Endogenous Nitric Oxide Synthase Research Articles

Evaluation of Oxidative Stress and Endothelial Dysfunction in COVID-19 Patients.

Background and Objectives: Heat shock proteins (HSPs) are stress proteins. The endogenous nitric oxide (NO) synthase inhibitor asymmetric dimethyl arginine (ADMA) is a mediator of endothelial dysfunction. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes endothelial dysfunction and coagulopathy through severe inflammation and oxidative stress. Using these markers, we analyzed the prognostic value of serum ADMA and HSP-90 levels for early prediction of severe coronavirus disease (COVID-19) patients. Materials and Methods: A total of 76 COVID-19 patients and 35 healthy control subjects were included in this case-control study. COVID-19 patients were divided into two groups: mild and severe. Results: Serum ADMA and HSP-90 levels were significantly higher in the COVID-19 patients compared to the control subjects (p < 0.001). Additionally, serum ADMA and HSP-90 levels were determined to be higher in a statistically significant way in severe COVID-19 compared to mild COVID-19 (p < 0.001). Univariable logistic regression analysis revealed that ADMA and HSP-90, respectively, were independent predictors of severe disease in COVID-19 patients (ADMA (OR = 1.099, 95% CI = 1.048-1.152, p < 0.001) and HSP-90 (OR = 5.296, 95% CI = 1.719-16.316, p = 0.004)). When the cut-off value for ADMA was determined as 208.94 for the prediction of the severity of COVID-19 patients, the sensitivity was 72.9% and the specificity was 100% (AUC = 0.938, 95%CI = 0.858-0.981, p < 0.001). When the cut-off value for HSP-90 was determined as 12.68 for the prediction of the severity of COVID-19 patients, the sensitivity was 88.1% and the specificity was 100% (AUC = 0.975, 95% CI= 0.910-0.997, p < 0.001). Conclusions: Increased levels of Heat shock proteins-90 (HSP-90) and ADMA were positively correlated with increased endothelial damage in COVID-19 patients, suggesting that treatments focused on preventing and improving endothelial dysfunction could significantly improve the outcomes and reduce the mortality rate of COVID-19. ADMA and HSP-90 might be simple, useful, and prognostic biomarkers that can be utilized to predict patients who are at high risk of severe disease due to COVID-19.

Oxygen-Evolving Radiotherapy-Radiodynamic Therapy Synergized with NO Gas Therapy by Cerium-Based Rare-Earth Metal-Porphyrin Framework.

The efficacy of traditional radiotherapy (RT) has been severely limited by its significant side effects, as well as tumor hypoxia. Here, the nanoscale cerium (Ce)-based metaloxo clusters (Ce(IV)6)-porphyrin (meso-tetra (4-carboxyphenyl) porphyrin, TCPP) framework loaded with L-arginine (LA) (denoted as LA@Ce(IV)6-TCPP) is developed to serve as a multifarious radio enhancer to heighten X-ray absorption and energy transfer accompanied by O2/NO generation for hypoxia-improved RT-radiodynamic therapy (RDT) and gas therapy. Within tumor cells, LA@Ce(IV)6-TCPP will first react with endogenous H2O2 and inducible NO synthase (iNOS) to produce O2 and NO to respectively increase the oxygen supply and reduce oxygen consumption, thus alleviating tumor hypoxia. Then upon X-ray irradiation, LA@Ce(IV)6-TCPP can significantly enhance hydroxyl radical (•OH) generation from Ce(IV)6 metaloxo clusters for RT and synchronously facilitate singlet oxygen (1O2) generation from adjacently-coordinated TCPP for RDT. Moreover, both the •OH and 1O2 can further react with NO to generate more toxic peroxynitrite anions (ONOO-) to inhibit tumor growth for gas therapy. Benefitting from the alleviation of tumor hypoxia and intensified RT-RDT synergized with gas therapy, LA@Ce(IV)6-TCPP elicited superior anticancer outcomes. This work provides an effective RT strategy by using low doses of X-rays to intensify tumor suppression yet reduce systemic toxicity.

Comparing Nitric Oxide Generation Pathways using Dietary L-Arginine and Dietary Nitrate: A 15N tracer study in Wistar Rats

Mammals generate nitric oxide (NO) through two main pathways. In the direct production pathway, endogenous nitric oxide synthase (NOS) enzymes catalyze the reaction of L-arginine with oxygen, producing nitric oxide and citrulline. When oxygen accessibility is low, the reductive pathway supplies nitric oxide through the nitrate-nitrite-NO reduction cascade. Additional research is needed to understand whether the two NO generation pathways interact and what effects the two pathways have on the body and each other. We investigated how dietary supplementation of 15N-L-arginine (0.5 g/kg), 15N-nitrate (0.013 g/kg), or a combination of L-arginine and nitrate influences nitrate concentrations in plasma, skeletal muscle, and liver tissue from Wistar rats. We administered treatments via oral gavage. Two hours after treatment administration, we euthanized the rats and collected tissue samples. The nitrate concentrations were measured using chemiluminescence, and future mass spectrometry analysis for 15N/14N ratios will be performed. The calculated concentrations were compared between treatment groups using single factor analysis of variance. Firstly, we observed that nitrate administration significantly increased total nitrate levels in plasma, liver, and muscle tissues compared to the control group. Interestingly, L-arginine alone did not result in a significant rise in total nitrate concentrations. Notably, liver tissue from the L-arginine-treated group had slightly lower concentrations of nitrate than the control group. The combination of L-arginine and nitrate showed no statistically significant difference when compared with the nitrate-treated group in all three tissues. These results suggest that a bolus of L-arginine does not have a substantial short-term effect (within 2 hours after ingestion) on total nitrate concentrations in rat tissues. Future mass spectrometry analysis will indicate how the tissues handle acute ingestion of two different NO sources: L-arginine and nitrate. Further research must be performed for a more detailed understanding of the kinetics of arginine and nitrate, as well as for the effects of these dietary supplements on improving NO homeostasis. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

RBC eNOS Transduces Regional O2 Gradients to Orchestrate RBC O2 Delivery Phenotype during Circulatory Transit

BACKGROUND: The role of endogenous endothelial NO synthase (eNOS) in mature RBCs is poorly understood. We have previously demonstrated functionally O2-dependent eNOS migration in RBCs, mediating (via s-nitrosylation) glycolytic metabolon assembly/disassembly during circulation in support of antioxidant systems during stress. We have further explored eNOS migration and now propose the following mechanism along with its functional significance. We (and others) suggest that hemoglobin (Hb) conformational change resulting from deoxygenation leads to its docking at the cytoplasmic domain of Band 3 (cdB3). We propose that (1) cdB3 consequently undergoes a conformational change that is transduced by Piezo1 (a mechanosensitive ion channel located in the RBC membrane); (2) Piezo1 activation results in calcium entry into the RBC, which (3) aids in the disassembly of the RBC cytoskeleton, (4) facilitates eNOS migration to the membrane (where calmodulin is mainly localized - thereby bringing these two important proteins together), and (5) binds to calmodulin, activating eNOS to generate nitric oxide (NO) - which then influences RBC phenotype that facilitates O 2 delivery, specifically related to RBC deformability and regulation of Hb~O 2 release, via enhanced Bohr effect. METHODS: Heparinized human, eNOS (-/-), or wt murine (C57BL/6J) red blood cells (RBCs) were imaged (Nikon) for the analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the Piezo1 agonist Yoda1, or a membrane tension buffer grammostola mechanotoxin #4 (GSMTx4; decoupling Piezo1), or carbon monoxide (CO) to maintain R-state Hb conformation upon RBC deoxygenation. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, protein 4.1, or eNOS), then with the secondary antibody prior to visualization. Interaction between eNOS and cytoskeleton proteins, as well as calmodulin, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader, and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. To assess the functional significance of this mechanism, Hb-O 2 affinity was determined from murine blood (eNOS (-/-) or wt). Oxygen dissociation curves (ODCs), were constructed at 2 fixed CO 2 levels (thin film rotating tonometer; in conjunction with a gas blender; with samples measured by arterial blood gas machine).Bohr effect was assessed by determining the area between the ODCs (ABC) at the 2 fixed CO2 levels (~40mmHg and ~70mmHg). RESULTS: We observed several significant findings related to red RBC deoxygenation. Upon RBC deoxygenation, there is an increase in calcium levels indicated by the Fluo3 signal, which is Piezo1 dependent. Cytoskeleton proteins, such as Actin, Spectrin, and protein 4.1, undergo disassembly upon RBC deoxygenation. This disassembly process is contingent on both Piezo1-mediated calcium flux and the conformational changes in hemoglobin (Hb) (and not pO 2). We found that eNOS is associated with 4.1 throughout the oxy/deoxy cycle and that eNOS exhibits the same migratory pattern as cytoskeleton proteins to the RBC membrane, where calmodulin is located, irrespective of the RBC's oxygenation state. Additionally, when eNOS localizes in the membrane, it exhibits interaction with calmodulin. The generation of nitric oxide (NO) by eNOS, as indicated by the DAF-FM signal, increases in a manner dependent on both calcium, Piezo1 and Hb conformational transition. The functional consequence of this mechanism appears to be related to O 2 delivery phenotype during circulatory transit, with cell deformability being affected in addition to an inhibition of the Bohr effect in eNOS (-/-) compared to wt mice (2006 ± 61 vs 1892 ± 69 ABC in WT (n = 3) vs eNOS (-/-) (n = 4), respectively; p = 0.0747). SUMMARY: Upon circulatory transit, RBC deoxygenation triggers the activation of eNOS. This activated eNOS is associated with Piezo1-mediated calcium flux, which is facilitated by calmodulin. These events are coordinated with the migration of eNOS within the red blood cell between the cytosol and the cell membrane. The migration of eNOS is influenced by cytoskeleton proteins.

Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Metabolic model for laboratory control of anti-ischaemic therapy effectiveness: a case study of nicorandil

Scientific relevance. A key anti-ischaemic mechanism of some medicinal products involves their effects on the metabolism of endothelial vasodilators, particularly the synthesis of nitric oxide from arginine and its precursor citrulline.Aim. The study was aimed to determine whether the plasma time course of guanidine derivatives (arginine precursors) is applicable to laboratory control of anti-ischaemic therapy effectiveness using a single oral dose of nicorandil in patients with coronary heart disease as a case study.Materials and methods. The authors used high-performance liquid chromatography to determine metabolites. Blood samples for analysis were obtained from 30 patients with angina pectoris (Grade II–III, Canadian Cardiovascular Society) and 30 healthy donors. All the study participants received a single oral dose of 20 mg nicorandil after 10 h of fasting.Results. At baseline, patients showed significantly higher plasma citrulline levels than donors. However, the elevated levels decreased to the healthy range after nicorandil administration. Plasma arginine levels in patients showed a statistically significant increase following nicorandil administration. Plasma homoarginine levels in patients remained reduced both before and after dosing. Nicorandil did not influence elevated levels of the endogenous nitric oxide synthase inhibitor (asymmetrical dimethylarginine).Conclusions. In addition to the established mechanisms responsible for altering cell metabolism, nicorandil enhances the contribution of citrulline to arginine resynthesis. It is reasonable to include citrulline and arginine, which are involved in the vasodilator response, in model schemes for laboratory control of the effectiveness of anti-ischaemic therapy.

Dimethylarginine Dimethylaminohydrolase - 1 expression is increased under tBHP-induced oxidative stress regulates nitric oxide production in PCa cells attenuates mitochondrial ROS-mediated apoptosis

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression is frequently elevated in different cancers including prostate cancer (PCa) and enhances nitric oxide (NO) production in tumor cells by metabolising endogenous nitric oxide synthase (NOS) inhibitors. DDAH1 protects the PCa cells from cell death and promotes survival. In this study, we have investigated the cytoprotective role of DDAH1 and determined the mechanism of DDAH1 in protecting the cells in tumor microenvironment. Proteomic analysis of PCa cells with stable overexpression of DDAH1 has identified that oxidative stress-related activity is altered. Oxidative stress promotes cancer cell proliferation, survival and causes chemoresistance. A known inducer of oxidative stress, tert-Butyl Hydroperoxide (tBHP) treatment to PCa cells led to elevated DDAH1 level that is actively involved in protecting the PCa cells from oxidative stress induced cell damage. In PC3-DDAH1— cells, tBHP treatment led to higher mROS levels indicating that the loss of DDAH1 increases the oxidative stress and eventually leads to cell death. Under oxidative stress, nuclear Nrf2 controlled by SIRT1 positively regulates DDAH1 expression in PC3 cells. In PC3-DDAH1+ cells, tBHP induced DNA damage is well tolerated compared to wild-type cells while PC3-DDAH1— became sensitive to tBHP. In PC3 cells, tBHPexposure has increased the production of NO and GSH which may be acting as an antioxidant defence to overcome oxidative stress. Furthermore, in tBHP treated PCa cells, DDAH1 is controlling the expression of Bcl2, active PARP and caspase 3. Taken together, these results confirm that DDAH1 is involved in the antioxidant defence system and promotes cell survival.

Decreased peripheral vascular function in patients with stage 3 and 4 chronic kidney disease who have elevated symmetric dimethylarginine but not asymmetric dimethylarginine

Cardiovascular disease is a leading cause of death in chronic kidney disease (CKD), yet the mechanisms remain unclear. Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous nitric oxide synthase inhibitors shown to be independent risk markers for cardiovascular disease. Previous research has predominantly focused on ADMA as a primary risk marker of reduced vascular function and increased cardiovascular mortality in end-stage (stage 5) CKD. However, limited research has explored the potential roles of ADMA and SDMA in relation to vascular function in the middle stages (stages 3 and 4) of CKD. Therefore, we tested the hypothesis that patients with stage 3-4 CKD would have elevated circulating ADMA and SDMA, which would correspond to a reduced vascular function compared to controls. We studied 17 patients with stage 3-4 CKD (age: 66 ± 7 years, 5 males, eGFR: 44 ± 16 ml/min/1.73m 2 ) and 8 controls (age: 59 ± 7 years; 3 males, eGFR: 81 ± 14 ml/min/1.73m 2 ). ADMA and SDMA were assessed in whole blood serum using liquid chromatography/tandem mass spectrometry by a commercial laboratory (Quest diagnostics). Macro- and micro-vascular function were assessed as brachial artery flow-mediated dilation (FMD; %) and peak reactive hyperemia blood velocity, respectively, using duplex Doppler ultrasound. Arterial stiffness was assessed as carotid-femoral pulse wave velocity (cfPWV) using arterial tonometry. Data are expressed as mean ± standard deviation. Interestingly, we found that SDMA (control: 103 ± 14, CKD: 143 ± 42 ng/mL, p=0.01) but not ADMA (control: 109 ± 7; CKD: 111 ± 28 ng/mL, p=0.40) was significantly elevated in patients with CKD compared to controls. FMD was significantly reduced in the CKD group (control: 5.04 ± 2.59.; CKD: 3.18 ± 1.98 %, p=0.03), while peak blood velocity was similar between groups (control: 53.47 ± 10.40, CKD: 59.72 ± 23.99 cm/s, p=0.25). There tended to be a negative relationship between SDMA concentrations and FMD (R= -0.31, p=0.07), whereas there was no relationship between ADMA and FMD (R= 0.22, p=0.14). Arterial stiffness was also significantly greater in the patient with CKD compared to controls (control: 6.74 ± 0.91, CKD: 7.95 ± 1.60 m/s, p=0.034). These data suggest that patients with stage 3-4 CKD who have elevated circulating SDMA but not ADMA exhibit an impaired macro-vascular function. Moreover, an augmented cfPWV also suggests increased arterial stiffness in patients with CKD with elevated SDMA. Collectively, these preliminary findings suggest that SDMA, but not ADMA, may play a more predominant role in mediating impaired vascular function in the middle stages of CKD. This project was supported by the National Heart, Lung, and Blood Institute R01 HL-127071. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Asymmetric Dimethylarginine as a Biomarker in Coronary Artery Disease.

As atherosclerosis remains a leading cause of morbidity and mortality worldwide despite the advances in its medical and interventional management, the identification of markers associated with its incidence and prognosis constitutes an appealing prospect. In this regard, asymmetric dimethylarginine (ADMA), a well-studied endogenous endothelial nitric oxide synthase inhibitor, represents a core mediator of endothelial dysfunction in atherosclerotic diseases. Given the pathophysiologic background of this molecule, its importance in the most frequent atherosclerotic manifestation, coronary artery disease (CAD), has been extensively studied in the past decades. The available evidence suggests elevation of ADMA in the presence of common cardiovascular risk factors, namely diabetes mellitus, arterial hypertension, and hypertriglyceridemia, being related to endothelial dysfunction and incident major adverse cardiovascular events in these groups of patients. Moreover, ADMA is associated with CAD occurrence and severity, as well as its prognosis, especially in populations with renal impairment. Interestingly, even in the absence of obstructive CAD, increased ADMA may indicate coronary endothelial dysfunction and epicardial vasomotor dysfunction, which are prognostication markers for incident cardiovascular events. In the case of acute coronary syndromes, high ADMA levels signify an augmented risk of incomplete ST-segment elevation resolution and poorer prognosis. Abnormal ADMA elevations may indicate adverse outcomes following percutaneous or surgical coronary revascularization, such as in-stent restenosis, graft patency, and hard cardiovascular endpoints. Finally, since its association with inflammation is significant, chronic inflammatory conditions may present with coronary endothelial dysfunction and subclinical coronary atherosclerosis by means of increased coronary artery calcium, with augmented ADMA acting as a biomarker.

Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism

The enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays a pivotal role in the regulation of nitric oxide levels by degrading the main endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Growing evidence highlight the potential implication of DDAH/ADMA axis in the etiopathogenesis of several neuropsychiatric and neurological disorders, yet the underlying molecular mechanisms remain elusive. In this study, we sought to investigate the role of DDAH1 in behavioral endophenotypes with neuropsychiatric relevance. To achieve this, a global DDAH1 knock-out (DDAH1-ko) mouse strain was employed. Behavioral testing and brain region-specific neurotransmitter profiling have been conducted to assess the effect of both genotype and sex. DDAH1-ko mice exhibited increased exploratory behavior toward novel objects, altered amphetamine response kinetics and decreased dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) level in the piriform cortex and striatum. Females of both genotypes showed the most robust amphetamine response. These results support the potential implication of the DDAH/ADMA pathway in central nervous system processes shaping the behavioral outcome. Yet, further experiments are required to complement the picture and define the specific brain-regions and mechanisms involved.

The relationship between reduced choroidal thickness due to high plasma asymmetrical dimethylarginine level and increased severity of diabetic retinopathy.

To evaluate the relationship between subfoveal choroidal thickness and plasma asymmetrical dimethylarginine level and the severity of diabetic retinopathy in patients with type 2 diabetes mellitus. A total of 68 cases, including 15 patients without diabetic retinopathy, 17 patients with nonproliferative diabetic retinopathy, 16 patients with type 2 diabetes mellitus and proliferative diabetic retinopathy, and 20 healthy patients (control group), were enrolled in this study. Subfoveal choroidal thickness was measured manually using the enhanced depth imaging optical coherence tomography scanning program, and plasma asymmetrical dimethylarginine level was measured using a commercial micro enzyme-linked immunosorbent assay kit. The subfoveal choroidal thickness values and plasma asymmetrical dimethylarginine levels were significantly different between the four groups (p<0.001 and p<0.001). The subfoveal choroidal thickness values were significantly lower in the proliferative diabetic retinopathy group than in the other three groups (no diabetic retinopathy, nonproliferative diabetic retinopathy, and control groups; p<0.001, p=0.045, and p<0.001, respectively). The plasma asymmetrical dimethylarginine levels were significantly higher in the proliferative diabetic retinopathy group than in the other three groups (p<0.001, p<0.04, and p<0.001, respectively). In addition, a significant negative correlation was also found between plasma asymmetrical dimethylarginine level and subfoveal choroidal thickness (p<0.001, r=-0.479). Asymmetrical dimethylarginine is an important marker of endothelial dysfunction and endogenous endothelial nitric oxide synthase inhibitor. The severity of diabetic retinopathy was related to increased plasma asymmetrical dimethylarginine level and reduced subfoveal choroidal thickness in type 2 diabetic patients with diabetic retinopathy.

Endogenous Endothelial Nitric Oxide Synthase (eNOS) Modulates Oxygen (O2) Responsive Control of Red Blood Cell (RBC) Energy Metabolism and Antioxidant Systems

Endogenous Endothelial Nitric Oxide Synthase (eNOS) Modulates Oxygen (O2) Responsive Control of Red Blood Cell (RBC) Energy Metabolism and Antioxidant Systems

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.

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.

Heart-targeted amelioration of sepsis-induced myocardial dysfunction by microenvironment responsive nitric oxide nanogenerators in situ

BackgroundA balanced endogenous level of bioavailable nitric oxide (NO) plays a key role in maintaining cardiovascular homeostasis. The bioactive NO level in the cardiomyocytes was much reduced during sepsis. However, it is clinically challenging for the NO gas therapy due to the lack of spatial and temporal release system with precise control. The purpose of this study is to design a NO-releasing biomaterial with heart-targeted capability responsive to the infectious microenvironment, thus ameliorating lipopolysaccharide (LPS)-induced cardiac dysfunction.ResultsThe heart-targeted NO delivery and in situ releasing system, PCM-MSN@LA, was synthesized using hollow mesoporous silica nanoparticles (MSN) as the carrier, and L-arginine (LA) as the NO donor. The myocardial delivery was successfully directed to heart by specific peptide (PCM) combined with low-intensity focused ultrasound (LIFU) guidance. The myocardial system synthesized NO from the LA released from PCM-MSN@LA in the presence of increased endogenous nitric oxide synthase (NOS) activity induced by LPS. This targeted NO release in situ achieved extraordinary protective effects against LPS-challenged myocardial injury by reducing the recruitment of inflammatory cells, inhibiting oxidative stress and maintaining the mitochondria integrity. In particular, this protection was not compromised by simultaneous circulation collapse as an adverse event in the context.ConclusionsPCM-MSN@LA + LIFU exhibited extraordinary cardioprotective effects against severe sepsis in the hearts of LPS-treated animals without the side effect of NO diffusion. This technology has great potential to be served as a novel therapeutic strategy for sepsis-induced myocardial injury.

Skeletal Muscle Contractile Function in Heart Failure With Reduced Ejection Fraction-A Focus on Nitric Oxide.

Despite advances over the past few decades, heart failure with reduced ejection fraction (HFrEF) remains not only a mortal but a disabling disease. Indeed, the New York Heart Association classification of HFrEF severity is based on how much exercise a patient can perform. Moreover, exercise capacity—both aerobic exercise performance and muscle power—are intimately linked with survival in patients with HFrEF. This review will highlight the pathologic changes in skeletal muscle in HFrEF that are related to impaired exercise performance. Next, it will discuss the key role that impaired nitric oxide (NO) bioavailability plays in HFrEF skeletal muscle pathology. Lastly, it will discuss intriguing new data suggesting that the inorganic nitrate ‘enterosalivary pathway’ may be leveraged to increase NO bioavailability via ingestion of inorganic nitrate. This ingestion of inorganic nitrate has several advantages over organic nitrate (e.g., nitroglycerin) and the endogenous nitric oxide synthase pathway. Moreover, inorganic nitrate has been shown to improve exercise performance: both muscle power and aerobic capacity, in some recent small but well-controlled, cross-over studies in patients with HFrEF. Given the critical importance of better exercise performance for the amelioration of disability as well as its links with improved outcomes in patients with HFrEF, further studies of inorganic nitrate as a potential novel treatment is critical.

Asymmetric Dimethylarginine (ADMA) in Pediatric Renal Diseases: From Pathophysiological Phenomenon to Clinical Biomarker and Beyond.

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) synthase inhibitor, inhibits NO synthesis and contributes to the pathogenesis of many human diseases. In adults, ADMA has been identified as a biomarker for chronic kidney disease (CKD) progression and cardiovascular risk. However, little attention is given to translating the adult experience into the pediatric clinical setting. In the current review, we summarize circulating and urinary ADMA reported thus far in clinical studies relating to kidney disease in children and adolescents, as well as systematize the knowledge on pathophysiological role of ADMA in the kidneys. The aim of this review is also to show the various analytical methods for measuring ADMA and the issues tht need to be addressed before transforming to clinical practice in pediatric medicine. The last task is to suggest that ADMA may not only be suitable as a diagnostic or prognostic biomarker, but also a promising therapeutic strategy to treat pediatric kidney disease in the future.

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Melatonin Prevents Chronic Kidney Disease-Induced Hypertension in Young Rat Treated with Adenine: Implications of Gut Microbiota-Derived Metabolites.

Melatonin, a signaling hormone with pleiotropic biofunctions, has shown health benefits. Trimethylamine-N-oxide (TMAO) and asymmetric dimethylarginine (ADMA) are uremic toxins involved in the development of hypertension. TMAO originates from trimethylamine (TMA), a gut microbial product. ADMA is an endogenous nitric oxide (NO) synthase inhibitor. We examined whether melatonin therapy could prevent hypertension and kidney disease by mediating gut microbiota-derived metabolites and the NO pathway using an adenine-induced chronic kidney disease (CKD) young rat model. Six-week-old young Sprague Dawley rats of both sexes were fed a regular diet (C group), a diet supplemented with 0.5% adenine (CKD group), or adenine plus 0.01% melatonin in their drinking water (CKD + M group) for three weeks (N = 8/group). Adenine-fed rats developed renal dysfunction, hypertension, renal hypertrophy and increased uremic toxin levels of TMAO and ADMA. Melatonin therapy prevented hypertension in both sexes and attenuated kidney injury in males. Melatonin reversed the changes to the plasma TMAO-to-TMA ratio induced by CKD in both sexes. Besides, the protective effects of melatonin were associated with restoration of gut microbiota alterations, including increased α-diversity, and enhancement of the abundance of the phylum Proteobacteria and the genus Roseburia in male rats. Melatonin therapy also partially prevented the increases in ADMA in male CKD rats. Melatonin sex-specifically protected young rats against hypertension and kidney injury induced by CKD. The results of this study contribute toward a greater understanding of the interaction between melatonin, gut microbiota-derived metabolites, and the NO pathway that is behind CKD, which will help to prevent CKD-related disorders in children.