Dimethylarginine Dimethylaminohydrolase Research Articles

Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway.

miR-21 has been confirmed to be overexpressed in neonatal rat lungs with hyperoxia-mediated bronchopulmonary dysplasia (BPD). The specific function of miR-21 in BPD is still unclear. We established the hyperoxia-induced BPD rat model in vivo and the hyperoxia-induced pulmonary microvascular endothelial cells (PMVECs) model in vitro. Transwell assay was utilized to detect the migratory capability of PMVECs. Tube formation assay was utilized to measure angiogenesis ability. ELISA was utilized to test nitric oxide (NO) production and the intracellular and extracellular Asymmetric Dimethylarginine (ADMA) concentration. Furthermore, the interaction between miR-21 and dimethylarginine dimethylaminohydrolase 1 (DDAH1) was evaluated using luciferase reporter assay. We found that miR-21 expression in PMVECs was increased by hyperoxia stimulation. Inhibition of miR-21 improved the migratory and angiogenic activities of PMVECs and overexpression of miR-21 exerted the opposite effects. Furthermore, knockdown of miR-21 increased NO production and decreased intracellular and extracellular ADMA concentration in hyperoxia-treated PMVECs. Next we proved that miR-21 could bind to DDAH1 and negatively regulate its expression. Rescues assays showed that DDAH1 knockdown reversed the effects of miR-21 depletion on hyperoxia-mediated PMVEC functions, NO production, and ADMA concentration. Importantly, miR-21 downregulation restored alveolarization and vascular density in BPD rats. This study demonstrates that inhibition of miR-21 improves pulmonary vascular responses in BPD by targeting the DDAH1/ADMA/NO pathway.

Asymmetric dimethylarginine accumulation under hyperglycemia facilitates β-cell apoptosis via inhibiting nitric oxide production

Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) has been shown to suppress apoptosis in pancreatic β-cells. In the present study, the influence of asymmetric dimethylarginine (ADMA), the major endogenous inhibitor of NOS, on the apoptosis-suppressive effect of NO was investigated. The expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), an ADMA-metabolizing enzyme, in INS-1 β-cells and in mouse pancreatic islets was drastically reduced by in vitro exposure to high-concentration glucose (20 mM) and by in vivo treatment of mice with the insulin receptor blocker S661, which resulted in hyperglycemia, respectively. In line with this, a higher ADMA level was observed in INS-1 cells exposed to 20 mM glucose. The treatment of INS-1 cells with ADMA, similarly to with the NOS inhibitor NG-nitro-L-arginine methyl ester, significantly facilitated 20 mM glucose-induced increase in cleaved caspase-3 protein expression. Furthermore, increased protein expression of cleaved caspase-3 and CHOP was observed in INS-1 cells with knockdown of DDAH2. These results suggest that ADMA accumulation through a decrease in DDAH2 expression in β-cells, which is induced under hyperglycemic conditions, facilitates β-cell apoptosis through suppression of cNOS-mediated NO production.

Maternal use of methamphetamine induces sex-dependent changes in myocardial gene expression in adult offspring.

Methamphetamine is a commonly abused illicit stimulant that has prevalent use among women of child-bearing age. While there are extensive studies on the neurological effects of prenatal methamphetamine exposure, relatively little is known about the effect of prenatal methamphetamine on the adult cardiovascular system. Earlier work demonstrated that prenatal methamphetamine exposure sex dependently (females only) sensitizes the adult heart to ischemic injury. These data suggest that prenatal exposure to methamphetamine may induce sex-dependent changes in cardiac gene expression that persist in adult offspring. The goal of this study was to test the hypothesis that prenatal methamphetamine exposure induces changes in cardiac gene expression that persist in the adult heart. Hearts of prenatally exposed female offspring exhibited a greater number of changes in gene expression compared to male offspring (184 changes compared with 74 in male offspring and 89 changes common between both sexes). Dimethylarginine dimethylaminohydrolase 2 and 3-hydroxybutyrate dehydrogenase 1 (genes implicated in heart failure) were shown by Western Blot to be under expressed in adult females that were prenatally exposed to methamphetamine, while males were deficient in 3-Hydroxybutyrate Dehydrogenase 1 only. These data indicate that prenatal methamphetamine exposure induces changes in gene expression that persist into adulthood. This is consistent with previous findings that prenatal methamphetamine sex dependently sensitizes the adult heart to ischemic injury and may increase the risk of developing cardiac disorders during adulthood.

Dimethylarginine dimethylaminohydrolase 1 protects PM2.5 exposure-induced lung injury in mice by repressing inflammation and oxidative stress

BackgroundAirborne fine particulate matter with aerodynamic diameter ≤ 2.5 μm (PM2.5) pollution is associated with the prevalence of respiratory diseases, including asthma, bronchitis and chronic obstructive pulmonary disease. In patients with those diseases, circulating asymmetric dimethylarginine (ADMA) levels are increased, which contributes to airway nitric oxide deficiency, oxidative stress and inflammation. Overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme degrading ADMA, exerts protective effects in animal models. However, the impact of DDAH1/ADMA on PM2.5-induced lung injury has not been investigated.MethodsDdah1−/− and DDAH1-transgenic mice, as well as their respective wild-type (WT) littermates, were exposed to either filtered air or airborne PM2.5 (mean daily concentration ~ 50 µg/m3) for 6 months through a whole-body exposure system. Mice were also acutely exposed to 10 mg/kg PM2.5 and/or exogenous ADMA (2 mg/kg) via intratracheal instillation every other day for 2 weeks. Inflammatory response, oxidative stress and related gene expressions in the lungs were examined. In addition, RAW264.7 cells were exposed to PM2.5 and/or ADMA and the changes in intracellular oxidative stress and inflammatory response were determined.ResultsDdah1−/− mice developed more severe lung injury than WT mice after long-term PM2.5 exposure, which was associated with greater induction of pulmonary oxidative stress and inflammation. In the lungs of PM2.5-exposed mice, Ddah1 deficiency increased protein expression of p-p65, iNOS and Bax, and decreased protein expression of Bcl-2, SOD1 and peroxiredoxin 4. Conversely, DDAH1 overexpression significantly alleviated lung injury, attenuated pulmonary oxidative stress and inflammation, and exerted opposite effects on those proteins in PM2.5-exposed mice. In addition, exogenous ADMA administration could mimic the effect of Ddah1 deficiency on PM2.5-induced lung injury, oxidative stress and inflammation. In PM2.5-exposed macrophages, ADMA aggravated the inflammatory response and oxidative stress in an iNOS-dependent manner.ConclusionOur data revealed that DDAH1 has a marked protective effect on long-term PM2.5 exposure-induced lung injury.

The ADMA–DDAH1 axis in ovarian apoptosis of polycystic ovary syndrome

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) mainly degrades asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor. Emerging evidence suggested that plasma ADMA is accumulated in patients with polycystic ovary syndrome (PCOS). However, ADMA–DDAH1 involvement in PCOS pathogenesis is unclear. Here, we used dehydroepiandrosterone (DHEA)-induced PCOS rats and the ovarian granulosa cell line KGN to investigate the effect of the ADMA–DDAH1 pathway on ovarian apoptosis. Moreover, we also quantified the ADMA levels and redox status in human serum specimens, Sprague Dawley rats and KGN cells to investigate the effect of ADMA–DDAH1 on redox status and ovarian apoptosis in PCOS. We enrolled 19 women with PCOS and 17 healthy women (controls) in this study. The women with PCOS had increased serum ADMA levels and decreased glutathione peroxidase (GSH-PX) compared with the controls. In Sprague Dawley rats, 21-day DHEA treatment established PCOS and the rat contained higher ADMA levels in serum and lower DDAH1 expression in ovaries. Moreover, the PCOS rat serum and ovaries exhibited increased levels of the oxidative stress marker malondialdehyde (MDA). ADMA treatment of the KGN cells induced reactive oxygen species accumulation and led to apoptosis. Contrastingly, overexpressing DDAH1 in the KGN cells significantly decreased ADMA levels, enhanced cell viability, and inhibited oxidative stress, while the effect was inverse in DDAH1 knockdown cells. Overall, our results demonstrated that PCOS involves elevated ADMA levels and redox imbalance. The ADMA–DDAH1 pathway exerted a marked effect on oxidative stress and ovarian apoptosis in PCOS. Our findings suggested that strategies for increasing DDAH1 activity in ovarian cells may provide a novel approach for ameliorating PCOS.

Assessment of DDAH1 and DDAH2 Contributions to Psychiatric Disorders via In Silico Methods.

The contribution of nitric oxide synthases (NOSs) to the pathophysiology of several neuropsychiatric disorders is recognized, but the role of their regulators, dimethylarginine dimethylaminohydrolases (DDAHs), is less understood. This study’s objective was to estimate DDAH1 and DDAH2 associations with biological processes implicated in major psychiatric disorders using publicly accessible expression databases. Since co-expressed genes are more likely to be involved in the same biologic processes, we investigated co-expression patterns with DDAH1 and DDAH2 in the dorsolateral prefrontal cortex in psychiatric patients and control subjects. There were no significant differences in DDAH1 and DDAH2 expression levels in schizophrenia or bipolar disorder patients compared to controls. Meanwhile, the data suggest that in patients, DDAH1 and DDHA2 undergo a functional shift mirrored in changes in co-expressed gene patterns. This disarrangement appears in the loss of expression level correlations between DDAH1 or DDAH2 and genes associated with psychiatric disorders and reduced functional similarity of DDAH1 or DDAH2 co-expressed genes in the patient groups. Our findings evidence the possible involvement of DDAH1 and DDAH2 in neuropsychiatric disorder development, but the underlying mechanisms need experimental validation.

Dimethylarginine dimethylaminohydrolase (DDAH-2) regulates the macrophage innate response and could be a target for atherosclerotic therapy: High-throughput RNA-seq approach

Background and Aims: Atherosclerosis is a chronic inflammatory disease characterised by the build-up of arterial lipid plaques. M1 macrophages have a crucial role in progression and pathogenesis of atherosclerosis. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes metabolise the endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), an independent risk factor for atherosclerosis. Two isoforms are known for DDAH, each with distinct tissue specificity. In mice atherosclerotic models, DDAH-1 overexpression reduced plaque formation through ADMA.

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling

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

HIF-1α and Nrf2 regulates hypoxia induced overexpression of DDAH1 through promoter activation in prostate cancer.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is overexpressed in prostate cancer (PCa) and promotes PCa progression in in vivo through the ADMA-NO pathway by degrading nitric oxide synthase (NOS) inhibitors such as asymmetric dimethylarginine (ADMA) and monomethylamine arginine (L-NMMA). In this study, we investigated the molecular mechanism involved in the overexpression of DDAH1 in PCa and examined its potential role as a therapeutic target. We observed that DDAH1expression is elevated in PCa (PC3, LNCaP, and DU145) cell lines under hypoxia. ChIP and reporter assay results confirmed that DDAH1 expression is positively regulated by HIF-1α through directly binding to the hypoxia response elements (HRE) located within the promoter region between -1242/-1238 upstream of its transcription start site (TSS). Under hypoxia, HIF-1α is translocated into the nucleus and activates its target gene expression in PC3 cells. Interestingly, in the event of HIF-1α inhibition or siRNA-mediated knockdown, an alternative transcription factor Nrf2 promotes DDAH1 expression through antioxidant response elements (AREs) on its promoter. ChIP assay results showed that Nrf2 binds to AREs located between -1016 / -1008bp from the TSS of DDAH1. Furthermore, knockdown of PCa therapeutic target HSP90, an essential co-factor for both HIF-1α and Nrf2 causes attenuation of hypoxia induced DDAH1 overexpression in PCa cells. These results demonstrate that hypoxia induced upregulation of DDAH1 expression is positively regulated by HIF-1α and Nrf2 in association with HSP90. Therefore, targeting tumor angiogenesis promoting DDAH1 along with standard androgen receptor (AR) targeted therapy may offer an effective strategy to prevent PCa progression.

Role of ADMA/DDAH-1 and iNOS/eNOS signaling in the gastroprotective effect of tadalafil against indomethacin-induced gastric injury.

Non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastric ulcers represent a significant clinical concern and adversely affect the quality of life. Inducible nitric oxide synthase/endothelial nitric oxide synthase (iNOS/eNOS) and asymmetric dimethylarginine/ dimethylarginine dimethylaminohydrolase-1 (ADMA/DDAH-1) signaling are key players in gastric ulcer pathogenesis. This work was planned to explore the role of iNOS/eNOS and ADMA/DDAH-1 signaling in rats with indomethacin-induced gastric ulcer, as potential pathways for the gastro-protective effect of tadalafil. Split into 5 separate groups, rats were assigned to control, tadalafil (10mg/kg, p.o), indomethacin (single oral dose of 60mg/kg), indomethacin + pantoprazole (40mg/kg, p.o), and indomethacin + tadalafil (10mg/kg, p.o). The results indicated that pretreatment with tadalafil significantly reduced ulcer index (UI), increased preventive index (PI), and counteracted indomethacin-induced histopathological aberrations. Tadalafil significantly reduced the gastric content of NO while it significantly elevated that of GSH and enhanced SOD activity. It significantly reduced the gastric expression of TNF-α and ADMA while it significantly elevated that of COX-2, PGE-2, and DDAH-1. Western blot analysis revealed that pretreatment with tadalafil significantly reduced iNOS protein expression while it significantly elevated that of eNOS. Collectively, these data suggest that tadalafil exerts potential protective effect against indomethacin-induced ulcer through suppression of inflammation, attenuation of oxidative stress, and boosting of antioxidants. Moreover, tadalafil protective effects are mediated via upregulation of PGE-2 with modulating the signaling pathways of ADMA/DDAH-1, and iNOS/eNOS. As a result, the current evidence corroborates the use of tadalafil in controlling gastric ulcers and preventing NSAID gastric side effects.

DDAH1 Promotes Lung Endothelial Barrier Repair by Decreasing Leukocyte Transendothelial Migration and Oxidative Stress in Explosion-Induced Lung Injury

Explosion-induced injury is the most commonly encountered wound in modern warfare and incidents. The vascular inflammatory response and subsequent oxidative stress are considered the key causes of morbidity and mortality among those in blast lung injury. It has been reported dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays important roles in regulating vascular endothelial injury repair and angiogenesis, but its role in explosion-induced injury remains to be explained. To explore the mechanism of vascular injury in blast lung, 40 C57BL/6 wild type mice and 40 DDAH1 knockout mice were randomly equally divided into control group and blast group, respectively. Body weight, lung weight, and dry weight of the lungs were recorded. Diffuse vascular leakage was detected by Evans blue test. The serum inflammatory factors, nitric oxide (NO) contents, and ADMA level were determined through ELISA. Hematoxylin-eosin staining and ROS detection were performed for histopathological changes. Western blot was used to detect the proteins related to oxidative stress, cell adhesion molecules and leukocyte transendothelial migration, vascular injury, endothelial barrier dysfunction, and the DDAH1/ADMA/eNOS signaling pathway. We found that DDAH1 deficiency aggravated explosion-induced body weight reduction, lung weight promotion, diffuse vascular leakage histopathological changes, and the increased levels of inflammatory-related factors. Additionally, DDAH1 deficiency also increased ROS generation, MDA, and IRE-1α expression. Regarding vascular endothelial barrier dysfunction, DDAH1 deficiency increased the expression of ICAM-1, Itgal, Rac2, VEGF, MMP9, vimentin, and N-cadherin, while lowering the expression of occludin, CD31, and dystrophin. DDAH1 deficiency also exacerbated explosion-induced increase of ADMA and decrease of eNOS activity and NO contents. Our results indicated that explosion could induce severe lung injury and pulmonary vascular insufficiency, whereas DDAH1 could promote lung endothelial barrier repair and reduce inflammation and oxidative stress by inhibiting ADMA signaling which in turn increased eNOS activity.

On the kinetic mechanism of dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18) catalyzes the hydrolysis of asymmetric Nω,Nω-dimethyl-l-arginine (ADMA), an endogenous inhibitor of human nitric oxide synthases. The active-site cysteine residue has been proposed to serve as the catalytic nucleophile, forming an S-alkylthiourea reaction intermediate, and serving as a target for covalent inhibitors. Inhibition can lead to ADMA accumulation and downstream inhibition of nitric oxide production. Prior studies have provided experimental evidence for formation of this covalent adduct but have not characterized it kinetically. Here, rapid quench-flow is used with ADMA and the DDAH from Pseudomonas aeruginosa to determine the rate constants for formation (k2 = 17 ± 2 s−1) and decay (k3 = 1.5 ± 0.1 s−1) of the covalent S-alkylthiourea adduct. A minimal kinetic mechanism for DDAH is proposed that supports the kinetic competence of this species as a covalent reaction intermediate and assigns the rate-limiting step in substrate turnover as hydrolysis of this intermediate. This work helps elucidate the different reactivities of S-alkylthiourea intermediates found among the mechanistically diverse pentein superfamily of guanidine-modifying enzymes and provides information useful for inhibitor development.

Recovery of hyperoxia‐induced DDAH1 suppression with FXR agonist GW4064 in human pulmonary microvascular endothelial cells

ObjectivePulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production.MethodshPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA.ResultsIn hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC.ConclusionsChronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.

DDAH1 Protects against Acetaminophen-Induced Liver Hepatoxicity in Mice.

In many developed countries, acetaminophen (APAP) overdose-induced acute liver injury is a significant therapeutic problem. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme for asymmetric dimethylarginine (ADMA) metabolism. Growing evidence suggests that liver dysfunction is associated with increased plasma ADMA levels and reduced hepatic DDAH1 activity/expression. The purpose of this study was to investigate the involvement of DDAH1 in APAP-mediated hepatotoxicity using Ddah1-/- and DDAH1 transgenic mice. After APAP challenge, Ddah1-/- mice developed more severe liver injury than wild type (WT) mice, which was associated with a greater induction of fibrosis, oxidative stress, inflammation, cell apoptosis and phosphorylation of JNK. In contrast, overexpression of DDAH1 attenuated APAP-induced liver injury. RNA-seq analysis showed that DDAH1 affects xenobiotic metabolism and glutathione metabolism pathways in APAP-treated livers. Furthermore, we found that DDAH1 knockdown aggravated APAP-induced cell death, oxidative stress, phosphorylation of JNK and p65, upregulation of CYP2E1 and downregulation of GSTA1 in HepG2 cells. Collectively, our data suggested that DDAH1 has a marked protective effect against APAP-induced liver oxidative stress, inflammation and injury. Strategies to increase hepatic DDAH1 expression/activity may be novel approaches for drug-induced acute liver injury therapy.

DDAH1/ADMA Regulates Adiponectin Resistance in Cerebral Ischemia via the ROS/FOXO1/APR1 Pathway.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cerebral ischemia injury via regulating the level of asymmetric dimethylarginine (ADMA). This study is aimed at exploring the effect of adiponectin resistance on ADMA-induced neuronal loss in ischemic stroke (IS) and the underlying mechanism. DDAH1 knockout (DDAH1−/−) and wild-type (DDAH1+/+) rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). Plasma and brain adiponectin levels and the expressions of adiponectin receptor 1 (APR1), adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK were determined after 24 h, 3 days, and 7 days. Neurological behavior, infarct volume, and adiponectin signaling were evaluated using adiponectin peptide or AdipoRon. The levels of reactive oxygen species (ROS) and Forkhead box O1 (FOXO1) (a transcription factor for APR1) were also assessed. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in primary neurons. DDAH1 was overexpressed in neurons, after which FOXO1 expression, ROS production, adiponectin resistance, and cell viability were detected. DDAH1−/− rats showed no significant difference in adiponectin level in either plasma or brain after MCAO/R in DDAH1+/+ rats, but downregulated APR1 expression and suppressed adiponectin signaling were observed. AdipoRon, but not adiponectin peptide, attenuated the neurological deficits and adiponectin resistance in DDAH1−/− rats. ROS accumulation and phosphorylated FOXO1 expression also increased with DDAH1 depletion. Following DDAH1 overexpression, decreased cell viability and inhibited adiponectin signaling induced by OGD/R were alleviated in primary neurons, accompanied by reduced ROS production and phosphorylated FOXO1 expression. Our study elucidated that in IS, DDAH1 protected against adiponectin resistance in IS via the ROS/FOXO1/APR1 pathway.

Esomeprazole covalently interacts with the cardiovascular enzyme dimethylarginine dimethylaminohydrolase: Insights into the cardiovascular risk of proton pump inhibitors

BackgroundProton pump inhibitors (PPIs) are widely prescribed drugs for the treatment of gastroesophageal reflux disease (GERD). Several meta-analysis studies have reported associations between prolonged use of PPIs and major adverse cardiovascular events. However, interaction of PPIs with biological molecules involved in cardiovascular health is incompletely characterized. Dimethylarginine dimethylaminohydrolase (DDAH) is a cardiovascular enzyme expressed in cardiomyocytes, and other somatic cell types in one of two isotypes (DDAH1 and DDAH2) to metabolize asymmetric dimethylarginine (ADMA); a cardiovascular risk factor and competitive inhibitor of nitric oxide synthases (NOSs). MethodsWe performed high throughput drug screening of over 130,000 small molecules to discover human DDAH1 inhibitors and found that PPIs directly inhibit DDAH1. We expressed and purified the enzyme for structural and mass spectrometry proteomics studies to understand how a prototype PPI, esomeprazole, interacts with DDAH1. We also performed molecular docking studies to model the interaction of DDAH1 with esomeprazole. X-ray crystallography was used to determine the structure of DDAH1 alone and bound to esomeprazole at resolutions ranging from 1.6 to 2.9 Å. ResultsAnalysis of the enzyme active site shows that esomeprazole interacts with the active site cysteine (Cys273) of DDAH1. The structural studies were corroborated by mass spectrometry which indicated that cysteine was targeted by esomeprazole to inactivate DDAH1. ConclusionsThe inhibition of this important cardiovascular enzyme by a PPI may help explain the reported association of PPI use and increased cardiovascular risk in patients and the general population. General significanceOur study calls for pharmacovigilance studies to monitor adverse cardiovascular events in chronic PPI users.

Cysteine-Rich Whey Protein Isolate (CR-WPI) Ameliorates Erectile Dysfunction by Diminishing Oxidative Stress via DDAH/ADMA/NOS Pathway.

Nitric oxide synthase- (NOS-) dependent endothelial dysfunction induced by oxidative stress (OS) is assumed to play a pivotal role in the pathogenesis and progression of diabetes mellitus-related erectile dysfunction (DMED). Cysteine-rich whey protein isolate (CR-WPI) is a widely used protein supplement and has been confirmed to reduce reactive oxygen species (ROS) by increasing cellular antioxidant glutathione (GSH). However, it is currently unknown whether CR-WPI elicits therapeutic effects in DMED. Here, we provide diabetic rats with CR-WPI to determine its effect on DMED and the underlying mechanisms. The results suggest that CR-WPI supplementation increased GSH biosynthesis and reduced ROS content and simultaneously upregulated the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/nitric oxide synthase (NOS) metabolic pathway. Evaluation of intracavernous pressure (ICP) also showed an improvement of penile erectile function in CR-WPI-treated rats. The results of the vitro cell culture showed that glutathione pretreatment protected corpus cavernosum smooth muscle cells (CCSMC) from H2O2-induced apoptosis by decreasing Caspase 9 and Caspase 3 expressions. These results augur well for the potential therapeutic application of dietary CR-WPI supplementation for treating diabetic erectile dysfunction.

A Therapeutic Extracorporeal Device for Specific Removal of Pathologic Asymmetric Dimethylarginine from the Blood

Introduction: Blood levels of uremic toxin, asymmetric dimethylarginine (ADMA), are strongly associated with mortality in sepsis, renal failure, and cardiovascular and renal disease patients. Methods: An extracorporeal approach to reduce pathological ADMA was developed. The dimethylarginine dimethylaminohydrolase (DDAH) was immobilized on agarose beads to prepare a cartridge. The efficacy of cartridge for ADMA lowering in blood was investigated. Results: The DDAH beads and cartridge reduced ADMA from solution or plasma. The magnitude of ADMA removal was dependent on the quantity of DDAH linked to the beads and the flow rate. When tested in association with plasmapheresis, the DDAH-cartridge was highly effective in ADMA removal from the blood and improved the arginine/ADMA ratio in a pig model. Conclusion: A new, safe, and effective extracorporeal approach to lower ADMA was developed which may have application in improving outcomes in patients with vascular complications and risk of mortality associated with high ADMA.

Pharmacokinetic Characterization of the DDAH1 Inhibitors ZST316 and ZST152 in Mice Using a HPLC-MS/MS Method.

The pharmacokinetic profile of ZST316 and ZST152, arginine analogues with inhibitory activity towards human dimethylarginine dimethylaminohydrolase-1 (DDAH1), was investigated in mice using a newly developed HPLC-MS/MS method. The method proved to be reproducible, precise, and accurate for the measurement of the compounds in plasma and urine. Four-week-old female FVB mice received a single dose of ZST316 and ZST152 by intravenous bolus (30 mg/Kg) and oral gavage (60 mg/Kg). ZST316 Cmax was 67.4 µg/mL (intravenous) and 1.02 µg/mL (oral), with a half-life of 6 h and bioavailability of 4.7%. ZST152 Cmax was 24.9 µg/mL (intravenous) and 1.65 µg/mL (oral), with a half-life of 1.2 h and bioavailability of 33.3%. Urinary excretion of ZST152 and ZST316 was 12.5%–22.2% and 2.3%–7.5%, respectively. At least eight urinary metabolites were identified. After chronic intraperitoneal treatment with the more potent DDAH1 inhibitor, ZST316 (30 mg/Kg/day for three weeks), the bioavailability was 59% and no accumulation was observed. Treatment was well tolerated with no changes in body weight vs. untreated animals and no clinical signs of toxicity or distress. The results of this study show that ZST316 has a favorable pharmacokinetic profile, following intraperitoneal administration, to investigate the effects of DDAH1 inhibition in mice.

Curcumin improves cardiac fibrosis by inhibiting endothelial-mesenchymal transition through NRF2-DDAH-ADMA-NO pathway

The present study analyzed the correlations between curcumin(Cur), nuclear factor E2 related factor 2(NRF2)-dimethylarginine dimethylaminohydrolase(DDAH)-asymmetric dimethylarginine(ADMA)-nitric oxide(NO) pathway, and endothelial-mesenchymal transition(EndMT) based on SD rats with cardiac fibrosis, and explored the effect and mechanism of Cur in resisting cardiac fibrosis to provide an in-depth theoretical basis for its clinical application in the treatment of heart failure. The cardiac fibrosis model was induced by subcutaneous injection of isoprenaline(Iso) in rats. Thirty-two rats were randomly divided into a control group, a model group, a low-dose Cur group(100 mg·kg~(-1)·d~(-1)), and a high-dose Cur group(200 mg·kg~(-1)·d~(-1)), with eight in each group. After 21 days of treatment, cardiac function was detected by echocardiography, degree of cardiac fibrosis by Masson staining, expression of CD31 and α-SMA by pathological staining, expression of VE-cadherin, vimentin, NRF2, and DDAH by Western blot, and ADMA level by HPLC. Compared with the model group, the Cur groups showed alleviated cardiac fibrosis, accompanied by increased CD31 and VE-cadherin expression and decreased α-SMA and vimentin expression, indicating relieved EndMT. Additionally, DDAH and NRF2 levels were elevated and ADMA and NO expression declined. Cur improves cardiac fibrosis by inhibiting EndMT presumedly through the NRF2-DDAH-ADMA-NO pathway.