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Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

Objective: Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency.Approach and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling.Conclusions: Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Chronic β-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested β-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to β-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in β-alanine homeostasis.Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma β-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg β-alanine.Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides ( 70% decrease; p 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, β-alanine remained unaffected in the circulation and in muscle (p 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p 0.05). Additionally, following an acute dose of β-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p 0.05). Interestingly, urinary β-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p 0.05).The data suggest that in vivo, β-alanine is a substrate of AGXT2; however, its importance in the metabolism of β-alanine and histidine-containing dipeptides seems small.

Dimethylguanidino valeric acid (DMGV) is a marker of fatty liver disease, incident coronary artery disease, cardiovascular mortality, and incident diabetes. Recently, it was reported that circulating DMGV levels correlated positively with consumption of sugary beverages and negatively with intake of fruits and vegetables in three Swedish community-based cohorts. Here, we validate these results in the Framingham Heart Study Third Generation Cohort. Furthermore, in mice, diets rich in sucrose or fat significantly increased plasma DMGV concentrations. DMGV is the product of metabolism of asymmetric dimethylarginine (ADMA) by the hepatic enzyme AGXT2. ADMA can also be metabolized to citrulline by the cytoplasmic enzyme DDAH1. We report that a high-sucrose diet induced conversion of ADMA exclusively into DMGV (supporting the relationship with sugary beverage intake in humans), while a high-fat diet promoted conversion of ADMA to both DMGV and citrulline. On the contrary, replacing dietary native starch with high-fiber-resistant starch increased ADMA concentrations and induced its conversion to citrulline, without altering DMGV concentrations. In a cohort of obese nondiabetic adults, circulating DMGV concentrations increased and ADMA levels decreased in those with either liver or muscle insulin resistance. This was similar to changes in DMGV and ADMA concentrations found in mice fed a high-sucrose diet. Sucrose is a disaccharide of glucose and fructose. Compared with glucose, incubation of hepatocytes with fructose significantly increased DMGV production. Overall, we provide a comprehensive picture of the dietary determinants of DMGV levels and association with insulin resistance.

Background and aims Cardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. Methods Streptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. Results STZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. Conclusions In summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model.

The endogenous methylated derivative of ʟ-arginine, Nω,Nω′-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent risk factor in many diseases, inhibits the activity of nitric oxide synthases and, consequently, modulates the availability of nitric oxide. While most studies on the biological role of ADMA have focused on endothelial and inducible nitric oxide synthases modulation and its contribution to cardiovascular, metabolic, and renal diseases, a role in regulating neuronal nitric oxide synthases and pathologies of the central nervous system is less understood. The two isoforms of dimethylarginine dimethylaminohydrolase (DDAH), DDAH1 and DDAH2, are thought to be the main enzymes responsible for ADMA catabolism. A current impediment is limited knowledge on specific tissue and cellular distribution of DDAH enzymes within the brain. In this study, we provide a detailed characterization of the regional and cellular distribution of DDAH1 and DDAH2 proteins in the adult murine and human brain. Immunohistochemical analysis showed a wide distribution of DDAH1, mapping to multiple cell types, while DDAH2 was detected in a limited number of brain regions and exclusively in neurons. Our results provide key information for the investigation of the pathophysiological roles of the ADMA/DDAH system in neuropsychiatric diseases and pave the way for the development of novel selective therapeutic approaches.

Summary Vascular endothelial cell growth factor (VEGF) is a key regulator of vascular permeability. Herein we aim to understand how acute and chronic exposures of VEGF induce different levels of vascular permeability. We demonstrate that chronic VEGF exposure leads to decreased phosphorylation of VEGFR2 and c-Src as well as steady increases of nitric oxide (NO) as compared to that of acute exposure. Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques for quantification, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, was shown to play essential roles in both acute and chronic vascular permeability in cultured human cells, zebrafish model, and Miles assay. Taken together, our data reveal acute and chronic VEGF exposures induce divergent signaling pathways and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis., Graphical abstract, Highlights • Chronic VEGF exposure induces a different signaling pattern in endothelial cells • A novel algorithm can precisely quantify the vascular permeability in zebrafish model • DDAH1 acts as a novel mediator of VEGF-induced vascular hyperpermeability, Cardiovascular medicine; Molecular genetics

Background The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of Agxt2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methods We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. Results We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. Conclusions Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.

The small free radical gas nitric oxide (NO) plays a key role in various physiological and pathological processes through enhancement of endothelial cell survival and proliferation. In particular, NO has emerged as a molecule of interest in carcinogenesis and tumor progression due to its crucial role in various cancer-related events including cell invasion, metastasis, and angiogenesis. The dimethylarginine dimethylaminohydrolase (DDAH) family of enzymes metabolize the endogenous nitric oxide synthase (NOS) inhibitors, asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), and are thus key for maintaining homeostatic control of NO. Dysregulation of the DDAH/ADMA/NO pathway resulting in increased local NO availability often promotes tumor growth, angiogenesis, and vasculogenic mimicry. Recent literature has demonstrated increased DDAH expression in tumors of different origins and has also suggested a potential ADMA-independent role for DDAH enzymes in addition to their well-studied ADMA-mediated influence on NO. Inhibition of DDAH expression and/or activity in cell culture models and in vivo studies has indicated the potential therapeutic benefit of this pathway through inhibition of both angiogenesis and vasculogenic mimicry, and strategies for manipulating DDAH function in cancer are currently being actively pursued by several research groups. This review will thus provide a timely discussion on the expression, regulation, and function of DDAH enzymes in regard to angiogenesis and vasculogenic mimicry, and will offer insight into the therapeutic potential of DDAH inhibition in cancer based on preclinical studies.

The elucidation of the metabolic pathways of the amino acid arginine and their role in health and disease have been an intensive focus of basic and clinical research for over a century. The recent advent of robust analytical techniques for biomarker assessment in large population cohorts has allowed the investigation of the pathophysiological role of specific arginine metabolites in key chronic disease states in old age, particularly those characterised by a reduced synthesis of endothelial nitric oxide, with consequent vascular disease and atherosclerosis. Two arginine metabolites have been increasingly studied in regard to their potential role in risk stratification and in the identification of novel therapeutic targets: the methylated arginine asymmetric dimethylarginine (ADMA) and the arginine analogue homoarginine. Higher circulating concentrations of ADMA, a potent inhibitor of nitric oxide synthesis, have been shown to predict adverse cardiovascular outcomes. By contrast, there is emerging evidence that homoarginine might exert cardioprotective effects. This review highlights recent advances in the biological and clinical role of ADMA and homoarginine in cardiovascular disease and other emerging fields, particularly chronic obstructive pulmonary disease, dementia, and depression. It also discusses opportunities for future research directions with the ultimate goal of translating knowledge of arginine metabolism, and its role in health and disease, into the clinical care of older adults.

Background: Dimethylarginine dimethylaminohydrolase 1 (DDAH1) hydrolyzes the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA). DDAH1 is also suggested to have ADMA-independent effects. DDAH1 overexpression lowers ADMA levels and protects from renal interstitial fibrosis and vascular oxidative stress in angiotensin-II-induced hypertension. The current study was designed to test the hypothesis that DDAH1 overexpression protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling. Methods: Angiotensin II (AngII) was infused in the doses of 0.75 and 1.5 mg/kg/day, respectively, in DDAH1 transgenic mice (TG) and wild type (WT) littermates via osmotic minipumps. Echocardiography was performed in the first and fourth week after start of the infusion. Systolic blood pressure was measured by the tail-cuff method. Cardiac hypertrophy and vascular remodeling was assessed by histology after 4 weeks of AngII infusion. Results: TG mice had decreased plasma and tissue ADMA. Infusion of Ang II resulted in an increase in systolic blood pressure, which was similar between TG and WT mice at week 1, however, TG mice were protected from a further increase in blood pressure. After 4-weeks infusion of AngII TG mice had significantly higher left ventricular lumen to wall ratio, smaller size of cardiomyocytes and reduced myocardial collagen expression compared to WT littermates. TG mice had lower left ventricular posterior wall thickness in systole and diastole as compared to WT controls. The vasomotor function of aortic rings in response to acetylcholine was improved in the TG mice as compared to the WT mice. TG mice had less aortic hypertrophy and fibrosis and more elastin in aorta as compared to WT mice. Aortic infiltration of CD45 + , CD3 + , CD8 + and CD4 + T-cells was significantly lower in TG than in WT mice. Conclusion: This study shows that upregulation of DDAH1 protects from AngII-induced cardiac hypertrophy and vascular remodeling. Upregulation of DDAH1 might be a potential therapeutic approach for protection from AngII – induced end organ damage. We are currently investigating, whether protective effects of DDAH1 are ADMA-dependent or ADMA-independent.

Background Increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are associated with cardiovascular and renal diseases. We and others have shown that both ADMA and SDMA can be Nα-acetylated to form asymmetric and symmetric Nα-acetyldimethylarginine (Ac-ADMA and Ac-SDMA). The current study further investigated this undercharacterized metabolic pathway. Methods ADMA and SDMA were infused in C57/BL6 mice for 3 days using osmotic minipumps. Half of the mice underwent bilateral nephrectomy 24 h before completion of the infusion. Plasma and tissue levels of Ac-ADMA and Ac-SDMA were detected by liquid chromatography-tandem mass spectrometry. Results ADMA and SDMA infusion resulted in a 3.6-fold increase in plasma Ac-ADMA and a 21-fold increase in plasma Ac-SDMA levels, respectively. Plasma Ac-ADMA and Ac-SDMA levels were dramatically increased after bilateral nephrectomy. The highest baseline tissue concentrations of Ac-ADMA and Ac-SDMA in wild-type mice were detected in the liver, kidney, small intestine, pancreas and spleen. Incubation of the tissue lysates with ADMA and SDMA resulted in increased levels of the corresponding Nα-acetylated products only in the liver, kidney and small intestine. Conclusions Our results show that overload of ADMA or SDMA leads to an increase in plasma Ac-ADMA and Ac-SDMA levels. This observation is consistent with the hypothesis that Ac-ADMA and Ac-SDMA are formed directly from ADMA and SDMA in vivo. The increase in plasma Ac-ADMA and Ac-SDMA concentrations after bilateral nephrectomy suggests that both compounds are predominantly eliminated via the kidneys. We demonstrated that acetylation of ADMA and SDMA occurs primarily in the liver, kidney and small intestine.

Cardiovascular disease is the major cause of morbidity and mortality in the world. Diabetes and its complications, such as diabetic nephropathy, dramatically increase cardiovascular risk. Association studies suggest that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, plays a role in the pathogenesis of diabetic nephropathy. The major pathway of ADMA catabolism is hydrolysis by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that lowering ADMA by overexpression of DDAH1 protects from development of diabetic nephropathy.Diabetes was induced with streptozotocin (STZ) in wild type and DDAH1 transgenic mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. ADMA levels were assessed by isotope-dilution tandem mass spectrometry, creatinine by standard laboratory methods and albumin by ELISA. Kidney tissues were stained for markers of glomerular cells, cell matrix, inflammation and cell proliferation.STZ led to development of diabetes in all injected mice. Transgenic overexpression of DDAH1 led to a decrease in plasma ADMA levels in healthy animals. Diabetic state itself did not lead to elevation of plasma ADMA levels. Diabetic mice of both genotypes developed albuminuria (27 and 25 vs. 9 and 6 μg albumin/mg creatinine) (p 0.01). There were no changes in glomerular matrix expansion, podocyte injury, inflammatory or proliferative response.STZ-induced diabetes led to the development of early features of diabetic nephropathy. Overexpression of DDAH1 and lowering of systemic ADMA levels did not prevent these changes, indicating that ADMA is not the major mediator of the early diabetic changes reflected by this experimental model.

Alanine-glyoxylate aminotransferase 2 (AGXT2) is a multifunctional mitochondrial aminotransferase that was first identified in 1978. The physiological importance of AGXT2 was largely overlooked for three decades because AGXT2 is less active in glyoxylate metabolism than AGXT1, the enzyme that is deficient in primary hyperoxaluria type I. Recently, several novel functions of AGXT2 have been 'rediscovered' in the setting of modern genomic and metabolomic studies. It is now apparent that AGXT2 has multiple substrates and products and that altered AGXT2 activity may contribute to the pathogenesis of cardiovascular, renal, neurological, and hematological diseases. This article reviews the biochemical properties and physiological functions of AGXT2, its unique role at the intersection of key mitochondrial pathways, and its potential as a drug target.

Asymmetric dimethylarginine (ADMA) is produced by protein methylation, a common mechanism of posttranslational protein modification. Elevated levels of ADMA lead to impaired endothelial nitric oxide production and subsequently to a range of cardiovascular and other diseases related to decreased nitric oxide production. Knowledge of the elimination pathways of ADMA and the possibility of influencing them is therefore of major clinical interest. One of these pathways is the N-acetylation and subsequent renal elimination of ADMA in the form of asymmetric Nα-acetyldimethylarginine (Ac-ADMA). In this work, we describe the first method to quantitatively determine Ac-ADMA in human plasma and urine. Ac-ADMA was separated by HPLC on a porous graphitic carbon column and selectively analyzed by tandem mass spectrometry. Ac-ADMA and the internal standard D7-Ac-ADMA were synthesized in-house. Precision and accuracy of the method were better than 5% in plasma and urine quality control samples. First results obtained with this method in samples of healthy volunteers showed plasma levels of 0.643±0.454 nmol/L and urine levels of 152.7±76.7 nmol/L or 13.0±8.9 nmol/mmol creatinine. The method is a suitable tool for investigating this currently mostly neglected ADMA elimination pathway.

Formaldehyde (FA) is toxic to the respiratory system, and nitric oxide (NO) dysfunction stimulates the onset of respiratory diseases. The involvement of dimethylarginine dimethylaminohydrolase (DDAH), the l-arginine analogue asymmetric dimethylarginine (ADMA) degrading enzyme, in FA-induced cell death in lung epithelial cells has not been investigated. In this study, we assessed the effect of FA on DDAH expression and endoplasmic reticulum (ER) stress in A549 cells. We also investigated the preventive effect of DDAH overexpression on ER stress and apoptosis in FA-induced cell death. FA decreased viability in A549 cells and decreased DDAH1 and DDAH2 mRNA and protein expression in a time-dependent manner (>4h). This coincided with increased phosphorylation of the ER stress proteins IRE1α, PERK, and eIF-2α, as well as increased expression of pro-apoptotic proteins such as Bax, C/EPB homologous protein (CHOP), cleaved PARP, and cleaved caspase-3, but decreased expression of the anti-apoptotic protein Bcl-2. ADMA treatment mimicked the effect of FA. Overexpression of DDAH1, but not DDAH2, prevented FA-induced decreases in cell viability, phosphorylation of IRE1α, PERK, and eIF2α, and expression of CHOP. Effects of DDAH1 overexpression, but not DDAH2 overexpression, restored FA-induced increases in Bax, CHOP, cleaved PARP, cleaved caspase-3 and decreases in Bcl-2. In conclusion, FA induces apoptosis of lung epithelial cells via a decrease of DDAH1 through ER stress.

Background: Elevation of the endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been shown to be associated with increased risk of cardiovascular diseases. There are two major pathways of ADMA catabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is poorly characterized. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of systemic levels of ADMA and improvement of vasomotor function. Methods and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. There were no developmental or phenotypic changes in the TG animals. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels were decreased by 15% (p Conclusion: In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation in vivo. AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.

Background: Numerous epidemiological studies have demonstrated an association between elevated levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) with cardiovascular diseases. ADMA can be hydrolysed by dimethylarginine dimethylaminohydrolase (DDAH). It can also be metabolized through a much less explored alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2), which converts ADMA to α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). It has been shown in previous Northern Blot and in-situ RNA-hybridisation experiments that the kidney is the main organ of Agxt2 expression in rats, while RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels. The goal of the current study was to analyse the expression of the enzyme in human tissues. Methods and Results: We performed immunohistochemical staining using two rabbit polyclonal anti-AGXT2 antibodies in both frozen and paraformaldehyde-fixed samples from a normal tissue bank. We saw the strongest expression of AGXT2 using both of the antibodies in the proximal convoluted tubule of the kidney and in the liver with a homogenous pattern throughout all the samples. We also observed weak staining in the skeletal and heart muscle. Aorta, small intestine and lungs were negative for AGXT2 expression. We also confirmed our immunohistochemistry data by RT-PCR. Conclusions: Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We are currently designing experiments to show the direct localization of AGXT2 in a human cell.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH ( Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine β-synthase ( Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by ∼50% in Cbs+/− mice fed the HM/LF diet compared with Cbs+/+ mice fed the control diet ( P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddah1 mRNA in the liver ( P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney ( P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity ( S-adenosylmethionine-to- S-adenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction.

Background and Purpose— Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). An elevation of plasma ADMA levels is associated with cardiovascular disease. ADMA is hydrolyzed by dimethylarginine dimethylaminohydrolases (DDAHs). The goal of this study was to determine whether overexpression of human DDAH-1 in transgenic (DDAH-1–Tg) mice inhibits the vascular effects of ADMA. Methods— Using nontransgenic (non-Tg) and DDAH-1–Tg mice, we compared responses of the carotid artery and aorta (in vitro) and of the cerebral arterioles (in vivo) in the absence or presence of ADMA. DDAH-1 expression and plasma levels of ADMA were also measured. Results— Western blotting indicated that vascular expression of DDAH-1 was increased markedly in DDAH-1–Tg mice. Plasma levels of ADMA were reduced by ≈50% in DDAH-1–Tg mice compared with non-Tg mice (0.19±0.02 vs 0.37±0.04 μmol/L, P l -arginine methyl ester (an inhibitor of NOS), an index of basal production of NO, was increased in DDAH-1–Tg mice compared with controls (50±4% vs 34±4%, P P P Conclusions— These findings provide the first evidence that overexpression of DDAH-1 increases basal levels of vascular NO and protects against ADMA-induced endothelial dysfunction in the cerebral circulation.

Introduction: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, which has been proposed to play a direct role in the pathogenesis of cardiovascular disease. There are two enzymatic pathways for degradation of ADMA: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The first pathway is well characterized, whereas the physiological role of AGXT2 is still poorly understood. The goal of our study was to test the hypothesis that transgenic overexpression of AGXT2 would lead to lowering of systemic levels of ADMA and improved vasomotor function. Methods and results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. Ubiquitous overexpression of the transgene was confirmed by qPCR and Western Blot. TG animals had normal weight and no observed developmental abnormalities. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels in AGXT2 TG animals were decreased by 15% (p Conclusions: Our findings show that upregulation of AGXT2 results in lower ADMA levels and improved endothelial-dependent relaxation in vivo. AGXT2 thereby may be a therapeutic target for long-term reduction of systemic ADMA levels and improvement of vascular function in vivo. This is especially important, because all the efforts to develop ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far. Our data suggest that AGXT2 might be a promising drug target for cardiovascular pathologies associated with elevated ADMA levels.

Background: Endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been proposed as a risk factor and mediator of cardiovascular diseases. ADMA can undergo hydrolysis by dimethylarginine dimethylaminohydrolase or be processed through an alternative pathway by alanine:glyoxylate aminotransferase 2. We and other have also shown that ADMA can be N-acetylated to form asymmetrical Nα-acetyldimethylarginine (Ac-ADMA). The goal of the study was to characterize this novel pathway in vivo and identify the organs, which are responsible for the ADMA-acetylating activity. Results: We infused ADMA in mice for three days. Control mice received saline infusion. Half of the mice underwent bilateral nephrectomy 24 hours before completion of the infusion, the rest had a sham surgery. ADMA infusion resulted in a 3.5 fold rise in plasma Ac-ADMA levels in the sham-operated mice (1.35 ± 0.45 nmol/L vs. 4.77 ± 0.88 nmol/L). In nephrectomized mice plasma Ac-ADMA levels were markedly increased even after saline infusion (45.08 ± 9.25 nmol/L) and further raised 5-fold after ADMA infusion (229.65 ± 91.30 nmol/L). We assessed tissue distribution of Ac-ADMA in mice and found the highest levels in pancreas, small intestine, liver and kidney. We incubated the lysates of those organs with ADMA for 24 hours and estimated production of Ac-ADMA. Only liver and kidney lysates showed significant increase in Ac-ADMA levels upon addition of ADMA (from 0.03 ± 0.01 nmol/μg protein to 0.18 ± 0.06 nmol/μg protein and from 0.06 ± 0.01 nmol/μg protein to 0.11 ± 0.02 nmol/μg protein). Conclusions: We showed that overload of ADMA leads to increase in plasma Ac-ADMA levels. This observation is consistent with the hypothesis that Ac-ADMA is formed from ADMA in vivo. The striking rise in plasma Ac-ADMA concentrations after bilateral nephrectomy suggests that Ac-ADMA is predominantly eliminated via the kidneys. We demonstrated that liver and kidney are the organs, in which acetylation of ADMA takes place. The high levels of Ac-ADMA in pancreas and intestine may be due to accumulation of Ac-ADMA in those organs. In the next step we want to identify the enzyme responsible for acetylation of ADMA and explore the clinical implications of this novel pathway of ADMA metabolism.

Background: Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. Methods: ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. Results: Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. Conclusions: Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.

There is little doubt that elevation of plasma total homocysteine is associated with increased cardiovascular risk. Over the past 2 decades, many large prospective studies have established that hyperhomocysteinemia predicts for an increased relative risk of coronary events, stroke, venous thromboembolism, and death.1,2 Hyperhomocysteinemia also has been shown to produce abnormalities of vascular structure and function in animal models.3 Paradoxically, however, several intervention trials have failed to demonstrate any clinical benefit from homocysteine-lowering therapy.4–8 What is the explanation for this paradox? See accompanying article on page 1158 One possibility is that hyperhomocysteinemia is a clinically important risk factor only when plasma total homocysteine is elevated to extremely high levels. The hypothesis that homocysteine is a cardiovascular risk factor first arose from clinical and pathological observations in children and young adults with hereditary homocystinuria.9 If untreated, these individuals develop severe hyperhomocysteinemia, with plasma total homocysteine levels greater than 100 μmol/L, and they have a high risk of developing pathological vascular lesions and thromboembolic events at a young age.10 When placed on homocysteine-lowering therapy (high doses of vitamin B6, vitamin B12, folic acid, or betaine, along with dietary methionine restriction), their risk of adverse vascular events decreases markedly.11 Improvement in vascular outcome occurs despite a moderate level of residual hyperhomocysteinemia. In contrast to the clear clinical benefit of homocysteine-lowering therapy in severe hyperhomocysteinemia, its potential role in mild hyperhomocysteinemia remains unproven. All of the recent intervention trials of homocysteine-lowering therapy have been performed in subjects with relatively mild hyperhomocysteinemia (plasma total homocysteine levels between 10 and 30 μmol/L). The negative …

There is an obvious contrast between the data from the epidemiological studies on hyperhomocysteinemia and the negative results of the homocysteine-lowering clinical trials. Moderate hyperhomocysteinemia might only be relevant in certain subgroups of subjects. The current study was focused on lipoprotein apheresis patients; the study goals were to determine the prevalence of hyperhomocysteinemia, to identify the association between homocysteine levels and cardiovascular events and to test the effects of lipoprotein apheresis and of the conventional homocysteine-lowering therapy.Sixty patients from our Lipoprotein Apheresis Center (37 males, 23 females, age 63.1 ± 10.8 years) were included in the study. All patients' records were reviewed with respect to age, sex, BMI, dyslipidemias, arterial hypertension, diabetes mellitus and incidence of vascular events in coronaries, carotids and lower extremities. Homocysteine was measured before and immediately after the apheresis procedure. We also observed the effects of conventional homocysteine-lowering therapy.The prevalence of hyperhomocysteinemia was 50%. Homocysteine levels correlated positively with number of cardiovascular events (p0.03) and serum creatinine (p0.0001) and negatively with serum HDL-cholesterol (p0.03). Neither oral nor intravenous medication with vitamin B and folic acid showed a significant homocysteine lowering effect. The median relative change value of homocysteine after apheresis session was -12% but was not statistically significant.The prevalence of hyperhomocysteinemia in lipoprotein apheresis patients is high. Neither apheresis nor more conventional methods appear to markedly influence homocysteine serum levels.

Elevated plasma concentrations of the asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse cardiovascular clinical outcomes. Both dimethylarginines can be degraded by alanine-glyoxylate aminotransferase 2 (Agxt2), which is also the key enzyme responsible for the degradation of endogenously formed β-aminoisobutyrate (BAIB). In the present study we wanted to investigate the effect of BAIB on Agxt2 expression and Agxt2-mediated metabolism of dimethylarginines. We infused BAIB or saline intraperitoneally for 7days in C57/BL6 mice via minipumps. Expression of Agxt2 was determined in liver and kidney. The concentrations of BAIB, dimethylarginines and the Agxt2-specific ADMA metabolite α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) was determined by LC-MS/MS in plasma and urine. As compared to controls systemic administration of BAIB increased plasma and urine BAIB levels by a factor of 26.5 (p

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.

Rationale : Hyperhomocysteinemia is a cardiovascular risk factor that is associated with elevation of the nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA). Objective : Using mice transgenic for overexpression of the ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1), we tested the hypothesis that overexpression of DDAH1 protects from adverse structural and functional changes in cerebral arterioles in hyperhomocysteinemia. Methods and Results : Hyperhomocysteinemia was induced in DDAH1 transgenic (DDAH1 Tg) mice and wild-type littermates using a high methionine/low folate (HM/LF) diet. Plasma total homocysteine was elevated approximately 3-fold in both wild-type and DDAH1 Tg mice fed the HM/LF diet compared with the control diet ( P P P P P P Conclusions : Overexpression of DDAH1 protects from hyperhomocysteinemia-induced alterations in cerebral arteriolar structure and vascular muscle function.

Elevated blood concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric-oxide (NO) synthase, are found in association with diabetes, hypertension, congestive heart failure, and atherosclerosis. ADMA levels are controlled by dimethylarginine dimethylaminohydrolases (DDAHs), cytosolic enzymes that hydrolyze ADMA to citrulline and dimethylamine. ADMA also has been proposed to be regulated through an alternative pathway by alanine-glyoxylate aminotransferase 2 (AGXT2), a mitochondrial aminotransferase expressed primarily in the kidney. The goal of this study was to define the subcellular localization of human AGXT2 and test the hypothesis that overexpression of human AGXT2 protects from ADMA-induced inhibition in nitric oxide (NO) production. AGXT2 was cloned from human kidney cDNA and overexpressed in COS-7 cells and human umbilical vein endothelial cells with a C-terminal FLAG epitope tag. Mitochondrial localization of human AGXT2 was demonstrated by confocal microscopy and a 41-amino acid N-terminal mitochondrial cleavage sequence was delineated by N-terminal sequencing of the mature protein. Overexpression of human AGXT2 in the liver of C57BL/6 mice using an adenoviral expression vector produced significant decreases in ADMA levels in plasma and liver. Overexpression of human AGXT2 also protected endothelial cells from ADMA-mediated inhibition of NO production. We conclude that mitochondrially localized human AGXT2 is able to effectively metabolize ADMA in vivo resulting in decreased ADMA levels and improved endothelial NO production.

In Response: In our editorial,1 we listed several potential explanations for the homocysteine paradox, including the possibility that mild hyperhomocysteinemia is a marker of another deleterious factor. Klevay proposes that copper deficiency might be such a factor. He notes that dietary deficiency of B vitamins (particularly folate and vitamin B12) is a frequent …

Hyperhomocysteinemia is an emerging risk factor for cardiovascular disease and stroke. The mechanisms underlying the pathophysiology of hyperhomocysteinemia are not completely defined, but endothelial dysfunction resulting from impaired bioavailability of nitric oxide is a consistent finding in experimental models. One potential mechanism for decreased nitric oxide bioavailability is inhibition of endothelial nitric oxide synthase by its endogenous inhibitor, asymmetric dimethylarginine (ADMA). Elevated plasma levels of ADMA have been found in association with hyperhomocysteinemia and endothelial dysfunction in both animals and humans. Additional studies are required to determine the mechanisms by which ADMA accumulates in hyperhomocysteinemia and to define the importance of ADMA in the endothelial dysfunction of hyperhomocysteinemia in vivo.