Endogenous Nitric Oxide Synthase Inhibitor Research Articles

Enhanced Nitric Oxide (NO) and Decreased ADMA Synthesis in Pediatric ADHD and Selective Potentiation of NO Synthesis by Methylphenidate.

Attention deficit hyperactivity disorder (ADHD) is a common pediatric psychiatric disorder, frequently treated with methylphenidate (MPH). Recently, MPH’s cardiovascular safety has been questioned by observational studies describing an increased cardiovascular risk in adults and blood pressure alterations in children. We considered members of the L-arginine (Arg)/nitric oxide (NO) pathway as possible early cardiovascular risk factors in pediatric ADHD children. They include the NO metabolites, nitrite and nitrate, the NO precursor Arg, and asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor and a cardiovascular risk factor in adults. We conducted a prospective clinical trial with 42 ADHD children (aged 6–16 years) with (n = 19) and without (n = 23) MPH treatment. Age-matched children without ADHD (n = 43) served as controls. All plasma and urine metabolites were determined by gas chromatography-mass spectrometry. We observed higher plasma nitrite and lower plasma ADMA concentrations in the ADHD children. MPH-treated ADHD children had higher plasma nitrite concentrations than MPH-untreated ADHD children. As NOS activity is basally inhibited by ADMA, MPH treatment seems to have decreased the inhibitory potency of ADMA. Percentiles of systolic blood pressure were higher in MPH-treated ADHD children. The underlying mechanisms and their implications in the MPH therapy of pediatric ADHD with MPH remain to be elucidated in larger cohorts.

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

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

DDAH-2 alleviates contrast medium iopromide-induced acute kidney injury through nitric oxide synthase.

Contrast medium-induced acute kidney injury (CI-AKI) is one of the most common causes of hospital-acquired acute renal failure. However, the pathogenesis of CI-AKI remains unclear. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that is largely metabolised by dimethylarginine dimethylaminohydroxylase (DDAH) in humans. Two isoforms of DDAH exist, namely, DDAH-1 and DDAH-2. In the present study, we examined whether the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI. Exposure to the contrast medium iopromide led to increase in creatinine and blood urea nitrogen (BUN) levels, accumulation of ADMA, increase in reactive oxygen species (ROS) generation, and an inflammatory response in mice kidney tissue. The injection of adenovirus-harbouring DDAH-2 lowered renal ADMA levels and had a reno-protective effect against contrast-medium injury by decreasing cell apoptosis, ROS, and fibrosis. By contrast, contrast medium-induced renal injury was exacerbated in heterozygous DDAH-2 knockout mice. In the in vitro study, overexpression of DDAH-2 increased the levels of nitrite and intracellular cGMP, while the DDAH-2 knockdown induced the opposite effect. These findings were also observed in the in vivo sample. Our findings provide the first evidence that the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI and that the protective effect of DDAH-2 probably arises from the modulation of NOS activity, oxidative stress, and the inflammatory process.

Dimethylarginine Dimethylaminohydrolase 1 Polymorphisms and Venous Intimal Hyperplasia in Hemodialysis Patients

Background: After angioplasty, veins are more prone to intimal hyperplasia than arteries. Veins tend to produce less nitric oxide (NO), which could lead to endothelial dysfunction. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase and contributes to cardiovascular disease. In humans, dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme for ADMA degradation. In this study, we aim to determine whether venous intimal hyperplasia in hemodialysis (HD) vascular access is influenced by common polymorphisms in the DDAH1 genes. Methods: This is a prospective observational cohort study. A total of 473 HD patients referred for the angioplasty of vascular access were enrolled. There were 190 arteriovenous grafts (AVG) and 283 arteriovenous fistulas (AVF). The follow-up lasted for 2 years after the interventions. Seven single nucleotide polymorphisms (SNPs) in DDAH1 were genotyped and ADMA were measured at baseline. The primary outcome was restenosis after angioplasty. Results: Among the 7 SNPs, plasma ADMA levels were significantly different in DDAH1 rs233112 (GA + GG vs. AA, 0.86 ± 0.23 vs. 0.82 ± 0.19 μM, p = 0.03) and rs1498373 (CT + TT vs. CC, 0.87 ± 0.23 vs. 0.82 ± 0.20 μM, p = 0.02) genotypes. The AVF group with GG + GA genotype of rs233112 and CT + TT genotype of rs1498373 had higher risks of early restenosis at 3 months. In the AVG group, only GG + GA genotype of rs233112 was associated with early restenosis. A combined analysis of AVG and AVF groups showed that patients with rs233112 GA + GG genotype and rs1498373 CT + TT genotype had higher risks of early restenosis (both p < 0.001). The multivariate analysis results showed that the association of these genotypes with early restenosis is independent of clinical, access, or biochemical factors. Conclusions: Our findings suggest that certain DDAH1 polymorphisms modulate circulating ADMA levels and are associated with venous intimal hyperplasia.

Interleukin enhancement binding factor 3 inhibits cardiac hypertrophy by targeting asymmetric dimethylarginine-nitric oxide

Persistent cardiac hypertrophy eventually leads to deterioration of heart function and changes to normal morphology. Decreased nitric oxide (NO) production plays a critical role in modulating cardiac hypertrophy. Interleukin enhancement binding factor 3 (ILF3), a member of the double-stranded RNA-binding protein family, is known to regulate the transcription and stability of mRNA. Therefore, the major aim of the present study was to determine the role of ILF3 in reduction of NO production in cardiac hypertrophy. Cardiac hypertrophy models of neonatal rat cardiomyocytes (NRCMs) and adult rats were induced by angiotensin II (Ang II) in this study. First, it was found that ILF3 expression, NO production, and nitric oxide synthase (NOS) activity was decreased in cultured cardiomyocytes and adult rats treated with Ang II, compared with NRCMs treated with vehicle and rats treated with saline infusion, respectively. These effects induced by Ang II were significantly exacerbated by specific ILF3 knockdown. Moreover, the level of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS, was increased significantly in the Ang II-induced hypertrophic NRCMs and adult rats. Additionally, decreased protein expression and mRNA level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) were observed. Furthermore, specific ILF3 knockdown further aggravated these effects, but didn't reduce the expression level of NOS isoforms. In conclusion, our data show that ADMA accumulation-mediated decrease in NO production plays an important role in cardiomyocyte remodeling, which may be associated with ILF3-mediated DDAH1 reduction.

The detrimental effect of asymmetric dimethylarginine on cholesterol efflux of macrophage foam cells: Role of the NOX/ROS signaling

BackgroundAsymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor and has been proposed to be an independent risk factor for cardiovascular diseases. However, little is known about its role in the regulation of lipid metabolism. In this study, we investigated the effect of ADMA on cholesterol metabolism and its underlying molecular mechanism. MethodsOxidized low-density lipoprotein (oxLDL)-induced macrophage foam cells were used as an in vitro model. Apolipoprotein E-deficient (apoE−/−) hyperlipidemic mice were used as an in vivo model. Western blot analysis was used to evaluate protein expression. Luciferase reporter assays were used to assess the activity of promoters and transcription factors. Conventional assay kits were used to measure the levels of ADMA, cholesterol, triglycerides, and cytokines. ResultsTreatment with oxLDL decreased the protein expression of dimethylarginine dimethylaminohydrolase-2 (DDAH-2) but not DDAH-1. Incubation with ADMA markedly increased oxLDL-induced lipid accumulation in macrophages. ADMA impaired cholesterol efflux following oxLDL challenge and downregulated the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 by interfering with liver X receptor α (LXRα) expression and activity. Additionally, this inhibitory effect of ADMA on cholesterol metabolism was mediated through the activation of the NADPH oxidase/reactive oxygen species pathway. In vivo experiments revealed that chronic administration of ADMA for 4 weeks exacerbated systemic inflammation, decreased the aortic protein levels of ABCA1 and ABCG1, and impaired the capacity of reverse cholesterol transport, ultimately, leading to the progression of atherosclerosis in apoE−/− mice. ConclusionOur findings suggest that the ADMA/DDAH-2 axis plays a crucial role in regulating cholesterol metabolism in macrophage foam cells and atherosclerotic progression.

Asymmetric Dimethylarginine Exacerbates Lipid Accumulation in Macrophage Foam Cells by Impairing LXRα/ABCA1-Dependent Cholesterol Efflux

Background: Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor and has been proposed to be an independent risk factor for cardiovascular diseases. However, little is known about its role in the regulation of lipid metabolism. In this study, we investigated the effect of ADMA on cholesterol metabolism and its underlying molecular mechanism. Methods: Oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cells were used as an in vitro model. Apolipoprotein E-deficient (ApoE−/−) hyperlipidemic mice were used as an in vivo model. Western blot analysis was used to evaluate protein expression. Luciferase reporter assays were used to assess the activity of promoters and transcription factors. Conventional assay kits were used to measure the levels of ADMA, cholesterol, triglycerides, and cytokines. Findings: Treatment with oxLDL decreased the protein expression of dimethylarginine dimethylaminotransferase-2 (DDAH-2) but not DDAH-1. Incubation with ADMA markedly increased oxLDL-induced lipid accumulation in macrophages. ADMA impaired cholesterol efflux following oxLDL challenge and downregulated the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 by interfering with liver X receptor α (LXRα) expression and activity. Additionally, this inhibitory effect of ADMA on cholesterol metabolism was mediated through the activation of the NADPH oxidase/reactive oxygen species pathway. In vivo experiments revealed that chronic administration of ADMA for 4 weeks exacerbated systemic inflammation, decreased the aortic protein levels of ABCA1 and ABCG1, and impaired the capacity of reverse cholesterol transport, ultimately, leading to the progression of atherosclerosis in apoE−/− mice. Interpretation: Our findings suggest that the ADMA/DDAH-2 axis plays a crucial role in regulating cholesterol metabolism in macrophage foam cells and atherosclerotic progression. Funding Statement: This study was supported by grants from the Ministry of Science and Technology of Taiwan (105-2320-B-010-036, 105-2811-B-010-022, 106-2320-B-002-057-MY3, 106-2320-B-002-056, and 106-2811-B-002-146). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University (No. 990803).

Association between asymmetric dimethylarginine and in-stent restenosis tissue characteristics assessed by optical coherence tomography

BackgroundImpaired bioavailability of endothelium-derived nitric oxide (NO) and endothelial dysfunction may play a pivotal role in the pathogenesis of in-stent restenosis (ISR) after coronary stenting. We aimed to investigate the relation between asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor, and the ISR lesions tissue characteristics assessed by optical coherence tomography (OCT). Methods and resultsForty-five patients with symptomatic ISR lesions (17 bare metal stents, 28 drug-eluting stents, medium implantation duration: 58.0 months) were evaluated by OCT for in-stent tissue characteristics and calcification. We defined neoatherosclerosis as the presence of lipid or calcified neointima in ISR lesions, and 12 (26.7%), 33 (73.3%) ISR lesions were classified as with homogenous neointima and neoatherosclerosis respectively. The patients with neoatherosclerosis have significantly higher plasma ADMA levels compared to those of patients with homogenous neointima (1.12 ± 0.21 μmol/l versus 0.83 ± 0.08 μmol/l, p < 0.001). Furthermore, the plasma ADMA level of ISR lesions with intra-stent calcification (n = 24, 53.3%) was also significantly higher than those of ISR lesions without (n = 21, 46.7%; p < 0.001). There was a highly significant association between plasma ADMA level and intra-stent relative calcium index (mean calcium arc × calcium length)/(360 × analyzed length) (p < 0.001, r = 0.702). In multivariate analyses adjusted for age, sex, diabetes, eGFR, plasma ADMA level remained the only significant predictor for the presence of neoatherosclerosis (p = 0.008) and intra-stent calcification (p < 0.001). In contrast, plasma ADMA level correlated with intra-stent relative lipid core index (mean lipid core arc × lipid core length)/(360 × analyzed length) only in subgroup without intra-stent calcification (p = 0.004, r = 0.596, multivariate-adjusted p = 0.022). ConclusionsIncreased plasma ADMA levels were associated with the development of in-stent neoatherosclerosis and calcification.

DDAH-2 Alleviates Contrast Medium Iopromide-Induced Acute Kidney Injury Through Nitric Oxide Synthase

Background: Contrast-induced acute kidney injury (CI-AKI) is one of the most common causes of hospital-acquired acute renal failure. However, the pathogenesis of CI-AKI remains unclear. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that is largely metabolized by dimethylarginine dimethylaminohydroxylase (DDAH) in humans. Two isoforms of DDAH exist, namely, DDAH-1 and DDAH-2. In this study, we examined whether the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI. Methods and Findings: Exposure to the contrast medium iopromide led to increases of creatinine and blood urea nitrogen levels, accumulation of ADMA, increase of reactive oxygen species (ROS) generation, and an inflammatory response in mice kidney tissue. The injection of adenovirus-harbouring DDAH-2 lowered renal ADMA levels and had a reno-protective effect against contrast-medium injury by decreasing cell apoptosis and fibrosis. By contrast, contrast-medium-induced renal injury was exacerbated in heterozygous DDAH-2 knockout mice. In the in vitro study, overexpression of DDAH-2 increased the levels of nitrite and intracellular cGMP, while the DDAH-2 knockdown induced the opposite effect. These findings were also observed in the in vivo sample. Interpretation: Our findings provide the first evidence that the DDAH-2/ADMA/NOS pathway might be involved in the pathogenesis of CI-AKI and that the protective effect of DDAH-2 probably is due to the modulation of NOS activity, oxidative stress, and the inflammatory process. Funding Statement: This work was supported by grants from the Ministry of Science and Technology of Taiwan [MOST-103-2314-B-075-046, 106-2314-B-075-044, 107-2314-B-075-060] and Veterans General Hospitals and University System of Taiwan Joint Research Program [VGHUST102-G7-5, 104-G7-5; 105-G7-3]. Declaration of Interests: The authors state: None. Ethics Approval Statement: All animal experiments were performed in accordance with the approved guidelines by the Institutional Animal Care and Use Committee (IACUC) of Taipei Veterans General Hospital (#2016-124).

Plasma ADMA, urinary ADMA excretion, and late mortality in renal transplant recipients

Cardiovascular disease (CVD) is the leading cause of death in renal transplant recipients (RTR). Elevated plasma asymmetric dimethylarginine (pADMA), an endogenous nitric oxide synthase inhibitor produced from the turnover of methylated arginine moieties in proteins, is a risk factor for CVD and mortality. It is unknown how urinary ADMA excretion (uADMA), one of the main ADMA elimination routes, is associated with long-term survival. Furthermore, the association of pADMA and uADMA with markers for turnover of arginine-methylated proteins is unknown. We analyzed ADMA using a validated GC–MS/MS method in plasma and 24-h urine samples of 685 RTR, included ≥ 1 year after transplantation. We also analyzed urine symmetric dimethylarginine (uSDMA) using the same method. Urinary creatinine and urea excretions were used as markers for turnover of muscle protein and amino acids, respectively. We applied Cox regression analyses to study associations of pADMA, uADMA, and uSDMA with all-cause and CVD mortality. Mean pADMA was 0.61 ± 0.12 µM, uADMA was 31 ± 13 µmol/24 h, and uSDMA was 52 ± 19 µmol/24 h. Over median follow-up of 5.4 [4.9–6.1] years, 147 RTR died, of which 58 (39%) from CVD. High pADMA was associated with high all-cause mortality (HR per SD [95% CI]: 1.45 [1.26–1.67], P < 0.001), while high uADMA was associated with low all-cause and CVD mortality (HR per SD [95% CI]: 0.57 [0.47–0.69], P < 0.001, and 0.55 [0.40–0.74], P < 0.001, respectively). The associations were independent of adjustment for potential confounders. Creatinine excretion was associated with both pADMA (st. β:− 0.21, P = 0.003) and uADMA (st. β: 0.49, P < 0.001), and urea excretion was associated with uADMA (st. β: 0.56, P < 0.001). Associations of uSDMA with outcomes and with creatinine excretion and urea excretion were comparable to those of uADMA. The associations of pADMA, uADMA and uSDMA with mortality were strongly affected by adjustment for creatinine excretion and urea excretion. We found for the first time that high uADMA and high uSDMA are associated with less risk of all-cause and CVD mortality. The links of uADMA and uSDMA with markers of muscle protein and amino acid turnover may serve to further understand ADMA and SDMA homeostasis and their clinical implications.

Functional sympatholysis is impaired in end-stage renal disease.

Patients with end-stage renal disease (ESRD) have decreased exercise capacity and exercise intolerance that contribute to cardiovascular risk. One potential mechanism underlying exercise intolerance in ESRD is impaired ability to oppose sympathetically mediated vasoconstriction within exercising skeletal muscle (i.e., functional sympatholysis, FS). We hypothesized that ESRD patients have impaired FS compared with healthy (CON) and hypertensive (HTN) controls and that impaired FS is related to circulating levels of the uremic toxin asymmetric dimethyl arginine (ADMA), an endogenous nitric oxide synthase inhibitor. Near-infrared spectroscopy-derived oxygen tissue saturation index (TSI) of the forearm muscle was measured continuously in 33 participants (9 CON, 14 HTN, 10 ESRD) at rest and during low-dose (-20 mmHg) lower body negative pressure (LBNP), moderate rhythmic handgrip exercise, and LBNP with concomitant handgrip exercise (LBNP+handgrip). Resting muscle TSI was lower in ESRD than in CON and HTN groups (CON = 67.8 ± 1.9%, HTN = 67.2 ± 1.1%, ESRD = 62.7 ± 1.5%, P = 0.03). Whereas CON and HTN groups had an attenuation in sympathetically mediated reduction in TSI during LBNP + handgrip compared with LBNP alone (P ≤ 0.05), this response was not present in ESRD (P = 0.71), suggesting impaired FS. There was no difference in plasma [ADMA] between groups (CON = 0.47 ± 0.05 µmol/l, HTN = 0.42 ± 0.06 µmol/l, ESRD = 0.63 ± 0.14 µmol/l, P = 0.106) and no correlation between plasma [ADMA] and resting muscle TSI (P = 0.84) or FS (P = 0.75). Collectively, these findings suggest that ESRD patients have lower muscle perfusion at rest and impaired FS but that these derangements are not related to circulating [ADMA].

Effect of renal function on homeostasis of asymmetric dimethylarginine (ADMA): studies in donors and recipients of renal transplants.

Asymmetric dimethylarginine (ADMA) is a methylated form of arginine and an endogenous nitric oxide synthase inhibitor. Renal function decline is associated with increase of plasma ADMA in chronic kidney disease populations. It is yet unknown how isolated renal function impairment affects ADMA homeostasis in healthy humans. Here, we measured plasma concentrations and urinary excretion of ADMA using GC-MS/MS in 130 living kidney donors before and at 1.6 (1.6-1.9) months after donation. We additionally analyzed 201 stable renal transplant recipients (RTR) that were included > 1year after transplantation, as a model for kidney disease in the context of single kidney state. We measured true glomerular filtration rate (mGFR) using 125I-iothalamate. To study enzymatic metabolism of ADMA, we also measured L-citrulline as primary metabolite. Mean age was 52 ± 10years in donors and 54 ± 12years in RTR. Renal function was significantly reduced from pre- to post-donation (mGFR: 104 ± 17 vs. 66 ± 10ml/min per 1.73m2 BSA, - 36 ± 7%, P < 0.001). Urinary ADMA excretion strongly and significantly decreased from pre- to post-donation (60.6 ± 16.0 vs. 40.5 ± 11.5µmol/24h, - 31.5 ± 21.5%, P < 0.001), while plasma ADMA increased only slightly (0.53 ± 0.08 vs. 0.58 ± 0.09µM, 11.1 ± 20.1%, P < 0.001). Compared to donors post-donation, RTR had significantly worse renal function (mGFR: 49 ± 18ml/min/1.73m2, - 25 ± 2%, P < 0.001) and lower urinary ADMA excretion (30.9 ± 12.4µmol/24h, - 23.9 ± 3.4%, P < 0.001). Plasma ADMA in RTR (0.60 ± 0.11µM) did not significantly differ from donors post-donation (2.9 ± 1.9%, P = 0.13). Plasma citrulline was inversely associated with mGFR (st. β: - 0.23, P < 0.001), consistent with increased ADMA metabolism to citrulline with lower GFR. In both groups, the response of urinary ADMA excretion to renal function loss was much larger than that of plasma ADMA. As citrulline was associated with GFR, our data indicate that with renal function impairment, a decrease in urinary ADMA excretion does not lead to a corresponding increase in plasma ADMA, likely due to enhanced metabolism, thus allowing for lower renal excretion of ADMA.

Small molecule inhibition of DDAH1 significantly attenuates triple negative breast cancer cell vasculogenic mimicry in vitro

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a key enzyme involved in the metabolism of the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA). Increased DDAH1 expression and subsequent increased NO production have been recently linked to cancer. Specifically, DDAH1 is implicated in establishment of a vascular network by tumour cells, vasculogenic mimicry (VM), which is strongly associated with tumour progression and poor patient prognosis. The use of DDAH1 inhibitors as potential therapeutic agents thus represents a growing field of interest. Here we describe a UPLC-MS assay to quantify stability and intracellular concentration of two small molecule DDAH1 inhibitors synthesised by our group, ZST316 and ZST152, following incubation with MDA-MB-231 breast cancer cells. In an in vitro assay of VM, both DDAH1 inhibitors significantly attenuated formation of capillary-like tube structures in a dose-dependent fashion. This was not due to cell toxicity or altered cell proliferation, but may be due in part to inhibition of cell migration. Mechanistically, we demonstrate significant modulation of the endogenous DDAH/ADMA/NO pathway following exposure of 100 μM ZST316 or ZST152: a 40% increase in the DDAH1 substrate ADMA, and a 38% decrease in the DDAH1 product l-citrulline. This study represents the first evidence for therapeutic inhibition of DDAH1 by small molecules in breast cancer.

Cerebrovascular reactivity and cerebral perfusion of rats with acute liver failure: role of L-glutamine and asymmetric dimethylarginine in L-arginine-induced response.

Cerebral blood flow (CBF) is impaired in acute liver failure (ALF), however, the complexity of the underlying mechanisms has often led to inconclusive interpretations. Regulation of CBF depends at least partially on variations in the local brain L-arginine concentration and/or its metabolic rate. In ALF, other factors, like an increased concentration of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor and elevated level of L-glutamine, may contribute to CBF alteration. This study demonstrated strong differences in the reactivity of the middle cerebral arteries and their response to extravascular L-arginine application between vessels isolated from rats with thioacetamide (TAA)-induced ALF and control animals. Our results also showed the decrease in the cerebral perfusion in TAA rats measured by arterial spin labeling perfusion magnetic resonance. Subsequently, we aimed to investigate the importance of balance between the concentration of ADMA and L-arginine in the CBF regulation. In vivo, intraperitoneal L-arginine administration in TAA rats corrected: (i) decrease in cerebral perfusion, (ii) decrease in brain extracellular L-arginine/ADMA ratio and (iii) increase in brain L-glutamine concentration. Our study implicates that impaired vascular tone of cerebral arteries is most likely associated with exposure to high ADMA and L-glutamine levels resulting in limited availability of L-arginine and might be responsible for reduced cerebral perfusion observed in ALF.

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;

026 Testosterone deficiency causes endothelial dysfunction via elevation of asymmetric dimethylarginine (ADMA) and oxidative stress in castrated rats

Testosterone is thought to mediate the erectile function by producing an adequate nitric oxide (NO), and therefore testosterone deficiency results in erectile dysfunction via reduced NO bioavailability. However, the mechanisms underlying endothelial dysfunction in testosterone deficiency remain unclear. We investigated the mechanism of endothelial dysfunction in castrated rats by focusing on the relationship between testosterone deficiency and asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. Male rats were categorised into the following groups: castrated (Cast), castrated with testosterone (3 mg/kg/day) (Cast+T), and sham (Sham). Erectile function using ICP measurements, endothelial function using isometric tension, ADMA levels using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) and oxidative stress (NADPH oxidase and p22phox), and inflammatory biomarker expression (IL-6, NF-κB) and ADMA generated (DDAH) were assessed 4 weeks postoperatively. Oxidative stress analysis was also performed by quantifying the 8-OHdG levels of DNA. The ICP/MAP ratios were 0.61 ± 0.03 in Sham, 0.26 ± 0.04 in Cast (p < 0.01 vs. Sham), and 0.56 ± 0.06 in Cast+T (p < 0.01 vs. Cast). Relaxation responses induced by acetylcholine were decreased in Cast compared to Sham and Cast+T (p < 0.05). Cast rats also had significantly lower serum NO metabolite (NOx) levels than Sham counterparts (Sham, 4.20 ± 0.54 μmol/L; Cast, 2.87 ± 0.28 μmol/L; p < 0.01). However, the Cast+T and Sham rats had similar serum NOx levels (Cast+T, 4.39 ± 0.77 μmol/L; p > 0.05). Serum ADMA levels were significantly higher in Cast (297.3 ± 22.9 ng/mL) than in Sham (200.8 ± 9.4 ng/mL) or Cast+T (198.1 ± 27.7 ng/mL; both p < 0.05). Oxidative stress and inflammatory marker messenger RNA expression were significantly higher in Cast than in Sham and Cast+T (p < 0.05). Expression of the ADMA generated biomarker was significantly higher in Cast than in Sham or Cast+T (both, p < 0.01). The 8-OHdG levels were significantly higher in Cast (18.7 ± 0.9 ng/DNA 1 ng) than in Sham (13.9 ± 1.4 ng/DNA 1 ng) or Cast+T (13.3 ± 1.7 ng/DNA; both p < 0.05).

Systemic concentrations of asymmetric dimethylarginine (ADMA) in chronic obstructive pulmonary disease (COPD): state of the art.

Experimental evidence suggests that oxidative stress (OS) may increase the activity of arginine methylating enzymes that produce the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). In addition, it is well documented that OS can significantly decrease the synthesis and/or activity of ADMA degrading enzymes, thus causing ADMA accumulation in biological fluids. Recent reports have focused on circulating methylated arginine concentrations in chronic obstructive pulmonary disease, a disease characterized by a significant increase in OS. This review discusses the results of these studies and the opportunities for further research in this area.

Ascorbic acid: a promising agent in chronic kidney disease?

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is related with impaired endothelial function and poor outcomes in chronic kidney disease (CKD) patients. In addition, oxidative stress and inflammation are also a common problem in CKD patients, and both have an important role in the development and progression of atherosclerosis and increased cardiovascular events in CKD. Ascorbic acid might be a promising agent in CKD for improving outcome by decreasing oxidative stress and ADMA levels.

Fourteen-Day Study of Praliciguat, a Soluble Guanylate Cyclase Stimulator, in Patients with Diabetes and Hypertension

Background: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, has been shown to augment nitric oxide (NO) signaling and reduce fasting plasma glucose and proteinuria in an animal model of diabetic nephropathy. In healthy adults, repeated oral doses of 15-40 mg were tolerated and lowered blood pressure (BP). Methods: This Phase 2a, double-blind, placebo (PBO)-controlled study assessed the effects of oral praliciguat in 26 patients with type 2 diabetes mellitus (T2DM) and hypertension on stable glycemic and BP-lowering therapy. Most (69%) were also on statins. Two praliciguat regimens were tested: (1) 40 mg QD for days 1-14 (N=10), and (2) 20 mg BID for days 1-7 then 40 mg QD for days 8-14 (N=10). Assessments included serum chemistry, fasting plasma glucose, BP and heart rate, platelet function, reactive hyperemia index (RHI), plasma biomarkers, and adverse events (AEs). Results: Results were similar for both praliciguat regimens and are here combined. After 14 days of treatment, praliciguat-treated patients had notable differences in least squares mean change from baseline (95% CI) relative to PBO in 24-hour mean arterial pressure [-4.7 mmHg (-10, 0.8)], heart rate [2.9 bpm (-0.7, 6.5)], fasting glucose [-13 mg/dL (-32, 7)], HOMA-IR [-26 (-58, 7)], total serum cholesterol [-26 mg/dL (-44, -7)], and LDL cholesterol [-20 mg/dL (-37, -3)]. Praliciguat had no effect on platelet function or RHI. Among the exploratory biomarkers tested, levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were lower in praliciguat vs. PBO recipients. Both praliciguat dose regimens were generally well tolerated. Except for one serious AE of upper gastrointestinal hemorrhage, AEs in praliciguat-treated patients were mild. Conclusion: In this initial trial, praliciguat had beneficial effects on BP and metabolic parameters in T2DM patients with hypertension on standard therapies. Further clinical investigation of praliciguat in T2DM patients is warranted. Disclosure J.P. Hanrahan: Employee; Self; Ironwood. J.D. Wakefield: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. P.J. Wilson: None. P. Miller: None. J. Chickering: None. L. Morrow: Stock/Shareholder; Self; ProSciento. Employee; Spouse/Partner; Eli Lilly and Company. M.L. Hall: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. A.T. Profy: Other Relationship; Self; Ironwood Pharmaceuticals.

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.