Dimethylarginine Dimethylaminohydrolase Research Articles

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Metabolism of asymmetric dimethylarginine in hypoxia: from bench to bedside.

Acute hypoxia and chronic hypoxia induce pulmonary vasoconstriction. While hypoxic pulmonary vasoconstriction is a physiological response if parts of the lung are affected, global exposure to hypoxic conditions may lead to clinical conditions like high-altitude pulmonary hypertension. Nitric oxide is the major vasodilator released from the vascular endothelium. Nitric oxide-dependent vasodilation is impaired in hypoxic conditions. Inhibition of nitric oxide synthesis is the most rapid and easily reversible molecular mechanism to regulate nitric oxide-dependent vascular function in response to physiological and pathophysiological stimuli. Asymmetric dimethylarginine is an endogenous, competitive inhibitor of nitric oxide synthase and a risk marker for major cardiovascular events and mortality. Elevated asymmetric dimethylarginine has been observed in animal models of hypoxia as well as in human cohorts under chronic and chronic intermittent hypoxia at high altitude. In lowlanders, asymmetric dimethylarginine is high in patients with pulmonary hypertension. We have recently shown that high asymmetric dimethylarginine at sea level is a predictor for high-altitude pulmonary hypertension. Asymmetric dimethylarginine is a highly regulated molecule, both by its biosynthesis and metabolism. Methylation of L-arginine by protein arginine methyltransferases was shown to be increased in hypoxia. Furthermore, the metabolism of asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) is decreased in animal models of hypoxia. Whether these changes are caused by transcriptional or posttranslational modifications remains to be elucidated. Current data suggest a major role of asymmetric dimethylarginine in regulating pulmonary arterial nitric oxide production in hypoxia. Further studies are needed to decipher the molecular mechanisms regulating asymmetric dimethylarginine in hypoxia and to understand their clinical significance.

Urinary Dimethylamine (DMA) and Its Precursor Asymmetric Dimethylarginine (ADMA) in Clinical Medicine, in the Context of Nitric Oxide (NO) and Beyond.

Asymmetric protein-arginine dimethylation is a major post-translational modification (PTM) catalyzed by protein-arginine methyltransferase (PRMT). Regular proteolysis releases asymmetric dimethylarginine (ADMA). Of the daily produced ADMA, about 10% are excreted unchanged in the urine. The remaining 90% are hydrolyzed by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and dimethylamine (DMA), which is readily excreted in the urine. The PRMT/DDAH pathway is almost the exclusive origin of urinary ADMA and the major source of urinary DMA. Dietary fish and seafood represent additional abundant sources of urinary DMA. The present article provides an overview of urinary ADMA and DMA reported thus far in epidemiological, clinical and pharmacological studies, in connection with the L-arginine/nitric oxide (NO) pathway and beyond, in neonates, children and adolescents, young and elderly subjects, males and females. Discussed diseases mainly include those relating to the renal and cardiovascular systems such as peripheral arterial occlusive disease, coronary artery disease, chronic kidney disease, rheumatoid arthritis, Becker muscular disease, Duchenne muscular disease (DMD), attention deficit hyperactivity disorder (ADHD), and type I diabetes. Under standardized conditions involving the abstinence of DMA-rich fresh and canned fish and seafood, urinary DMA and ADMA are useful as measures of whole-body asymmetric arginine-dimethylation in health and disease. The creatinine-corrected excretion rates of DMA range from 10 to 80 µmol/mmol in adults and up to 400 µmol/mmol in children and adolescents. The creatinine-corrected excretion rates of ADMA are on average 10 times lower. In general, diseases are associated with higher urinary DMA and ADMA excretion rates, and pharmacological treatment, such as with steroids and creatine (in DMD), decreases their excretion rates, which may be accompanied by a decreased urinary excretion of nitrate, the major metabolite of NO. In healthy subjects and in rheumatoid arthritis patients, the urinary excretion rate of DMA correlates positively with the excretion rate of dihydroxyphenylglycol (DHPG), the major urinary catecholamines metabolite, suggesting a potential interplay in the PRMT/DDAH/NO pathway.

Dimethylarginine dimethylaminohydrolase-1 contributes to exercise-induced cardiac angiogenesis in mice.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) maintains nitric oxide (NO) bioavailability by degrading asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase (NOS). It has been well established that DDAH1 and exercise play crucial roles in promoting cardiac angiogenesis under pathological conditions. However, the role of DDAH1 in exercise-induced cardiac angiogenesis remains unclear. In this study, we focused on the change in DDAH1 in response to moderate exercise and the underlying mechanism of exercise-induced cardiac angiogenesis. Eight-week-old male DDAH1 global knockout (KO) mice and DDAH1flox/flox mice (wild-type) were randomly divided into sedentary groups (control) and swimming groups (exercise). After eight weeks of swimming at five days per week, all the mice were anesthetized and sacrificed. Histological examination and Western blot analysis were performed. There were low levels of myocardial capillaries in DDAH1 KO mice under control and exercise conditions. Notably, exercise elevated DDAH1 protein expression, as observed by Western blot analysis. The common cardiac angiogenesis biomarkers vascular endothelial growth factor (VEGF) and Caveolin-1 were increased during exercise. A significant difference in VEGF was observed between the DDAH1 KO and wild-type groups. Similarly, increased Caveolin-1 expression was abrogated in DDAH1 KO mice. Furthermore, we tested the R-Ras/AKT/GSK3β signaling pathway to study the underlying molecular mechanism. DDAH1 may regulate the R-Ras/AKT/GSK3β pathway due to distinct protein changes in this pathway in the DDAH1 KO and wild-type groups. Our findings suggest that DDAH1 plays an important role in exercise-induced cardiac angiogenesis by regulating the R-Ras/AKT/GSK3βsignaling pathway.

Protection of the transplant kidney during cold perfusion with doxycycline: proteomic analysis in a rat model

BackgroundIt has been previously shown that doxycycline (Doxy) protects the kidney from preservation injury by inhibition of matrix metalloproteinase. However, the precise molecular mechanism involved in this protection from injury is not known. We used a pharmaco-proteomics approach to identify potential molecular targets associated with kidney preservation injury.MethodsRat kidneys were cold perfused with or without doxycycline (Doxy) for 22 h. Kidneys perfusates were analyzed for the presence of injury markers such as lactate dehydrogenase (LDH), and neutrophil-gelatinase associated lipocalin (NGAL). Proteins extracted from kidney tissue were analyzed by 2-dimensional gel electrophoresis. Proteins of interest were identified by mass spectrometry.ResultsTriosephosphate isomerase, PGM, dihydropteridine reductase-2, pyridine nucleotide-disulfide oxidoreductase, phosphotriesterase-related protein, and aminoacylase-1A were not affected by cold perfusion. Perfusion with Doxy increased their levels. N(G),N(G)-dimethylarginine dimethylaminohydrolase and phosphoglycerate kinase 1 were decreased after cold perfusion. Perfusion with Doxy led to an increase in their levels.ConclusionsThis study revealed specific metabolic enzymes involved in preservation injury and in the mechanism whereby Doxy protects the kidney against injury during cold perfusion.

Dihydromyricetin increases endothelial nitric oxide production and inhibits atherosclerosis through microRNA-21 in apolipoprotein E-deficient mice.

Natural products were extracted from traditional Chinese herbal emerging as potential therapeutic drugs for treating cardiovascular diseases. This study examines the role and underlying mechanism of dihydromyricetin (DMY), a natural compound extracted from Ampelopsis grossedentata, in atherosclerosis. DMY treatment significantly inhibits atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4‐positive T cells in the vessel wall and hepatic inflammation, whereas increases nitric oxide (NO) production and improves lipid metabolism in apolipoprotein E‐deficient (Apoe− / −) mice. Yet, those protective effects are abrogated by using NOS inhibitor L‐NAME in Apoe− / − mice received DMY. Mechanistically, DMY decreases microRNA‐21 (miR‐21) and increases its target gene dimethylarginine dimethylaminohydrolase‐1 (DDAH1) expression, an effect that reduces asymmetric aimethlarginine (ADMA) levels, and increases endothelial NO synthase (eNOS) phosphorylation and NO production in cultured HUVECs, vascular endothelium of atherosclerotic lesions and liver. In contrast, systemic delivery of miR‐21 in Apoe− / − mice or miR‐21 overexpression in cultured HUVECs abrogates those DMY‐mediated protective effects. These data demonstrate that endothelial miR‐21‐inhibited DDAH1‐ADMA‐eNOS‐NO pathway promotes the pathogenesis of atherosclerosis which can be rescued by DMY. Thus, DMY may represent a potential therapeutic adjuvant in atherosclerosis management.

A single nucleotide polymorphism in DDAH1 leads to decreased nitric oxide production in neonatal cord blood‐derived stimulated lymphoblastoid cells

Bronchopulmonary dysplasia (BPD) is chronic lung disease in preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD and is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made by nitric oxide synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Translational studies from our group identified a single nucleotide polymorphism (SNP) in the DDAH1 gene, rs480414, that was associated with a decreased risk for PH in BPD patients. Therefore, we aim to determine if the DDAH1 SNP rs480414 affects DDAH1 function in cell culture. Neonatal cord blood specimens were treated with Epstein‐Barr virus to select for B‐lymphocytes and transform into lymphoblastoid cell lines (LCLs) that were genotyped (SNP or wild‐type) at the rs480414 locus. We tested the hypothesis that in LCLs the SNP rs480414 results in a gain of function mutation in DDAH1, which would lead to greater NO‐mediated apoptosis as compared to DDAH1 wild‐type (WT). LCLs (WT, n=3 and SNP, n=3) were stimulated with phorbol myristate (PMA), IL‐4, and IL‐13 for 48 hours, protein was analyzed by western blot analysis for DDAH1, cleaved and total caspase‐3 and ‐8, and β‐actin. RT‐PCR was performed and expression of DDAH1 and iNOS mRNA was evaluated. Cell media was assayed for concentrations of nitrite using a chemiluminescence NO analyzer. Spectrophotometry was used to measure the conversion of ADMA to L‐citrulline. A standard L‐citrulline curve was used to calculate DDAH activity after normalization to protein concentration. LCLs with the DDAH1 SNP had similar levels of DDAH1 protein expression (n=3, p=0.58), similar mRNA expression (n=3, p=0.1), and similar DDAH activity (p=0.35) compared to DDAH1 WT. We found that LCLs with the DDAH1 SNP had similar iNOS mRNA expression (p=0.6), lower nitrite levels (p=0.04), lower cleaved caspase‐3 levels (p=0.04), and similar cleaved caspase‐8 levels (p=0.2) than DDAH1 WT LCLs. Contrary to our hypothesis, these finding suggests that the DDAH1 SNP rs480414 may result in a loss of function mutation, resulting in lower NO production and lower apoptosis in LCLs. This is the first evidence that the DDAH1 SNP rs480414 has an effect on cell function, however we speculate that pulmonary vascular endothelial cells will likely have a different physiology.Support or Funding InformationThis work was supported by NIH‐K08 HL129080.

Elucidation of the Metabolic Pathways of Methylarginines in Saccharomyces cerevisiae

BACKGROUNDThe methylation of arginine residues in proteins is a ubiquitous post‐translational modification that is predominantly metabolized during protein catabolism. It has been postulated that methylarginines play a functional role in the mammalian stress response due to their presence in stress granules, although this has not been largely explored. Saccharomyces cerevisiae is a suitable model organism to study the role of methylarginines in the eukaryotic stress response due to the high degree of similarity between the yeast and mammalian stress pathways and methyltransferasomes. Free methylarginines need to be tightly regulated because they inhibit the production of nitric oxide, a cell signalling molecule that is crucial in eukaryotic stress responses. Mammals metabolize free methylarginine into citrulline and methylamine by dimethylarginine dimethylaminohydrolase (DDAH) to circumvent nitric oxide synthase inhibition. Conversely, yeast lack classical DDAH activity and therefore cannot counteract this inhibition in the same way. The purpose of this research is to explore how yeast cope with free methylarginine in response to stress.METHODSWe applied different stress conditions to yeast cells, including heat shock, osmotic shock, rapamycin treatment, nutrient deprivation, and stationary phase and observed the effect on methylarginine concentration in proteins and in spent culture media. Methylarginine species were quantified using a tandem mass spectrometry assay. To explore possible metabolic pathways of yeast methylarginines, we also monitored the expression levels and subcellular localization of a constitutively expressed autophagy marker GFP‐Atg8, a key component in the formation of the phagophore assembly site, via Western Blot and fluorescence microscopy. We also observed the effect of gene deletions involved in autophagy and the proteasome on methylarginine processing.RESULTSThe condition that elicited the most significant change in methylarginine concentration response was stationary phase, a nutrient‐depriving growth condition. Specifically, we observed a substantial release of free methylarginine species into the culture media, accompanied by cleavage of GFP‐Atg8 and localization of GFP‐Atg8 to the vacuole, consistent with functional autophagy. In yeast mutants lacking functional autophagy, we fail to see the expelled free methylarginine species. However, in proteasome‐deficient yeast, methylarginine efflux is still observed.CONCLUSIONSWe postulate that autophagy serves as the major metabolic pathway for degradation of methylarginine‐containing proteins in yeast. We predict that methylarginine efflux is a crucial coping mechanism to avoid methylarginine‐mediated cell toxicity due to inhibition of nitric oxide synthesis. This work examines yeast methylarginines under stress for the first time and reveals new effects of methylarginine. This will contribute to the knowledge of how cells respond to stress and impacts what we know about autophagy and amino acid metabolism.Support or Funding InformationWe thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support (RGPIN‐2015–04450 Discovery Grant to A.F., USRA to E.Z., and Alexander Graham Bell CGS‐D to J.I.B.).

DDAH1 Overexpression in Human Pulmonary Vascular Endothelial Cells Co‐Cultured with Human Pulmonary Artery Smooth Muscle Cells

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients as a result of pathologic vascular remodeling, and PH is an independent predictor of death in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the majority of ADMA in vivo and there are two isoforms, DDAH1 and DDAH2. Translational studies from our group analyzed a cohort of BPD patients and identified a potential biomarker, DDAH1 single nucleotide polymorphism (rs480414), that decreased the risk of PH in BPD by 61%. In the present study, we hypothesized that overexpression of DDAH1 in human pulmonary microvascular endothelial cells (hPMVEC) would lead to greater apoptosis and lower proliferation of human pulmonary artery smooth muscle cells (hPASMC) via effects in hPMVEC. We utilized a co‐culture system where hPMVEC were transfected with AdDDAH1 or AdGFP control for 24h and then incubated in co‐culture with hPASMC for 24h. Analysis included 1) western blot analysis for cleaved and total caspase‐3, ‐8, ‐9, p21, PCNA, and β‐actin and 2) trypan blue exclusion for viable cell numbers. Transfection of hPMVEC with AdDDAH1 resulted in greater DDAH1 protein expression (n=6, p=0.003) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found greater cleaved caspase‐3 (n=6, p=0.01), greater cleaved caspase‐8 (n=6, p=0.03), similar cleaved caspase‐9 (n=6, p=0.94), similar p21 (n=6, p=0.7), and similar PCNA (n=3, p=0.82) protein expression as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found lower hPASMC viable cell numbers (n=3, p=0.008) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC treated with DETA NONOate 250μM or DETA NONOate 250μM + Hemoglobin 100μM, the hPASMC viable cell numbers were lower compared to control (n=3, p=0.001; n=3, p=0.003, respectively). There was no difference detected between DETA NONOate 250μM and DETA NONOate 250μM + Hemoglobin 100μM groups (n=3, p=0.429). In conclusion, DDAH1 overexpression in hPMVEC resulted in greater apoptosis and lower proliferation in hPASMC, consistent with our hypothesis. We propose that DDAH1 is a potential new therapy for BPD‐PH via relieving pulmonary vascular endothelial ADMA‐inhibition of eNOS, resulting in greater endogenous NO production, greater pulmonary vascular wall apoptosis, and potentially less pathologic vascular remodeling. Future studies will determine if DDAH1 endothelial knockout mice have worse lung function as compared to wild‐type, after exposure to BPD‐PH model of LPS and hyperoxia.Support or Funding InformationK08 HL129080 (PI: Trittmann)

Curcumin attenuates endothelial cell fibrosis through inhibiting endothelial-interstitial transformation.

Curcumin (Cur) has various pharmacological activities, including anti‐inflammatory, antiapoptotic and anticancer effects. However, there is no report on the effect of Cur on endothelial cell fibrosis. This study was designed to investigate the effect and mechanism of Cur on endothelial cell fibrosis. An endothelial cell fibrosis model was established by using transforming growth factor (TGF) induction. Proliferation assays, qRT‐PCR, western blotting and immunostaining were performed to investigate the effects and mechanism of Cur on endothelial cell fibrosis. We found that in human umbilical vein endothelial cells (HUVECs), TGF‐β1 treatment significantly decreased the expression of nuclear factor erythroid‐2‐related factor 2 (NRF‐2), dimethylarginine dimethylaminohydrolase‐1 (DDAH1), and VE‐cadherin, the secretion of cellular nitric oxide (NO) and the activity of nitrous oxide synthase (NOS), while asymmetric dimethylarginine (ADMA) and the release of inflammatory factors were elevated. Immunofluorescence showed decreased CD31 and increased α‐smooth muscle actin (α‐SMA). Overexpression of NRF‐2 significantly attenuated the effects of TGF‐β1, while downregulation of DDAH1 potently counteracted the effect of NRF‐2. In addition, ADMA treatment resulted in similar results to those of TGF‐β1, and Cur significantly attenuated the effect of TGF‐β1, accompanied by increased VE‐cadherin, DDAH1 and NRF‐2 and decreased matrix metalloproteinase‐9 (MMP‐9) and extracellular regulated protein kinases 1/2 (ERK1/2) phosphorylation. The NRF‐2 inhibitor ML385 had the opposite effect as that of Cur. These results demonstrated that Cur inhibits TGF‐β1‐induced endothelial‐to‐mesenchymal transition (EndMT) by stimulating DDAH1 expression via the NRF‐2 pathway, thus attenuating endothelial cell fibrosis.

SAT-252 The association of genotype polymorphisms of endothelial function-related genes with restenosis of arteriovenous access after angioplasty in hemodialysis patients

Venous intimal hyperplasia is more aggressive than arterial intimal hyperplasia after angioplasty. Veins tend to produce less nitric oxide, which could predispose to endothelial dysfunction. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and contributes to vascular disease. In human, dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme degrading ADMA. We aimed to determine whether venous intimal hyperplasia in dialysis vascular access is influenced by common polymorphisms in the DDAH1 genes. This is a prospective observational cohort study. A total of 473 hemodialysis patients referred for angioplasty of hemodialysis vascular accesses were enrolled. There were 190 grafts and 283 native fistulas. Follow-up lasted for two years after interventions. Seven single nucleotide polymorphisms in DDAH1 were genotyped and ADMA were measured at baseline. Primary outcome was restenosis after angioplasty. Among the seven single nucleotide polymorphisms, plasma ADMA levels were significantly different in DDAH1 rs233112 (GA+GG vs. AA, 0.86 ± 0.23 vs. 0.82 ± 0.19μM, P=0.03) and rs1498373 (CT+TT vs. CC, 0.87 ± 0.23 vs. 0.82 ± 0.20 μM, P=0.02) genotypes. The native-access group with GG+GA genotype of rs233112 and CT+TT genotype of rs1498373 had higher risks of early restenosis at 3 months. In graft-access groups, only GG+GA genotype of rs233112 was associated with early restenosis. Combined analysis of graft- and native- access groups showed that patients with rs233112 GA+GG genotype and rs1498373 CT+TT genotype had higher risks of early restenosis (both p <0.001). In the multivariate analysis, the association of these genotypes with early restenosis is independent of clinical, access or biochemical Our findings suggest that certain DDAH1 polymorphisms modulate circulating ADMA levels and are associated with venous intimal hyperplasia.

Transcriptional and Metabolomic Analysis of L-Arginine/Nitric Oxide Pathway in Inflammatory Bowel Disease and Its Association with Local Inflammatory and Angiogenic Response: Preliminary Findings

L-arginine/nitric oxide pathway in Crohn’s disease (CD) and ulcerative colitis (UC) is poorly investigated. The aim of current study is to quantify pathway serum metabolites in 52 CD (40 active), 48 UC (33 active), and 18 irritable bowel syndrome patients and 40 controls using mass spectrometry and at determining mRNA expression of pathway-associated enzymes in 91 bowel samples. Arginine and symmetric dimethylarginine decreased (p < 0.05) in active-CD (129 and 0.437 µM) compared to controls (157 and 0.494 µM) and active-UC (164 and 0.52 µM). Citrulline and dimethylamine increased (p < 0.05) in active-CD (68.7 and 70.9 µM) and active-UC (65.9 and 73.9 µM) compared to controls (42.7 and 50.4 µM). Compared to normal, CD-inflamed small bowel had downregulated (p < 0.05) arginase-2 by 2.4-fold and upregulated dimethylarginine dimethylaminohydrolase (DDAH)-2 (1.5-fold) and arginine N-methyltransferase (PRMT)-2 (1.6-fold). Quiescent-CD small bowel had upregulated (p < 0.05) arginase-2 (1.8-fold), DDAH1 (2.9-fold), DDAH2 (1.5-fold), PRMT1 (1.5-fold), PRMT2 (1.7-fold), and PRMT5 (1.4-fold). Pathway enzymes were upregulated in CD-inflamed/quiescent and UC-inflamed colon as compared to normal. Compared to inflamed, quiescent CD-colon had upregulated DDAH1 (5.7-fold) and ornithine decarboxylase (1.6-fold). Concluding, the pathway is deregulated in CD and UC, also in quiescent bowel, reflecting inflammation severity and angiogenic potential. Functional analysis of PRMTs and DDAHs as potential targets for therapy is warranted.

Inhibition of Dimethylarginine Dimethylaminohydrolase (DDAH) Enzymes as an Emerging Therapeutic Strategy to Target Angiogenesis and Vasculogenic Mimicry in Cancer.

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.

Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

Long non-coding RNA H19 confers resistance to gefitinib via miR-148b-3p/DDAH1 axis in lung adenocarcinoma.

Epidermal growth factor receptor tyrosine kinase inhibitors therapy, such as gefitinib, have proven to be effective for lung adenocarcinoma with epidermal growth factor receptor-sensitive mutations. However, drug resistance remains inevitable and the underlying mechanisms are still elusive and poorly understood. In order to explore the mechanisms underlying tyrosine kinase inhibitors resistance, we used long non-coding RNA microarray analysis and found that long non-coding RNA H19 was highly expressed in gefitinib-resistant cell lines. In addition, knockdown of long non-coding RNA H19 was found to be able to decrease cell proliferation, half maximal inhibitory concentration (IC50) of gefitinib, migration and invasion. Mechanistically, we demonstrated that long non-coding RNA H19 positively regulated dimethylarginine dimethylaminohydrolase-1 expression via sponging miR-148b-3p. Furthermore, overexpression or inactivation of miR-148b-3p could enhance or reverse the inhibitory effect of long non-coding RNA H19 inhibition in lung adenocarcinoma cells, respectively. High expression of either long non-coding RNA H19 or dimethylarginine dimethylaminohydrolase-1 was associated with poorer overall survival in patients with lung adenocarcinoma, while high expression of miR-148b was associated with better overall survival. Overall, our data revealed that long non-coding RNA H19 confers resistance to gefitinib via miR-148b/dimethylarginine dimethylaminohydrolase-1 axis in lung adenocarcinoma, which offers a new insight into the epidermal growth factor receptor tyrosine kinase inhibitors therapy resistance.

DDAH-1 via HIF-1 target genes improves cerebral ischemic tolerance after hypoxic preconditioning and middle cerebral artery occlusion-reperfusion

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) as an indirect regulator of nitric oxide (NO) metabolism, its role in hypoxic preconditioning (HPC) and ischemic tolerance (IT) of ischemic stroke has still been unknown and needs to be elucidated. Herein, DDAH-1 knock-out (KO) and wild-type (WT) rats underwent HPC and middle cerebral artery occlusion/reperfusion (MCAO/R) model. After 24 h, neurological severity scores, TTC staining and TUNEL assay were used to evaluate neurological damages. To explore the mechanism, the expression of hypoxia inducible factor (HIF-1α) and its target genes were assessed by Western blot and RT-qPCR. NO and ADMA contents were also tested. In addition, supplementation of l-arginine to DDAH-1 KO rats was used to explore the role of DDAH-1 in regulating NO. After HPC the ischemic outcome improved in both KO and WT rats, while KO rats showed attenuated IT exhibiting less expression of HIF-1α and its target genes, lower NO but higher ADMA content. The supplement of l-arginine to KO rats partly alleviated neurological damages accompanied with higher expression of HIF-1α. To sum up, DDAH-1 could regulate the level of NO and enhance IT following HPC and MCAO model via activating the expression of HIF-1α and its target genes.

Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and μOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and μ-opioid receptor (μOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased μOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of μOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. μOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and μOR, and α2A-AR and μOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and μOR heterodimers in the NTS, leading to progressive hypertension.

Inhibition of Dimethylarginine Dimethylaminohydrolase 1 Improves the Outcome of Sepsis in Pregnant Mice.

Sepsis is one of the most important causes of maternal mortality. In our previous work, we established a polymicrobial sepsis (cecal ligation and puncture [CLP]) model in murine pregnancy and found that pregnant mice had a greater susceptibility to septic shock. In this model, mortality seemed to be associated with the development of early hemodynamic dysfunction and although circulating cytokine levels were similar, "off target" lung inflammatory cell numbers were greater in pregnant mice.Here, we have used the same CLP model to test the hypothesis that inhibiting the metabolism of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine would improve the outcome of sepsis in pregnancy. We used a dimethylarginine dimethylaminohydrolase 1-selective inhibitor (L-257), which reduces vascular nitric oxide synthesis without impairing immune cell function, in combination with a broad-spectrum antibiotic (Imipenem) and studied the outcome of septic shock in pregnant mice. Treatments were administered 3 h after CLP and samples were taken 3 h later. Both Imipenem and L-257 treatment alone slightly improved mortality rates from 13% (NaCl) to 20% (Imipenem) and 33% (L-257), whereas the combination of Imipenem and L-257 significantly improved survival to 50%. Imipenem and L-257 together prevented cardiovascular collapse and improved both organ function and bacterial killing, but did not reduce lung inflammatory cell numbers and actually increased lung cytokine levels.These data suggest that conventional management in combination with selective inhibition of DDAH1 may have therapeutic potential in the management of sepsis in pregnancy.

ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus

Background and aimsCardiovascular 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. MethodsStreptozotocin (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. ResultsSTZ 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. ConclusionsIn 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.

Exploring the role of dimethylarginine dimethylaminohydrolase-mediated reduction in tissue asymmetrical dimethylarginine levels in cardio-protective mechanism of ischaemic postconditioning in rats.

Objective(s):Reperfusion of ischaemic myocardium results in reduced nitric oxide (NO) biosynthesis by endothelial nitric oxide synthase (eNOS) leading to endothelial dysfunction and subsequent tissue damage. Impaired NO biosynthesis may be partly due to increased levels of asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of eNOS. As dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme responsible for degradation of ADMA, the present study was designed to explore the role of DDAH/ADMA/NO pathway in cardio-protective mechanism of ischaemic postconditioning.Materials and Methods:Isolated rat hearts were subjected to myocardial ischaemia for 30 min followed by reperfusion for 2 hours in control group. Myocardial injury was assessed by measurement of infarct size, left ventricular developed pressure (LVDP), lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes in coronary effluents. The reperfused hearts were homogenised and tissue concentration of nitrite, ADMA level and DDAH enzyme activity was determined.Results:A significant increase in infarct size, LDH, CK release in coronary effluents and ADMA level in myocardial tissue was observed in control group. The increase in tissue ADMA coincided with reductions of NO tissue concentrations and DDAH activity. Ischaemic postconditioning significantly attenuated ischaemia-reperfusion induced myocardial injury manifested in the terms of decreased infarct size, LDH, CK, tissue ADMA along with increase in NO levels and DDAH enzyme activity. Pretreatment with L-Homocysteine (300 µM), a competitive inhibitor of DDAH, and L-NG-nitroarginine methyl ester (L-NAME; 100 µM), an inhibitor of eNOS, completely abolished ischaemic postconditioning-induced myocardial protection.Conclusion:Enhancing DDAH activity by postconditioning may be a novel target to reduce ADMA level and increase NO bioavailability to prevent myocardial ischaemia-reperfusion injury.