Impaired Nitric Oxide Research Articles

Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. The pathogenesis of this syndrome is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, and nitric oxide (NO) deficiency. NO deficiency in MELAS syndrome is likely to be multifactorial in origin with the decreased availability of the NO precursors, arginine and citrulline, playing a major role. In this study we used stable isotope infusion techniques to assess NO production in children with MELAS syndrome and healthy pediatric controls. We also assessed the effect of oral arginine and citrulline supplementations on NO production in children with MELAS syndrome. When compared to control subjects, children with MELAS syndrome were found to have lower NO production, arginine flux, plasma arginine, and citrulline flux. In children with MELAS syndrome, arginine supplementation resulted in increased NO production, arginine flux, and arginine concentration. Citrulline supplementation resulted in a greater increase of these parameters. Additionally, citrulline supplementation was associated with a robust increase in citrulline concentration and flux and de novo arginine synthesis rate. The greater effect of citrulline in increasing NO production is due to its greater ability to increase arginine availability particularly in the intracellular compartment in which NO synthesis takes place. This study, which is the first one to assess NO metabolism in children with mitochondrial diseases, adds more evidence to the notion that NO deficiency occurs in MELAS syndrome, suggests a better effect for citrulline because of its greater role as NO precursor, and indicates that impaired NO production occurs in children as well as adults with MELAS syndrome. Thus, the initiation of treatment with NO precursors may be beneficial earlier in life. Controlled clinical trials to assess the therapeutic effects of arginine and citrulline on clinical complications of MELAS syndrome are needed.

Cryptotanshinone inhibits TNF-α-induced early atherogenic events in vitro

Endothelial dysfunction has been implicated in the pathogenesis of atherosclerosis. Salvia miltiorrhiza (danshen) is a traditional Chinese medicine that has been effectively used to treat cardiovascular disease. Cryptotanshinone (CTS), a major lipophilic compound isolated from S. miltiorrhiza, has been reported to possess cardioprotective effects. However, the anti-atherogenic effects of CTS, particularly on tumor necrosis factor-α (TNF-α)-induced endothelial cell activation, are still unclear. This study aimed to determine the effect of CTS on TNF-α-induced increased endothelial permeability, monocyte adhesion, soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), monocyte chemoattractant protein 1 (MCP-1) and impaired nitric oxide production in human umbilical vein endothelial cells (HUVECs), all of which are early events occurring in atherogenesis. We showed that CTS significantly suppressed TNF-α-induced increased endothelial permeability, monocyte adhesion, sICAM-1, sVCAM-1 and MCP-1, and restored nitric oxide production. These observations suggest that CTS possesses anti-inflammatory properties and could be a promising treatment for the prevention of cytokine-induced early atherogenesis.

The Vascular Endothelium in Chronic Kidney Disease: A Novel Target for Aerobic Exercise.

Endothelial dysfunction occurs in chronic kidney disease (CKD) and increases the risk for cardiovascular disease. The mechanisms of endothelial dysfunction seem to evolve throughout kidney disease progression, culminating in reduced L-arginine transport and impaired nitric oxide bioavailability in advanced disease. This review examines the hypothesis that aerobic exercise may reverse endothelial dysfunction by improving endothelial cell L-arginine uptake in CKD.

Vascular hyperpolarization in human physiology and cardiovascular risk conditions and disease.

Hyperpolarization causing smooth muscle relaxation contributes to the maintenance of vascular homeostasis, particularly in small-calibre arteries and arterioles. It may also become a compensatory vasodilator mechanism upregulated in states with impaired nitric oxide (NO) availability. Bioassay of vascular hyperpolarization in the human circulation has been hampered by the complexity of mechanisms involved and the limited availability of investigational tools. Firm evidence, however, supports the notion that hyperpolarization participates in the regulation of resting vasodilator tone and vascular reactivity in healthy subjects. In addition, an enhanced endothelium-derived hyperpolarization contributes to both resting and agonist-stimulated vasodilation in a variety of cardiovascular risk conditions and disease. Thus, hyperpolarization mediated by epoxyeicosatrienoic acids (EETs) and H2 O2 has been observed in coronary arterioles of patients with coronary artery disease. Similarly, ouabain-sensitive and EETs-mediated hyperpolarization has been observed to compensate for NO deficiency in patients with essential hypertension. Moreover, in non-hypertensive patients with multiple cardiovascular risk factors and in hypercholesterolaemia, KCa channel-mediated vasodilation appears to be activated. A novel paradigm establishes that perivascular adipose tissue (PVAT) is an additional regulator of vascular tone/function and endothelium is not the only agent in vascular hyperpolarization. Indeed, some PVAT-derived relaxing substances, such as adiponectin and angiotensin 1-7, may exert anticontractile and vasodilator actions by the opening of KCa channels in smooth muscle cells. Conversely, PVAT-derived factors impair coronary vasodilation via differential inhibition of some K+ channels. In view of adipose tissue abnormalities occurring in human obesity, changes in PVAT-dependent hyperpolarization may be relevant for vascular dysfunction also in this condition.

Homocysteine, hyperhomocysteinemia and vascular contributions to cognitive impairment and dementia (VCID)

Homocysteine is produced physiologically in all cells, and is present in plasma of healthy individuals (plasma [HCy]: 3–10μM). While rare genetic mutations (CBS, MTHFR) cause severe hyperhomocysteinemia ([HCy]: 100–200μM), mild-moderate hyperhomocysteinemia ([HCy]: 10–100μM) is common in older people, and is an independent risk factor for stroke and cognitive impairment. As B-vitamin supplementation (B6, B12 and folate) has well-validated homocysteine-lowering efficacy, this may be a readily-modifiable risk factor in vascular contributions to cognitive impairment and dementia (VCID).Here we review the biochemical and cellular actions of HCy related to VCID. Neuronal actions of HCy were at concentrations above the clinically-relevant range. Effects of HCy <100μM were primarily vascular, including myocyte proliferation, vessel wall fibrosis, impaired nitric oxide signalling, superoxide generation and pro-coagulant actions. HCy-lowering clinical trials relevant to VCID are discussed. Extensive clinical and preclinical data support HCy as a mediator for VCID. In our view further trials of combined B-vitamin supplementation are called for, incorporating lessons from previous trials and from recent experimental work. To maximise likelihood of treatment effect, a future trial should: supply a high-dose, combination supplement (B6, B12 and folate); target the at-risk age range; and target cohorts with low baseline B-vitamin status. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

New-onset atrial fibrillation and thromboembolic risk: Cardiovascular syzygy?

Atrial fibrillation (AF) is a condition that confers increased thromboembolic risk. Oral anticoagulant (OAC) therapy can attenuate this risk. However, use of OAC therapy is determined largely by the presence of additional clinical factors (encapsulated by the CHA2DS2VASc score) that incrementally elevate stroke risk. Currently, there is no specific recommendation regarding urgency of initiation of OAC therapy in the presence of new-onset AF, except where cardioversion is being considered. Recently, it has become increasingly apparent that there is a period immediately following the onset of AF of particularly accentuated thromboembolic risk (with respect to chronic AF): the physiological bases for this risk are as yet incompletely understood. However, given that both inflammation and impaired nitric oxide signaling are pivotally involved in the pathogenesis of AF, these factors may also mediate thrombotic risk in the context of new-onset AF. Advances in OAC therapy have recently been achieved, with development of agents that are comparable or superior to warfarin for mitigation of stroke risk, but with a safety profile similar to aspirin therapy. Thus, the incremental increase in thromboembolic risk experienced by new-onset AF patients constitutes a previously widely neglected case in favor of the rapid application of OAC therapy to such individuals. This review seeks to summarize the thromboembolic risk observed in new-onset AF and the emerging understanding of the physiological bases for this risk.

Abstract 18673: JNK Inhibition Restores Endothelial Function in Type 2 Diabetes Mellitus

Introduction: Diabetes mellitus type 2 is an increasingly public health problem and it is a major cause in the development of cardiovascular diseases. Endothelial dysfunction is a key mechanism that contributes to the pathogenesis of cardiovascular diseases and is a well-known feature of clinical diabetes. Prior studies have demonstrated an impaired nitric oxide bioavailability and a reduced endothelium-dependent vasodilation under diabetic conditions and in animal models, JNK activity has been widely described to be involved in systemic insulin resistance. Hypothesis: Our study aimed to evaluate the involvement of JNK in endothelial dysfunction, studying its potential role in altered eNOS activation and NO synthesis in diabetic patients. Methods: We measured endothelial function and JNK activity in freshly isolated endothelial cells from diabetic patients (n=38) and nondiabetic controls (n=40). Results: ECs from diabetic patients displayed impaired eNOS activation and reduced NO release after insulin and A23187 stimulation, consistent with the presence of endothelial dysfunction. JNK activation was higher in diabetic (**P=0.003), and was associated with lower flow-mediated dilation (r=-0.53, *P=0.02). In endothelial cells from diabetic patients, treatment with JNK chemical inhibitor (SP600125) restored eNOS activation and insulin response (***P&lt;0.001). Nitric oxide bioactivity after A23187 stimuli with diabetes was also recovered in endothelial cells from patients with diabetes. Conclusions: In summary, our data suggest that JNK activation contributes to vascular insulin resistance and endothelial dysfunction in patients with type 2 diabetes and may represent a target in novel therapeutic opportunities.

Regulation of skeletal muscle blood flow during exercise in ageing humans.

The regulation of skeletal muscle blood flow and oxygen delivery to contracting skeletal muscle is complex and involves the mechanical effects of muscle contraction; local metabolic, red blood cell and endothelium-derived substances; and the sympathetic nervous system (SNS). With advancing age in humans, skeletal muscle blood flow is typically reduced during dynamic exercise and this is due to a lower vascular conductance, which could ultimately contribute to age-associated reductions in aerobic exercise capacity, a primary predictor of mortality in both healthy and diseased ageing populations. Recent findings have highlighted the contribution of endothelium-derived substances to blood flow control in contracting muscle of older adults. With advancing age, impaired nitric oxide availability due to scavenging by reactive oxygen species, in conjunction with elevated vasoconstrictor signalling via endothelin-1, reduces the local vasodilatory response to muscle contraction. Additionally, ageing impairs the ability of contracting skeletal muscle to blunt sympathetic vasoconstriction (i.e. 'functional sympatholysis'), which is critical for the proper regulation of tissue blood flow distribution and oxygen delivery, and could further reduce skeletal muscle perfusion during high intensity and/or large muscle mass exercise in older adults. We propose that initiation of endothelium-dependent hyperpolarization is the underlying signalling event necessary to properly modulate sympathetic vasoconstriction in contracting muscle, and that age-associated impairments in red blood cell adenosine triphosphate release and stimulation of endothelium-dependent vasodilatation may explain impairments in both local vasodilatation and functional sympatholysis with advancing age in humans.

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice

Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2Akita+/−), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2Akita+/−) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2Akita+/− and TgeNOS-Ins2Akita+/− were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2Akita+/− mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2Akita+/− was significantly higher compared with the Ins2Akita+/− and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target.

Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.

Development, validation, and comparison of four methods to simultaneously quantify l-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry.

To understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality.

Sodium Nitrite Improves Exercise Hemodynamics and Ventricular Performance in Heart Failure With Preserved Ejection Fraction

BackgroundThere is no effective medical treatment for heart failure with preserved ejection fraction (HFpEF). Increases in pulmonary capillary wedge pressure (PCWP) develop in patients with HFpEF during exercise coupled with impaired nitric oxide (NO) signaling. Nitrite can be reduced to bioactive NO in vivo, particularly under conditions of tissue hypoxia, as with exercise. ObjectivesThis study sought to determine whether acute nitrite administration improves exercise hemodynamics and cardiac reserve in HFpEF. MethodsIn a double-blind, randomized, placebo-controlled, parallel-group trial, subjects with HFpEF (N = 28) underwent invasive cardiac catheterization with simultaneous expired gas analysis at rest and during exercise, before and 15 min after treatment with either sodium nitrite or matching placebo. ResultsBefore the study drug infusion, HFpEF subjects displayed an increase in PCWP with exercise from 16 ± 5 mm Hg to 30 ± 7 mm Hg (p < 0.0001). After study drug infusion, the primary endpoint of exercise PCWP was substantially improved by nitrite compared with placebo (adjusted mean: 19 ± 5 mm Hg vs. 28 ± 6 mm Hg; p = 0.0003). Nitrite-enhanced cardiac output reserve improved with exercise (+0.5 ± 0.7 l/min vs. −0.4 ± 0.7 l/min; p = 0.002) and normalized the increase in cardiac output relative to oxygen consumption. Nitrite improved pulmonary artery pressure-flow relationships in HFpEF and increased left ventricular stroke work with exercise versus placebo, indicating an improvement in ventricular performance with stress. ConclusionsAcute sodium nitrite infusion favorably attenuates hemodynamic derangements of cardiac failure that develop during exercise in individuals with HFpEF. Prospective trials testing long-term nitrite therapy in this population are warranted. (Acute Effects of Inorganic Nitrite on Cardiovascular Hemodynamics in Heart Failure With Preserved Ejection Fraction; NCT01932606)

Impaired endothelium-dependent vasodilation does not initiate the development of sunitinib-associated hypertension.

Tyrosine kinase inhibitors targeting angiogenesis have become an important part of the treatment of patients with several types of cancer. One of the most reported side effects of vascular endothelial growth factor receptor (VEGFR)-targeted therapies is hypertension. In this study, we hypothesized that the development of hypertension in patients treated with sunitinib, a multitargeted tyrosine kinase inhibitor, is preceded by reduced endothelium-dependent vasodilation. Moreover, we hypothesized that this endothelial dysfunction is a result of impaired nitric oxide release. In a placebo-controlled experiment, we determined vascular responses in isolated mesenteric arteries of rats (n = 26) after 7 days of sunitinib treatment. Sunitinib reduced endothelium-dependent vasodilation, but not endothelium-independent vasodilation. Moreover, we observed that the difference in endothelium-dependent vasodilation between controls and sunitinib-treated animals disappeared in the presence of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide antagonist. In patients with metastatic renal cell carcinoma, before and 1 week after start of sunitinib, the endothelium-dependent vasodilator response to intra-arterial acetycholine and the endothelium-independent vasodilator response to intra-arterial sodium nitroprusside was assessed with venous occlusion plethysmography. No changes in forearm blood flow ratios were observed. Mean arterial pressure did significantly increase from 101.9 ± 3.8 to 106.1 ± 2.6 mmHg after 1 week and further to 115.8 (±4.9) mmHg after 2 weeks of treatment. In animals, this study confirms that exposure to high concentrations of sunitinib reduces endothelium-dependent vasodilation by reducing endothelial release of nitric oxide. In humans, however, reduced endothelium-dependent vasodilation does not precede the development of hypertension in patients treated with sunitinib.

Evaluation of pulmonary artery pressure and resistance by pulsed Doppler echocardiography in patients with end-stage renal disease on dialysis therapy

BackgroundPulmonary hypertension (PH) is one of the most important comorbidities in patients undergoing hemodialysis (HD). The goal of the present work is to determine the possible etiologic factors for its occurrence. MethodsThe prevalence of PH was estimated by Doppler echocardiography in a cohort of 100 patients aged 49.3±13.9years on regular HD. Mean pulmonary artery pressure was estimated from pulmonary acceleration time by Mahan’s regression equation. Pulmonary vascular resistance and pulmonary capillary wedge pressure were calculated. We focused on the effect of HD on left and right ventricle diastolic and systolic function. Right ventricle systolic function was assessed by tricuspid annular systolic excursion and pulsed Doppler myocardial performance index. Since impaired endothelial function was postulated as an underlying cause of PH, we studied the effects of HD on brachial artery endothelial function. ResultsThe current study found that pulmonary hypertension was prevalent in 70% of patients on dialysis. Left atrium diameter, left ventricle mass indexed to body surface area, and mitral E/E′ were increased in the dialysis group (4.4±0.2cm, 126.5±24.6g/m2, and 16.9±4.4, respectively, p<0.001 for all). Pulmonary artery systolic pressure was positively correlated to duration of dialysis and negatively correlated to glomerular filtration rate (p<0.001 and r=−0.991). Pulmonary vascular resistance was significantly increased in dialysis patients (1.9±0.2 Wood units vs. 1.2 Wood units in controls, p<0.001). Endothelial dysfunction, defined as brachial artery flow mediated dilatation <6%, was found in 46% of dialysis group. ConclusionIncreased pulmonary artery systolic pressure in the HD population could be attributed to left atrium dilatation and left ventricle diastolic dysfunction. Pulmonary vascular resistance was significantly increased in dialysis group. This might be explained by impaired endothelial nitric oxide synthesis that not only caused systemic vasoconstriction but also affected the pulmonary vasculature.

Soluble guanylate cyclase stimulator BAY 41-8543 and female sex ameliorate uremic aortic remodeling in a rat model of mild uremia.

Cardiovascular disease is the leading comorbidity in renal patients and has been related to impaired nitric oxide signaling. Estrogens exert protective effects on the vascular system. This study investigates the effects of biological sex and nitric oxide-independent soluble guanylate cyclase (sGC) stimulator BAY 41-8543 on aortic remodeling in experimental mild uremia. Age-matched male and female Wistar rats were assigned for 18 weeks into sham-operated, subtotally nephrectomized (SNX), SNX + BAY 41-8543 and SNX + hydralazine. Analysis involved functional, histological, and molecular kidney and thoracic aorta parameters. SNX significantly increased SBP, which was comparably reduced to control levels by BAY 41-8543 and hydralazine. In SNX males, uremic aortic remodeling was characterized by marked media thickening and increased media-to-lumen ratio (P < 0.01), vascular smooth muscle cell (VSMC) proliferation, macrophage infiltration, extracellular matrix turnover, decreased aortic elastin-to-collagen ratio (P < 0.01) and endothelial nitric oxide-synthase (eNOS) mRNA expression (P < 0.05). No significant alterations of aortic media-to-lumen ratio, VSMC proliferation, macrophage infiltration, matrix metalloproteinase-2, and eNOS mRNA expressions were seen in female uremic animals. BAY 41-8543 significantly ameliorated uremic aortic remodeling and stiffening involving reduced VSMC proliferation, collagen I-deposition, extracellular matrix turnover, and increased elastin content and eNOS mRNA expression. Hydralazine treatment did not substantially alter aortic remodeling. Experimental mild uremia leads to pronounced aortic hypertrophic remodeling and stiffening with sex-dependent alternations, and these are more severe in male rats. BAY 41-8543 ameliorates uremic aortic remodeling in a blood pressure-independent manner. The results suggest that sGC-stimulators may offer a novel treatment mode for pathological arterial wall remodeling in patients with impaired renal function.

Abstract P608: Role Of Nitric Oxide In The Pathophysiology Of Hypertension Associated With Low Nephron Endowment.

A low nephron endowment is a strong predictor for future risk of hypertension, kidney and cardiovascular disease. Previously, we have shown that fetal uninephrectomy (uni-x) results in elevated arterial pressure, reduced renal function and low plasma renin activity in adulthood in sheep. However, the mechanisms via which a reduction in renal mass in early life contributes to the development of hypertension and renal disease are not well understood. Nitric oxide (NO) plays an important role in driving the fall in renal vascular resistance (RVR) and rise in glomerular filtration rate (GFR) that characterizes the normal postnatal maturation of renal function in the newborn. We hypothesized that the early onset of renal dysfunction in individuals with a congenital reduction in renal mass is due to a reduced renal contribution of NO. Fetal uni-x (n=9) or sham operation (n=7) was performed at 100d of gestation (term=150 days). In conscious female lambs at 6 months of age, mean arterial pressure (MAP) was measured via an indwelling carotid arterial catheter and GFR and renal blood flow (RBF) were measured via clearance methods before and after nitric oxide synthase inhibition (L-NAME; 20mg/kg/h iv). Basal MAP was elevated in uni-x as compared to the sham sheep (sham: 80 ± 2 mmHg, uni-x: 86 ± 1 mmHg; P &lt; 0.01). In response to systemic administration of L-NAME MAP increased similarly in both groups (sham: 13 ± 3 mmHg, uni-x: 13 ± 4 mmHg). Basal RBF (sham: 933 ± 29 ml/kg/h; uni-x 757 ± 43 ml/kg/h; P&lt;0.01) and GFR (sham: 144 ± 3 ml/kg/h; uni-x 107 ± 3 ml/kg/h; P&lt;0.01) was lower in the uni-x as compared to the sham sheep. In response to L-NAME the decline in RBF (sham: 73 ± 3 %; uni-x: 47 ± 3%; P&lt;0.001) and GFR (sham: 54 ± 4 %; uni-x: 43 ± 1%; P&lt;0.03) was less in uni-x as compared to sham sheep. Filtration fraction decreased significantly in the sham (P&lt;0.001) but not the uni-x sheep in response to L-NAME. Renal dysfunction and hypertension in young hypertensive sheep born with a low nephron endowment maybe underpinned by impaired nitric oxide bioavailability. Targeting the nitric oxide system may offer therapeutic potential in children born with a reduced renal mass, in terms of treatment interventions and as a biomarker of prognosis.

Aging effects on pedicled fasciocutaneous flap survival in rats.

Poorer surgical outcomes in older patients undergoing locoregional head and neck reconstruction have raised questions about tolerance of aging tissue to iatrogenic ischemic insults. We examined the effects of aging on viability of pedicled composite flaps in 2-month and 6-month old Sprague-Dawley male rats and correlated flap survival with vascular endogenous growth factor (VEGF) and VEGF receptor 2-mediated signaling events. Flap segments were assessed for gross/cellular necrosis by optical microscopy and for proangiogenic, apoptotic, and proliferative protein-marker content. Flap necrosis significantly increased with age (4.2% in young vs 49.17% in old), correlating with reduced expression of VEGF, inhibition of signal transducer and activator of transcription 3 (STAT3), and Akt activation, impaired Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation, elevated Bax/Bcl-2 ratio, activation of Caspase-3, upregulated nuclear poly (ADP-ribose) polymerase-1 (PARP-1) cleavage and lower proliferating cell nuclear antigen (PCNA) levels. Pedicled flap survival is higher in younger rats in part because of unhindered expression of VEGF and enhanced activity of cell survival and promigratory signaling pathways. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1152-E1162, 2016.

Mechanisms Of Hypoxia-Induced Immune Escape In Cancer And Their Regulation By Nitric Oxide

The acquired ability of tumour cells to avoid destruction by immune effector mechanisms (immune escape) is important for malignant progression. Also associated with malignant progression is tumour hypoxia, which induces aggressive phenotypes such as invasion, metastasis and drug resistance in cancer cells. Our studies revealed that hypoxia contributes to escape from innate immunity by increasing tumour cell expression of the metalloproteinase ADAM10 in a manner dependent on accumulation of the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α). Increased ADAM10 expression leads to shedding of the NK cell-activating ligand, MICA, from the surface of tumour cells, thereby resulting in resistance to NK cell-mediated lysis. Our more recent studies demonstrated that hypoxia, also via HIF-1α accumulation, increases the expression of the inhibitory co-stimulatory ligand PD-L1 on tumour cells. Elevated PD-L1 expression leads to escape from adaptive immunity via increased apoptosis of CD8+ cytotoxic T lymphocytes. Accumulating evidence indicates that hypoxia-induced acquisition of malignant phenotypes, including immune escape, is in part due to impaired nitric oxide (NO)-mediated activation of cGMP signalling and that restoration of cGMP signalling prevents such hypoxic responses. We have shown that NO/cGMP signalling inhibits hypoxia-induced malignant phenotypes likely in part by interfering with HIF-1α accumulation via a mechanism involving calpain. These findings indicate that activation of NO/cGMP signalling may have useful applications in cancer therapy.

Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.

NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.

Platelet hyperaggregability in patients with atrial fibrillation. Evidence of a background proinflammatory milieu.

Atrial fibrillation (AF) is a condition where platelet hyperaggregability is commonly present. We examined potential physiological bases for platelet hyperaggregability in a cohort of patients with acute and chronic AF. In particular, we sought to identify the impact of inflammation [myeloperoxidase (MPO) and C-reactive protein (CRP)] and impaired nitric oxide (NO) signaling. Clinical and biochemical determinants of adenosine diphosphate (ADP)-induced platelet aggregation were sought in patients (n = 106) hospitalized with AF via univariate and multivariate analysis. Hyper-responsiveness of platelets to ADP was directly (r = 0.254, p < 0.01) correlated with plasma concentrations of thrombospondin-1 (TSP-1), a matricellular protein that impairs NO responses and contributes to development of oxidative stress. In turn, plasma TSP-1 concentrations were directly correlated with MPO concentrations (r = 0.221, p < 0.05), while MPO concentrations correlated with those of asymmetric dimethylarginine (ADMA, r = 0.220, p < 0.05), and its structural isomer symmetric dimethylarginine (SDMA, r = 0.192, p = 0.05). Multivariate analysis identified TSP-1 (β = 0.276, p < 0.05) concentrations, as well as female sex (β = 0.199, p < 0.05), as direct correlates of platelet aggregability, and SDMA concentrations (β = - 0.292, p < 0.05) as an inverse correlate. We conclude that platelet hyperaggregability, where present in the context of AF, may be engendered by impaired availability of NO, as well as via MPO-related inflammatory activation.