COMMENTARIES

Dimethylated L-Arginine Analogs and Atherogenesis

Andrzej Surdacki, 2^nd Department of Cardiology, Jagiellonian University, 17 Kopernika Street, 31-501 Cracow, Poland

Origin and Elimination of Methylated L-Arginine Analogs

Asymmetric N^G,N^G-dimethyl-L-arginine (ADMA), an endogenous competitive inhibitor of nitric oxide (NO) synthesis, is formed by methylation of the guanidine nitrogens of arginine residues within proteins through the action of S-adenosylmethionine-dependent protein arginine N-methyltransferases (PRMTs) type I (PRMTs-I), which catalyze formation of ADMA and NG-monomethyl-L-arginine (L-NMMA) [1,2]. In contrast, symmetric N^G,N'^G-dimethyl-L-arginine (SDMA) (a stereoisomer of ADMA) and L-NMMA are products of PRMTs type II (PRMTs-II) [1,2]. Preferential substrates of PRMTs-I are heterogeneous nuclear ribonucleoproteins A1 and A2, some nucleolar ribonuclear proteins, basic fibroblast growth factor, and probably also histones and heat shock proteins, whereas PRMTs-II methylate myelin basic protein, one of the main protein components of the meylin sheath [2]. Functional consequences of arginine methylation are a fascinating topic [2], yet beyond the scope of this mini-review. ADMA and SDMA are liberated during breakdown of proteins containing dimethylated arginine residues [1]. Urinary excretion is the only route of elimination of SDMA, whereas over 80% of ADMA generated daily is degraded by dimethylarginine dimethylaminohydrolase (DDAH) which also decomposes L-NMMA [1,3]. The expression of PRMTs-I is stimulated by native low-density lipoproteins (nLDL) and especially their oxidized forms (oxLDL), whereas DDAH activity is inhibited by oxLDL, glucose, homocysteine, tumor necrosis factor ?, all of which act presumably via raised oxidative stress [1]. This suggests that an imbalance between ADMA synthesis and degradation with consequent excessive ADMA accumulation may be precipitated by intracellular oxidative stress and proinflammatory cytokines, both of which are implicated in the pathogenesis of atherosclerosis, an inflammatory disease.

ADMA Levels in Blood of Patients with Atherosclerosis or Risk Factors

Vallance et al. [4] were the first to observe elevated ADMA and SDMA levels (almost 8-fold) in plasma of patients with end-stage renal disease in 1992. During the next 20 years, excessive ADMA accumulation and/or increased ADMA/L-arginine ratio have been reported in patients with peripheral [5], carotid [6], and coronary [7,8] atherosclerosis, as well as in subjects without clinical evidence of atherosclerosis, yet with the presence of such atherosclerotic risk factors as hypercholesterolemia [9], essential hypertension [10], diabetes mellitus [11], insulin resistance [12] and hyperhomocysteinemia [13]. Moreover, associations between ADMA levels and the extent or severity of atherosclerosis, degree of endothelial dysfunction and biochemical indices of impaired NO generation have been found. Finally, ADMA emerged as an independent predictor of major adverse cardiovascular events in coronary artery disease (CAD) [14-16] and end-stage renal disease [17].

Mechanisms of ADMA Ability to Influence Endothelial Function and Atherogenesis

Strong arguments in favor of pathophysiological relevance of ADMA accumulation have come from papers who reported either increases or decreases in NO generation on over-expression [18] or inhibition [19], respectively, of DDAH, the ADMA-degrading enzyme. This suggests that intracellular ADMA and DDAH activity continuously modulate endothelial NO formation, thus affecting endothelial function and atherogenesis. On the other hand, it remains an intriguing issue how ADMA is able to exert any biological effects in the presence of saturating L-arginine concentrations which are well above the Michaelis-Menten constant (K_m) of endothelial NO synthase for L-arginine (2.9 µmol/l) [20]. For the same mechanistic reason, the ability of exogenous L-arginine to produce NO-dependent vascular effects is a surprising phenomenon, so-called "L-arginine paradox" [20]. It can be speculated that both these phenomena may be interrelated. Indeed, in cell cultures intracellular ADMA levels were about 10-fold higher than in the medium [21]. Moreover, after ADMA or L-NMMA had been added into the surrounding medium, they were concentrated in endothelial cells [22,23], which indicates their active uptake with possible competition between L-arginine and some of its methylated analogs for the transport. Additionally, the ADMA/L-arginine ratio averaged 1:70 in the rabbit plasma [24], 1:7 in the rabbit endothelial cells [25], and even 1:1.5 in the regenerated dysfunctional endothelium of the rabbit carotid artery after experimental endothelial injury [25]. The latter might be of particular clinical relevance at acute or chronic endothelial injury (e.g. acute coronary syndromes, balloon angioplasty with stent implantation, or clustering of risk factors in stable angina) when the rate of endothelial renewal is crucial for prognosis. Moreover, endothelial NO synthase is colocalized with the y⁺ L-arginine transmembrane carriers in plasmalemmal caveolae, which suggests that within selected compartments in the vicinity of endothelial NO synthase L-arginine/ADMA ratio might be different from those in the extracellular fluid or in whole-cell homogenates [20]. In agreement with this hypothesis, exogenous ADMA levels required to inhibit NO formation in endothelial cells or isolated vascular segments are much lower than those capable to inhibit purified endothelial NO synthase [23]. Similarly, significant in vitro vasoconstrictive responses were observed at concentrations of ADMA added into the organ baths beginning from levels observed in pathophysiological conditions [1,4]. Moreover, at decreased bioavailability of L-arginine (i.e. L-arginine deficiency or ADMA excess) not only is NO release depressed but NO synthases can be converted into sources of superoxide anion [26] with consequent formation of peroxynitrite and nitrosative stress. Accordingly, ADMA stimulated oxidant-sensitive nuclear factor ?B-regulated transcription pathway with consequent expression of monocyte chemoattractant protein-1 [21], a chemokine which augments the affinity of monocytic integrins for adhesion molecules on endothelial surface with subsequent firm adhesion of monocytes to the endothelium, a key early event in atherogenesis [27,28].

Novel Aspects of Proatherogenic ADMA Activity

Novel concepts of proatherogenic ADMA activity has come from studies dealing with angiogenesis and vasculogenesis. As NO participates in endothelial cells proliferation and migration and prevents endothelial apoptosis [29], the ability to counteract these effects is accountable for antiangiogenic ADMA activity [30]. In addition, ADMA accelerates replicative senescence of endothelial cells, i.e. their inability to divide [31]. This occurs in part via NO deficiency-dependent inhibition of telomerase activity with consequent potentiated rate of telomere attrition, considered a molecular clock that triggers cellular senescence [31,32]. Moreover, ADMA modulates the number and function of circulating endothelial progenitor cells (EPCs). EPCs originate from the bone marrow and participate in neovascularization of ischemic tissues [33], reendothelization after endothelial injury [34], and continuous renewal of the endothelium [35]. Low numbers of EPCs in the blood had been reported in conditions also associated with elevated ADMA levels (coronary [36,37] and carotid [38] atherosclerosis and traditional risk factors [36,39]) and ? similarly to ADMA ? is an independent marker of adverse prognosis in CAD [40,41]. The mobilization of EPCs from the bone marrow into the blood is dependent on the local L-arginine-NO pathway in vascular endothelial cells of the bone marrow stroma [42]. In 2005 Thum et al. [37] have described blood EPCs depletion in proportion to elevated plasma ADMA and severity of coronary atherosclerosis. They have also reported an ability of ADMA to inhibit differentiation of EPCs in vitro with a significant effect on the size of EPCs-derived endothelial cells colonies at as low ADMA concentrations as 1 µmol/l, i.e. close to the normal range [37]. This appears to reflect a novel aspect of proatherogenic ADMA activity which does not appear to be limited to CAD. In fact, we have recently described elevated ADMA levels associated with blood EPCs depletion and early carotid atherosclerosis in rheumatoid arthritis [43], a disease associated with accelerated atherogenesis.

SDMA - More Than Biologically Inactive Stereoisomer of ADMA?

SDMA - which had long been considered a biologically inactive ADMA stereoisomer - has recently been found to decrease NO synthesis in endothelial cells beginning from 2 µmol/l [44]. This may have resulted from the SDMA-induced competitive inhibition of the y⁺ carriers-dependent L-arginine influx into the cells as well as from the exchange of SDMA against intracellular L-arginine [45]. Additionally, SDMA (but not ADMA) was an independent predictor of one of angiographic parameters of coronary atherosclerosis in 147 patients with angiographically proven CAD [44]. This can be of particular relevance in mild-to-moderate renal dysfunction (affecting about 13 millions subjects in the United States) because in these patients ADMA increases are relatively mild as compared to elevations of SDMA [46] which - in contrast to ADMA - is not decomposed by DDAH and whose only route of elimination is urinary excretion [1,3]. This contrasts with preferential accumulation of ADMA in the presence of other atherosclerotic risk factors, the latter being consistent with the predominantly endothelial origin of circulating ADMA as endothelial cells release more ADMA than SDMA [21]. Therefore it does not seem implausible to assume that not only ADMA but also SDMA may contribute to high cardiovascular morbidity and mortality in chronic kidney disease, a major public health problem.

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