA in cardiovascular medicine [6,89], the introduction of a sensitive and reliable standardized measurement into routine diagnostic should be soon accomplished.

Conclusion

  1. Top of page
  2. Abstract
  3. The ADMA–NO connection
  4. Conclusion
  5. References

ADMA is a potent and long-lasting endogenous NOS inhibitor that is thought to be a key player in the process of chronic vascular disease. Future research must therefore elucidate in detail the relationship between ADMA, DDAH, NOS and NO in human disease. In addition, large controlled trials on therapeutic interventions that reduce plasma ADMA levels should assess outcome in populations at risk.

References

  1. Top of page
  2. Abstract
  3. The ADMA–NO connection
  4. Conclusion
  5. References
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