Endothelium-derived Relaxing Factor Research Articles

Developmental conditioning of endothelium-derived hyperpolarizing factor-mediated vasorelaxation.

Objectives:The endothelium maintains vascular homeostasis through the release of endothelium-derived relaxing factors (EDRF) and endothelium-derived hyperpolarization (EDH). The balance in EDH : EDRF is disturbed in cardiovascular disease and may also be susceptible to developmental conditioning through exposure to an adverse uterine environment to predispose to later risk of hypertension and vascular disease.Methods:Developmentally conditioned changes in EDH : EDRF signalling pathways were investigated in cremaster arterioles (18–32 μm diameter) and third-order mesenteric arteries of adult male mice offspring of dams fed either a fat-rich (high fat, HF, 45% energy from fat) or control (C, 10% energy from fat) diet. After weaning, offspring either continued on high fat or were placed on control diets to give four dietary groups (C/C, HF/C, C/HF, and HF/HF) and studied at 15 weeks of age.Results:EDH via intermediate (IKCa) and small (SKca) conductance calcium-activated potassium channels contributed less than 10% to arteriolar acetylcholine-induced relaxation in in-situ conditioned HF/C offspring compared with ∼60% in C/C (P < 0.01). The conditioned reduction in EDH signalling in HF/C offspring was reversed in offspring exposed to a high-fat diet both before and after weaning (HF/HF, 55%, P < 0.01 vs. HF/C). EDH signalling was unaffected in arterioles from C/HF offspring. The changes in EDH : EDRF were associated with altered endothelial cell expression and localization of IKCa channels.Conclusion:This is the first evidence that EDH-mediated microvascular relaxation is susceptible to an adverse developmental environment through down-regulation of the IKCa signalling pathway. Conditioned offspring exposed to a ‘second hit’ (HF/HF) exhibit adaptive vascular mechanisms to preserve dilator function.

Endothelial dysfunction and vascular disease - a 30th anniversary update.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries.

12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity. Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3 , trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK). All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3 , C3 and hepoxilin A3 (3μm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9±3.2, 29.7±4.6, 82.2±5.0 and 88.0±2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3 , C3 and hepoxilin A3 (10μm) inhibited U46619 (10nM)-induced increases in intracellular calcium by 53.0±7.2%, 32.8±5.0% and 37.9±13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

Diffuse coronary vasospasm in a patient with hypothyroidism

Coronary vasospasm is an important etiology of ischemic heart disease. Sometimes thyroid disorders may be associated with cardiovascular disease. The major cardiovascular changes that occur in hypothyroidism include a decrease in cardiac output and cardiac contractility, a reduction in heart rate, and an increase in peripheral vascular resistance. There are also significant changes in modifiable atherosclerotic risk factors, including hypercholesterolemia, diastolic hypertension, carotid intimal media thickness, and endothelial derived relaxation factor (nitric oxide), which accompany overt hypothyroidism. Here, we present a case of diffuse coronary arterial vasospasm in a patient with hypothyroidism.

In Search of a New Screening Test for Osteoarthritis - Is Urinary Nitrate the Answer?

Introduction: Even as early as 1916 it was suggested that the nitric oxide is synthetized in the mammals. This nitric oxide, synthetized in the body, was responsible for vasodilation by the endothelium derived relaxing factors and also by the stimulation of the guanylyl cyclase. Inflammatory conditions increase the production of nitrates and nitrites which is seen in the response to endotoxin and is accompanied with nitrosamine formation. Nitric oxide, hence, has been thought to be the physiological messenger and is increases in the pathological processes. This present study was conducted to find the association between the urinary secretion of nitrates and the osteoarthritis. Design: A total of 200 patients coming to the hospital out of 100 were patients with osteoarthritis of the knee joint and 100 were randomly selected. All the patients were screened for the causes of secondary osteoarthritis. Urine sample from all the patients was obtained along with X-ray of bilateral knees was taken to assess the grade of osteoarthritis. The osteoarthritic patients were assigned Group A and the randomly chosen patients were assigned the Group B. Both groups were compared and the results were evaluated. Results: In group A there were 5 cases with urinary nitrate positive as compared to 4 cases with positive urinary nitrate in group B, which was not statistically significant (p > 0.05). Conclusion: The current study clearly shows that there is no association between the urinary nitrate levels and OA.

Endothelium-Dependent Vasorelaxant Effects of Dealcoholized Wine Powder of Wild Grape (Vitis coignetiae) in the Rat Thoracic Aorta.

The vasorelaxant effects of dealcoholized wild grape (Vitis coignetiae) wine were investigated with isolated rat thoracic aorta. In our present study, we demonstrate that wild grape wine powder (WGWP) induced relaxation of aortic rings preconstricted with norepinephrine in a dose-dependent manner (at concentrations ranging from 0.1 to 1 mg/mL). The vasorelaxant effect of WGWP was dependent on intact endothelia, which was attenuated by incubation with inhibitors of endothelium-derived relaxing factors, such as NG-nitro-L-arginine (nitric oxide synthase inhibitor), methylene blue (guanylate cyclase inhibitor), and indomethacin (cyclooxygenase inhibitor). Moreover, treatment with WGWP and atropine (muscarinic receptor antagonist) or diphenylhydramine (histamine receptor antagonist) significantly inhibited endothelium-dependent vasorelaxation. Our results suggest that WGWP induces relaxation in rat aortic rings in an endothelium-dependent manner. Results further indicate that this effect occurs via nitric oxide-cGMP pathway and prostacyclin-cAMP pathway through a muscarinic receptor and histamine receptor.

The Role of Endothelium-Derived Relaxing Factor in The Regulation of SNP Induced Vasorelaxation: Sodium-Potassium-Chloride Cotransporter

Vascular endothelium plays a key role in the local regulation of vascular tone and vascular architecture. However, it is unclear whether the combination of NKCC cotransporter with endothelial mediated factor and potassium channel in rat aortic ring contributes in regulation of the vascular activity. In the current study, the potential role of endothelium-mediated relaxing factors and potassium channel in SNP induced vasorelaxation in precontracted isolated rat aortic rings in the presence of Bumetanide as NKCC blocker were investigated. The maximum vasorelaxation induced by SNP significantly blocked by the presence BUM. SNP induced relaxation was affected by combination of BUM with Indomethacin, Clotrimazole. TEA and BaCl2 significantly blocked of SNP induced relaxation in aorta preincubated with all blockers used, but BaCl2 showed a more potent effect as compared with others. These results indicate that SNP – induced vasorelaxation was mediated via EETs, Prostaglandin, BKCa and KIR pathways which ultimately enhanced the contribution of chloride transporter in aortic smooth muscle cells hyperpolarization and subsequent relaxation.

Nitric Oxide: A Regulator of Cellular Function in Health and Disease

Nitric oxide (NO) is a gaseous messenger molecule synthesized from L-arginine and molecular oxygen by three different NO synthases, that is, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) form [1]. Since its discovery in the early 1980s by the three Nobel Laureates Furchgott, Ignarro & Murad [2], NO has been widely recognised as an important signalling molecule in many physiological processes. The initial identification of NO as an endothelium-derived relaxing factor (EDRF) [3] generated great interest in its function in vascular biology. Over the following years, however, the focus on NO research rapidly expanded from the vascular system to its role in immunity and inflammation, the nervous system, pregnancy, aging, and cell death. Many studies suggest that excessive or abnormal production of NO plays a crucial role in neuronal cell death associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, as well as various conditions of vascular dysfunction. At physiological levels, NO is essential for neuronal function, differentiation and survival through activation of signalling pathways that include the cyclic guanosine monophosphate (cGMP)/soluble guanylyl cyclase pathway [4] and S-nitrosylation, in which NO reversibly binds to thiol groups of proteins [5]. The vast network of NO-associated signalling paradigms further includes acetylation/deacetylation and methylation/demethylation modifications and peroxynitrite formation (leading to nitrotyrosination of protein residues), as well as modulation of gene expression via epigenetic changes [5–8]. How controlled S-nitrosylation/nitrotyrosination of proteins and activation of the NO/cGMP signalling pathway promote cellular survival and induce epigenetic changes while uncontrolled signalling promotes cell death and dysfunction remains to be elucidated. The aim of this Special Issue is to gather information encapsulating the above signalling pathways. The articles published in this Special Issue largely cover (1) NO signalling in neuronal function and disease as well as (2) vascular targets in endothelial function and dysfunction, both of which involve the broad range of actions of this signalling molecule. In order to understand the contribution of NO to neuronal dysfunction, one has to consider that NO is a crucial molecule in cellular physiology. Numerous studies show the involvement of NO in neuronal development, plasticity, excitability, and transmission [8–12]. However, the common mechanisms across several neurodegenerative disorders relate to the neurotoxicity of NO and its downstream reactive nitrogen species (RNS). Enhanced nitrotyrosine immunoreactivity is evident in brains from patients with Alzheimer's and Parkinson's disease. Nitrated proteins are associated with β-amyloid deposition and nitrotyrosination of Tau protein and synaptophysin has been reported in brain samples from patients with Alzheimer's disease. The potential downstream signalling pathways of these posttranslational modifications are discussed in the review by S. A. Bradley and J. R. Steinert in this Issue. The cellular roles of NO in neurodegenerative disease, such as Alzheimer's, are reviewed by R. Balez and L. Ooi with a focus on neurotoxicity versus neuroprotection while the role of aberrant NO signalling in neurodevelopmental disorders such as Fragile X syndrome is investigated in the study by E. Lima-Cabello et al. Other topics of this issue focus on aspects of vascular NO signalling with particular interest in fetoplacental dysfunction caused by abnormal eNOS regulation (as discussed by A. Leiva et al.). Endothelial function and eNOS regulation are essential for healthy cardiovascular responses but are also critical for adaptations during pregnancy. Several diseases associated with vascular dysfunction such as atherosclerosis, diabetes mellitus, hypertension, or preeclampsia involve altered NO signalling [13–15]. The paper by A. Leiva et al. reviews the mechanisms leading to abnormal NO signalling, which include reduced bioavailability of L-arginine (NOS substrate) and tetrahydrobiopterin (BH4), abnormal calcium-calmodulin signalling, and activation and inhibition of eNOS activity through phosphorylation of Ser1177 or Thr495, respectively [16]. NO has also been reported to play a crucial role in the transition of fetoplacental endothelial cells from a mitogenic to a metabolic phenotype in the macrocirculation, compared with a change from a metabolic to a mitogenic phenotype in the microcirculation in response to insulin in gestational diabetes mellitus [17]. Thus, the actions of NO are not only important during vascular adaptations to pregnancy and related dysfunctions but also essential during vascular signalling induced by physical training resulting in elevated nitrite and nitrate levels as reported in the study by A. M. Jacomini et al. Together, this Special Issue highlights the tremendous diversity of NO signalling pathways with regard to its function in health and disease. All contributing publications have emphasised that NO represents an important signalling molecule and future research required in this field will extend the understanding of the broad actions of NO. Luis Sobrevia Lezanne Ooi Scott Ryan Joern R. Steinert

“Endothelial Protector Drugs” and Diabetes: Is there a Role for these Drugs?

Diabetic vasculopathy, including macro and micro vascular disorders, is the leading cause of morbidity and mortality in patients with type 1 (T1) and type 2 (T2) diabetes mellitus (DM) [1]. A lot of researches pointed out that endothelial dysfunction, characterized by an imbalance between Endothelium-Derived Relaxing Factors (EDRFs) and endothelium-derived contracting factors (EDCFs) play a central role on the development and progression of diabetic vasculopathy [2-5]. Endothelial dysfunction and inflammation, as indicated by abnormal flow-dependent vasodilatation and by increased circulating levels of adhesion molecules (ICAM-1 and E-selectin) are known to occur in T2DM and seems to be an important predictor in systemic atherogenesis [6]. Both hyperglycemia and insulin administration increasing circulating levels of endothelin-1 (ET-1), an endothelial cell (EC)-derived potent vasoconstrictor peptide with mitogenic, pro-oxidative and pro-inflammatory properties that have shown to be extremely relevant to the pathophysiology of diabetic vasculopathy [7-10]. Circulating and local levels of ET-1 are increased in diabetic animal models and diabetic patients [1,11,12].

Development of nitric oxide catalytic coatings by conjugating 3,3-disulfodipropionic acid and 3,3-diselenodipropionic acid for improving hemocompatibility.

Nitric oxide (NO), discovered as an endothelium-derived relaxing factor, has been found to have multiple intracellular effects in vascular diseases including vasorelaxation regulation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet activation. In the work described here, the authors have developed a NO-catalytic bioactive coating for improving hemocompatibility. The authors first prepared a dopamine and hexamethylendiamine (PDAM/HD) amine-rich adherent copolymer coating to introduce amine groups onto 316L stainless steel, followed by covalently conjugating 3,3-disulfodipropionic acid (S-S) and 3,3-diselenodipropionic acid (Se-Se), which mimic glutathione peroxidase-like catalytic production of NO. S-S and Se-Se were immobilized on the PDAM/HD surface via carbodiimide coupling chemistry. X-ray photoelectron spectroscopy analysis revealed clear S2p and Se3d signals, confirming the immobilization of S-S and Se-Se on the PDAM/HD surface. The NO release behavior of different samples was investigated. In detail, two species of thionitrites (RSNO), S-nitrosoglutathione (GSNO, endogenous NO donors) and S-nitrosoacetylpenicillamine (SNAP) were chosen as NO donors to investigate the NO catalytic properties of S-S and Se-Se modified PDAM/HD surfaces. Not only Se-Se@PDAM/HD but also S-S@PDAM/HD coatings showed the ability to continuously catalyze RSNO to generate NO in the presence of proper thiol reducing agent. For the Se-Se@PDAM/HD coating, the NO release amount and rate were greater than S-S@PDAM/HD in both GSNO and SNAP conditions. The results showed that organosulfide species possesses NO catalytic ability as well as organoselenium species. The authors demonstrated that both S-S@PDAM/HD and Se-Se@PDAM/HD coatings exhibited outstanding inhibition effect on platelet adhesion, aggregation and activation via the cyclic guanylate monophosphate signal pathway. Thus these results suggested that NO catalytic coatings based on organoselenium and organosulfide species immobilization can help to improve hemocompatibility. NO-catalytic strategies possess huge potential applications in blood-contacting devices.

Abstract 109: Protective Effects of Nuclear Factor E2-related Factor 2 Activation on Angiotensin II Induced Microvascular Endothelial Dysfunction

Background: Nuclear factor E2-related factor 2 (Nrf2) is a regulator of the cellular adaptive response to oxidative stress, but its effects on microvascular function are poorly characterized. Our previous studies in human glomerular endothelial cells found that tert-butylhydroquinone (tBHQ; a Nrf2 activator) reduced ROS, asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor) and increased eNOS. We hypothesized that tBHQ would prevent microvascular endothelial dysfunction via activation of Nrf2 / antioxidant responses in angiotensin II (ANG II) induced hypertension. Methods: Mesenteric resistance arterioles (MRAs) were isolated from mice infused for 14 days with ANG II (400 ng/kg/min) or vehicle and given oral tBHQ (0.1% of water) or vehicle (n=6 mice/group). Acetylcholine-induced endothelium dependent relaxation (EDR), endothelial derived relaxation factor (EDRF) and contracting factor (EDCF) of MRAs were assessed by myograph. Vascular nitric oxide (NO), and ROS were assessed by RatioMaster. ROS biomarkers (urinary excretion of malondialdehyde [MDA]) and 8-isoprotane [8-Iso]), ADMA and protein expressions of MRAs were measured. Results: tBHQ prevented the effects (all P&lt;0.05) of ANG II infusion-induced the increases of urinary MDA (50±3 vs 83±11 nmol/mg creatine) and 8-Iso (1.4±0.1 vs 3.8±0.3 vs ng/mg creatinine) and decreases of EDR (75 ± 3 vs 53±5%), EDRF (23± 2 vs 17± 3%) and NO (0.3 ± 0.02 vs 0.2 ± 0.03 units), and enhanced EDCF (7 ± 1 vs 13 ± 2%) and associated ROS production (0.11 ± 0.03 vs 0.3 ± 0.08 units) and microvascular ADMA (18 ± 1 vs 13 ± 1 nmol/mg protein). ANG II increased the MRAs protein expressions (of β-actin, all P&lt;0.05) for NOX1 (0.60 ± 0.03 vs 0.38± 0.04) and NOX2 (0.80 ± 0.04 vs 0.36± 0.05) and reduced phosphorylated eNOS (0.26 ±0.07 vs 0.53±0.09), whereas tBHQ increased Nrf2 expression (0.44 ±0.03 vs 0.73±0.04) and prevented these changes with ANG II infusion. Conclusions: tBHQ upregulates Nrf2 and thereby engages the antioxidant responses to protect microvessels of ANG II-infused mice from the adverse effectors of ROS, NO deficiency and ADMA. This is a novel potential therapeutic target that activates Nrf2/antioxidant response element to protect against vascular oxidative stress, endothelial dysfunction and CVD.

AB005. The role of serendipity in sexual medicine

The word “serendipity” is considered very difficult to define; it may be considered “A talent for making fortunate discoveries while searching for other things”. The history of sexual medicine teaches us the importance of serendipity along with research. Serendipity in the year 1988 was initiated by a slow elevator. Dr. Jacob Rajfer, Professor of Urology, had attended a meeting on the 2nd floor of the Univ. California LA and was waiting for an elevator to take him to his office on the 6th floor. The elevator was very slow to arrive, so Dr. Rajfer began looking around. There was a sign on a door across from the elevator that read: “Vascular smooth muscle lab”. Dr. Rajfer knew that an unknown substance, Endothelium-derived Relaxing Factor (EDRF) dilated blood vessels by relaxing smooth muscles. A penile erection resulted from vasodilation and the penis was full of vascular smooth muscle. The head of the vascular smooth muscle lab was Dr. Louis Ignarro who had just published that EDRF might be Nitric Oxide (NO). Drs. Rajfer and Ignarro began collaborating, and in 1990 published that ‘NO’ mediates erections: a discovery started by a slow elevator and leading to Dr. Ignarro receiving a Nobel Prize in 1998 and Pfizer scientists developing Viagra! Viagra, which was able to dilate smooth muscle, was being developed by Pfizer researchers to treat angina when increased nocturnal and spontaneous erections were noted. The FDA approved Viagra as the first oral treatment for erectile dysfunction in 1998. Serendipity, research and Viagra impacted how we look at aging and intimacy. The language of sexual medicine changed as well: the old term, ‘impotence’ (lacking in power) was changed to ‘erectile dysfunction’ or ED. Serendipity has played a role in the discovery of more than 24% of all drugs on the market. We would all do well to heed the wise words of Louis Pasteur: “Chance Favors the Prepared Mind”.

Development and therapeutic applications of nitric oxide-releasing materials.

Prior to the truly seismic revelation that nitric oxide (NO) was the elusive endothelium-derived relaxing factor, NO was considered simply a reactive pollutant for the vast majority of the biology and biomedical research community – there was essentially no linkage to actual in vivo functional processes, let alone therapeutic applications. Even the relationship between NO and the therapeutic efficacy of nitroglycerin was at this point still unknown. The abrupt change in the research status of NO, initiated by the monumental discovery of the NO-elusive endothelium-derived relaxing factor connection, heralded a new era of investigation that continues to dramatically expand every year. Research on NO has now expanded to include: mechanisms of physiological and biochemical action; the importance of temporal and site-specific NO generation under both normal and pathological conditions; the nature, properties and synthesis of longer lived NO-related molecules that preserve and extend NO bioactivity; the role of nitrite as a stable source of physiological levels of NO; potential therapeutic targets for NO and other NO-related molecules; food and supplements to enhance levels of NO in the circulation; and development of topical and systemic delivery vehicles for therapeutic levels of NO and NO-releasing molecules. In fact, it is becoming ever more apparent that many of the current drugs being developed to address numerous disease conditions are in effect elegant methods of stimulating NO in the appropriate locations and amount. What is also clear is with every new discovery and shift of the research landscape, new NO-related questions emerge at an equally steady pace. Issues seemingly as fundamental as to whether NO promotes or inhibits cancer growth remain controversial. Limitations in addressing not only basic questions relating to how NO participates in both physiological and pathophysiological processes but also how to utilize NO therapeutically arise from an inability to appropriately and effectively deliver and monitor NO in vivo. This issue is an attempt to update overviews of where we are with respect to several of these rapidly evolving NO associated foci of research with an emphasis on delivery and detection strategies. We are fully cognizant that with the level of activity and the rate at which new insights are emerging, we can at best provide but a transient depiction that represents at least a breath catching pause for researchers to take stock of what has been accomplished and where we might be headed. The contributors to this effort are all at the cutting edge of NO research and represent a cohesive effort to encompass many of the important issues that pertain to the ultimate biomedical objective of harnessing the extraordinary therapeutic potential of this seemingly simple molecule.

Constrictor prostanoids and uridine adenosine tetraphosphate: vascular mediators and therapeutic targets in hypertension and diabetes.

Vascular dysfunction plays a pivotal role in the development of systemic complications associated with arterial hypertension and diabetes. The endothelium, or more specifically, various factors derived from endothelial cells tightly regulate vascular function, including vascular tone. In physiological conditions, there is a balance between endothelium-derived factors, that is, relaxing factors (endothelium-derived relaxing factors; EDRFs) and contracting factors (endothelium-derived contracting factors; EDCFs), which mediate vascular homeostasis. However, in disease states, such as diabetes and arterial hypertension, there is an imbalance between EDRF and EDCF, with a reduction of EDRF signalling and an increase of EDCF signalling. Among EDCFs, COX-derived vasoconstrictor prostanoids play an important role in the development of vascular dysfunction associated with hypertension and diabetes. Moreover, uridine adenosine tetraphosphate (Up4 A), identified as an EDCF in 2005, also modulates vascular function. However, the role of Up4 A in hypertension- and diabetes-associated vascular dysfunction is unclear. In the present review, we focused on experimental and clinical evidence that implicate these two EDCFs (vasoconstrictor prostanoids and Up4 A) in vascular dysfunction associated with hypertension and diabetes.

Abstract 642: Wy14643 and Fenofibrate Reduce Acetylcholine-induced Contractions in Spontaneously Hypertensive Rat Aortae

Introduction and hypothesis: Hypertension is associated with endothelial dysfunction, which is defined as an imbalanced release of endothelium-derived relaxing factors (EDRF) and endothelium-derived contracting factors (EDCF). Oxidative stress contributes to endothelial dysfunction by increasing the production of EDCF. Research studies have demonstrated that peroxisome proliferator-activated receptor alpha agonists, Wy14643 (WY) and fenofibrate (FF), improve endothelial function. Therefore, we assessed the hypothesis that WY and FF inhibited acetylcholine-induced endothelium-dependent contractions through reduction of oxidative stress in aortae of spontaneously hypertensive rats (SHR). Method: Thoracic aortic rings, with and without endothelium, were isolated from 40-44 weeks old male SHR and their normotensive counterparts, Wistar Kyoto rats (WKY). They were suspended in organ chambers for isometric tension recording. Responses to the endothelium-dependent vasoactive agent, acetylcholine (10 -8 - 10 -4 M), and to the oxidizing agent, hydrogen peroxide (10 -8 - 10 -4 M), were examined in the presence of N ω -nitro-L-arginine methyl ester (L-NAME, 3*10 -4 M; nitric oxide synthase inhibitor). Results: Acetylcholine caused significantly greater contractions in aortic rings, with endothelium, of SHR than in those of WKY. The contraction was significantly inhibited by the combination of Tiron (10 -3 M; intracellular superoxide anion scavenger) and DETCA (10 -4 M; superoxide dismutase inhibitor), suggesting that the reactive oxygen species, hydrogen peroxide, was involved in vascular dysfunction in hypertension. Both WY (10 -5 M) and FF (10 -5 M) significantly inhibited contractions to acetylcholine in SHR aorta, suggesting that they may reduce the amount of hydrogen peroxide or inhibit its downstream signaling. Moreover, they inhibited contractions induced by hydrogen peroxide in SHR aortic rings with, but not in those without, endothelium, indicating that their inhibitory effect was endothelium-dependent. Conclusion: The peroxisome proliferator-activated receptor alpha agonists act on the endothelium to reduce acetylcholine-induced contractions that are mediated by hydrogen peroxide in aortae of hypertensive rats.

Nitric oxide influences potassium currents in inner hair cells isolated from guinea-pig cochlea.

Nitric oxide (NO) is a diffusible second messenger, which regulates neurotransmission, serving as the principal endothelium-derived relaxing factor. NO also acts as an ion channel modulator. Nitric oxide synthase (NOS) has been identified in the inner ear, although its physiological role remains unclear. In the present study, the effects of NO onto K currents in inner hair cells (IHCs) were investigated. IHCs were acutely isolated and K currents were recorded by conventional whole-cell voltage-clamp recordings. NO donors sodium nitroprusside (SNP) were applied directly to the cells. In 1mM SNP solutions, the amplitude of outward K currents (IK,f and IK,s) reversely decreased; however, fast activation kinetics was preserved. In the current-voltage relationship curves, the maximal slope conductances were 53.2nS and 44.2nS in control solutions and 1mM SNP solutions, respectively. At the membrane potential of +110mV, the amplitudes of outward currents were 9.2±2.9nA in control solutions and 7.3±2.7nA in 1mM SNP solutions, showing a significant difference. NO acts as a K channel modulator in IHCs. A fast K current suppression may account for the high-frequency hearing impairment by the prevention of fast repolarization.

The Role of Endothelial Dysfunction in the Pathogenesis of Vascular Complications of Diabetes Mellitus - A High Priority Area of Investigation

Abstract Endothelium, the inner layer of the vasculature, represents the interface between blood and organ systems and it is active in the process of contraction and relaxation of vascular smooth muscle and in functions like secretion of vasoactive substances. Endothelial dysfunction is an important cause of cardiovascular disease. The function of the endothelium can be assessed by invasive and noninvasive methods. Endothelial cells produce vasoactive substances like endothelium derived relaxing factor, prostacyclin, nitric oxide, and endothelium derived hyperpolarizing factor. Diabetes mellitus is associated with an increased risk of cardiovascular diseases. Hyperglycemia leads to cardiovascular damage through different pathways, including the polyol and hexosamine pathways, generation of advanced glycation end products, and activation of protein kinase C. Together with hyperglycemia induced mitochondrial dysfunction and endoplasmic reticulum stress, all these can promote the accumulation of reactive oxygen species. The oxidative stress induced by hyperglycemia promotes endothelial dysfunction with an important role in micro and macro vascular disease. Insulin-resistance could be independently predictive of cardiovascular disease. Life style modification and pharmacotherapy could possibly ameliorate the effect of insulin resistance

Bone Marrow Mesenchymal Stem Cells Alleviate Extracellular Kynurenine Levels, as Detected by High-Performance Liquid Chromatography.

Endothelial cell dysfunction plays an important role in the occurrence and development of sepsis, which is a consequence of the interaction between coagulation and inflammation. Kynurenine (KYN) is an endothelium-derived relaxing factor that makes a large contribution to sepsis pathophysiology. In this study, we investigated the influence of bone marrow mesenchymal stem cells (BMSCs) on KYN production by cultured endothelial cells. KYN and tryptophan (TRP) concentrations in cell supernatants were simultaneously measured with a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector (FLD) and an ultraviolet detector (UVD). Our results revealed that lipopolysaccharide-stimulated endothelial cells produced more KYN, which was accompanied by a parallel decrease in TRP. When co-cultured with BMSCs, KYN and TRP production were significantly decreased compared to lipopolysaccharide (LPS)-induction alone. Our results suggest that BMSCs can attenuate endothelial cell damage by decreasing KYN as detected with HPLC. This method is the first to be capable of capturing functional changes in the cells and is simple, fast, and suitable for cellular level research purposes.

Molecular Basis for Mercury-Induced Alteration in Endothelial Function: NO and its Modulators

Epidemiological and animal studies have suggested a strong association between the environmental and occupational exposure to mercury and risk of cardiovascular diseases (CVD). One of the triggering factors of CVD is endothelial dysfunction. The endothelium can evoke relaxations and contractions of the underlying smooth muscle, by releasing vasoactive agents. Nitric oxide (NO), formed by endothelial NO synthase (eNOS), is the best characterized endothelium derived relaxing factor (EDRF). The release of NO is down regulated/upregulated by factors like oxidative stress, estrogen and diseases like diabetes and hypercholelestrolemia, etc. The inhibition/ activation of eNOS by mercury, affecting the NO release is one of the proposed mechanisms for mercury-induced vascular diseases. In addition, during exposure to mercury, overproduction of reactive oxygen species (ROS) can occur, resulting in oxidative stress, which is another major risk factor for endothelial dysfunction. In this article, molecular basis for mercury-mediated alteration in endothelium derived vasodilator (NO) and factors modulating the release of NO are being reviewed.

H2S and Blood Vessels: An Overview.

The physiological and biomedical importance of hydrogen sulfide (H2S) has been fully recognized in the cardiovascular system as well as in the rest of the body. In blood vessels, cystathionine γ-lyase (CSE) is a major H2S-producing enzyme expressed in both smooth muscle and endothelium as well as periadventitial adipose tissues. Regulation of H2S production from CSE is controlled by a complex integration of transcriptional, posttranscriptional, and posttranslational mechanisms in blood vessels. In smooth muscle cells, H2S regulates cell apoptosis, phenotypic switch, relaxation and contraction, and calcification. In endothelial cells, H2S controls cell proliferation, cellular senescence, oxidative stress, inflammation, etc. H2S interacts with nitric oxide and acts as an endothelium-derived relaxing factor and an endothelium-derived hyperpolarizing factor. H2S generated from periadventitial adipose tissues acts as an adipocyte-derived relaxing factor and modulates the vascular tone. Extensive evidence has demonstrated the beneficial roles of the CSE/H2S system in various blood vessel diseases, such as hypertension, atherosclerosis, and aortic aneurysm. The important roles signaling in the cardiovascular system merit further intensive and extensive investigation. H2S-releasing agents and CSE activators will find their great applications in the prevention and treatment of blood vessel-related disorders.