Dimethylarginine Dimethylaminohydrolase Research Articles

Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline: results and overview.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthase. SDMA is excreted in the urine without major metabolization. About 10% of daily produced ADMA are excreted unchanged in the urine. The major elimination route of ADMA (about 90%) involves its hydrolysis to dimethylamine (DMA) and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and excretion of DMA in the urine. High circulating and low excretory concentrations of ADMA are considered risk factors. Experimentally, DDAH activity can be inhibited by SH-specific agents such as inorganic and organic mercury compounds, and by S-nitrosothiols which block the SH group of a particular cysteine moiety of DDAH that is essential for its hydrolytic activity. Alternatively, DDAH activity can be inhibited by organic compounds that compete with the substrate ADMA for DDAH. Arginine analogs that contain substituents on guanidine nitrogen atom(s) (NG) represent a class of DDAH inhibitors. In the present study, we investigated the effects of physiological and natural amino acid derivatives of L-arginine and L-citrulline as well as of nitrate and nitrite, the major circulating and excretory metabolites of NO and NO donating drugs. Here, we report for the first time that the physiological NG-hydroxy-L-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC50 ≈ 100µM). Two plant L-citrulline derivatives, i.e., Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline (connatin), were found to inhibit almost completely hepatic DDAH activity in vitro in rat homogenate at a concentration of 100µM each. At pharmacological concentrations (i.e., 1mM), inorganic nitrate, but not inorganic nitrite, was found to increase rat liver DDAH activity. In urine of 18 patients with Becker's muscular dystrophy, nitrate was found to correlate closely with DMA (Spearman, r = 0.73, p = 0.002). In summary, NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline are novel inhibitors of DDAH activity. This article provides an overview of amino acid-based DDAH inhibitors and discusses potential underlying inhibition mechanisms.

Evidence for a protective role for the rs805305 single nucleotide polymorphism of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in septic shock through the regulation of DDAH activity

BackgroundDimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the synthesis of nitric oxide (NO) through the metabolism of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA). Pilot studies have associated the rs805305 SNP of DDAH2 with ADMA concentrations in sepsis. This study explored the impact of the rs805305 polymorphism on DDAH activity and outcome in septic shock.MethodsWe undertook a secondary analysis of data and samples collected during the Vasopressin versus noradrenaline as initial therapy in septic shock (VANISH) trial. Plasma and DNA samples isolated from 286 patients recruited into the VANISH trial were analysed. Concentrations of L-Arginine and the methylarginines ADMA and symmetric dimethylarginine (SDMA) were determined from plasma samples. Whole blood and buffy-coat samples were genotyped for polymorphisms of DDAH2. Clinical data collected during the study were used to explore the relationship between circulating methylarginines, genotype and outcome.ResultsPeak ADMA concentration over the study period was associated with a hazard ratio for death at 28 days of 3.3 (95% CI 2.0–5.4), p < 0.001. Reduced DDAH activity measured by an elevated ADMA:SDMA ratio was associated with a reduced risk of death in septic shock (p = 0.03). The rs805305 polymorphism of DDAH2 was associated with reduced DDAH activity (p = 0.004) and 28-day mortality (p = 0.02). Mean SOFA score and shock duration were also reduced in the less common G:G genotype compared to heterozygotes and C:C genotype patients (p = 0.04 and p = 0.02, respectively).ConclusionsPlasma ADMA is a biomarker of outcome in septic shock, and reduced DDAH activity is associated with a protective effect. The polymorphism rs805305 SNP is associated with reduced mortality, which is potentially mediated by reduced DDAH2 activity.Trial registrationISRCTN Registry, ISRCTN20769191. Registered on 20 September 2012.

Using clinical and genetic data to predict pulmonary hypertension in bronchopulmonary dysplasia.

Pulmonary hypertension significantly increases morbidity and mortality in infants with bronchopulmonary dysplasia. The frequency of single nucleotide polymorphisms in arginase-1 (ARG1 rs2781666) and dimethylarginine dimethylaminohydrolase-1 (DDAH1 rs480414) genes has been found to differ in a cohort of bronchopulmonary dysplasia patients with pulmonary hypertension (cases) and without pulmonary hypertension (controls). Therefore, we tested the hypothesis that combining these genotypes with phenotypic data would better predict pulmonary hypertension in bronchopulmonary dysplasia patients. Bronchopulmonary dysplasia patients (n=79) born at <35weeks gestation were studied. Pulmonary hypertension was diagnosed by echocardiographic criteria (n=20). ROC curves to predict pulmonary hypertension in bronchopulmonary dysplasia were generated from genotype and/or clinical data. Cases were born at an earlier gestation and weighed less at birth than did controls. ROC curves for rs2781666 had an AUC of 0.61, while rs480414 had an AUC of 0.66. Together, the AUC was 0.70. When clinical data were added to the genetic model, AUC was 0.73. These findings demonstrate that ROC predictive modelling of pulmonary hypertension in bronchopulmonary dysplasia improves with inclusion of both genotypic and phenotypic data. Further refinement of these types of models could facilitate the implementation of precision medicine approaches to pulmonary hypertension in bronchopulmonary dysplasia.

Insights into hepatic and renal FXR/DDAH-1/eNOS pathway and its role in the potential benefit of rosuvastatin and silymarin in hepatic nephropathy

ObjectivesThe repression of renal Farnesoid X Receptor (FXR) had been shown to result from lack of bile acid production from cirrhotic liver. We hypothesized that silymarin and rosuvastatin (Rvs) could have a hepatorenal therapeutic effects in hepatic nephropathy through induction of FXR. MethodsForty two male Wistar rats were used; naïve (n = 12); six of them were sacrificed after 4 weeks and six continued till the end of the experiment. Thirty rats were treated as follows: Rvs, silymarin, thioacetamide (TAA), TAA + Rvs and TAA + silymarin. Liver and kidney function tests as well as the renal and hepatic expression of transforming growth factor β1 (TGFβ1), FXR, dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and eNOS were performed. Histological and immuno-histochemical studies of liver and kidney were also done. ResultsTAA-inducted liver cirrhosis was associated with significant deterioration of liver and renal functions together with increasing expression of hepatic and renal TGFβ1 and decreasing expression of hepatic and renal FXR, DDAH-1 and eNOS. Giving silymarin or Rvs induced hepatic and renal improvement which was evidenced biochemically and histologically. Significant positive correlation was detected between all the investigated biomarkers except for the correlation between FXR and TGFβ1 which was negative. ConclusionsIn conclusion, liver cirrhosis is associated with deterioration of renal functions. Silymarin and Rvs have a potential hepatorenal therapeutic benefit through simultaneous enhancement of FXR/DDAH-1/eNOS pathway in both organs.

PO-214 DDAH1 knockout has a protective effect on muscle damage caused by downhill running

Objective Purpose：Downhill running can causes muscle damage, called delayed muscle damage and induced oxidative stress and inflammatory reaction, causing abnormity of skeletal muscle morphology, changing in blood biochemical indexes, and decreasing in function of skeletal muscle systolic. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is degraded by dimethylarginine dimethylaminohydrolase 1 (DDAH1). There were new evidences demonstrated that DDAH1 is an important regulator of cell redox state and apoptosis. In summary, the study shown that DDAH1 is an important regulator of cell redox state and apoptosis. Emerging evidences suggests that DDAH1 controls cellular oxidative stress and apoptosis via a miR-21-dependent pathway. However, the effect and mechanism of DDAH1 on damage of skeletal muscle caused by downhill running is not clear enough. Thus，the purpose of this experiment was to determine the effect and mechanism of DDAH1 in downhill running.&#x0D; Keys: downhill running; delayed onset muscle soreness(DOMS); eccentric exercise; skeletal muscle.&#x0D; Methods Method: The experimental mice were 24 female C57 mice of 10 weeks old and 24 female DDAH1 hybrid knockout mice of 10 weeks old. DDAH1 KO mice used for this study was knockout of dimethylarginine dimethylaminohydrolase 1 compared with WT mice. Animals were fed standard laboratory chow and had access to water ad libitum. C57 mice were divided into 3 groups: C57 control, C57 48H, C57 120H; DDAH1 KO mice were divided into 3 groups: DDAH1 control, DDAH1 48H, DDAH1 120H. C57 and DDAH1 KO mice used for this study completed a single bout of downhill running exercise (20°, 17 m/min, 60 min), and gastrocnemius muscle, soleus muscle and quadriceps femoris muscle were collected 48 and 120 hours (H) postexercise (PE). C57control group and DDAH1 KO control group dose not exercise. Speed on the treadmill was gradually increased from 10 to 17m/min during a 7-min warm-up period (increased of 1m/min every minute). All experiments were conducted at approximately the same time of day. Maximal grip strength was measured ifor each groups. Grip strength testing was completed to detect post-eccentric exercise injury in C57 and DDAH1 KO mice. All results were analyzed by means of methods of histological and molecular biological.&#x0D; Results Method: The experimental mice were 24 female C57 mice of 10 weeks old and 24 female DDAH1 hybrid knockout mice of 10 weeks old. DDAH1 KO mice used for this study was knockout of dimethylarginine dimethylaminohydrolase 1 compared with WT mice. Animals were fed standard laboratory chow and had access to water ad libitum. C57 mice were divided into 3 groups: C57 control, C57 48H, C57 120H; DDAH1 KO mice were divided into 3 groups: DDAH1 control, DDAH1 48H, DDAH1 120H. C57 and DDAH1 KO mice used for this study completed a single bout of downhill running exercise (20°, 17 m/min, 60 min), and gastrocnemius muscle, soleus muscle and quadriceps femoris muscle were collected 48 and 120 hours (H) postexercise (PE). C57control group and DDAH1 KO control group dose not exercise. Speed on the treadmill was gradually increased from 10 to 17m/min during a 7-min warm-up period (increased of 1m/min every minute). All experiments were conducted at approximately the same time of day. Maximal grip strength was measured ifor each groups. Grip strength testing was completed to detect post-eccentric exercise injury in C57 and DDAH1 KO mice. All results were analyzed by means of methods of histological and molecular biological.&#x0D; Conclusions Conclusion: The DDAH1 knockout has a protective effect on delayed onset muscle soreness(DOMS) caused by downhill running, and accelerate the injury recovery. &#x0D;

Arginine bioavailability and endothelin-1 system in the regulation of vascular function of umbilical vein endothelial cells from intrauterine growth restricted newborns

Background and aimsIntrauterine growth restriction (IUGR) is a major risk factor for perinatal morbidity and mortality, leading to long-term adverse cardiovascular outcomes. The present study aimed to investigate the potential mechanisms in IUGR-associated vascular endothelial dysfunction. Methods and resultsHuman umbilical vein endothelial cells (HUVECs) were derived from IUGR or normal newborns. We found that the proliferation of IUGR-derived HUVECs was accelerated compared to those from normal subjects. Gene profiles related to vascular function including vasomotion, oxidative stress, and angiogenesis were dysregulated in IUGR-HUVECs. Compared with HUVECs from normal newborns, nitric oxide (NO) production was reduced, with imbalance between endothelial nitric oxide synthase (eNOS) and arginase-2 (Arg-2) in IUGR. Meanwhile, intracellular asymmetric dimethylarginine (ADMA) level was elevated with diminished dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression in IUGR-HUVECs. Furthermore, endothelin-1 (ET-1) and hypoxia-inducible factor 1α (HIF-1α) expression were increased, and endothelin receptor type-B (ETBR) was reduced in the IUGR group. IUGR-HUVECs exposed to hypoxia increased the ratio of ADMA to l-arginine, HIF-1α and protein arginine methyltransferase 1 (PRMT1) expression compared to controls. ConclusionsThe present study demonstrated that the reduction of NO bioavailability and release results from elevated Arg-2, accumulation of intracellular ADMA, and imbalance of ET-1 and ETBR, further leading to IUGR-associated vascular endothelial dysfunction. Our study provides novel evidence on the mechanism underlying fetal programming associated with IUGR, which will serve as potential therapeutic targets in the prevention of adverse cardiovascular consequences in adulthood.

Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin-1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA-mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen-inducible endothelial cell-specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR-219-5p in HUVECs. Although the endothelial cell-specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low-dose angiotensin II infusion-induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low-dose angiotensin II-induced increases in blood pressure.-Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

SZSJ protects against insomnia by a decrease in ADMA level and an improvement in DDAH production in sleep-deprived rats

AimsRecent investigations have shown that dimethylarginine dimethylaminohydrolase (DDAH) may be related to sleep disorder. The present study was conducted to test the hypnotic effect of jujubosides from Semen Ziziphi Spinosae (SZSJ) on sleep deprivation and the mechanisms involved. Materials and methodsForty rats were randomly divided into 4 groups (n = 10): two groups of sleep-deprived rats treated with SZSJ (10 or 30 mg/kg per day), sleep-deprived rats group, and control group. At the end of experiment, cerebral DDAH expression, cerebral asymmetric dimethylarginine (ADMA) level, sleep parameters, behavioral activities, and cerebral neurotransmitters level brain was examined. Key findingsSix days after treatment, SZSJ treatment up-regulated cerebral expression of DDAH I and DDAH II concomitantly with elevated parameters of total sleep time, cerebral γ‑aminobutyric acid and 5‑hydroxytryptamine, and reduced values of forelimb lifting-up frequency and walking time, cerebral 8-isoprostane, ADMA, noradrenaline, and glutamic acid in sleep deprivation of rats. SignificanceThe present results suggest that SZSJ Protects against insomnia by a decrease in ADMA level and an improvement in DDAH production in sleep-deprived rats.

Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N5-(1-imino-2-chloroethyl)-l-ornithine ( KI = 1.3 μM, kinact = 0.34 min-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline ( Ki = 470 μM) and 2-chloroacetamidine ( KI = 310 μM, kinact = 4.0 min-1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N5-(1-imino-2-chloroisopropyl)-l-ornithine ( kinact /KI = 208 M-1 s-1) and N5-(1-imino-2-chlorisopropyl)-l-lysine ( kinact /KI = 440 M-1 s-1), and one that lengthens the linker beyond that found in the substrate, N5-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, KI = 0.19 μM, kinact = 0.22 min-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 104 M-1 s-1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 102 M-1 s-1), and has a partition ratio of 1 with a >100 000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 μM) with cytotoxicity appearing only at higher concentrations (ED50 = 118 μM). A 1.91 Å resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.

Accumulation of Endogenous Asymmetric Dimethylarginine Contributes to Diabetic Cardiomyopathy via Endoplasmic Reticulum Stress in Diabetic Rats

Diabetic cardiomyopathy is a major cause of morbidity and mortality among diabetic patients. Endoplasmic reticulum (ER) stress has been involved in diabetic cardiomyopathy. Accumulation of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) is associated with diabetic cardiovascular complications. This study was to determine whether ADMA accumulation contributes to diabetic cardiomyopathy and whether its mechanism is related to ER stress in streptozotocin-induced diabetic rats and cultured rat cardiomyocytes. Cardiac function was measured by M-echocardiography. ER stress was reflected by transcription of glucose-regulated protein78, phosphorylation of eukaryotic initiation factor 2α kinase, splicing of X box-binding protein-1 mRNA and expression of C/EBP homologues protein. Changes of ADMA signal pathway were evaluated by ADMA and nitric oxide contents, NOS and dimethylarginine dimethylaminohydrolase activities, as well as their protein expression. The levels of ADMA in serum and myocardium were significantly elevated in diabetic rats compared to control rats. This elevation was associated with left ventricular diastolic and systolic dysfunction of diabetic rats in comparison with control rats. Furthermore, ADMA accumulation was not only coupled with the disorder of ADMA signal pathway, but also tied to ER stress and oxidative stress in myocardium of diabetic rats. Treatment of diabetic rats with antioxidant N-acetylcysteine could reduce oxidative stress and ER stress, but also ameliorate cardiac dysfunction of diabetic rats. Similar results to diabetic rats were observed in the exogenous NOS inhibitor-treated normal rats and ADMA-incubated cardiomyocytes. These results indicate that endogenous ADMA accumulation plays a pivotal role in the development of diabetic cardiomyopathy, and the underlying mechanism is close related to ER stress and oxidative stress. Disclosure Y. Xiong: None. Y. He: None. W. Fang: None. Y. Lei: None. Y. Lin: None. Z. He: None.

Cardiomyocyte dimethylarginine dimethylaminohydrolase1 attenuates left-ventricular remodeling after acute myocardial infarction: involvement in oxidative stress and apoptosis.

Asymmetric dimethylarginine (ADMA) is a risk factor for heart diseases. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes are key proteins for ADMA degradation. Endothelial DDAH1 is a vital regulator of angiogenesis. DDAH1 is also expressed in cardiomyocytes. However, the role of DDAH1 in cardiomyocytes needs further clarification. Herein, we used an inducible cardiac-specific DDAH1 knockdown mouse (cardiac DDAH1-/-) to investigate the role of cardiomyocyte DDAH1 in left-ventricular (LV) remodeling after acute myocardial infarction (AMI). DDAH1flox/flox and α-MHCMerCreMer mice were used to generate cardiac DDAH1-/- mice. Deletion of DDAH1 in cardiomyocytes was confirmed by Western blotting. No significant differences were observed in plasma ADMA levels and LV function between cardiac DDAH1-/- mice and control mice. Cardiac DDAH1-/- mice showed aggravated LV remodeling 4weeks after AMI, as demonstrated by a large infarct area and impaired LV function. The rate of cardiomyocyte apoptosis and level of oxidative stress were higher in the LV tissue of cardiac DDAH1-/- mice than in that of control mice. However, treatment of cardiomyocytes with exogenous ADMA had no effect on reactive oxygen species (ROS) levels or apoptosis sensitivity. Cardiac DDAH1-/- LV tissue showed downregulated superoxide dismutase2 (SOD2) expression, and treatment of DDAH1-/- cardiomyocytes with the SOD mimic tempol significantly attenuated apoptosis and ROS levels under hypoxic conditions. Tempol administration also attenuated oxidative stress and apoptosis in cardiac DDAH1-/- LV tissue and partially alleviated LV remodeling after AMI. DDAH1 in cardiomyocytes plays a vital role in attenuating LV remodeling after AMI by regulating intracellular ROS levels and apoptosis sensitivity via a SOD2-dependent pathway.

COMPARATIVE PROTEOMIC ANALYSIS IN MICRODISSECTED RENAL VESSELS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Objective: Systemic hypertension has a profound impact on the renal vascular physiology and functionality. Our goal was to identify the biological pathways and macromolecules of the renal arteriolar wall, which are involved with the development of hypertrension. Design and method: Tissue derived exclusively from renal vessels of 4 Spontaneously Hypertensive Rat (SHR) and 4 normotensive controls (Wistar Kyoto,WKY) at 20 weeks using Laser Capture Microdissection on 14 micrometer cryosections was used. High sensitivity proteomic analysis was performed in the microdissected homogenized material in order to detect early molecular alterations associated with hypertension of the renal vessels before the onset of vascular damage. Results: Proteomic analysis revealed 688 proteins; 550 proteins were found in both groups, of which 58 proteins were differentially expressed (15 proteins were up-regulated and 43 proteins were down-regulated in SHR). 71 proteins were found exclusively in control WKY rats and 67 exclusively in SHR rats. Pathway enrichment analysis revealed 114 and 111 pathways in WKY and SHR, respectively and 106 common pathways in both groups. Many of the interesting differentially expressed proteins identified in our study are relevant to vascular tone regulation. Thus proteins involved with NO and vasodilation and affecting eNOS include Xaa-Pro aminopeptidase 1 (XPP1), N (G) N (G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), Dehydropteridine reductase (DHPR), whereas proteins involved with blood pressure regulation by the renin-angiotensin system include Glutamyl aminopeptidase / Aminopeptidase A (AMPE) and Aminopeptidase N (AMPN). Moreover, pathway enrichment analysis revealed that the eNOS activation pathway is disregulated only in the hypertensive SHR animals. Conclusions: Our study demonstrates that hypertension causes early proteomic changes in the renal vessels of SHR compared to WKY. These changes are relevant to vascular tone regulation and consequently may be involved in the development of vascular damage and hypertensive nephrosclerosis. Further studies are required to explore whether these pathways and molecules are involved with hypertensive nephrosclerosis and to identify components that could be considered new therapeutic targets

The effects of proton pump inhibitor on hepatic vascular responsiveness and hemodynamics in cirrhotic rats

BackgroundLiver cirrhosis is associated with increased intrahepatic resistance due to hepatic fibrosis and exaggerated vasoconstriction. Recent studies have indicated that proton pump inhibitors (PPIs), in addition to acid suppression, modulate vasoactive substances and vasoresponsiveness. PPIs are frequently prescribed in patients with cirrhosis due to a higher prevalence of peptic ulcers, however other impacts are unknown. MethodsLiver cirrhosis was induced in Sprague–Dawley rats with common bile duct ligation (BDL). On the 29th day after BDL and after hemodynamic measurements, the intrahepatic vascular responsiveness to high concentrations of endothelin-1 (ET-1) was evaluated after preincubation with (1) Krebs solution (vehicle), (2) esomeprazole (30 μM), or (3) esomeprazole plus Nω-nitro l-arginine (NNA, a non-selective NO synthase (NOS) inhibitor, 10−4 M). After perfusion, the hepatic protein expressions of endothelial NOS (eNOS), inducible NOS (iNOS), cyclooxygenase (COX)-1, COX-2, endothelin-1, DDAH-1 (dimethylarginine dimethylaminohydrolase-1, ADMA inhibitor), DDAH-2, ADMA (asymmetrical dimethyl arginine, NOS inhibitor) were evaluated. In the chronic model, the BDL rats received (1) vehicle; or (2) esomeprazole (3.6 mg/kg/day, oral gavage) from the 1st to 28th day after BDL. On the 29th day and after hemodynamic measurements, plasma liver biochemistry and liver fibrosis were evaluated. ResultsEsomeprazole did not affect hepatic ET-1 vasoresponsiveness. The hepatic protein expressions of the aforementioned factors were not significantly different among the groups. There were no significant differences in hemodynamics, liver biochemistry and hepatic fibrosis after chronic esomeprazole administration. ConclusionPPIs did not affect hepatic vasoresponsiveness or the release of vasoactive substances. Furthermore, they did not influence hemodynamics, liver biochemistry or severity of hepatic fibrosis in the cirrhotic rats.

Assessment of the direct effects of DDAH I on tumour angiogenesis in vivo

Nitric oxide (NO) has been strongly implicated in glioma progression and angiogenesis. The endogenous inhibitors of NO synthesis, asymmetric dimethylarginine (ADMA) and N-monomethyl-l-arginine (l-NMMA), are metabolized by dimethylarginine dimethylaminohydrolase (DDAH), and hence, DDAH is an intracellular factor that regulates NO. However, DDAH may also have an NO-independent action. We aimed to investigate whether DDAH I has any direct role in tumour vascular development and growth independent of its NO-mediated effects, in order to establish the future potential of DDAH inhibition as an anti-angiogenic treatment strategy. A clone of rat C6 glioma cells deficient in NO production expressing a pTet Off regulatable element was identified and engineered to overexpress DDAH I in the absence of doxycycline. Xenografts derived from these cells were propagated in the presence or absence of doxycycline and susceptibility magnetic resonance imaging used to assess functional vasculature in vivo. Pathological correlates of tumour vascular density, maturation and function were also sought. In the absence of doxycycline, tumours exhibited high DDAH I expression and activity, which was suppressed in its presence. However, overexpression of DDAH I had no measurable effect on tumour growth, vessel density, function or maturation. These data suggest that in C6 gliomas DDAH has no NO-independent effects on tumour growth and angiogenesis, and that the therapeutic potential of targeting DDAH in gliomas should only be considered in the context of NO regulation.

Abstract 238: Biological Role and Therapeutic Potential of Nitric Oxide Signaling in Aortic Valve Stenosis: Insights From Genetic Modulation of DDAH1

The balance between local asymmetric dimethylarginine (ADMA) levels and dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity is a major determinant of local nitric oxide synthase activity. Previous studies suggested that increased plasma ADMA levels are strongly associated with the aortic valve stenosis, causal or mechanistic links have yet to be elucidated. Thus, the aim of this study was to investigate whether reduction or overexpression of DDAH1 protects aortic valve dysfunction in mice. To experimentally alter DDAH1 levels, we used low density lipoprotein receptor deficient, apolipoprotein B100-only (LA) mice that were either DDAH1 wild-type (D +/+ or D 0/0 ), heterozygous (D +/- ), or transgenic/overexpressing (D Tg/0 ) littermates. All mice were fed a western diet for 6 months. Reducing DDAH1 increased plasma ADMA levels (LA-D +/+ =0.89 ± 0.02 μmol/L, LA-D +/- =1.1 ± 0.02 μmol/L, p&lt; 0.05) and blood pressure (LA-D +/+ = 125 ± 4 mmHg, LA-D +/- = 141 ± 3 mmHg, p&lt; 0.05) following 6 months of western diet feeding. Critically, aortic valve function was significantly worse in DDAH1-deficient mice (LA-D +/+ 2.20±0.09 m/sec versus LA-D +/- =2.41±0.11 m/sec, p&lt; 0.05) and was associated with higher levels of valve cusp calcification (Alizarin Red, LA-D +/+ = 10 ± 3% versus LA-D +/- = 20 ± 4%). Reciprocally, overexpression of DDAH1 in hypercholesterolemic littermates reduced ADMA levels (LA-D 0/0 = 1.4 ± 0.2 μmol/L, LA-D Tg/0 = 0.7 ± 0.1μmol/L, p&lt; 0.05) and systolic blood pressure (LA-D 0/0 = 136 ± 6 mmHg, LA-D Tg/0 = 124 ± 4 mmHg, p&lt; 0.05). Interestingly, aortic valve function was not improved by overexpression of DDAH1 (LA-D 0/0 = 2.36 ± 0.06 m/sec versus LA-D Tg/0 mice (2.43 ± 0.21 m/sec), nor was valvular calcification. These results suggest that while endogenous levels of DDAH1 expression protect against increases in circulating ADMA, blood pressure, valvular calcification, and valvular dysfunction. Interestingly, overexpression of DDAH1 does not appear to confer additional protection against progression of valvular stenosis. Collectively, our data suggest effective therapeutic harnessing of the DDAH1/ADMA/NOS axis will require targeting of downstream effector molecules—such as oxidation state of soluble guanylate cyclase—known to be pathologically altered and rate limiting in a variety of cardiovascular diseases.

Abstract 287: Transgenic Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Protects From Angiotensin II - Induced Cardiac Hypertrophy and Vascular Remodeling

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.

Abstract 128: Disruption of Methylarginine Metabolism Impairs Vascular Homeostasis During Pregnancy

Endogenously produced asymmetrically methylated forms of L-arginine (ADMA) competitively inhibit all three isoforms of nitric oxide synthase enzymes and therefore have the potential to exert significant cardiovascular effects. Elevated circulating ADMA concentrations have been reported in a number of cardiovascular disease states, including preeclampsia, suggesting that impaired ADMA metabolism may contribute to pathology. In order to test the causal relationship between ADMA concentrations and hemodynamics in pregnancy we employed radiotelemetric measurement of hemodynamic function in mice with either global or fetal-specific deletion of the predominant ADMA metabolising enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1). Global deletion of DDAH1 in non-pregnant female mice caused an elevation in circulating ADMA concentrations (2.851±0.289 vs 1.216±0.072 μM p&lt;0.01) and blood pressure (118±2.28 vs 110.36±0.82 mmHg p&lt;0.05). During the first and second trimesters of pregnancy DDAH1 -/- mice showed no further elevation in ADMA and normal pregnancy related changes in blood pressure were observed. However, during the third trimester ADMA levels in DDAH1 -/- mice increased further and this was associated with increased concentrations of sEng and sFlt1 (2288±301.58 vs 1181.35 ± 161.83 pg/ml p&lt;0.01 and 153.31 ± 8.57 vs 114.42 ± 8.79 ng/ml p&lt;0.001) and an exaggerated hypertensive response (115.773 ± 3.7 vs 101.93 ± 3.27 mmHg p&lt;0.05). Fetal-specific deletion of DDAH1 elevated fetal ADMA concentrations (4.97 ± 0.29 μM vs 1.7 ± 0.12 μM p&lt;0.001) but did not impact on maternal ADMA concentrations or blood pressure during early pregnancy. However in the third trimester a significant elevation in maternal ADMA concentrations was detected (2.49 ± 0.17 vs 1.75 ± 0.13 μM p&lt;0.01) and was associated with an elevation in maternal blood pressure. No changes in sEng or sFLt1 were detectable in fetal-specific DDAH1 -/- mice. Our data indicate that elevated ADMA is sufficient to increase levels of sFLlt1 and sEng and disrupt maternal hemodynamics. The fetus is a significant source of ADMA in late pregnancy suggesting that dysfunctional fetal ADMA metabolism is sufficient to impair maternal hemodynamic function.

Sulfatase 1 mediates the attenuation of Ang II-induced hypertensive effects by CCL5 in vascular smooth muscle cells from spontaneously hypertensive rats

Extracellular sulfatases, sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling and carcinogenesis. Chemokine CCL5 inhibits Ang II-induced hypertensive mediators via angiotensin II (Ang II) type 2 receptor (AT2 R) pathway in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). In this study, we investigated the effect of Sulfs on anti-hypertensive effects of CCL5 in SHR VSMCs. CCL5 attenuated Ang II-induced inhibition of sulfatase activity in SHR VSMCs. Inhibition of Ang II-induced 12-lipoxygenase (12-LO) and endothelin-1 (ET-1) expression by CCL5 was reduced in Sulf1 small interfering RNA (siRNA)-transfected SHR VSMCs. In addition, attenuation of Ang II-induced dimethylarginine dimethylaminohydrolase-1 (DDAH-1) inhibition by CCL5 was reduced in Sulf1 siRNA-transfected SHR VSMCs. Downregulation of Sulf2 did not affect inhibitory effects of CCL5 on Ang II-induced 12-LO and ET-1 expression and Ang II-induced inhibition of DDAH-1 expression in SHR VSMCs. Downregulation of Sulf1 abrogated the expression of CCL5-induced AT2 R messenger RNA (mRNA) and synergistic effect of CCL5 on Ang II-induced AT2 R expression in SHR VSMCs. These findings suggest that Sulf1 is a potential up-regulatory factor in anti-hypertensive actions of CCL5 via AT2 R pathway on Ang II-induced hypertensive effects in SHR VSMCs.

Nephroprotective effects of nebivolol in 2K1C rats through regulation of the kidney ROS-ADMA-NO pathway.

To evaluate the protective effect of nebivolol against kidney damage and elucidate the underlying mechanism in a two-kidney, one-clip (2K1C) rat model. 2K1C rats were obtained by clipping left renal artery of male Wistar rats and were considered hypertensive when systolic blood pressure (SBP) was ≥160mmHg 4 weeks after surgery. The 2K1C hypertensive rats were divided into untreated, nebivolol (10mg/kg, ig), and atenolol (80mg/kg, ig) treatment groups. The treatments lasted for 8 weeks. SBP, kidney structure and function, plasma and kidney angiotensin (Ang) II, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and the oxidant status were examined. Kidney protein expression of NADPH oxidase (Nox) isoforms and its subunit p22phox, nitric oxide synthase (NOS) isoforms, protein arginine N-methyltransferase (PRMT) 1, and dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 was tested by western blotting. Nebivolol and atenolol exerted similar hypotensive effects. However, atenolol had little effect while nebivolol significantly ameliorated the functional decline and structural damage in the kidney, especially in non-clipped kidney (NCK), which was associated with the reduction of Ang II in NCK. Moreover, nebivolol inhibited the NCK production of reactive oxygen species (ROS) by decreasing Nox2, Nox4, and p22phox expression. Further, nebivolol reduced the plasma and kidney ADMA levels by increasing DDAH2 expression and decreasing PRMT1 expression. Nebivolol also increased the NCK NO level by ameliorating the expression of kidney NOS isoforms. Our results demonstrated that long-term treatment with nebivolol had renoprotective effect in 2K1C rats partly via regulation of kidney ROS-ADMA-NO pathway.

DDAH‐1 Regulates NO‐Mediated Apoptosis and Cell Proliferation in Human Pulmonary Microvascular Endothelial Cells

BACKGROUNDNitric oxide (NO) is an endogenous pulmonary vasodilator produced by endothelial NO synthase (eNOS), and decreased eNOS‐derived NO underlies many types of pulmonary hypertension (PH). Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, is elevated in the plasma of patients with bronchopulmonary dysplasia (BPD) prior to the onset of PH. ADMA is hydrolyzed to citrulline by dimethylarginine dimethylaminohydrolase (DDAH), and our group has previously found that a DDAH1 SNP is differentially expressed in BPD‐PH patients compared to BPD control patients.OBJECTIVETo test the hypothesis that siRNA‐mediated knockdown of DDAH‐1 will attenuate NO‐mediated apoptosis in human pulmonary microvascular endothelial cells (hPMVEC).DESIGN/METHODShPMVEC were transfected with siRNA against DDAH‐1 or scramble control for 24h, washed and fresh media placed on them and incubated in 21% O2/5% CO2 for 24h. Protein was analyzed by western blot for DDAH‐1, eNOS, arginase I, arginase II, cleaved caspase‐3, cleaved caspase‐8, and cleaved caspase‐9, and loading controls (β‐actin, total cleaved caspase‐3, total cleaved caspase‐8, and total cleaved caspase‐9). Cell media was assayed for nitrite concentration using chemiluminescence. In a separate set of experiments after transfection, hPMVECs were washed, trypsinized, and viable cells were counted after trypan blue exclusion. In a third set of studies after transfection, hPMVECs were treated with 0.1μM of the NO donor DETA NONOate for 24h or 0.1μM DETA NONOate and 100μM of the caspase‐3 inhibitor Z‐DEVD‐FMK for 24h, and viable cell numbers were counted after trypan blue exclusion.RESULTSThere was no difference in protein levels of eNOS, arginase II, or cleaved caspase‐9 between hPMVEC treated with DDAH‐1 siRNA and scramble controls. However, hPMVEC treated with DDAH‐1 siRNA had less DDAH‐1, arginase I, cleaved caspase‐3, and cleaved caspase‐8 protein levels than did scramble controls (p&lt;0.05). hPMVEC treated with DDAH‐1 siRNA had lower media nitrite concentration than did scramble controls (p&lt;0.05). hPMVEC treated with DDAH‐1 siRNA had greater viable cell numbers than did scramble controls (p&lt;0.05). hPMVEC treated with DDAH‐1 siRNA and DETA NONOate had viable cell numbers that were less than DDAH‐1 siRNA alone (p&lt;0.05) and similar to that seen in scramble control hPMVECs. When hPMVEC were treated with DDAH‐1 siRNA, DETA NONOate, and Z‐DEVD‐FMK, viable cell numbers were increased three fold from scramble control (p&lt;0.05).CONCLUSIONSWe found that knockdown of DDAH‐1 with siRNA in hPMVEC resulted in less NO production, less caspase‐3 activation and more viable cells. These data suggest that greater levels of ADMA are associated with lower levels of NO‐mediated apoptosis in hPMVEC. We speculate that enhancement of DDAH‐1 activity may be a viable therapeutic target to decrease pulmonary vascular wall proliferation and thereby ameliorate the vascular remodeling that occurs in PH.Support or Funding InformationThis research was funded by the National Heart, Lung, and Blood Institute (Grant K08HL129080), and The Research Institute, Nationwide Children's Hospital.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.