Administration Of Nitric Oxide Donor Research Articles

Blood pressure lowering enhances cerebrospinal fluid efflux to the systemic circulation primarily via the lymphatic vasculature

BackgroundInside the incompressible cranium, the volume of cerebrospinal fluid is directly linked to blood volume: a change in either will induce a compensatory change in the other. Vasodilatory lowering of blood pressure has been shown to result in an increase of intracranial pressure, which, in normal circumstances should return to equilibrium by increased fluid efflux. In this study, we investigated the effect of blood pressure lowering on fluorescent cerebrospinal fluid tracer absorption into the systemic blood circulation.MethodsBlood pressure lowering was performed by an i.v. administration of nitric oxide donor (sodium nitroprusside, 5 µg kg−1 min−1) or the Ca2+-channel blocker (nicardipine hydrochloride, 0.5 µg kg−1 min−1) for 10, and 15 to 40 min, respectively. The effect of blood pressure lowering on cerebrospinal fluid clearance was investigated by measuring the efflux of fluorescent tracers (40 kDa FITC-dextran, 45 kDa Texas Red-conjugated ovalbumin) into blood and deep cervical lymph nodes. The effect of nicardipine on cerebral hemodynamics was investigated by near-infrared spectroscopy. The distribution of cerebrospinal fluid tracers (40 kDa horse radish peroxidase,160 kDa nanogold-conjugated IgG) in exit pathways was also analyzed at an ultrastructural level using electron microscopy.ResultsNicardipine and sodium nitroprusside reduced blood pressure by 32.0 ± 19.6% and 24.0 ± 13.3%, while temporarily elevating intracranial pressure by 14.0 ± 7.0% and 18.2 ± 15.0%, respectively. Blood pressure lowering significantly increased tracer accumulation into dorsal dura, deep cervical lymph nodes and systemic circulation, but reduced perivascular inflow along penetrating arteries in the brain. The enhanced tracer efflux by blood pressure lowering into the systemic circulation was markedly reduced (− 66.7%) by ligation of lymphatic vessels draining into deep cervical lymph nodes.ConclusionsThis is the first study showing that cerebrospinal fluid clearance can be improved with acute hypotensive treatment and that the effect of the treatment is reduced by ligation of a lymphatic drainage pathway. Enhanced cerebrospinal fluid clearance by blood pressure lowering may have therapeutic potential in diseases with dysregulated cerebrospinal fluid flow.

Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation. We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L). Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Myocardial stretch-induced compliance is abrogated under ischemic conditions and restored by cGMP/PKG-related pathways.

Introduction: Management of acute myocardial infarction (MI) mandates careful optimization of volemia, which can be challenging due to the inherent risk of congestion. Increased myocardial compliance in response to stretching, known as stretch-induced compliance (SIC), has been recently characterized and partly ascribed to cGMP/cGMP-dependent protein kinase (PKG)-related pathways. We hypothesized that SIC would be impaired in MI but restored by activation of PKG, thereby enabling a better response to volume loading in MI. Methods: We conducted experiments in ex vivo rabbit right ventricular papillary muscles under ischemic and non-ischemic conditions as well as pressure-volume hemodynamic evaluations in experimental in vivo MI induced by left anterior descending artery ligation in rats. Results: Acutely stretching muscles ex vivo yielded increased compliance over the next 15min, but not under ischemic conditions. PKG agonists, but not PKC agonists, were able to partially restore SIC in ischemic muscles. A similar effect was observed with phosphodiesterase-5 inhibitor (PDE5i) sildenafil, which was amplified by joint B-type natriuretic peptide or nitric oxide donor administration. In vivo translation revealed that volume loading after MI only increased cardiac output in rats infused with PDE5i. Contrarily to vehicle, sildenafil-treated rats showed a clear increase in myocardial compliance upon volume loading. Discussion: Our results suggest that ischemia impairs the adaptive myocardial response to acute stretching and that this may be partly prevented by pharmacological manipulation of the cGMP/PKG pathway, namely, with PDE5i. Further studies are warranted to further elucidate the potential of this intervention in the clinical setting of acute myocardial ischemia.

The NOS/NO System in Renal Programming and Reprogramming.

Nitric oxide (NO) is a gaseous signaling molecule with renoprotective properties. NO can be produced in NO synthase (NOS)-dependent or -independent manners. NO deficiency plays a decisive role in chronic kidney disease (CKD). Kidney development can be affected in response to adverse intrauterine conditions that induce renal programming, thereby raising the risk of developing CKD in adulthood. Conversely, detrimental programming processes could be postponed or halted prior to the onset of CKD by early treatments, namely reprogramming. The current review provides an overview of the NOS/NO research performed in the context of renal programming and reprogramming. NO deficiency has been increasingly found to interact with the different mechanisms behind renal programming, such as oxidative stress, aberrant function of the renin-angiotensin system, disturbed nutrient-sensing mechanisms, dysregulated hydrogen sulfide signaling, and gut microbiota dysbiosis. The supplementation of NOS substrates, the inhibition of asymmetric dimethylarginine (ADMA), the administration of NO donors, and the enhancement of NOS during gestation and lactation have shown beneficial effects against renal programming in preclinical studies. Although human data on maternal NO deficiency and offspring kidney disease are scarce, experimental data indicate that targeting NO could be a promising reprogramming strategy in the setting of renal programming.

NO-IL-6/10-IL-1β axis: a new pathway in steatotic and non-steatotic liver grafts from brain-dead donor rats.

Brain death (BD) and steatosis are both risk factors for organ dysfunction or failure in liver transplantation (LT). Here, we examine the role of interleukin 6 (IL- 6) and IL-10 in LT of both non-steatotic and steatotic liver recovered from donors after brain death (DBDs), as well as the molecular signaling pathways underlying the effects of such cytokines. BD reduced IL-6 levels only in nonsteatotic grafts, and diminished IL-10 levels only in steatotic ones. In both graft types, BD increased IL-1β, which was associated with hepatic inflammation and damage. IL-6 administration reduced IL-1β only in non-steatotic grafts and protected them against damage and inflammation. Concordantly, IL-1β inhibition via treatment with an IL-1 receptor antagonist caused the same benefits in non-steatotic grafts. Treatment with IL-10 decreased IL-1β only in steatotic grafts and reduced injury and inflammation specifically in this graft type. Blockading the IL-1β effects also reduced damage and inflammation in steatotic grafts. Also, blockade of IL-1β action diminished hepatic cAMP in both types of livers, and this was associated with a reduction in liver injury and inflammation, then pointing to IL-1β regulating cAMP generation under LT and BD conditions. Additionally, the involvement of nitric oxide (NO) in the effects of interleukins was evaluated. Pharmacological inhibition of NO in LT from DBDs prompted even more evident reductions of IL-6 or IL-10 in non-steatotic and steatotic grafts, respectively. This exacerbated the already high levels of IL-1β seen in LT from DBDs, causing worse damage and inflammation in both graft types. The administration of NO donors to non-steatotic grafts potentiated the beneficial effects of endogenous NO, since it increased IL-6 levels, and reduced IL-1β, inflammation, and damage. However, treatment with NO donors in steatotic grafts did not modify IL-10 or IL-1β levels, but induced more injurious effects tan the induction of BD alone, characterized by increased nitrotyrosine, lipid peroxidation, inflammation, and hepatic damage. Our study thus highlights the specificity of new signaling pathways in LT from DBDs: NO-IL-6-IL-1β in non-steatotic livers and NO-IL-10-IL-1β in steatotic ones. This opens up new therapeutic targets that could be useful in clinical LT.

Nitric Oxide Donor Administration For improvement of Uterine Blood Flow in Unexplained Recurrent Pregnancy Loss

Background: Recurrent pregnancy loss is still a challenging obstetric condition. It linked to impaired uterine blood, and vasodilators seem to prevent recurrent pregnancy loss. The aim of the work: The current study aimed to compare between investigate the Doppler uterine artery indices in females with recurrent pregnancy loss [unexplained]. It also aimed to study the effect of intravaginal isosorbide mononitrate, on the pattern of uterine blood flow. Materials and Methods: This observational, nested case control study had been completed at Al-Azhar University Hospitals. Two groups of females [each 50 females] were studied. The control group [females with no previous pregnancy loss and have a viable child or more]. The study group [females with history of unexplained recurrent pregnancy loss]. Vaginal color Doppler ultrasound was done to assess pulsation and resistance indices of uterine arteries in the mid-luteal phase of the cycle. Then, 20 mg of isosorbide mononitrate was applied vaginally and measurement of the vascular indices were registered after two hours again. Both groups were compared and the effect of isosorbide mononitrate on the uterine artery vascular indices the study group was documented. Results: Higher uterine artery resistance and pulsation indices are noticed in females with recurrent pregnancy. A significant reduction in these indices was found after vaginal application of Isosorbide mononitrate. Both groups were comparable regarding patient demographics. Doppler indices were significantly lower in control than study group. The endometrial thickness was negatively correlated with right RI and left PI (r = -0.328, -0.342, p = 0.039 and 0.031, respectively). Conclusion: Poor uterine perfusion is associated with spontaneous abortion. The vaginal application of isosorbide mononitrate, nitric oxide donor could be an effective treatment for the correction of uterine vasculature in patients with recurrent pregnancy loss.

A Paradoxical Vasodilatory Nutraceutical Intervention for Prevention and Attenuation of Migraine—A Hypothetical Review

Studies suggest that migraine pain has a vascular component. The prevailing dogma is that peripheral vasoconstriction activates baroreceptors in central, large arteries. Dilatation of central vessels stimulates nociceptors and induces cortical spreading depression. Studies investigating nitric oxide (NO) donors support the indicated hypothesis that pain is amplified when acutely administered. In this review, we provide an alternate hypothesis which, if substantiated, may provide therapeutic opportunities for attenuating migraine frequency and severity. We suggest that in migraines, heightened sympathetic tone results in progressive central microvascular constriction. Suboptimal parenchymal blood flow, we suggest, activates nociceptors and triggers headache pain onset. Administration of NO donors could paradoxically promote constriction of the microvasculature as a consequence of larger upstream central artery vasodilatation. Inhibitors of NO production are reported to alleviate migraine pain. We describe how constriction of larger upstream arteries, induced by NO synthesis inhibitors, may result in a compensatory dilatory response of the microvasculature. The restoration of central capillary blood flow may be the primary mechanism for pain relief. Attenuating the propensity for central capillary constriction and promoting a more dilatory phenotype may reduce frequency and severity of migraines. Here, we propose consideration of two dietary nutraceuticals for reducing migraine risk: L-arginine and aged garlic extracts.

Improving tumor hypoxia and radiotherapy resistance via in situ nitric oxide release strategy

Radiation therapy remains one of the main treatments for cancer. However, conventional radiotherapy not only manifests a low radiation accumulation in the tumor site, but also displays numerous negative effects. The most serious clinical problem is the radiotherapy resistance leading to cancer deterioration. As an important gaseous signal molecule, nitric oxide (NO) has been widely studied for its role in regulating angiogenesis, improving hypoxia, and inhibiting tumor growth. However, due to the unstable characteristic, the application of NO in cancer therapy is still limited. Here, we designed a micellar system formed by a NO donor, D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS)-NO, for enabling sustained NO release to efficiently deliver NO into the tumor area. TPGS-NO could accumulate in the tumor site for extended circulation, thereby releasing NO to exert antitumor effects and enhance radiotherapy effects under low-oxygen conditions. It demonstrated the increased sensitivity of radiotherapy through enhancing tumor angiogenesis appropriately reducing tumor area hypoxia, which significantly induced tumor cell apoptosis and inhibited its repair during radiation. This work may show great potential in synergistic radiotherapy against cancer by facile NO donor administration.

Biochemical parameters of rat blood in the models of chronic heart failure and chronic kidney disease at the administration of nitric oxide donor

In modern veterinary medicine, the simultaneous occurrence of chronic heart failure and chronic kidney disease is often found. However, the cause and effect often exchange places, which creates great difficulties in the animals’ treatment. Chlofusan acts on both systems. It improves cardiac and renal functions by means of providing cardioprotective and nephroprotective effects. Models of chronic heart failure and chronic kidney disease in rats provide important information on the pathophysiology of these diseases in other animal species, and the assessment of changes in the biochemical analysis of blood makes it possible to assess the state of the heart and kidneys in the study. In the course of research, the results of rat biochemical analysis were studied on models of chronic heart and kidney failure with the introduction of an exogenous nitric oxide donor. Chlofuzan contributes to a partial balance restoration of biochemical blood parameters in rats, which indicates the restoration of the mutual work of the heart and kidneys.

Factors affecting the pathophysiology of sepsis, an inflammatory disorder: Key roles of oxidative and nitrosative stress

Sepsis, one of the primary causes of mortality in the intensive care units, occurs due to the host’s dysregulated immune responses to an infection. Consequently, persistent systemic inflammation along with suppressed adaptive immunity ensues, resulting in deranged metabolism, recurrent infections, tissue damage and multi-organ failure. The uncontrolled oxidative stress mediated by the imbalance between the generation of reactive oxygen species and their neutralization by the host’s antioxidant system is involved in inflammation-induced damage. The profound deleterious effects in the host range from mitochondrial dysfunction and endothelial damage to reduced cardiac output. Therefore, antioxidant therapy was actively considered to have therapeutic benefits in sepsis patients. Although some success has been obtained with the use of antioxidants in sepsis patients, considerable ambiguity persists that prevents their routine use. Another key molecule that may dictate the outcome and prognosis during sepsis is nitric oxide (NO). This pleiotropic molecule plays a central role in inflammation and in leukocyte recruitment at the site of inflammation. NO is synthesized by three different isoforms of nitric oxide synthases (NOS) and significantly high and sustained levels of NOS2 have been reported in sepsis. Abundant literature supports the protective roles of NO during sepsis; however, there is uncertainty in various reports. The administration of NO donors in clinical trials for sepsis treatment has encountered limited success. NO, during sepsis, acts like a double-edged sword: increased NO levels can result in hypotension, whereas reduced levels contribute to poor organ perfusion and an elevated susceptibility to infection. Therefore, several parameters need to be evaluated, while considering the potential of antioxidant and NO-based therapy during sepsis.

The effect of the release of exogenous nitric oxide on the responses of the pregnant human myometrium to oxytocin.

Currently there is insufficient evidence to support the routine administration of nitric oxide donors in the treatment of threatened preterm labor. An understanding of the role that nitric oxide plays in the management of threatened preterm labor may lead to more effective treatment and prevention. This is why the aim of our study was to examine the involvement of exogenous nitric oxide release in regulating responses of the human pregnant myometrium to oxytocin. Biopsies of human myometrial tissue during pregnancy were obtained from 8 pregnant women, aged 21-35 years. The responses of the specimens to oxytocin in the absence and presence of a DETA/NO were recorded under isometric conditions. Preincubation with exogenous nitric oxide significantly (p<0.001) attenuated the contractile response of the uterine strips to oxytocin in concentrations higher than 10-8 mol/L. The inhibition of nitric oxide synthesis alone or in combination with DETA/NO incubation did not significantly change the oxytocin contractile effect in the concentration-response curve. Moreover, there was no significant variation in the mean value for log EC50 for oxytocin between the group with oxytocin alone and other groups. We present evidence in support of the hypothesis that continuous nitric oxide supply to the human pregnant myometrium environment attenuates its response to oxytocin but only when endogenous production of nitric oxide is not impaired.

Reversal of cerebrovascular constriction in experimental cerebral malaria by L-arginine

Vascular dysfunction associated with low nitric oxide (NO) biavailability and low plasma L-arginine levels is observed in both human and experimental cerebral malaria (ECM). In ECM, cerebrovascular constriction results in decreased pial blood flow and hypoxia, and administration of NO donors reverses constriction and increases survival. Supplementation of L-arginine, the substrate for NO synthesis by NO synthases, has been considered as a strategy to improve vascular health and act as adjunctive therapy in human severe malaria. We investigated the effect of L-arginine supplementation on pial vascular tonus of mice with ECM after direct superfusion on the brain surface or systemic delivery. Pial arteriolar diameters of Plasmodium berghei-infected mice with implanted cranial windows were measured using intravital microscopy methods, before and after L-arginine administration. Systemic delivery of L-arginine was performed intravenously, at 10, 50, 100 and 200 mg/kg, as bolus injection or slowly through osmotic pumps, combined or not with artesunate. Direct superfusion of L-arginine (10−7M, 10−5M and 10−3M) on the brain surface of mice with ECM resulted in immediate, consistent and dose-dependent dilation of pial arterioles. ECM mice showed marked cerebrovascular constriction that progressively worsened over a 24 h-period after subcutaneous saline bolus administration. L-arginine administration prevented the worsening in pial constriction at all the doses tested, and at 50 mg/kg and 100 mg/kg it induced temporary reversal of vasoconstriction. Slow, continuous delivery of L-arginine by osmotic pumps, or combined bolus administration of artesunate with L-arginine, also prevented worsening of pial constriction and resulted in improved survival of mice with ECM. L-arginine ameliorates pial vasoconstriction in mice with ECM.

Nitroglycerin application and coronary arteriogenesis.

BackgroundIn the presence of a coronary occlusion, pre-existing small collateral vessels (arterioles) develop into much larger arteries (biological bypasses) that have the potential to allow a certain level of perfusion distal to the blockage. Termed arteriogenesis, this phenomenon proceeds via a complex combination of events, with nitric oxide (NO) playing an essential role. The aim of this study was to investigate the effects of supplemental administration of NO donors, i.e., short-acting nitroglycerin (NTG) or slow-release pelleted isosorbide dinitrate (ISDN), on collateral development in a repetitive coronary artery occlusion model in rats.MethodsCoronary collateral growth was induced via a repetitive occlusion protocol (ROP) of the left anterior descending coronary artery (LAD) in rats. The primary endpoints were the histological evaluation of rat heart infarct size and ST-segment elevation (ECG-analysis) upon final permanent occlusion of the LAD (experimentally induced myocardial infarction). The effects of NTG or ISDN were also evaluated by administration during 5 days of ROP. We additionally investigated whether concomitant application of NTG can compensate for the anti-arteriogenic effect of acetylsalicylic acid (ASA).ResultsAfter 5 days of ROP, the mean infarct size and degree of ST-elevation were only slightly lower than those of the SHAM group; however, after 10 days of the protocol, the ROP group displayed significantly less severe infarct damage, indicating enhanced arteriogenesis. Intermittent NTG application greatly decreased the ST-elevation and infarct size. The ISDN also had a positive effect on arteriogenesis, but not to the same extent as the NTG. Administration of ASA increased the infarct severity; however, concomitant dosing with NTG somewhat attenuated this effect.ConclusionIntermittent treatment with the short-acting NTG decreased the size of an experimentally induced myocardial infarct by promoting coronary collateral development. These new insights are of great relevance for future clinical strategies for the treatment of occlusive vascular diseases.

Critical Roles of Carbon Monoxide and Nitric Oxide in Ca2+ Signaling for Insulin Secretion in Pancreatic Islets.

Glucagon-like peptide-1 (GLP-1) increases intracellular Ca2+ concentrations, resulting in insulin secretion from pancreatic β-cells through the sequential production of Ca2+ mobilizing messengers nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR). We previously found that NAADP activates the neuronal type of nitric oxide (NO) synthase (nNOS), the product of which, NO, activates guanylyl cyclase to produce cyclic guanosine monophosphate (cGMP), which, in turn, induces cADPR formation. Our aim was to explore the relationship between Ca2+ signals and gasotransmitters formation in insulin secretion in β-cells upon GLP-1 stimulation. We show that NAADP-induced cGMP production by nNOS activation is dependent on carbon monoxide (CO) formation by heme oxygenase-2 (HO-2). Treatment with exogenous NO and CO amplifies cGMP formation, Ca2+ signal strength, and insulin secretion, whereas this signal is impeded when exposed to combined treatment with NO and CO. Furthermore, CO potentiates cGMP formation in a dose-dependent manner, but higher doses of CO inhibited cGMP formation. Our data with regard to zinc protoporphyrin, a HO inhibitor, and HO-2 knockdown, revealed that NO-induced cADPR formation and insulin secretion are dependent on HO-2. Consistent with this observation, the administration of NO or CO donors to type 2 diabetic mice improved glucose tolerance, but the same did not hold true when both were administered concurrently. Our research reveals the role of two gas transmitters, CO and NO, for Ca2+ second messengers formation in pancreatic β-cells. These results demonstrate that CO, the downstream regulator of NO, plays a role in bridging the gap between the Ca2+ signaling messengers during insulin secretion in pancreatic β-cells.

Effect of a nitric oxide donor on maternal hemodynamics in fetal growth restriction

To evaluate the effect on maternal cardiovascular parameters of treatment with a nitric oxide (NO) donor and plasma volume expansion in pregnancies complicated by fetal growth restriction (FGR). Twenty-six pregnant women with a diagnosis of FGR were treated with transdermal patches of a NO donor and plasma volume expansion by co-administration of oral fluids. We compared the treated group to a historical control group of untreated FGR patients. Hemodynamic indices were obtained using the UltraSonic Cardiac Output Monitor system. At diagnosis, the two groups were similar in terms of maternal and hemodynamic characteristics. In the treated group, we found a significant increase in maternal cardiac output and stroke volume and a decrease in systemic vascular resistance after 2 weeks of therapy. No significant differences were found 2 weeks after diagnosis in the untreated group. The treated group delivered infants with higher birth-weight centile than did the untreated control group. The combined therapeutic approach of NO donor administration and plasma volume expansion in FGR apparently improves significantly maternal hemodynamic indices. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Effect of Transdermal Nitroglycerine on Doppler Velocity Waveforms of the Uterine, Umbilical and Fetal Middle Cerebral Arteries in Patients with Chronic Placental Insufficiency: A Prospective RCT.

Increase in Nitric Oxide (NO) may be important in vascular adaptation needed to accommodate increased uteroplacental blood flow as pregnancy advances. Hence, in certain conditions like Pregnancy Induced Hypertension (PIH) and Fetal Growth Restriction (FGR), NO donors may play an effective role in increasing uteroplacental perfusion. Transdermal route appears to be a safe and effective route. To evaluate the effect of nitroglycerine patch on Doppler velocity waveforms of the uterine, umbilical and fetal middle cerebral arteries in patients with chronic placental insufficiency. A prospective randomized controlled clinical trial was conducted on eighty consecutive pregnant women with FGR with or without PIH and having evidence of altered waveform velocimetry in uterine, umbilical and fetal middle cerebral artery. They were divided into two groups- study and control group. Transdermal nitroglycerine patch (10 mg per 24 hours) was applied in study group for three consecutive days. Changes in various Doppler indices were noted after three days of patch application and compared between the two groups. Analysis was carried out using SPSS (Statistical Package for Social Studies) for Windows version 20.0 and online GraphPad software (Prism 5 for Windows) version 5.01. A significant fall in the systolic and diastolic ratio (S/D), Pulsatility Index (PI) and Resistivity Index (RI) of the uterine (3.07±0.52, 1.04±0.14 and 0.54±0.10 respectively, p<0.001) and umbilical artery (3.73±3.30, 1.18±0.21and 0.64±0.07 respectively, p<0.001) was noted after three days of patch application. No such significant change was observed in the middle cerebral artery indices. The therapeutic approach of NO donor administration via transdermal route in pregnant patients with chronic placental insufficiency, apparently improved both maternal and fetoplacental haemodynamics, thus may help in improving perinatal outcome.

F4. NO donors and haemodynamic changes in fetal growth restriction

Introduction: Pregnancies complicated by fetal growth restriction (FGR) are known to be associated with a reduced expansion of maternal intravascular space and a lower cardiac output. Therapy with nitric oxide (NO) donors, in addition to plasma volume expansion (PVE) could improve maternal haemodynamic indices and pregnancy outcome.Methods: To evaluate maternal cardiovascular effects of NO donors. We enrolled 52 women with the diagnosis of FGR. Patients were divided into those treated with transdermal patches of NO donors and PVE or a control group. We obtained haemodynamic indices using an USCOM system.Results: At enrolment, the two groups were similar in terms of maternal, fetal and haemodynamic characteristics. In the group treated with NO donors and PVE, we found a significant increase in cardiac output, stroke volume and a decrease of systemic vascular resistance after therapy (see table). At birth the treated group also gave birth to babies with higher birth weight centile.Conclusions: The combined therapeutic approach of NO donor administration and PVE in FGR significantly improves maternal hemodynamic indices. Despite the observation nature of the data, there is suggestion that the use of NO donors together with PVE may also improve pregnancy outcome by increasing the fetal growth.

Endothelial nitric oxide synthase: a potential therapeutic target for cerebrovascular diseases.

Endothelial nitric oxide (NO) is a significant signaling molecule that regulates cerebral blood flow (CBF), playing a pivotal role in the prevention and treatment of cerebrovascular diseases. However, achieving the expected therapeutic efficacy is difficult using direct administration of NO donors. Therefore, endothelial nitric oxide synthase (eNOS) becomes a potential therapeutic target for cerebrovascular diseases. This review summarizes the current evidence supporting the importance of CBF to cerebrovascular function, and the roles of NO and eNOS in CBF regulation.

Catherine Pasquier Memorial Award Lecture (shared): Critical role of oxidative and nitrosative stress in pathophysiology: Inflammation, cell injury and cancer

The presence of chronic inflammation associated with sustained cell death and proliferation turnover is a potential tissue environment for cancer development. In this situation, oxidative and nitrosative stress are the major driving force playing a key role either as a preventing or inducer/promoter of tumorigenesis. During inflammatory response, activated macrophages release a great variety of protease that degraded extracellular matrix, inflammatory mediators that plays a relevant role in chemotaxis and cell signaling, as well as oxygen and nitrogen reactive species (ROS and RNS, respectively) actively involved in cell defense through oxidation of essential biological molecules such as DNA, carbohydrates, proteins and lipids. Iron-loosely bound plays a relevant role in promoting oxidative-dependent reactions leading to cell damage. In this sense, we have shown that locally iron overload induced an accumulation of neutrophils with increased NADPH-derived superoxide anion generation in exudates from rats submitted to carrageenan-induced granuloma model. The increase of lipid peroxidation products was associated with the presence of iron-loosely bound, reduction of antioxidant status in inflammatory exudates and blood, as well as induction of hepatic acute phase response. A similar pro-inflammatory and oxidant reaction was observed by intraperitoneal iron-dextran administration in animals with Mycobacterium butyricum-induced adjuvant arthritis. The administration of a diet devoid of polyunsaturated fatty acids reduced the substrate for lipid peroxidation, and consequently a decrease of conjugated diennes and thiobarbituric acid-reactive substances in plasma and hepatic microsomal fraction were observed in animals with adjuvant arthritis. The acute phase response induced by different inflammatory mediators in hepatocytes has been studied in detail. The induction of carrageenan-induced granuloma drastically reduced the expression of drug metabolizing enzymes (DME) (CYP3A1, CYP4A, CYP2B1, CYP2D and CYP2E1) and their related-activities in rat livers. Cytokines (IL-1α, IL-6 and TNF-α) exerted a specific timely-dependent regulation of DME expression (CYP1A1, CYP1A2 and CYP3A4) and secretion of acute phase reactants (albumin, ferritin, fibrinogen and ceruloplasmin) in cultured primary human hepatocytes. Although TNF-α induces acute phase response and liver injury in different experimental settings such as sepsis, we have also shown that TNF-α-dependent induction of nitric oxide synthase type II (NOS-2) hepatic expression during PGE1 pre-administration drastically reduces D-galactosamine (D-GalN)-induced liver injury in rats. The pre-administration of PGE1 also reduced cell death induced by D-GalN in primary culture of human and rat hepatocytes. However, the cytoprotective properties of PGE1 do not involve a reduction of D-GalN-induced ROS production. Nitric oxide (NO) has demonstrated to exert pro- and anti-apoptotic properties according to local concentration, target cell, as well as the presence of other ROS such as superoxide anion generating high reactive peroxynitrite. We have observed that moderate increase of PGE1-dependent NO prevented further potent NF-kB-dependent NOS-2 expression and apoptosis induced by D-GalN in cultured rat hepatocytes. However, the administration of PGE1 at advanced stages of cell injury or high doses of NO donor increased cell death in D-GalN-treated human and rat hepatocytes. The induction of apoptosis by D-GalN was associated with ROS production, mitochondrial dysfunction and post-translation (S-nitrosylation and Tyrosine nitration) modifications of proteins involved in unfolded protein response, enzymatic antioxidants, cell metabolism and death in cultured human hepatocytes. In consequences, the administration of antioxidants such as N-acetylcysteine, Q10 and MnTBAB reduced ROS generation, mitochondrial dysfunction and apoptosis induced by D-GalN and glycochenodeoxycholate (GCDCA) in cultured human hepatocytes. The cytoprotective properties of α-tocopherol appears to mostly related to gene regulation (NOS-2, PPAR-α and carnitine palmitoyl transferase) rather than to its antioxidant activity in D-GalN-induced cell death in hepatocytes. The reduction of cell death by α-tocopherol was also associated with reduction of CYP7A1, Na+-taurocholate co-transporting polypeptide (NTCP), and heme oxygenase-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and Tyrosine nitration, and reduced Taurocholic acid uptake in hepatocytes. The pro-apoptotic properties of high NO donor concentration have been successfully applied to exert antitumoral activity in liver cancer cells. In this sense, the administration of NO donor or NOS-3 overexpression induces p53-dependent cell death receptor expression and apoptosis in hepatoma cell lines. The first generation adenovirus specifically designed to induce NOS-3 overexpression in liver cancer cells induced oxidative and nitrosative stress, DNA damage, p53, cell death receptors expression and their S-nitrosylation, and apoptosis that was correlated to a reduction of cell proliferation and growth of tumors orthotopically implanted in fibrotic livers. The S-nitrosylation of cell death receptors (TNF-α, CD95 and TRAIL-R1) plays a critical role on the shift from extrinsic to intrinsic cell death pathways during antitumoral molecular therapy (Sorafenib Tosylate) in liver cancer cells. In conclusion, new data are emerging that elucidate the extraordinary role that plays ROS- and RNS-dependent posttranslational target modifications in regulating intracellular cell signaling.

PP65 - Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells

Nitric oxide (NO) plays a relevant role during cell death regulation that limits the survival of tumor cells. We have recently shown that the overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoma cells. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in hepatoblastoma cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic markers in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced cell death in HepG2 cells. However, low concentration of the drug (10 nM) increased the expression of cell death receptors and extrinsic apoptotic pathway (caspase-8) that both diminished at higher concentrations of the drug (10 µM). High doses of Sorafenib correlated to a rise of caspase-9 and caspase-3 activities, as well as DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in cells treated with Sorafenib. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death shift from the extrinsic to intrinsic apoptotic signaling in hepatoblastoma cells.