Nitric Oxide Imbalance Research Articles

S-51-2: ROLE OF THE SYMPATHETIC NERVOUS SYSTEM IN HYPERTENSION: FOCUSED ON THE CENTRAL NERVOUS SYSTEM

Activation of the sympathetic nervous system plays a critical role in the pathogenesis of hypertension. In particular, the central nervous system (CNS) organizes the sympathetic outflow and various inputs from the periphery. These include afferent neural inputs, circulating hormone, cellular factors, and inflammatory and immune responses. Also, within the brain, several factors affect and determine the central sympathetic outflow. Interestingly, these factors are activated particularly in the autonomic nuclei such as the paraventricular nucleus of the hypothalamus and rostral ventrolateral medulla of the brainstem. I will review what and how these factors are altered in various stages of hypertension from the results of basic studies using animal models of hypertension including our own and other investigators. I will address topics such as imbalance of nitric oxide and reactive oxygen species, activation of the brain renin-angiotensin pathway, and altered immunity including Toll-like receptor activation, regulatory T cell inhibition, and perivascular macrophages. In addition, activation of the mineral corticoid receptors in the CNS could be responsible for salt-sensitive hypertension. Finally, I will introduce some new aspects of neural mechanisms of hypertension in the recent progress in the field of research. These experimental studies regarding CNS mechanisms of hypertension can be translated to clinical practice and novel therapies.

Pathophysiology of Pulmonary Hypertension in Sickle Cell Disease

The pathophysiology of pulmonary hypertension (PH) in sickle cell disease (SCD) is multifactorial: hemolysis, hypercoagulability, hypoxemia, oxidative stress, platelet activation, increased adhesiveness, inflammatory cell activation and genetic susceptibility, all contributing in varying degrees to endothelial dysfunction. Intravascular hemolysis is the main pathological process contributing to vasculopathy by releasing toxic red blood cell products that impair endothelial function, cause hypercoagulable state and drive oxidative and inflammatory stress. Hemolysis induced nitric oxide imbalance is one the most important contributors to high pulmonary artery pressures seen in SCD. Multi-faceted, targeted interventions, before irreversible vasculopathy develops, will allow for improved patient outcomes and life expectancy, stressing the need for a better understanding of the multiple pathophysiological mechanisms involved in the development of PH before considering those patients for targeted therapies. Hemolysis is still considered as the main contributor of PH in SCD but the mechanisms by which it causes PH are still not completely known. This review precisely presents the various pathophysiological mechanisms and factors that have been proposed till date to help the reader get an overview.

Say NO to ROS: Their Roles in Embryonic Heart Development and Pathogenesis of Congenital Heart Defects in Maternal Diabetes.

Congenital heart defects (CHDs) are the most prevalent and serious birth defect, occurring in 1% of all live births. Pregestational maternal diabetes is a known risk factor for the development of CHDs, elevating the risk in the child by more than four-fold. As the prevalence of diabetes rapidly rises among women of childbearing age, there is a need to investigate the mechanisms and potential preventative strategies for these defects. In experimental animal models of pregestational diabetes induced-CHDs, upwards of 50% of offspring display congenital malformations of the heart, including septal, valvular, and outflow tract defects. Specifically, the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) signaling is a major driver of the development of CHDs in offspring of mice with pregestational diabetes. NO from endothelial nitric oxide synthase (eNOS) is crucial to cardiogenesis, regulating various cellular and molecular processes. In fact, deficiency in eNOS results in CHDs and coronary artery malformation. Embryonic hearts from diabetic dams exhibit eNOS uncoupling and oxidative stress. Maternal treatment with sapropterin, a cofactor of eNOS, and antioxidants such as N-acetylcysteine, vitamin E, and glutathione as well as maternal exercise have been shown to improve eNOS function, reduce oxidative stress, and lower the incidence CHDs in the offspring of mice with pregestational diabetes. This review summarizes recent data on pregestational diabetes-induced CHDs, and offers insights into the important roles of NO and ROS in embryonic heart development and pathogenesis of CHDs in maternal diabetes.

Pharmacotherapy for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a condition associated with substantial morbidity and mortality. Over the last 25 years there has been a significant evolution in the therapies to treat PAH. These therapies are effective for patients with group I PAH and group IV PH [chronic thromboembolic pulmonary hypertension (CTEPH)]. PAH is characterized by an imbalance of nitric oxide, prostacyclin and endothelin levels, and current pharmacotherapy involves these three pathways. Earlier clinical trials involving PAH-specific therapies evaluated improvements in 6-minute walk time as a primary improvement whereas contemporary trials have been larger and focused on morbidity and mortality reductions. While there may be a role for monotherapy in disease management, most patients should be considered for dual or triple therapy.

Differential lethal and sublethal effects in embryonic zebrafish exposed to different sizes of silver nanoparticles

Various parameters can influence the toxic response to silver nanoparticles (Ag NPs), including the size and surface properties, as well as the exposure environment and the biological site of action. Herein, we assess the intestinal toxicity of three different sizes (10, 40, and 100 nm) of Ag NPs in embryonic zebrafish, and describe the relationship between the properties and behavior of Ag NPs in the exposure medium, and induction of lethal and sublethal effects. We find that the composition of the medium and the size contribute to differential NPs agglomeration, release of Ag ions, and subsequent effects during exposure. The exposure medium causes dramatic reduction in silver dissolution due to the presence of salts and divalent cations, which limits the lethal potential of silver ions. Lethality is observed primarily for embryos exposed to medium sized Ag NPs (40 nm), but not to the supernatant originated from particles, which suggests that the exposure to particulate silver is the main cause of mortality. On the other hand, the exposure to 10 nm and 100 nm NPs, as well as Ag ions, only causes sublethal developmental defects in skeletal muscles and intestine, and induces a nitric oxide imbalance.

Effect of Hypotensive Brain Death on the Donor Liver and Its Mechanism in an Improved Bama Miniature Pig (Sus scrofa domestica) Model

BackgroundWe aimed to observe the effect of hypotensive brain death on the donor liver and understand its pathophysiological mechanism in improved pig model. MethodsThe model was induced using the modified intracranial water sac inflation method in 16 Bama miniature pigs. Effects of hypotensive brain death on liver function and tissue morphology were evaluated via changes in liver function enzyme index, liver tissue alkaline phosphatase levels, hourly bile flow, and liver tissue pathology. Its pathophysiological mechanism was examined on the basis of changes in portal vein blood flow, hepatic artery blood flow, portal venous endotoxin level, and liver tissue cytokine levels. ResultsAfter model establishment, portal vein blood flow, hepatic arterial blood flow, hourly bile flow, and alkaline phosphatase content in hepatic tissue significantly decreased, and serum aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase levels significantly increased. Hematoxylin-eosin staining of liver tissue showed that after model establishment, hepatic tissue injury was gradually aggravated and hepatic cells were irreversibly damaged at 7 hours. Portal vein endotoxin levels significantly increased after brain death. Tumor necrosis factor α, interleukin 1, and endothelin 1 levels in liver tissues significantly increased at 3, 6, and 12 hours after brain death (P < .05), and hypoxia-inducible factor 1–α and nitric oxide levels significantly decreased (P < .05). ConclusionsHepatic injury was progressively aggravated under hypotensive brain death. The mechanism of donor liver injury under hypotensive brain death may involve low liver perfusion, release of intestinal endotoxin and inflammatory factors (eg, tumor necrosis factor α and interleukin 1), decreased hypoxia-inducible factor 1–α, and endothelin 1 and nitric oxide imbalance.

Atmospheric nanoparticles affect vascular function using a 3D human vascularized organotypic chip.

Inhaled atmospheric nanoparticles (ANPs) can migrate into human blood vessels. However, the exact pathogenesis has not yet been well elucidated. In this study, a perfusable 3D human microvessel network was constructed in a microfluidic device. This functional 3D micro-tissue partly mimicked the physiological response of human vessels. Intravascular nanoparticles tend to adsorb proteins to form a protein corona. Based on this pathological response, vessel permeability and vasoconstriction resulting from ANP stimulation might be related to vascular inflammation. It mediated abnormal expression of nuclear factor-κB (NF-κB) and an influx of intracellular Ca2+ ([Ca2+]i). This biological behavior disturbed the normal expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF). The imbalance of nitric oxide (NO) and endothelin-1 (ET-1) further resulted in endothelial cell contraction. All these bio-events induced the loss of tight junctions (ZO-1) which enhanced vessel permeability. Meanwhile, ANP induced-vascular toxicity was also found in mice. Our observations provide a plausible explanation for how the ANPs affect human vascular function. The vessel-on-chip provides a bridge between in vitro results and human responses. We aimed to use this human 3D functional microvascular model to mimic the physiological responses of human vessels. This model is suitable for the evaluation of vascular toxicity after the human vessel exposure to ANPs.

Differential Expression of Nitric Oxide Synthase Isoforms nNOS and iNOS in Patients with Non-Segmental Generalized Vitiligo.

Nitric oxide (NO) is involved in several biological processes, but its role in human melanogenesis is still not well understood. Exposure to UVA and UVB induces nitric oxide production in keratinocytes and melanocytes through the activation of constitutive nitric oxide synthase, increasing tyrosinase activity and melanin synthesis, whereas inducible nitric oxide synthase over expression might be involved in hypopigmentary disorders. The aim of this study was to evaluate whether inducible nitric oxide synthase and neuronal nitric oxide synthase expression were modified in vitiligo skin compared to healthy controls. Skin biopsies were obtained from inflammatory/lesional and white/lesional skin in 12 patients with active, non-segmental vitiligo; site-matched biopsies of normal skin from eight patients were used as controls. Nitric oxide synthase isoforms expression was evaluated by confocal laser scanning microscopy and Western Blot analysis. Inducible nitric oxide synthase expression was significantly increased in inflammatory/lesional skin compared to healthy skin; melanocytes showed a moderate neuronal nitric oxide synthase expression in white/lesional skin, demonstrating that metabolic function still goes on. The obtained data demonstrated that vitiligo lesions were characterized by modifications of nitric oxide synthase isoforms, thus confirming the hypothesis that nitric oxide imbalance is involved in vitiligo and supporting the idea that nitric oxide synthase inhibitors might be used as a possible therapeutic approach for the management of vitiligo.

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics.

Oxidative stress and generation of free radicals are fundamental in initiating pathophysiological mechanisms leading to an inflammatory cascade resulting in high rates of morbidity and death from many inherited point mutation-derived hemoglobinopathies. Hemoglobin (Hb)E is the most common point mutation worldwide. The βE-globin gene is found in greatest frequency in Southeast Asia, including Thailand, Malaysia, Indonesia, Vietnam, Cambodia, and Laos. With the wave of worldwide migration, it is entering the gene pool of diverse populations with greater consequences than expected. While HbE by itself presents as a mild anemia and a single gene for β-thalassemia is not serious, it remains unexplained why HbE/β-thalassemia (HbE/β-thal) is a grave disease with high morbidity and mortality. Patients often exhibit defective physical development, severe chronic anemia, and often die of cardiovascular disease and severe infections. Recent Advances: This article presents an overview of HbE/β-thal disease with an emphasis on new findings pointing to pathophysiological mechanisms derived from and initiated by the dysfunctional property of HbE as a reduced nitrite reductase concomitant with excess α-chains exacerbating unstable HbE, leading to a combination of nitric oxide imbalance, oxidative stress, and proinflammatory events. Additionally, we present new therapeutic strategies that are based on the emerging molecular-level understanding of the pathophysiology of this and other hemoglobinopathies. These strategies are designed to short-circuit the inflammatory cascade leading to devastating chronic morbidity and fatal consequences. Antioxid. Redox Signal. 26, 794-813.

Hepatic Arginase - Nitric oxide imbalance: Impact of carcinogenesis and therapeutic effect of sodium channel blockage in an in vivo rat model

Abstract Objective: Nitric oxide synthase and arginase are frequently antagonistic and interactive, although both use L-arginine as common substrate. Their balance is of potential functional importance. How the balance changes in cancer is unknown. Increasing evidence suggests that progression of carcinomas involves functional voltage-gated sodium channel (VGSC) activity. Methods: The present study extended this study to liver and aimed to determine whether (i) DMBA carcinogenesis would affect the activities of arginase and NOS and (ii) treatment with Na-channel blocker RS100642 would ameliorate the impact of the carcinogen on the arginase-NOS balance. Results: DMBA application significantly increased arginase activity and, correspondingly, the level of L-ornithine by 25-33%. In contrast, NOS activity decreased by 11%. Importantly, RS100642 treatment completely suppressed the effect on arginase. Conclusion: It is concluded (i) that DMBA carcinogenesis changes the hepatic arginase-NOS balance, increasing the overall dominance of arginase and (ii) that VGSC inhibition has a protective effect on liver.

Genetic Deletion of ACE2 Induces Vascular Dysfunction in C57BL/6 Mice: Role of Nitric Oxide Imbalance and Oxidative Stress

Accumulating evidence indicates that angiotensin-converting enzyme 2 (ACE2) plays a critical role in cardiovascular homeostasis, and its altered expression is associated with major cardiac and vascular disorders. The aim of this study was to evaluate the regulation of vascular function and assess the vascular redox balance in ACE2-deficient (ACE2-/y) animals. Experiments were performed in 20–22 week-old C57BL/6 and ACE2-/y male mice. Evaluation of endothelium-dependent and -independent relaxation revealed an impairment of in vitro and in vivo vascular function in ACE2-/y mice. Drastic reduction in eNOS expression at both protein and mRNA levels, and a decrease in •NO concentrations were observed in aortas of ACE2-/y mice in comparison to controls. Consistently, these mice presented a lower plasma and urine nitrite concentration, confirming reduced •NO availability in ACE2-deficient animals. Lipid peroxidation was significantly increased and superoxide dismutase activity was decreased in aorta homogenates of ACE2-/y mice, indicating impaired antioxidant capacity. Taken together, our data indicate, that ACE2 regulates vascular function by modulating nitric oxide release and oxidative stress. In conclusion, we elucidate mechanisms by which ACE2 is involved in the maintenance of vascular homeostasis. Furthermore, these findings provide insights into the role of the renin-angiotensin system in both vascular and systemic redox balance.

Human Ghrelin: A Gastric Hormone with Cardiovascular Properties.

Ghrelin is a growth hormone-releasing peptide, isolated from the stomach. Researches in progress documented that ghrelin participates in the stimulation of the hypothalamus-pituitary-adrenal axis at the hypothalamic level and in the regulation of energy balance. Growth hormone-independent functions have been ascribed to ghrelin. Among others, a large body of literature demonstrated the presence of specific receptors for ghrelin, distributed at the level of cardiomyocytes and endothelial cells. Therefore, a link between ghrelin and cardiovascular system has been hypothesized and, then, demonstrated in both experimental and clinical studies. Ghrelin has largely documented cardiac beneficial effects, including protection from ischemia/reperfusion injury, attenuation of left ventricular remodeling following myocardial infarction, and improvement of left ventricular function. Exercise level in patients with chronic heart failure had also been seen. Ghrelin exerts these effects through several mechanisms, including the inhibition of apoptosis. At the level of blood vessels, ghrelin exerts a significant impact on vascular function. In particular, acutely infused, ghrelin reverses endothelial dysfunction by increasing NO availability and restores the endothelin-1/nitric oxide imbalance in the peripheral microcirculation of patients with metabolic syndrome. Antioxidant/anti-inflammatory effects, and-or an ameliorated insulin sensitivity are proposed mechanisms whereby ghrelin exerts its vascular protective actions. At higher doses, ghrelin also decreases blood pressure, by mechanisms that involve the modulation of sympathetic nervous system. This finding highlights the ghrelin system as a promising candidate for cardiovascular drug discovery.

Bioelectrical Activity of the Amygdala of Rats under Conditions of Chronic Alcoholism and Imbalance of Nitric Oxide

Evaluation of biopotentials in the region of the amygdala of rats in the combined influence of alcohol and drugs, causing an imbalance of nitric oxide is described in this paper. Chronic alcoholism leads to structural morphological changes in the liver. A study of the amplitude of electrical activity showed that the electrical potential in the amygdala after 8 weeks chronic alcoholism was lower compared with normal rats. The frequency spectrum analysis showed decreased in the 2 times or more absolute spectral powers of all components. The relative spectral powers of its components: δ: θ: α: β was as a 2: 2: 2: 4. The use of sodium nitroprusside (donator of NO) together with the chronic consumption of ethanol reduces the effect of ethanol on low and high-frequence portion the performance of the bioelectrical activities spectrum in the amygdala. The use of non-selective blocker of NO-synthase – nitroarginine aggravated by the braking action of alcohol. The important role of nitric oxide in the development of adaptive-compensatory reactions of the damaged body is described. Our results may be useful to assess the contribution of NO in operation amygdala, conditions of chronic pathologies, and treatment of neuropsychiatric conditions, including diseases caused by alcohol abuse.

Bioelectrical Activity of the Amygdala of Rats under Conditions of Chronic Alcoholism and Imbalance of Nitric Oxide

Evaluation of biopotentials in the region of the amygdala of rats in the combined influence of alcohol and drugs, causing an imbalance of nitric oxide is described in this paper. Chronic alcoholism leads to structural morphological changes in the liver. A study of the amplitude of electrical activity showed that the electrical potential in the amygdala after 8 weeks chronic alcoholism was lower compared with normal rats. The frequency spectrum analysis showed decreased in the 2 times or more absolute spectral powers of all components. The relative spectral powers of its components: δ: θ: α: β was as a 2: 2: 2: 4. The use of sodium nitroprusside (donator of NO) together with the chronic consumption of ethanol reduces the effect of ethanol on low and high-frequence portion the performance of the bioelectrical activities spectrum in the amygdala. The use of non-selective blocker of NO-synthase – nitroarginine aggravated by the braking action of alcohol. The important role of nitric oxide in the development of adaptive-compensatory reactions of the damaged body is described. Our results may be useful to assess the contribution of NO in operation amygdala, conditions of chronic pathologies, and treatment of neuropsychiatric conditions, including diseases caused by alcohol abuse.

Mechanisms of enhanced vasoconstriction in the mouse model of atherosclerosis: the beneficial effects of sildenafil.

Sildenafil ameliorates aortic relaxations in apolipoprotein E knockout (apoE) mice. Now, we tested the hypothesis that endothelial dysfunction (ED) in this model is characterized by contractile hyperresponsiveness to phenylephrine (PE) and that this abnormality may be repaired using sildenafil. The aortic rings were evaluated in apoE mice treated with sildenafil (apoE-sil, 40 mg/kg/day) and compared with apoE and wild-type (WT) mice administered with vehicle (veh). The apoE-veh mice exhibited an imbalance of nitric oxide and reactive oxygen species (NO/ROS) levels and an increased maximum response (Rmax, 20%) and sensitivity (7%) to PE, which were not modified by endothelial removal. Under the prostanoids blockade, vasocontraction was decreased more in apoE-veh (-37%) than in WT (-27%) and apoE-sil (-30%) mice. NADPH-oxidase blockade abolished the enhanced contractile responsiveness in apoE-veh (-33%), without effects in WT and apoE-sil groups. The atherosclerotic lesions and the imbalance of NO/ROS were reduced (40%) in apoE-sil mice. In conclusion, ED in apoE mice was characterized by decreased NO-bioavailability and contractile hyperresponsiveness, due to thromboxane and oxidative stress, and was normalized by sildenafil. The beneficial effects of this phosphodiesterase-5 inhibitor on ED and lipid deposition provide new insights for its use as adjuvant in the treatment of atherosclerosis.

Mechanisms of disease: paracine effects of adipose tissue, progenitor cell function, and epigenetics of diabetic vascular disease.

![Graphic][1] Adipose tissue has been recognized as a source of cytokines that contributes to chronic inflammation in patients with metabolic syndrome and diabetes.1,2 Furthermore, perivascular epicardial adipose tissue has been related to cardiovascular risk factors and coronary artery calcification,3 as well as to the severity of coronary artery disease.4 Endothelial dysfunction is an early stage of atherosclerosis,5 and hence it is of interest to see how perivascular adipose tissue interferes with the control of vascular tone. The first paper in this issue, entitled ‘Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue’ 6 by Agostino Virdis from the University of Pisa in Italy assessed the impact of the cytokine tumour necrosis factor-alpha (TNF-α) produced by vascular and perivascular tissue on the endothelin-1 (ET-1)7 and nitric oxide (NO) system,8 which are both altered in many disease states. To that end, small arteries from 16 obese subjects and 14 controls were mounted in a pressurized myograph, and endogenous ET-1 activity was assessed by adding the ETA blocker BQ-123. The contributions of TNF-α and NO were tested using the anti-TNF-α antibody infliximab and the inhibitor of the l-arginine pathway l-NAME ( N ω-nitro-l-arginine methylester). Obese subjects showed a blunted l-NAME-induced vasoconstriction (reflecting a reduced basal NO release), which was augmented by infliximab, while the relaxation to BQ-123 (reflecting basal ET-1 production) was attenuated by infliximab but unaffected by l-NAME. Interestingly, removal of perivascular adipose tissue reversed these effects. Obese subjects also produced more superoxide, which was decreased by the NADPH oxidase inhibitor apocynin, l-NAME, and BQ-123, and abolished by infliximab. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular and … [1]: /embed/inline-graphic-1.gif

Studying nitrosative stress in Parkinson's disease.

Parkinson's disease (PD) is marked by a selective degeneration of dopaminergic neurons in the brain stem and it is the second most common neurodegenerative disorder. The pathogenic mechanism of PD is not completely known but it is believed that oxidative stress involving the imbalance of nitric oxide (NO) signaling is involved. Recent studies have suggested that NO, through the modification of protein's cysteine residues can contribute to the pathogenesis of PD. This NO modification, designated as S-nitrosylation, is emerging as an important signaling mechanism that regulates increasing number of cellular processes such as vesicle trafficking, receptor mediated signal transduction, gene transcription, and cell death. In our studies, we found that increased nitrosative stress promotes the S-nitrosylation of neuroprotective proteins and compromises their function which contributes to the development of PD. One of the obstacles in studying S-nitrosylation signaling is how to detect this modification in biological samples. Here, two simple and commonly used methods in detecting S-nitrosylated proteins are introduced for the study of this NO signaling mechanism.

Townes Transgenic Sickle Cell Mouse Model Displays Erectile Dysfunction

Introduction: Erectile function alterations result from an imbalance of nitric oxide (NO)-mediated relaxations and sympathetic-mediated vasoconstriction in the erectile tissue. The degree of contraction of corpus cavernosum (CC) smooth muscle determines the functional state of penile flaccidity, tumescence, erection, or detumescence. Patients with sickle-cell disease (SCD) display alterations in erectile function. Priapism is most frequently reported and untreated acute ischaemic priapism results in erectile dysfunction (ED). Previous studies have shown that patients with ED exhibit priapic activity, however, ED unassociated with priapism is still poorly investigated. Transgenic sickle cell mice have been employed to better understand the complex pathophysiology of SCD. The Berkeley murine model displays features of priapism and is associated with an upregulation of the NO-cGMP signaling pathway in the cavernosal tissue, reflecting in an uncontrolled erectile response. Townes mice express human sickle hemoglobin and exhibit the major features found in humans with SCD. However, no detailed study has investigated the pathophysiological alterations of corpus cavernosum (CC) in Townes SCD mice. Thus, the aim of this study was to characterize the erectile function in these animals, focusing on the role of the NO signaling pathway and contractile machinery.Methods: Townes transgenic sickle cell mice and C57BL/6 mice (control) aged 3 to 4 months-old were used (Wu et al Blood 2006). The intracavernous pressure (ICP) was assessed following electrical stimulation of cavernous nerve in anaesthetized mice. In separate protocols, strips of CC were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent responses) and sodium nitroprusside (SNP; endothelium-independent responses), as well as electrical-field stimulation (EFS; nitrergic relaxations) were obtained in cavernosal strips precontracted with the α1-adrenergic receptor agonist phenylephrine (3-10 µM). Contracting responses to phenylephrine and EFS were also obtained in both control and SCD mice.Results: The cavernous nervous stimulation caused frequency-dependent increases of ICP in control and SCD groups. However, ICP was 37% lower in SCD mice compared to the control group (P < 0.05). Phenylephrine (0.01 – 100 µM) induced concentration-dependent CC contractions in both control and SCD mice, but maximal contractile responses were significantly greater (P < 0.05) in SCD compared to control mice (1.32 ± 0.11 and 0.80 ± 0.04 mN, respectively). Likewise, EFS-induced neurogenic CC contractions in SCD mice were 50% higher (P < 0.05) compared to the control. The cumulative addition of ACh (0.001 – 10 µM) produced concentration-dependent CC relaxations in both groups, but maximal relaxations were significantly higher in SCD (78 ± 6%; P < 0.05) compared to control mice (50 ± 4%). Similarly, SNP (0.01 – 10 µM) produced concentration-dependent CC relaxations, but, again, the maximal relaxations elicited by this agent were significantly higher in SCD (96 ± 3; P < 0.05) compared to control mice (77 ± 5 %; n=9). The nitrergic relaxations induced by EFS were also significantly higher (P < 0.001) in SCD mice compared to control mice (8 Hz: 90 ± 6 and 65 ± 4 %, respectively; n=5).Conclusion: Our study shows that this type of SCD mouse exhibits enhanced α1-adrenoceptor-mediated vasoconstriction and erectile dysfunction. Interestingly, however, NO-mediated CC relaxations are greater in the SCD mice. It is likely, therefore, that CC vasoconstriction in SCD overcomes the NO-dependent erectile stimulus, making penile tumescence more difficult to occur. These results are in contrast with the data from the Berkeley SCD mice, which indicate exaggerated in vivo erectile responses. The reason to this difference among the two SCD models is not clear. Taken together our results indicate that ED, unassociated with priapism, as seen in the Townes mouse, should be investigated in SCD patients.Financial Support: FAPESP/CNPq. DisclosuresNo relevant conflicts of interest to declare.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue

We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia

ObjectivesEnhanced external counterpulsation (EECP) could improve endothelium-dependent vasodilatation of carotid artery and restore imbalance of nitric oxide and endothein-1 in patients with coronary artery disease. Our study was designed to test the hypothesis that long-term EECP may protect vascular endothelial cells from apoptosis by modifying apoptosis-related gene expression. MethodsEighteen male Yorkshire pigs were randomly assigned to three groups: usual diet (Normal), high cholesterol diet (HC) and high cholesterol diet plus EECP (HC+EECP). Vascular endothelial cells were isolated from the aortic endothelium and identified by CD31 staining and DiI-Ac-LDL reaction. Morphological changes were observed by both scanning and transmission electronic microscopes. TUNEL technique was applied to detect the apoptotic index of vascular endothelial cells. Two genes, Apaf-1 and BIRC2, were chosen for exploring the potential mechanisms of action at the molecular level. ResultsEECP brought a certain degree of alleviation from ultrastructural changes such as shrinking and blebbing of cytomembrane, marginalization, degeneration, and fragmentation of the nucleus. EECP also significantly reduced apoptotic indices while compared with that of control (177±12‰ vs. 237±23‰, P<0.05). The Apaf-1 expression at both protein and mRNA level in pigs of HC+EECP group was significantly decreased than those of the HC group (P<0.05), whereas the BIRC2 expression was significantly enhanced after EECP treatment, documented by immunostaining and semi-quantitative RT-PCR analysis, respectively (P<0.05). ConclusionsEECP could protect vascular endothelial cells from apoptosis, thereby delaying the progression of early atherosclerotic lesions possibly through transcriptional down-regulation of pro-apoptotic gene Apaf-1, and up-regulation of anti-apoptotic gene BIRC2.