Leiper J, Nandi M, Torondel B, Murray-Rust J, Malaki M, O'Hara B, et al. Disruption of methylarginine metabolism impairs vascular homeostasis. Nat Med 2007;13:198-203. (Available at: www.nature.com; Reprinted by permission.)
Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.
Decompensated cirrhosis is characterized by severe circulatory derangements, including progressive splanchnic vasodilatation and portal hypertension, which are reflected in its clinical manifestations. Splanchnic vasodilatation in turn results in relative arterial underfilling with consequent activation of the neurohumoral system, leading to vasoconstriction of numerous vascular beds, including the liver, kidneys, and brain, and increased cardiac output.1 An important mechanism influencing splanchnic vascular tone is believed to be excessive nitric oxide (NO) generation in response to the effects of shear stress and the modulation of cofactors for endothelial nitric oxide synthase (eNOS). Paradoxically, hepatic NO generation is reduced, and this is thought to be the result of reduced hepatic eNOS activity in animal models2 and humans,3, 4 despite the preservation of eNOS messenger RNA and protein.5, 6 The exact mechanisms underlying a reduction in eNOS activity are unclear, but recent studies have suggested roles for nitric oxide synthase (NOS) inhibitors such as caveolin-15 and eNOS trafficking inducer (NOSTRIN),4 in addition to disorders in guanylate cyclase activity7 and posttranslational eNOS modifications.8 Our recent studies have indicated that additional mechanisms may be involved and have focused on the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), and its relationship with the severity of portal hypertension during hepatic inflammation.9
NO plays an important role in the maintenance of vascular tone,10 and reduced local NO production has been shown to be associated with endothelial dysfunction in conditions varying from coronary atheromatous disease to diabetes mellitus and pre-eclampsia.11 Endothelium-derived NO is generated from the metabolism of L-arginine by eNOS. Since 1992, an endogenous inhibitor of NOS, ADMA, has been recognized as competing with L-arginine for the active binding site of NOS.12 The plasma levels of ADMA are increased in a number of disparate conditions and are thought to contribute to endothelial vasodilator dysfunction.13 ADMA and its stereoisomer, symmetric dimethylarginine (SDMA), are synthesized by the action of protein arginine methyltransferases and are released during proteolysis. Most of the ADMA that is generated is converted to citrulline by dimethylarginine dimethylaminohydrolases (DDAH),14 whereas SDMA does not undergo metabolism by this route. The liver is an important site of ADMA metabolism both in its production because of the high rate of protein turnover and, more importantly, in its elimination through the action of DDAH.15
There has been increasing interest in the liver literature with respect to ADMA, with data suggesting that patients with decompensated cirrhosis have higher ADMA levels than those with compensated disease and that these levels may increase further with evolving liver failure.16, 17 The presence of inflammation also increases ADMA levels and exacerbates vascular dysfunction by reducing NOS activity, as suggested by studies of cultured endothelial cells exposed to tumor necrosis factor α or oxidized low-density lipoprotein, which have demonstrated a reduction in DDAH activity and an increase in ADMA.18 This may be extrapolated to the incidence of posttransplantation rejection, when ADMA levels are also elevated,17 and also to the setting of acute liver failure, where, we have shown, ADMA levels correlate with indices of inflammation (Fig. 1).19 However, whether ADMA is causally related to these pathophysiological states has remained an unanswered question to date.
In their article, Leiper et al.20 have addressed the issue of causality by testing the hypothesis that the accumulation of endogenous ADMA alters vascular function by inhibiting ADMA metabolism through targeted gene deletion of the enzyme DDAH-1 and also through chemical inhibition of its active site. This article demonstrates that global homozygous deletion of DDAH-1 is lethal in utero, whereas heterozygote DDAH-1+/− animals have normal development and no overt phenotypic abnormalities. Plasma and tissue ADMA are also increased in DDAH-1+/− animals with no change in the SDMA concentrations (the inactive isomer, which is not believed to be metabolized by DDAH). The application of DDAH inhibitors yields results similar to those of knockouts, resulting in increased plasma ADMA concentrations at levels similar to those reported in cardiovascular disease patients (0.2–0.6 μmol/L) but with no direct inhibitory activity against NOS. Functional studies in isolated vessels confirmed reduced NO and increased ADMA generation, with impaired vasodilatation to acetylcholine, which was reversible with L-arginine but not D-arginine; this confirmed the specificity of effects on NOS by elevated ADMA. The mean arterial blood pressure, systemic vascular resistance, and right ventricular pressure were increased and the cardiac output was reduced in DDAH-1+/− mice in comparison with wild-type mice. An assessment of vessels of the pulmonary tree showed an increase in the wall-to-lumen ratio, suggesting that they may have undergone remodeling in the context of increased ADMA. The authors also studied a model of endotoxic shock in which treatment of cultured isolated blood vessels with bacterial lipopolysaccharide led to hyporeactivity to the contractile effects of phenylephrine, because of the effects of increased expression of inducible nitric oxide synthase (iNOS). These effects were reversed by a treatment with a DDAH inhibitor (increasing ADMA in the culture medium), and this response was attenuated by the addition of L-arginine. By an extension of this observation, the injection of a DDAH inhibitor into rats with falling blood pressure after lipopolysaccharide administration resulted in a stabilization of blood pressure.
The authors elegantly show that the loss of DDAH-1 function through genetic or chemical inhibition results in increased ADMA concentrations, decreased NO-dependent vascular effects, and consequent changes in the vascular resistance. This study is the first to clearly show a causal link between changes in DDAH expression and vascular dysfunction that may help to explain the significant number of studies suggesting associations between elevated ADMA levels and vascular effects in different organ beds. Specifically, we recently reported a study of patients with alcoholic cirrhosis,9 demonstrating that patients with additional inflammatory alcoholic hepatitis had higher plasma and hepatic tissue ADMA levels while also having higher portal pressures. Moreover, elevated plasma and tissue ADMA concentrations were predictive of the development of organ failure and outcome. Our study showed that patients with high ADMA and inflammatory alcoholic hepatitis also had decreased DDAH expression. The data by Leiper et al.20 support the notion that an alteration in DDAH expression and/or activity in liver disease leads to high ADMA along with resultant endothelial dysfunction and increased intrahepatic resistance.
It is important to note that the actions of DDAH may extend beyond the inhibition of NOS and that changes in DDAH protein levels may exert direct biological effects through protein-protein interactions. It has been demonstrated that pathophysiological concentrations of ADMA in coronary artery endothelial cells result in changes in the gene expression for several proteins, including bone morphogenic protein, and enzymes involved in arginine methylation (protein arginine N-methyltransferase 3).21 Neither a high dose of a NOS inhibitor nor SDMA replicated the effect of ADMA on gene expression, and this suggests the possibility of NO-independent actions, which may occur through a further increase in oxidative stress and superoxide generation.22 In contrast, a recent report has shown that overexpression of DDAH in a transgenic animal that has incurred traumatic vascular injury leads to reduced plasma ADMA levels and enhanced endothelial cell regeneration in comparison with the wild type, while also reducing neointima formation.23
Another aspect raised in the article by Leiper et al.20 is the potential for therapy for conditions in which there is an excess of NO production, such as endotoxic shock. In this condition, the NO liberated through iNOS generation in isolated blood vessels and in animals treated with lipopolysaccharide was shown to be blocked by DDAH inhibition, with resultant stabilization of the blood pressure. One might consider extending such an application of DDAH inhibition to the context of decompensated portal hypertension with the development of hepatorenal syndrome, in which renal perfusion is limited. This follows increased renal microvascular resistance through increased renin-angiotensin activation as a result of marked arterial underfilling.24 In this setting, reducing splanchnic NO generation may increase systemic vascular resistance and preload so that renal perfusion is enhanced. Such a notion requires formal testing but provides a novel application of the improved understanding of NOS regulation by ADMA and its importance in the evolution of increased vascular resistance in advanced liver disease.