Synthase Inhibitor Asymmetric Dimethylarginine Research Articles

Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol®) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

Asymmetric Dimethylarginine in Patients with Ascending Aortic Aneurysms.

Ascending thoracic aortic aneurysm (aTAA) is a heterogeneous group of disorders that involve impaired endothelial function. The nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) serves as an endothelial dysfunction marker. Thus, we investigated ADMA levels in patients with aTAA. Eighty-six patients with aTAA and 18 healthy individuals were enrolled. All patients underwent echocardiography. Plasma ADMA levels were measured using high-performance liquid chromatography. ADMA levels were higher in aTAA patients than in control patients (p = 0.034). According to the multivariable regression model, higher ADMA levels were associated with ascending aortic diameter (p = 0.017), smoking (p = 0.016), and log-transformed estimated glomerular filtration rate (eGFR, p = 0.005). This pilot study demonstrates an association of ADMA with ascending aortic dilatation; however, further studies are needed to investigate whether increased ADMA levels underlie aTAA development.

Arginines Plasma Concentration and Oxidative Stress in Mild to Moderate COPD.

BackgroundElevated plasma concentrations of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) have been observed in respiratory conditions such as asthma and cystic fibrosis. Since oxidative stress has been shown to increase the activity of arginine methylating enzymes, hence increased ADMA synthesis, and to reduce ADMA degrading enzymes, hence increased ADMA concentrations, we assessed methylated arginines concentrations in chronic obstructive pulmonary disease (COPD), a disease characterized by increased oxidative stress.MethodsPlasma arginine, ADMA and symmetric dimethylarginine (SDMA), oxidative stress markers (thiobarbituric acid reactive substances, TBARS, and plasma proteins SH, PSH) and antioxidants (taurine and paraoxonase 1, PON1, activity) were measured in 43 COPD patients with mild (n = 29) or moderate (n = 14) disease and 43 age- and sex-matched controls.ResultsTBARS significantly increased with COPD presence and severity (median 2.93 vs 3.18 vs 3.64 μmol/L, respectively in controls, mild and moderate group, p<0.0001 by ANOVA) whereas PSH decreased (6.69±1.15 vs 6.04±0.85 vs 5.33±0.96 μmol/gr prot, p<0.0001 by ANOVA). Increased ADMA/arginine ratio, primarily due to reduced arginine concentrations, was also observed with COPD presence and severity (median 0.0067 vs 0.0075 vs 0.0100, p<0.0001 by ANOVA). In multiple logistic regression analysis, only TBARS (OR 0.44, 95% CI 0.25–0.77; p = 0.0045) and ADMA/Arginine ratio (OR 1.72, 95% CI 2.27–13.05; p = 0.02) were independently associated with COPD severity.ConclusionCOPD presence and severity are associated with increased oxidative stress and alterations in arginine metabolism. The reduced arginine concentrations in COPD may offer a new target for therapeutic interventions increasing arginine availability.

Serum L-arginine and dimethylarginine levels in migraine patients with brain white matter lesions

Background/Aim Migraine is a risk factor for the formation of silent brain white matter lesions (WMLs) that are possibly ischemic in nature. Although dysfunction of the L-arginine/nitric oxide (NO) pathway has been associated with oxidative stress and endothelial dysfunction in migraine, its role in WML development has not been specifically investigated. Thus, this prospective study aimed to measure the serum concentrations of the NO substrate L-arginine, the NO synthase inhibitor asymmetric dimethylarginine (ADMA), and the L-arginine transport regulator symmetric dimethylarginine (SDMA) in migraine patients in a headache-free period. Methods All participants underwent MR imaging to assess for the presence of WMLs on fluid-attenuated inversion recovery imaging. Altogether 109 migraine patients (43 with lesions, 66 without lesions) and 46 control individuals were studied. High-performance liquid chromatography was used to quantify L-arginine, ADMA and SDMA serum concentrations. Migraine characteristics were investigated, and participants were screened for risk factors that can lead to elevated serum ADMA levels independent of migraine. Results Migraine patients and controls did not differ in regard to vascular risk factors. Migraineurs with WMLs had a longer disease duration ( p < 0.001) and a higher number of lifetime headache attacks ( p = 0.005) than lesion-free patients. Higher L-arginine serum levels were found in both migraine subgroups compared to controls ( p < 0.001). Migraine patients with WMLs showed higher ADMA concentrations than lesion-free patients and controls ( p < 0.001, for both). In migraineurs, the presence of WMLs, aura and increasing age proved to be significant predictors of increased ADMA levels ( p = 0.008, 0.047 and 0.012, respectively). SDMA serum levels of lesional migraineurs were higher than in nonlesional patients ( p < 0.001). The presence of lesions and increasing age indicated an increased SDMA level ( p = 0.017 and 0.001, respectively). Binary logistic regression analysis showed that ADMA level ( p = 0.006), increasing age ( p = 0.017) and the total number of lifetime migraine attacks ( p = 0.026) were associated with an increased likelihood of exhibiting WMLs. There was no significant effect of age on ADMA and SDMA concentrations in controls. Conclusions Elevated ADMA levels may impact the pathogenesis of migraine-related WMLs by influencing cerebrovascular autoregulation and vasomotor reactivity. Higher SDMA concentrations may indirectly influence NO synthesis by reducing substrate availability. Elevated L-arginine serum levels might reflect an increased demand for NO synthesis.

L-citrulline for protection of endothelial function from ADMA-induced injury in porcine coronary artery.

Endogenous nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a cardiovascular risk factor. We tested the hypothesis that L-citrulline may ameliorate the endothelial function altered by ADMA in porcine coronary artery (PCA). Myograph study for vasorelaxation, electrochemical measurement for NO, RT-PCR, and Western blot analysis for expression of eNOS, argininosuccinate synthetase (ASS), and p-eNOSser1177 were performed. cGMP was determined by enzyme immunoassay. Superoxide anion (O2.−) production was detected by the lucigenin-enhanced chemiluminescence method. Compare with controls (96.03% ± 6.2%), the maximal relaxation induced by bradykinin was significantly attenuated (61.55% ± 4.8%, p < 0.01), and significantly restored by L-citrulline (82.67 ± 6.4%, p < 0.05) after 24 hours of ADMA exposure. Expression of eNOS, p-eNOSser1177, and ASS in PCA significantly increased after L-citrulline incubation. L-citrulline also markedly restored the NO production, and cGMP level which was reduced by ADMA. The increased O2.− production by ADMA was also inhibited by L-citrulline. L-citrulline restores the endothelial function in preparations treated with ADMA by preservation of NO production and suppression of O2.− generation. Preservation of NO is attributed to the upregulation of eNOS expression along with activation of p-eNOSser1177. L-citrulline improves endothelium-dependent vasodilation through NO/ cGMP pathway.

Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs.

Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Asymmetric dimethyl-arginine metabolism in a murine model of cigarette smoke-mediated lung inflammation

There is increasing evidence that the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethyl-arginine (ADMA) is involved in the pathogenesis of chronic lung diseases. One important regulator of this molecule is the ADMA-metabolizing enzyme dimethyl-arginine dimethyl-aminohydrolase (DDAH). The objective of this study was to determine whether perturbation of the ADMA-DDAH pathway contributes to lung inflammation following exposure to cigarette smoke (CS). For these studies, wild-type and DDAH transgenic mice were sham or CS-exposed. Serum ADMA levels were determined by mass spectrometry. ADMA content and DDAH expression were also visualized in mouse lung tissue by immunohistochemistry. DDAH expression was determined by real-time quantitative PCR (qPCR). Inflammation was assessed by H&E staining and analyses of total cell counts and fluid tumor necrosis factor (TNF)-α levels (using ELISA) in lung lavage fluid. NF-κB binding activity in mouse lung epithelial (LA-4) cells was assessed by a transcription factor-binding assay. The results indicated that the concentration of serum ADMA was increased following exposure to CS, and this corresponded with increased ADMA content in bronchial epithelial cells in lung tissue. Total lung DDAH expression was significantly decreased in lung tissue and cultured LA-4 cells following CS exposure. Addition of exogenous ADMA increased CSE-mediated NF-κB binding activity and TNFα production in LA-4 cells more than 2-fold compared to that in CSE-exposed controls. CS-mediated lung inflammation was significantly attenuated in DDAH transgenic mice compared to in wild-type controls. These findings demonstrated that lung ADMA metabolism was altered in mice following CS exposure and suggested that ADMA played a role in CS-mediated inflammation through increasing the presence of inflammatory mediators in lung epithelial cells.

Effects of apocynin, an NADPH oxidase inhibitor, on levels of ADMA, MPO, iNOS and TLR4 induced by myocardial ischemia reperfusion.

In this study, the effects of apocynin, an NADPH oxidase inhibitor, on the levels of inducible nitric oxide synthase (iNOS) and the toll-like receptor 4 (TLR4), which are inflammatory mediators in myocardial ischemia-reperfusion (MIR) injury, and myeloperoxidase (MPO), which is the indicator of neutrophil infiltration and the endogenous nitric oxide synthase inhibitor asymmetric dimethyl arginine (ADMA) increasing with oxidative stress were investigated. MIR injury was accomplished by the application of occlusion for 30 minutes and reperfusion for 120 minutes in the left anterior descending artery (LAD). In the study, 21 Sprague-Dawley male rats were divided into three groups: a sham group (n = 7); a MIR group (n = 7); and a MIR + apocynin treatment group (n = 7, before the procedure, an intraperitoneal administration of 10 mg/kg of apocynin for 15 days). After reperfusion, iNOS, TLR4, MPO and ADMA levels in myocardial tissue were measured by ELISA. While myocardial TLR4, MPO and ADMA levels increased in the MIR group, these parameters were found to be decreased significantly in the group treated with apocynin. Although iNOS levels showed an increase in the MIR group compared to the sham group and a reduction in the MIR+apocynin group, there was no statistically significant difference between the groups. In our study, the effect of the treatment of apocynin in MIR on ADMA, MPO, iNOS and TLR4 levels in myocardial tissue was shown for the first time. It is thought that apocynin treatment may show a protective effect in MIR injury by affecting oxidative stress (ADMA) and inflammatory parameters (iNOS, MPO).

Effect of Lowering Asymmetric Dimethylarginine (ADMA) on Vascular Pathology in Atherosclerotic ApoE-Deficient Mice with Reduced Renal Mass

The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1), on atherosclerosis in subtotally nephrectomized (SNX) ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n = 39) and C57Bl/6J wild-type littermates (WT, n = 27) with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction) ApoE-competent DDAH1 transgenic and wild-type littermates (n = 11) served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD) in this mouse model.

Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment.

Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice

Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.

Biological Functional Relevance of Asymmetric Dimethylarginine (ADMA) in Cardiovascular Disease

There is growing evidence that increased levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) may contribute to endothelial dysfunction. Studies in animal models as well as in humans have suggested that the increase in ADMA occurs at a time when vascular disease has not yet become clinically evident. ADMA competitively inhibits NO elaboration by displacing l-arginine from NO synthase. In a concentration-dependent manner, it thereby interferes not only with endothelium-dependent, NO-mediated vasodilation, but also with other biological functions exerted by NO. The upshot may be a pro-atherogenic state. Recently, several studies have investigated the effect of various therapeutical interventions on ADMA plasma concentrations.

Letter by Kurtoglu et al Regarding Article, “Asymmetric Dimethylarginine in Response to Recombinant Tissue-Type Plasminogen Activator and Erythropoietin in Acute Stroke”

We read the article by Worthmann et al1 with interest. The authors investigated whether the combination of recombinant tissue-type plasminogen activator (rtPA) and erythropoietin (EPO) increases the release of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). The patients were divided into 4 groups according to treatment: placebo, EPO, rtPA+placebo, and EPO+rtPA. They found that ADMA levels increased during the observation time in the EPO, the EPO+rtPA, and the placebo groups, whereas a treatment effect on ADMA levels was revealed only in the rtPA+placebo group when adjusted for age and infection at …

Treatment with the Nitric Oxide Donor SNP Increases Triiodothyronine Levels in Hyper- and Hypothyroid Sprague-Dawley Rats

Nitric oxide pathway might play a crucial role in the pathophysiology of thyroid dysfunctions. This study aimed to investigate the impact of nitric oxide (NO) on hypothyroid and hyperthyroid Sprague-Dawley rats under controlled diet. Furthermore, the effects of the nitric oxide donor sodium nitroprusside (SNP) on thyroid dysfunctions were also assessed. Sprague-Dawley rats (n=107) were subdivided into normal diet and high-fat diet (HFD) groups and grouped into controls, hypothyroid, hyperthyroid, and SNP treated groups. Hypothyroidism was induced through propylthiouracil, whereas hyperthyroidism by triiodothyronine (T3). After 12 weeks of T3 treatment, serum nitric oxides (NOX), endogenous asymmetric dimethylarginine (ADMA), body weight and food intake were analyzed. Hypothyroid rats showed decreased serum T3 levels, hyperthyroid rats increased T3 compared to controls. Diet had no impact on T3. Thyroid dysfunctions were accompanied by changes in calorie intake and body weight. Serum NOX was significantly reduced in normal diet hypothyroid rats. SNP administration compensated the decrease and markedly increased T3. NO synthase inhibitor ADMA levels were significantly higher in the HFD control group than in the normal diet controls. ADMA was declined in both hypothyroid groups and increased in normal diet hyperthyroid rats. An association of thyroid dysfunctions with reduced bioavailability of NO and alterations of ADMA levels could be established. Treatment with the NO donor SNP resulted in an increase of serum T3 levels. These results demonstrate that the NO pathway is implicated in thyroid dysfunctions, which may be of clinical relevance.

ADMA and prognosis in patients with heart failure and preserved ejection fraction

Background: Elevated plasma levels of the endogenous nitric oxid synthase inhibitor asymmetric dimethylarginine (ADMA) contribute to endothelial dysfunction (ED) and seem to be a predictor for cardiovascular mortality. ED plays an important role in heart failure, but few studies have investigated the relation between ADMA, peripheral ED and prognosis in patients with heart failure and preserved ejection fraction (HFpEF). Purpose: This study was performed to determine whether ADMA and ED measured by flow mediated dilation (FMD) can predict prognosis of patients with HFpEF. Methods: We conducted a prospective study on 72 patients with HFpEF. We determined: left ventricular ejection fraction (LVEF), the ratio of early transmitral flow velocity to tissue Doppler early diastolic mitral annular velocity (E/e'), FMD, ADMA and NT-proBNP levels. All patients were in NYHA class II or III, had LVEF > 50%, and E/e' >15. Patients were prospectively followed-up for heart failure-related events, including heart failure-related death and hospitalization due to worsening of heart failure for 30 months. Results: Median values for ADMA were 0,54±0,13 μmol/l. During follow-up 26 of patients had an event: 20 patients were hospitalized due to worsening of heart failure, six patients died for cardiac reasons. There was a strong correlation between ADMA and NTproBNP (p=0, 0006, r=0, 51) and an inverse correlation between ADMA and FMD (p<0.0001, r= -0, 8) and between FMD and NT-proBNP (p<0, 0001, r= -0, 63). Heart failure related death correlated with ADMA (p=0,001, r=0,49), NT-proBNP (p=0,04, r=0,31)and inversely FMD (p=0,01, r= -0,38). In multiple regression analysis independent predictor for death remained only ADMA (p=0,03, r=0,49). Worsening of heart failure correlated with ADMA (p<0, 0001, r=0, 79), NT-proBNP (p<0, 0001, r=0, 69) and inversely with FMD (p<0, 0001, r= -0, 8). In multiple regression analysis the strongest predictor for worsening of heart failure was ADMA (p=0,003, r=0,9) followed by FMD (p=0,03, r= -0,8) and NT-proBNP (p=0,04, r=0,69). Conclusions: Endothelial dysfunction is a predictor of poor prognosis in patients with heart failure and preserved ejection fraction. ADMA provides the strongest independent prediction of disease progression and adverse long-term outcomes.

Asymmetric Dimethylarginine in Response to Recombinant Tissue-Type Plasminogen Activator and Erythropoietin in Acute Stroke

In the German Multicenter Erythropoietin (EPO) Stroke Trial, patients not receiving thrombolysis most likely benefited from EPO on clinical recovery, whereas a combination of rtPA and EPO was associated with increased mortality. We investigated whether the combination of rtPA and EPO increased release of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA), and thereby potentially deteriorated ischemic stroke outcome, as suggested from experimental data. ADMA was determined in serum samples from 90 patients of the German Multicenter EPO Stroke Trial taken at days 1 (within 6 hours after symptom onset), 2, 3, 4, and 7 after stroke using high-performance liquid chromatography-tandem mass spectrometry. ADMA was analyzed for the different treatment groups (EPO, n=25; placebo, n=30; rtPA+placebo, n=18; EPO+rtPA, n=17). Clinical outcome was expressed as difference between National Institutes of Health Stroke Scale at baseline and 90 days. ADMA levels significantly increased during the observation time in EPO, EPO+rtPA, and placebo groups (P<0.05). A treatment effect on ADMA levels was revealed by repeated measures ANOVA only in the rtPA+placebo group (P=0.027). Here, ADMA levels were decreased compared with the placebo group (P<0.05). Both the EPO and the rtPA+placebo groups in the Hannover subgroup of the EPO trial had better outcome than the placebo group (P<0.05). Our data underscore the potential benefit of EPO in ischemic stroke. The hypothesis from experimental data, that EPO treatment increases ADMA in stroke patients, was disproved. Further studies are needed to clarify whether decreased ADMA might contribute to therapeutic rtPA effects.

Transsulfuration Pathway Thiols and Methylated Arginines: The Hunter Community Study

BackgroundSerum homocysteine, when studied singly, has been reported to be positively associated both with the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine [ADMA, via inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity] and with symmetric dimethylarginine (SDMA). We investigated combined associations between transsulfuration pathway thiols, including homocysteine, and serum ADMA and SDMA concentrations at population level.MethodsData on clinical and demographic characteristics, medication exposure, C-reactive protein, serum ADMA and SDMA (LC-MS/MS), and thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione, and cysteinylglycine; capillary electrophoresis) were collected from a sample of the Hunter Community Study on human ageing [n = 498, median age (IQR) = 64 (60–70) years].ResultsRegression analysis showed that: a) age (P = 0.001), gender (P = 0.03), lower estimated glomerular filtration rate (eGFR, P = 0.08), body mass index (P = 0.008), treatment with beta-blockers (P = 0.03), homocysteine (P = 0.02), and glutamylcysteine (P = 0.003) were independently associated with higher ADMA concentrations; and b) age (P = 0.001), absence of diabetes (P = 0.001), lower body mass index (P = 0.01), lower eGFR (P<0.001), cysteine (P = 0.007), and glutamylcysteine (P<0.001) were independently associated with higher SDMA concentrations. No significant associations were observed between methylated arginines and either glutathione or taurine concentrations.ConclusionsAfter adjusting for clinical, demographic, biochemical, and pharmacological confounders the combined assessment of transsulfuration pathway thiols shows that glutamylcysteine has the strongest and positive independent associations with ADMA and SDMA. Whether this reflects a direct effect of glutamylcysteine on DDAH activity (for ADMA) and/or cationic amino acid transport requires further investigations.

Effects of chronic SDMA infusion on glomerular filtration rate, blood pressure, myocardial function and renal histology in C57BL6/J mice

Symmetrical dimethylarginine (SDMA), the structural isomer of the nitric oxide synthase inhibitor asymmetrical dimethylarginine, has long been regarded as an inert substance. Recent epidemiological and preclinical data suggest that it might be involved in the pathophysiology of renal and cardiovascular diseases. Therefore, we aimed to investigate the effect of chronic SDMA infusion on renal and cardiac function in mice. Eight-week-old male C57Bl/6 mice received vehicle-controlled infusion of SDMA (250 µmol/kg/days) for 28 days using osmotic minipumps (n = 24/group). The following parameters were monitored: glomerular filtration rate (GFR; fluoresceinyl thiocarbamoyl-inulin excretion kinetic), cardiac function (echocardiography) and blood pressure (tail cuff). Blood samples for SDMA determination were obtained at baseline, 2 and 4 weeks. Mice were euthanized at 4 weeks to obtain tissue for renal histology. Chronic SDMA infusion led to a significant increase of SDMA levels from 0.26 ± 0.10 to 3.49 ± 1.66 µmol/L (P < 0.001) at 4 weeks. Despite this SDMA increase, the GFR did not change (1224 ± 351 versus 1017 ± 345 mL/min/g body weight, n.s.) at 4 weeks, when compared with baseline. We did not find any histological changes, particularly no effect on fibrosis or endothelias nitric oxide synthase expression. There was neither an effect of SDMA on systolic blood pressure (106 ± 12 versus 111 ± 18 mmHg, n.s.) nor on ejection fraction (54.2 ± 1.7 versus 58.4 ± 1.9%, n.s.). Based on our experiments, it seems unlikely that chronically elevated SDMA alone has an effect on renal and cardiac function in otherwise healthy mice. Future studies have to clarify the potential pathophysiological role of SDMA in cardiovascular disease.

Effect of chronic elevated asymmetric dimethylarginine (ADMA) levels on granulopoiesis

The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is elevated in both animal models of chronic inflammatory disorders as well as in patients with chronic inflammatory disease. In vivo data suggest that ADMA can increase the number of circulating monocytes and possibly affect their adhesion potential in vitro. The aim of our study was to evaluate possible effects of chronically elevated levels of ADMA on white blood cell count (WBC), leukocyte subsets, and WBC distribution pattern using a model of chronic exogenous ADMA infusion. Male Sprague-Dawley rats (n = 20, 10 weeks of age) were randomized to receive either (1) isotonic saline or (2) ADMA applied by osmotic mini pumps. After 28 days of infusion, all animals were sacrificed for blood and tissue sampling. WBC count, flow cytometry for subtype assessment, and histological assessment were performed. Over a time period of 28 days, continuous ADMA infusion significantly increased mean plasma levels (1.26 ± 0.07 μmol/l) as compared to saline infusion (0.57 ± 0.02 μmol/l). Clinical side effects were not observed. Despite a physiologically relevant rise in ADMA plasma levels, measured by decrease of the L-arginine/AMDA ratio-a surrogate parameter of NO production capacity-there was no effect on WBC count or pattern of leukocyte subsets. Numbers and morphology of peripheral blood cells as well as number of NK-cells leveling liver and spleen were not affected by chronic ADMA infusion. Chronically elevated ADMA levels in otherwise healthy rats did not affect WBC counts or leukocyte subsets. Furthermore, anemia frequently found in patients with progressive renal failure and elevated ADMA levels, was not observed. In a chronic inflammatory state, elevated ADMA levels themselves are rather the result than the cause of the underlying inflammatory process.

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

BackgroundThe 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 FindingsTherefore, 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/SignificanceOur 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.