FXR Agonist INT-747 Upregulates DDAH Expression and Enhances Insulin Sensitivity in High-Salt Fed Dahl Rats

Aims

Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.

Methods and Results

In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.

Conclusion

Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.     

Dimethylarginine Dimethylaminohydrolase1 Is an Organ-Specific Mediator of End Organ Damage in a Murine Model of Hypertension

Background

The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension.

Methodology/Principal Findings

Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury.

Conclusion/Significance

Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.     

Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.     

Involvement of DDAH/ADMA/NOS pathway in nicotine-induced endothelial dysfunction

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is a key contributor for endothelial dysfunction. Decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), a major hydrolase of ADMA, causes accumulation of ADMA under cardiovascular abnormalities. The study was to determine whether nicotine-induced endothelial dysfunction is related to modulating DDAH/ADMA/NOS pathway. Four-week oral nicotine treatment (5 mg/kg/day) significantly increased the plasma level of ADMA and decreased aortic DDAH expression as well as impaired endothelial function in Sprague-Dawley rats. Similarly, the medium levels of both ADMA and lactate dehydrogenase were markedly elevated in umbilical vein endothelial cells (HUVECs) treated with nicotine (10 microM) for 48 h. Nicotine-induced endothelial damages were markedly attenuated by L-arginine or overexpression of DDAH-II. Nicotine greatly downregulated both mRNA and protein levels of DDAH-II, and decreased DDAH activity in HUVECs. HUVECs express alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), whose antagonists could block these effects of nicotine mentioned above. Intracellular Ca2+ chelator did not affect nicotine-induced decrease in DDAH-II mRNA level. In conclusion, nicotine modulates DDAH/ADMA/NOS pathway of endothelial cell via activation of alpha7 nAChR, which may be involved in endothelial dysfunction associated to smoking.     

Increased asymmetric dimethylarginine (ADMA) dimethylaminohydrolase (DDAH) activity in childhood hypercholesterolemia type II

Asymmetric dimethylarginine (ADMA) systemic concentrations are elevated in hypercholesterolemic adults and contribute to nitric oxide (NO) dependent endothelial dysfunction. Decreased activity of the key ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase (DDAH) may be involved. Yet, the ADMA/DDAH/NO pathway has not been investigated in childhood hypercholesterolemia. We studied 64 children with hypercholesterolemia type II (HCh-II) and 54 normocholesterolemic (NCh) children (mean ± SD; age, years: 11.1 ± 3.5 vs. 11.9 ± 4.6). Plasma and urine ADMA was measured by GC-MS/MS. Dimethylamine (DMA), the ADMA metabolite, creatinine, nitrite and nitrate in urine were measured by GC-MS. The DMA/ADMA molar ratio in urine was calculated to estimate whole body DDAH activity. ADMA plasma concentration (mean ± SD; nM: 571 ± 85 vs. 542 ± 110, P = 0.17) and ADMA urinary excretion rate (mean ± SD: 7.1 ± 2 versus 7.2 ± 3 μmol/mmol creatinine, P = 0.6) were similar in HCh-II and NCh children. Both DMA excretion rate [median (25th-75th percentile): 56.3 (46.4-109.1) vs. 45.2 (22.2-65.5) μmol/mmol creatinine, P = 0.0004] and DMA/ADMA molar ratio [median (25th-75th percentile): 9.2 (6.0-16.3) vs. 5.4 (3.8-9.4), P = 0.0004] were slightly but statistically significantly increased in HCh-II children compared to NCh children. Plasma and urinary nitrite and nitrate were similar in both groups. In HCh-II whole body DDAH activity is elevated as compared to NCh. HCh-II children treated with drugs for hypercholesterolemia had lower plasma ADMA levels than untreated HCh-II or NCh children, presumably via increased DDAH activity. Differences between treated and untreated HCh-II children were not due to differences in age. In conclusion, HCh-II children do not have elevated ADMA plasma levels, largely due to an apparent increase in DDAH activity. While this would tend to limit development of endothelial dysfunction, it is not clear whether this might be medication-induced or represent a primary change in HCh-II children.     

DDAH1 plays dual roles in PM2.5 induced cell death in A549 cells

BackgroundDimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that can degrade asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor. Emerging evidence suggests that alterations in the ADMA–DDAH1 pathway are involved in environmental pollution induced airway inflammation. However, the role of DDAH1 in protection against cytotoxicity of ambient airborne particulate matter is unclear.MethodsWe examined the influence of DDAH1 expression on oxidative stress and cell apoptosis in human type II alveolar epithelial A549 cells exposed to PM_2.5 (particulate matter with an aerodynamic diameter less than 2.5 μM).ResultsWe found that PM_2.5 exposure for 48 h significantly decreased DDAH1 expression. However, knockdown of DDAH1 prior to PM_2.5 exposure actually attenuated the cytotoxicity of PM_2.5. Cytoprotection in DDAH1 deficient cells was due to increased reactive oxygen species, activation of PI3K–AKT and mitogen-activated protein kinase (MAPK) pathways, subsequent activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and this caused a subsequent reduction in PM_2.5 induced oxidative stress relative to control. DDAH1 depletion also repressed the induction of inducible NOS (iNOS) in PM_2.5-exposed cells and knockdown of iNOS protected cells against PM_2.5 induced cell death. Interestingly, overexpression of DDAH1 also exerted a protective effect against the cytotoxicity of PM_2.5 and this was associated with a reduction in oxidative stress and upregulation of the anti-apoptotic protein Bcl-2.ConclusionsOur data indicate that DDAH1 plays dual roles in protection against cytotoxicity of PM_2.5 exposure, apparently by limiting PM_2.5 induced oxidative stress.General significanceOur findings reveal new insights into the role(s) of the DDAH1/ADMA in pulmonary protection against airborne pollutants. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis

NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis.Key words: asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), nitric oxide (NO), angiogenesis, endothelial, motilityNO is generated from L-arginine by NOS; a process which is competitively inhibited by the arginine analogues ADMA and L-NMMA. These endogenous factors are products of proteolytic degradation of methylated proteins. ADMA and L-NMMA are metabolised by DDAH I and II, thereby enhancing NO generation. Of relevance to vascular biology, dysfunctional DDAH activity and ADMA accumulation are risk factors for cardiovascular disorders, including hypertension, artherosclerosis, diabetes, insulin resistance, hypercholesterolemia and homocysteinemia (reviewed in ref. ¹).The DDAH I null mouse was generated recently by Leiper et al.² to facilitate investigation of the role of the DDAH/ADMA pathway in the pathology of cardiovascular disorders. While the absence of DDAH I causes a lethal phenotype, heterozygotes (HT) did not display any obvious abnormalities. However, ADMA levels were raised in tissues and plasma, in association with raised blood pressure and systemic vascular resistance, and reduced cardiac output and heart rate. Synthetic DDAH I inhibitors were designed by the authors and were shown by crystallography to bind to the active site of the enzyme and induce local distortions at this region. Confirming that loss of DDAH I was responsible for ADMA accumulation, these inhibitors enhanced ADMA levels in wildtype mice, and resulted in cardiovascular changes similar to those seen in the HT background. Inhibitor treatment also promoted ADMA release from wildtype blood vessels maintained ex vivo, indicating that the DDAH/ADMA pathway is directly responsible for maintaining cardiovascular function in this model.Evidence was also presented for a causal link between ADMA metabolism and reduced NO levels. In an ex vivo model, aortic rings from HT mice displayed enhanced phenylephrine-induced contraction and reduced acetylcholine-induced relaxation, while DDAH I inhibitors induced similar responses in aortic rings from wildtype mice; indicative of reduced levels of endothelial-derived NO. Further demonstrating an ADMA/NO-dependent mechanism, exogenous L-arginine restored a normal response to these vasomodulators in the HT model (by competing with ADMA for interaction with NOS). Similarly, cultured endothelial cells from HT vessels produced more ADMA and less NO than cells from wildtype vessels, and DDAH I inhibitors induced a similar phenotype in wildtype endothelial cells. The significance of DDAH I/ADMA and NO in vascular disease was tested in a disease model. Endotoxic shock was induced in rats by intravenous infusion of LPS, which induces excess NO production, resulting in systemic hypotension. After blood pressure had fallen by 20%, infusion of a DDAH I inhibitor was able to rapidly stabilise blood pressure, in accordance with inhibition of NO production through reduced ADMA metabolism. Thus, when DDAH I is reduced, ADMA is increased and endogenous NO inhibited, resulting in altered vascular function.Another related study investigated a mechanistic understanding of the role of ADMA/DDAH/NO in angiogenesis.³ The authors demonstrated that ADMA regulates endothelial cell motility and phenotype by inhibiting NO-dependent changes in activity of Rho-GTPases; key mediators of cytoskeletal dynamics and motility. Treatment of pulmonary artery endothelial cells with ADMA enhanced stress fibres and focal adhesion formation in conjunction with increased activity of RhoA in pull-down assays. In accordance with these observations, motility, tracked by time-lapse microscopy, was inhibited by ADMA treatment, and ADMA effects were reversed by a Rho kinase inhibitor (Y-27632) or by adenoviral-mediated gene transfer of a dominant negative RhoA mutant. RhoA activity is mediated by PKG, which mediates RhoA-Ser188 phosphorylation, preventing RhoA localization to the membrane and inhibiting its activity.⁴ In further support of a RhoA-dependent mechanism, ADMA reduced phosphorylation at RhoA-Ser188, while a PKG activator was also able to revert ADMA effects on motility. Further, a non-phosphorylatable mutant of RhoA, Ala188RhoA, or a specific PKG inhibitor, each inhibited cell motility to a similar level as ADMA treatment alone. Inhibition of NO production and endothelial cell motility by ADMA was also reversed by a NO donor, SNAP, or by DDAH I or II overexpression via adenovirus-mediated gene transfer. Thus, reduction of NO/PKG levels by ADMA reduces RhoA phosphorylation at Ser188 resulting in enhancement of RhoA activity and inhibition of cell motility.The significance of these molecular mechanisms to angiogenesis was demonstrated using endothelial cells and aortic ring explants from HT DDAH I and wildtype mice. HT endothelial cells, which secrete more ADMA and produce less NO than their wildtype counterparts, exhibit enhanced RhoA activity and stress fibre formation in conjunction with reduced motility. Reduced sprouting from ex vivo aortic rings was also observed in the HT model, which was mimicked by addition of exogenous ADMA in the wildtype background. These data demonstrate that in vivo, DDAH/ADMA levels are likely to play a key role in control of endothelial cell motility and angiogenesis by regulating NO production.     

Dimethylarginine Dimethylaminohydrolase-1 Transgenic Mice Are Not Protected from Ischemic Stroke

Background

Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).

Methodology/Principal Findings

We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.

Conclusion/Significance

Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.     

A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the hydrolysis of asymmetric dimethylarginine (ADMA) to L-citrulline and dimethylamine. DDAH is currently investigated as a promising target for therapeutic interventions, as ADMA has been found to be elevated in cardiovascular disease. In many tissues continuous endogenous formation of ADMA and L-citrulline poses considerable limitations to the presently used assays for DDAH activity, which are commonly based on the measurement of ADMA or L-citrulline. We therefore developed a stable-isotope-based assay suitable for 96-well plates to determine DDAH activity. Using deuterium-labeled ADMA ([(2)H(6)]-ADMA) as substrate and double stable-isotope labeled ADMA ([(13)C(5)-(2)H(6)]-ADMA) as internal standard we were able to simultaneously determine formation and metabolism of ADMA in renal and liver tissue of mice by LC-tandem MS. Endogenous formation of ADMA could largely be abolished by addition of protease inhibitors, while metabolism of [(2)H(6)]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [(2)H(6)]-ADMA was 2.4% and 4.8% in renal and liver tissue, respectively. The inter-assay coefficient of variation for DDAH activity based on degradation of [(2)H(6)]-ADMA determined in separate samples from the same organs was determined to be 8.9% and 10% for mouse kidney and liver, respectively. The present DDAH activity assay allows for the first time to simultaneously determine DDAH activity and endogenous formation of ADMA, SDMA, and L-arginine in tissue.     

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

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

Involvement of the endothelial DDAH/ADMA pathway in nitroglycerin tolerance: the role of ALDH-2

Previous studies have shown that nitroglycerin (GTN) tolerance is closely related to an oxidative stress-induced decrease in activity of mitochondrial isoforms of aldehyde dehydrogenase (ALDH-2), and prolonged GTN treatment causes endothelial dysfunction. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production and induce oxidative stress in endothelial cells. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endothelium function. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the development of GTN tolerance in endothelial cells. Tolerance, reflected by the decrease in cyclic GMP (cGMP) production, was induced by exposure of human umbilical vein endothelial cells (HUVECs) to GTN (10 microM) for 16 h. While the treatment increased reactive oxygen species (ROS) production/malondialdehyde (MDA) concentration and decreased ALDH-2 activity as well as cGMP production, it markedly increased the level of ADMA in culture medium and decreased DDAH activity in endothelial cells. Exogenous ADMA significantly enhanced ROS production/MDA concentration and inhibited ALDH-2 activity, and overexpression of DDAH2 could significantly suppress GTN-induced oxidative stress and inhibition of ALDH-2 activity, which is also attenuated by L-arginine. Therefore, our results suggest for the first time that the endothelial DDAH/ADMA pathway plays an important role in the development/maintenance of GTN tolerance.     

Asymmetric dimethylarginine exacerbates Aβ-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease

Growing evidence suggests a strong association between cardiovascular risk factors and incidence of Alzheimer disease (AD). Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, has been identified as an independent cardiovascular risk factor and is also increased in plasma of patients with AD. However, whether ADMA is involved in the pathogenesis of AD is unknown. In this study, we found that ADMA content was increased in a transgenic Caenorhabditis elegans β-amyloid (Aβ) overexpression model, strain CL2006, and in human SH-SY5Y cells overexpressing the Swedish mutant form of human Aβ precursor protein (APPsw). Moreover, ADMA treatment exacerbated Aβ-induced paralysis and oxidative stress in CL2006 worms and further elevated oxidative stress and Aβ secretion in APPsw cells. Knockdown of type 1 protein arginine N-methyltransferase to reduce ADMA production failed to show a protective effect against Aβ toxicity, but resulted in more paralysis in CL2006 worms as well as increased oxidative stress and Aβ secretion in APPsw cells. However, overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) to promote ADMA degradation significantly attenuated oxidative stress and Aβ secretion in APPsw cells. Collectively, our data support the hypothesis that elevated ADMA contributes to the pathogenesis of AD. Our findings suggest that strategies to increase DDAH1 activity in neuronal cells may be a novel approach to attenuating AD development.     

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels,VEGF expression,and cell phenotype

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high-throughput chemical screening

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.     

Tissue-specific downregulation of dimethylarginine dimethylaminohydrolase in hyperhomocysteinemia

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 beta-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 approximately 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.