Atherosclerosis is a chronic cardiovascular disease which increases risk of major cardiovascular events including myocardial infarction and stroke. Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) have long been recognised as a hallmark of cardiovascular disease and are associated with cardiovascular risk factors including hypertension, obesity and hypertriglyceridemia. In this review, we discuss the clinical literature that link ADMA concentrations to increased risk of the development of atherosclerosis. The formation of atherosclerotic lesions relies on the interplay between vascular dysfunction, leading to endothelial activation and the accumulation of inflammatory cells, particularly macrophages, within the vessel wall. Here, we review the mechanisms through which elevated ADMA contributes to endothelial dysfunction, activation and reactive oxygen species (ROS) production; how ADMA may affect vascular smooth muscle phenotype; and finally whether ADMA plays a regulatory role in the inflammatory processes occurring within the vessel wall.

Objective—: Asymmetric dimethylarginine is an endogenous inhibitor of NO synthesis that may mediate cardiovascular disease. Alanine-glyoxylate aminotransferase-2 (AGXT2) has been proposed to degrade asymmetric dimethylarginine. We investigated the significance of AGXT2 in methylarginine metabolism in vivo and examined the effect of this enzyme on blood pressure. Methods and Results—: In isolated mouse kidney mitochondria, we show asymmetric dimethylarginine deamination under physiological conditions. We demonstrate increased asymmetric dimethylarginine, reduced NO, and hypertension in an AGXT2 knockout mouse. We provide evidence for a role of AGXT2 in methylarginine metabolism in humans by demonstrating an inverse relationship between renal (allograft) gene expression and circulating substrate levels and an association between expression and urinary concentrations of the product. Finally, we examined data from a meta-analysis of blood pressure genome-wide association studies. No genome-wide significance was observed, but taking a hypothesis-driven approach, there was a suggestive association between the T allele at rs37369 (which causes a valine-isoleucine substitution and altered levels of AGXT2 substrate) and a modest increase in diastolic blood pressure ( P =0.0052). Conclusion—: Although the effect of variation at rs37369 needs further study, these findings suggest that AGXT2 is an important regulator of methylarginines and represents a novel mechanism through which the kidney regulates blood pressure.

Objective—: Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl- l -arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl- l -arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. Approach and Results—: Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P =0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) μmol/L ( P =0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P <0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. Conclusions—: Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.

Background—: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis and a risk factor for cardiovascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes are responsible for ADMA breakdown. It has been reported that endothelial DDAH1 accounts for the majority of ADMA metabolism. However, we and others have shown strong DDAH1 expression in a range of nonendothelial cell types, suggesting that the endothelium is not the only site of metabolism. We have developed a new endothelium-specific DDAH1 knockout mouse (DDAH1 ^En−/− ) to investigate the significance of endothelial ADMA in cardiovascular homeostasis. Methods and Results—: DDAH1 deletion in the DDAH1 ^En−/− mouse was mediated by Tie-2 driven Cre expression. DDAH1 deletion was confirmed through immunocytochemistry, whereas Western blotting showed that DDAH1 remained in the kidney and liver, confirming expression in nonendothelial cells. Plasma ADMA was unchanged in DDAH1 ^En−/− mice, and cultured aortas released amounts of ADMA to similar to controls. Consistent with these observations, vasoreactivity ex vivo and hemodynamics in vivo were unaltered in DDAH1 ^En−/− mice. In contrast, we observed significantly impaired angiogenic responses both ex vivo and in vivo. Conclusions—: We demonstrate that endothelial DDAH1 is not a critical determinant of plasma ADMA, vascular reactivity, or hemodynamic homeostasis. DDAH1 is widely expressed in a range of vascular and nonvascular cell types; therefore, the additive effect of DDAH1 expression in multiple organ systems determines plasma ADMA concentrations. Endothelial deletion of DDAH1 profoundly impairs the angiogenic capacity of endothelial cells, indicating that intracellular ADMA is a critical determinant of endothelial cell response.

Circulating levels of asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, are increased in patients with idiopathic pulmonary hypertension (IPAH). We hypothesized that ADMA abrogates gap junctional communication, required for the coordinated regulation of endothelial barrier function and angiogenesis, and so contributes to pulmonary endothelial dysfunction. The effects of ADMA on expression and function of gap junctional proteins were studied in human pulmonary artery endothelial cells; pulmonary endothelial microvascular cells from mice deficient in an enzyme metabolizing ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI); and blood-derived endothelial-like cells from patients with IPAH. Exogenous and endogenous ADMA inhibited protein expression and membrane localization of connexin 43 (Cx43) in a nitric oxide/soluble guanosine monophosphate/c-jun-dependent manner in pulmonary endothelial cells, resulting in the inhibition of gap junctional communication, increased permeability, and decreased angiogenesis. The effects of ADMA were prevented by overexpression of DDAHI or Cx43 and by treatment with rotigaptide. Blood-derived endothelial-like cells from IPAH patients displayed a distinct disease-related phenotype compared to cells from healthy controls, characterized by reduced DDAHI expression, increased ADMA production, and abnormal angiogenesis. In summary, we show that ADMA induces pulmonary endothelial dysfunction via changes in expression and activity of Cx43. Cells from IPAH patients exhibit abnormal DDAHI/Cx43 signaling as well as differences in gap junctional communication, barrier function, and angiogenesis. Strategies that promote DDAHI/Cx43 signaling may have an endothelium-protective effect and be beneficial in pulmonary vascular disease.