Dimethylarginine dimethylaminohydrolase overexpression ameliorates atherosclerosis in apolipoprotein E-deficient mice by lowering asymmetric dimethylarginine.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is increasingly recognized as a novel biomarker in cardiovascular disease. To date, it remains unclear whether elevated ADMA levels are merely associated with cardiovascular risk or whether this molecule is of functional relevance in the pathogenesis of atherosclerotic vascular disease. To clarify this issue, we crossed dimethylarginine dimethylaminohydrolase (DDAH) transgenic mice that overexpress the human isoform 1 of the ADMA degrading enzyme DDAH into ApoE-deficient mice to generate ApoE(-/-)/hDDAH1(+/-) mice. In these mice, as well as ApoE(-/-) wild-type littermates, atherosclerosis within the aorta as well as vascular function of aortic ring preparations was assessed. We report here that overexpression of hDDAH1 reduces plaque formation in ApoE(-/-) mice by lowering ADMA. The extent of atherosclerosis closely correlated with plasma ADMA levels in male but not female mice fed either a standard rodent chow or an atherogenic diet. Functional analysis of aortic ring preparations revealed improved endothelial function in mice overexpressing hDDAH1. Our findings provide proof-of-principle that ADMA plays a causal role as a culprit molecule in atherosclerosis and support recent evidence indicating a functional relevance of DDAH enzymes in genetic mouse models. Together, these results demonstrate that pharmacological interventions targeting the ADMA/DDAH pathway may represent a novel approach in the prevention and management of cardiovascular diseases.

Role of dimethylarginine dimethylaminohydrolases in the regulation of endothelial nitric oxide production.

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.

Role of the PRMT-DDAH-ADMA axis in the regulation of endothelial nitric oxide production.

There is abundant evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO), formed in healthy vascular endothelium from the amino acid precursor l-arginine. Endothelial dysfunction is increased by various cardiovascular risk factors, metabolic diseases, and systemic or local inflammation. One mechanism that has been implicated in the development of endothelial dysfunction is the presence of elevated levels of asymmetric dimethylarginine (ADMA). Free ADMA, which is formed during proteolysis, is actively degraded by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH) which catalyzes the conversion of ADMA to citrulline and dimethylamine. It has been estimated that more than 70% of ADMA is metabolized by DDAH (Achan et al. [1]). Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA mediated eNOS impairment. However, recent studies suggest that DDAH may regulate eNOS activity and endothelial function through both ADMA-dependent and -independent mechanisms. In this regard, elevated plasma ADMA may serve as a marker of impaired methylarginine metabolism and the pathology previously attributed to elevated ADMA may be manifested, at least in part, through altered activity of the enzymes involved in ADMA regulation, specifically DDAH and PRMT.

Regulation of eNOS-derived superoxide by endogenous methylarginines.

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and N (G)-monomethyl- l-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH 4) depletion eNOS also generates (*)O 2 (-); however, the effects of methylarginines on eNOS-derived (*)O 2 (-) generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on (*)O 2 (-) production from eNOS under conditions of BH 4 depletion. In the absence of BH 4, ADMA dose-dependently increased NOS-derived (*)O 2 (-) generation, with a maximal increase of 151% at 100 microM ADMA. L-NMMA also dose-dependently increased NOS-derived (*)O 2 (-), but to a lesser extent, demonstrating a 102% increase at 100 microM L-NMMA. Moreover, the native substrate l-arginine also increased eNOS-derived (*)O 2 (-), exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either l-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for l-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and (*)O 2 (-) generation from eNOS. These observations have important implications with regard to the therapeutic use of l-arginine and the methylarginine-NOS inhibitors in the treatment of disease.

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation.

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.