Endothelial Dimethylarginine Dimethylaminohydrolase 1 Is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis.

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.

Dimethylarginine dimethylaminohydrolase 2 regulates nitric oxide synthesis and hemodynamics and determines outcome in polymicrobial sepsis.

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.

Unexpected effect of proton pump inhibitors: elevation of the cardiovascular risk factor asymmetric dimethylarginine.

BACKGROUND: Proton pump inhibitors (PPIs) are gastric acid-suppressing agents widely prescribed for the treatment of gastroesophageal reflux disease. Recently, several studies in patients with acute coronary syndrome have raised the concern that use of PPIs in these patients may increase their risk of major adverse cardiovascular events. The mechanism of this possible adverse effect is not known. Whether the general population might also be at risk has not been addressed. METHODS AND RESULTS: Plasma asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Elevated plasma ADMA is associated with increased risk for cardiovascular disease, likely because of its attenuation of the vasoprotective effects of endothelial nitric oxide synthase. We find that PPIs elevate plasma ADMA levels and reduce nitric oxide levels and endothelium-dependent vasodilation in a murine model and ex vivo human tissues. PPIs increase ADMA because they bind to and inhibit dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA. CONCLUSIONS: We present a plausible biological mechanism to explain the association of PPIs with increased major adverse cardiovascular events in patients with unstable coronary syndromes. Of concern, this adverse mechanism is also likely to extend to the general population using PPIs. This finding compels additional clinical investigations and pharmacovigilance directed toward understanding the cardiovascular risk associated with the use of the PPIs in the general population.

Alanine-glyoxylate aminotransferase-2 metabolizes endogenous methylarginines, regulates NO, and controls blood pressure.

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). CONCLUSIONS: 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.

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus.

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo.