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This study aims to investigate the role of shear stress in cellular remodeling and angiogenesis with relation to nitric oxide (NO). We observed a 2-fold increase in endothelial cell (EC) migration in relation to actin re-arrangements under 15 dyne/cm(2) shear stress. Blocking NO production inhibited the migration and ring formation of ECs by 6-fold and 5-fold, respectively under shear stress. eNOS-siRNA knockdown technique also ascertained a 3-fold reduction in shear stress mediated ring formation. In ovo artery ligation model with a half and complete flow block for 30 min showed a reduction of angiogenesis by 50% and 70%, respectively. External stimulation with NO donor showed a 2-fold recovery in angiogenesis under both half and complete flow block conditions. NO intensity clustering studies by using Diaminofluorescein diacetate (DAF-2DA) probed endothelial monolayer depicted pattern-changes in NO distribution and cluster formation of ECs under shear stress. Immunofluorescence and live cell studies revealed an altered sub-cellular localization pattern of eNOS and phospho-eNOS under shear stress. In conclusion, shear-induced angiogenesis is mediated by nitric oxide dependent EC migration.

Since the detection of nitric oxide two and a half decades ago, there has been an incredible boost in endothelial function research, which is fascinating the research community. Physiologically, endothelial cells synthesize a number of vasoactive substances. In particular, several endothelium-derived relaxing factors (EDRFs) have been characterized, whereby nitric oxide is the most important. In humans, endothelial dysfunction is one of the first clinically detectable alterations in the development of atherosclerosis and is characterized by an imbalance in the release of vasoactive substances. Thus, it is the aim of this article to give an overview about endothelial function in humans, to summarize the different possibilities to assess endothelial function in this species, and to give an overview of the role of EDRFs in different cardiovascular diseases.

The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca(2+)] and the opening of endothelial SK(Ca) and IK(Ca) channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K(+) that effluxes through SK(Ca) activates myocytic and endothelial Ba(2+)-sensitive K(IR) channels leading to myocyte hyperpolarisation. K(+) effluxing through IK(Ca) activates ouabain-sensitive Na(+)/K(+)-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K(+) that effluxes through endothelial K(Ca) channels. During vessel contraction, K(+) efflux through activated myocyte BK(Ca) channels generates intravascular K(+) clouds. These compromise activation of Na(+)/K(+)-ATPases and K(IR) channels by endothelium-derived K(+) and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H(2)O(2) and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BK(Ca) and/or K(ATP), which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.

Background— The aim of this study was to compare the impact of transradial catheterization with hydrophilic-coated catheter sheaths versus uncoated sheaths on NO-mediated endothelial-dependent and -independent vasodilator function. Methods and Results— Thirty-five subjects undergoing transradial catheterization were recruited and assessed before and the day after catheterization. A subgroup was also assessed 3 to 4 months after catheterization. Subjects received hydrophilic-coated sheaths (n=15) or uncoated sheaths (n=20). Radial artery flow-mediated dilatation and endothelium- and NO-dependent arterial dilatation were assessed within the region of sheath placement. Glyceryl trinitrate endothelium-independent NO-mediated function was also assessed. The noncatheterized arm provided an internal control. Flow-mediated dilatation in the catheterized arm decreased from 10.3�3.8% to 5.3�3.3% and 8.1�2.4% to 5.2�3.7% in the coated and uncoated groups, respectively ( P P P P P Conclusions— Placement of a catheter sheath inside the radial artery disrupts vasodilator function, which recovers after 3 months. No differences were evident between hydrophilic-coated and uncoated sheaths.

Nitric oxide (NO) and carbon monoxide (CO) are well established as messenger molecules throughout the body, gasotransmitters, based on striking alterations in mice lacking the appropriate biosynthetic enzymes. Hydrogen sulfide (H(2)S) is even more chemically reactive, but until recently there was little definitive evidence for its physiologic formation. Cystathionine beta-synthase (EC 4.2.1.22), and cystathionine gamma-lyase (CSE; EC 4.4.1.1), also known as cystathionine, can generate H(2)S from cyst(e)ine. Very recent studies with mice lacking these enzymes have established that CSE is responsible for H(2)S formation in the periphery, while in the brain cystathionine beta-synthase is the biosynthetic enzyme. Endothelial-derived relaxing factor activity is reduced 80% in the mesenteric artery of mice with deletion of CSE, establishing H(2)S as a major physiologic endothelial-derived relaxing factor. H(2)S appears to signal predominantly by S-sulfhydrating cysteines in its target proteins, analogous to S-nitrosylation by NO. Whereas S-nitrosylation typically inhibits enzymes, S-sulfhydration activates them. S-nitrosylation basally affects 1-2% of its target proteins, while 10-25% of H(2)S target proteins are S-sulfhydrated. In summary, H(2)S appears to be a physiologic gasotransmitter of comparable importance to NO and carbon monoxide.

Background Peripheral conduit artery endothelium-dependent dilatation decreases with aging in humans. Lactotripeptides (LTPs) and regular exercise can improve endothelium-dependent dilatation, but combining these lifestyle modifications may be more effective than either treatment alone. We conducted a randomized, place-controlled trial with four different intervention arms. Methods A total of 43 postmenopausal women (50-65 years old) were randomly divided into placebo, LTP, exercise and placebo (Ex+placebo), or exercise and LTP (Ex+LTP) groups. LTP or placebo was administered orally for 8 weeks. The exercise groups completed an 8-week moderate aerobic exercise (walking or cycling) intervention. Results There were no statistically significant differences in baseline flow-mediated dilatation (FMD) and most other key dependent variables among the groups. FMD significantly increased in the LTP, Ex+placebo, and Ex+LTP groups whereas no such changes were observed in the placebo control group. The magnitude of increases in FMD was significantly greater in the Ex+LTP group than other intervention groups. Conclusion We concluded that LTP ingestion combined with regular aerobic exercise improves endothelium-dependent dilatation to a greater extent than monotherapy with either intervention alone in postmenopausal women.

The renal pressure-natriuresis mechanism is the dominant controller of body fluid balance and long-term arterial pressure. In recent years, it has become clear that the balance of reactive oxygen and nitrogen species within the renal medullary region is a key determinant of the set point of the renal pressure-natriuresis curve. The development of renal medullary oxidative stress causes dysfunction of the pressure-natriuresis mechanism and contributes to the development of hypertension in numerous disease models. The purpose of this review is to point out the known mechanisms within the renal medulla through which reactive oxygen and nitrogen species modulate the pressure-natriuresis response and to update the reader on recent advances in this field.

The endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive autacoids such as nitric oxide, prostacyclin, and a third factor or pathway termed 'endothelium-derived hyperpolarizing factor' (EDHF). Although the functional influence of NO and EDHF is sometimes reciprocal (i.e., their effects decrease or increase with the reduction in vessel diameter, respectively), recent insights led to the identification of caveolae and caveolin as common regulators of their production. In this review, we will first focus on the current understanding of the caveolin/eNOS paradigm and will then detail the most recent findings on the role of caveolae in driving EDHF-signaling pathways.

Robert Furchgott's discovery of the obligatory role that the endothelium plays in the regulation of vascular tone has proved to be a major advance in terms of our understanding of the cellular basis of diabetic vascular disease. Endothelial dysfunction, as defined by a reduction in the vasodilatation response to an endothelium-dependent vasodilator (such as acetylcholine) or to flow-mediated vasodilatation, is an early indicator for the development of the micro- and macroangipathy that is associated with diabetes. In diabetes, hyperglycaemia plays a key role in the initiation and development of endothelial dysfunction; however, the cellular mechanisms involved as well as the importance of dyslipidaemia and co-morbidities such as hypertension and obesity remain incompletely understood. In this review, we discuss the mechanisms whereby hyperglycaemia, oxidative stress and dyslipidaemia can alter endothelial function and highlight their effects on endothelial nitric oxide synthase (eNOS), the endothelium-dependent hyperpolarising factor (EDHF) pathway(s), as well as on the role of endothelium-derived contracting factors (EDCFs) and adipocyte-derived vasoactive factors such as adipose-derived relaxing factor (ADRF).

Endothelium-dependent changes in vasomotor tone, whether evoked by vasoactive agents or physical forces, are recognized as essential for the local hemodynamic control in various normal and pathological circumstances. They are based on a complex signaling network within the vascular wall. In recent years, substantial efforts have been made to analyze how such signals are generated and used in the endothelium-dependent control of vascular smooth muscle. The underlying mechanisms vary with species, age, sex, hormonal status, vascular bed studied, caliber of the blood vessels, triggering stimuli, pre-existing vascular tone, oxidative stress, and pathology. Such aspects and many others will be addressed specifically by the authors contributing to this volume.

Pharmacologic strategies to reduce pulmonary vascular tone and to treat pulmonary hypertension originally aimed to enrich vascular smooth muscle cyclic adenosine monophosphate levels. Alternatively, increasing cyclic guanosine monophosphate (cGMP) also reduces pulmonary vascular tone. Inhaled nitric oxide is extremely efficacious in increasing cGMP and selectively reducing mean pulmonary arterial pressure in pediatric cardiac patients. It is considered standard treatment in most centers. However, not all patients respond to inhaled nitric oxide and withdrawal is sometimes problematic. This has prompted investigation of alternative methods to increase intracellular vascular smooth muscle cGMP. Phosphodiesterase type 5 is particularly abundant in the lung vasculature of patients with severe pulmonary hypertension. Its inhibition with the sildenafil class of drugs is now commonplace. Drugs that affect cGMP metabolism in children with acute pulmonary hypertension are the subject of this review and consensus statement. Oral sildenafil is recommended in postoperative pulmonary hypertension after failed withdrawal of inhaled NO (class I, level of evidence B). The effectiveness of prolonged treatment with sildenafil in documented postoperative pulmonary hypertension is not well established (class IIb, level of evidence C). Sildenafil is indicated in idiopathic pulmonary hypertension, although data have been extrapolated mainly from adult trial (class I, level of evidence A, extrapolated). Recently, completed pediatric trials have seemed to support this recommendation. Longer-acting and intravenous forms of phosphodiesterase type 5 inhibitors, brain natriuretic peptides, and direct soluble guanylate cyclise activators all have appeal, but there is insufficient experience in children with acute pulmonary hypertensive disorders for recommendations on treatment.

The endothelium plays an important role in maintaining cardiovascular homeostasis by synthesizing and releasing several vasodilating substances, including vasodilator prostaglandins, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). Since the first report on the existence of EDHF, several substances/mechanisms have been proposed for the nature of EDHF, including epoxyeicosatrienoic acids (metabolites of arachidonic P450 epoxygenase pathway), K ions, and electrical communications through myoendothelial gap junctions. We have demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in animals and humans. For the synthesis of H(2)O(2)/EDHF, endothelial NO synthase system that is functionally coupled with Cu,Zn-superoxide dismutase plays a crucial role. Importantly, endothelium-derived H(2)O(2) plays important protective roles in the coronary circulation, including coronary autoregulation, protection against myocardial ischemia/reperfusion injury, and metabolic coronary vasodilatation. Indeed, our H(2)O(2)/EDHF theory demonstrates that endothelium-derived H(2)O(2), another reactive oxygen species in addition to NO, plays important roles as a redox-signaling molecule to cause vasodilatation as well as cardioprotection. In this review, we summarize our current knowledge on H(2)O(2)/EDHF regarding its identification and mechanisms of synthesis and actions.

Endothelial dysfunction is a common feature of hypertension, and it results from the imbalanced release of endothelium-derived relaxing factors (EDRFs; in particular, nitric oxide) and endothelium-derived contracting factors (EDCFs; angiotensin II, endothelins, uridine adenosine tetraphosphate, and cyclooxygenase-derived EDCFs). Thus, drugs that increase EDRFs (using direct nitric oxide releasing compounds, tetrahydrobiopterin, or L-arginine supplementation) or decrease EDCF release or actions (using cyclooxygenase inhibitor or thromboxane A2/prostanoid receptor antagonists) would prevent the dysfunction. Many conventional antihypertensive drugs, including angiotensin-converting enzyme inhibitors, calcium channel blockers, and third-generation beta-blockers, possess the ability to reverse endothelial dysfunction. Their use is attractive, as they can address arterial blood pressure and vascular tone simultaneously. The severity of endothelial dysfunction correlates with the development of coronary artery disease and predicts future cardiovascular events. Thus, endothelial dysfunction needs to be considered as a strategic target in the treatment of hypertension.

Background— Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (α and β). Deletion of the β 1 subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. Methods and Results— To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle–specific knockout mice for the β 1 subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. Conclusions— We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

Rationale: The cardiac inwardly rectifying K + current ( I K1 ) plays a critical role in modulating excitability by setting the resting membrane potential and shaping phase 3 of the cardiac action potential. Objective: This study aims to analyze the effects of nitric oxide (NO) on human atrial I K1 and on Kir2.1 channels, the major isoform of inwardly rectifying channels present in the human heart. Methods and Results: Currents were recorded in enzymatically isolated myocytes and in transiently transfected CHO cells, respectively. NO at myocardial physiological concentrations (25 to 500 nmol/L) increased inward and outward I K1 and I Kir2.1 . These effects were accompanied by hyperpolarization of the resting membrane potential and a shortening of the duration of phase 3 of the human atrial action potential. The I Kir2.1 increase was attributable to an increase in the open probability of the channel. Site-directed mutagenesis analysis demonstrated that NO effects were mediated by the selective S -nitrosylation of Kir2.1 Cys76 residue. Single ion monitoring experiments performed by liquid chromatography/tandem mass spectrometry suggested that the primary sequence that surrounds Cys76 determines its selective S -nitrosylation. Chronic atrial fibrillation, which produces a decrease in NO bioavailability, decreased the S -nitrosylation of Kir2.1 channels in human atrial samples as demonstrated by a biotin-switch assay, followed by Western blot. Conclusions: The results demonstrated that, under physiological conditions, NO regulates human cardiac I K1 through a redox-related process.

Female mice are protected from the cerebrovascular dysfunction induced by angiotensin II (Ang II), an effect attributed to estrogen. We examined whether such cerebrovascular protection from Ang II is related to the estrous cycle. Cerebral blood flow was monitored by laser-Doppler flowmetry in anesthetized (urethane-chloralose) C57BL/6 female mice equipped with a cranial window. The phase of the estrous cycle was determined by vaginal smear cytology and plasma estrogen measurement. Ang II (0.25 μg/kg per minute, IV, 30 to 45 minutes) elevated arterial pressure (15 to 20 mm Hg) equally across the estrous cycle. However, in proestrus and estrus, phases in which estrogen is relatively high, Ang II did not impair the increase in the cerebral blood flow induced by neural activity or by endothelium-dependent vasodilators ( P >0.05 from vehicle). In contrast, in diestrus (lower estrogen), Ang II induced a marked cerebrovascular dysfunction comparable to that of male mice. For example, the cerebral blood flow responses to whisker stimulation and to the endothelium-dependent vasodilator acetylcholine were attenuated by 41±12% and 49±12%, respectively ( P P P >0.05 from control). Topical treatment with ICI182,780 reestablished Ang II–induced oxidative stress in proestrus ( P >0.05 from diestrus). We conclude that the protection from the neurovascular dysfunction induced by acute administration of Ang II in females depends on the estrous cycle and may underlie the increased propensity to cerebrovascular damage associated with low estrogen states.

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nm peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10–20 nm). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nm being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pm provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.

ObjectivesThis study sought to explore the effect of glycemic-index dietary carbohydrates on endothelium-dependent flow-mediated dilation (FMD) in overweight and obese nondiabetic volunteers.BackgroundPost-prandial hyperglycemia has been recognized as a cardiovascular risk factor in both the diabetic and the general population. Endothelial dysfunction has been shown to occur in diabetic and hyperglycemic patients.MethodsWe prospectively assessed brachial artery FMD in 56 healthy overweight and obese nondiabetic volunteers (38 [67.9%] men, mean age 48 ± 6 years) on 4 separate mornings, 1 to 2 weeks apart. After overnight fasting, the percent FMD (%FMD) improvement and endothelium-independent nitroglycerin-mediated dilation (%NTG) were assessed, after which subjects received 1 of 4 group meals at each visit (placebo [water] or a carbohydrate meal of glucose, cornflakes, or high-fiber cereal). Meals were distributed in a rotating randomized fashion, such that each subject received all 4 meals once throughout the study period.ResultsFasting and 2-h post-prandial serum glucose levels were similar in all 3 meals, whereas at 30 to 90 min, serum glucose levels were significantly higher after glucose and cornflakes (high glycemic) compared with fiber (low glycemic). Baseline %FMD, not significantly different in the 3 carbohydrate-based meals, was reduced 2 h post-prandially in all groups, showing statistical significance in only high-glycemic index meals: glucose (15 ± 9% vs. 10 ± 8%, p < 0.01), cornflakes (13 ± 7% vs. 9 ± 7%, p < 0.01). No correlation was observed between the %FMD reduction rate and glucose levels throughout the study period.ConclusionsHigh- compared with low-glycemic carbohydrate consumption significantly suppresses FMD in nondiabetic overweight and obese volunteers, suggesting a mechanism whereby high-glycemic meals may enhance cardiovascular risk.

The endothelial cells control the tone of the underlying vascular smooth muscle by releasing vasoactive substances. Endothelium-derived relaxing factors (EDRF), in particular nitric oxide have received considerable attention, but much less is known about the ability of the endothelial cells to release endothelium-derived contracting factors (EDCF). The possible players of endothelium-dependent contractions and the underlying mechanisms leading to the release of EDCF will be discussed in the present review. EDCF is likely to consist of two components: 1) prostanoids (including endoperoxides, prostacyclin, thromboxane A(2), and prostaglandin E(2)) and 2) reactive oxygen species. The former directly activate thromboxane/prostaglandin endoperoxide (TP) receptors of the vascular smooth muscle cells which leads to their contraction, while the latter first stimulate the cyclooxygenase in the smooth muscle with subsequent stimulation of the TP receptors by the prostanoids produced. Dysfunction in calcium handling is the leading causal factor for the exacerbated occurrence of endothelium-dependent contractions in the aorta of the spontaneously hypertensive rat (SHR). The observed increased expressions of endothelial COX-1, prostacyclin synthase, thromboxane synthase and enhanced TP receptor sensitivity are not prerequisites for, but intensify the magnitude of endothelium-dependent contractions. Selective TP receptor antagonists are effective in preventing endothelium-dependent contractions in vitro which highlights the prospective use of such drugs in correcting the imbalanced release of endothelium-derived vasoactive substances that accompany vascular disease.

Background Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the conversion of l -arginine to the endothelium-derived vasodilator nitric oxide. We evaluated if circulating levels of l -arginine and ADMA, and the ratio thereof, were related to endothelium-dependent vasodilation in both resistance and conductance arteries in the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study. Methods and results In this population-based study, vascular function of 1016 subjects aged 70 was evaluated by the invasive forearm technique with intra-brachial infusion of acetylcholine (EDV), by measurement of flow-mediated dilation (FMD) of the brachial artery, and by change in reflectance index (RI) by pulse wave analysis. After adjustment for resting forearm blood flow, gender, Framingham risk score, and glomerular filtration rate, the l -arginine/ADMA ratio was positively related to EDV (P = 0.005). This association was mainly driven by l -arginine, because if both l -arginine and ADMA were entered as predictor variables in a single regression model, l -arginine was positively associated with EDV (P = 0.001), whereas the negative association between ADMA and EDV was not significant. In contrast, after adjustment for baseline brachial diameter, gender, Framingham risk score, and glomerular filtration rate, the l -arginine/ADMA ratio was negatively related to FMD (P = 0.004). Again, this association was mainly driven by l -arginine (P = 0.002), whereas ADMA was not significantly related to FMD. The change in RI was not related to l -arginine, ADMA or their ratio. Conclusions In elderly subjects the l -arginine/ADMA ratio and l -arginine, but not ADMA, are positively related to endothelium-dependent vasodilation in resistance arteries, but negatively in a conduit artery.

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K+ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with l-NAME, Nω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO•) with hydroxocobalamin (HXC) or HNO with l-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and l-cysteine reduced ACh-mediated relaxation, as did l-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with l-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO•, as HXC and l-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO• = HNO > EDHF and EDHF > HNO = NO• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels. British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009 This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

Accumulating evidence demonstrates that dietary intake of n -3 polyunsaturated fatty acids (PUFAs) is associated with a reduced incidence of several cardiovascular diseases that involve endothelial dysfunction. However, the molecular mechanism remains unclear. We previously reported that mesenteric arteries from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats exhibit endothelial dysfunction, leading to an imbalance between endothelium-derived vasodilators \[namely, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)] and vasoconstrictors \[endothelium-derived contracting factors (EDCFs)\] \[namely cyclooxygenase (COX)-derived prostanoids\\] ( Am J Physiol Heart Circ Physiol 293:H1480–H1490, 2007). We hypothesized that treating OLETF rats with eicosapentaenoic acid (EPA), a major n -3 PUFA, may improve endothelial dysfunction by correcting this imbalance. In OLETF rats [compared with age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: 1) acetylcholine (ACh)-induced (endothelium-dependent) relaxation was impaired, 2) NO- and EDHF-mediated relaxations and nitrite production were reduced, and 3) ACh-induced EDCF-mediated contraction, production of prostanoids, and the protein expressions of COX-1 and COX-2 were all increased. When OLETF rats received chronic EPA treatment long-term (300 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: 1) improvements in ACh-induced NO- and EDHF-mediated relaxations and COX-mediated contraction, 2) reduced EDCF- and arachidonic acid-induced contractions, 3) normalized NO metabolism, 4) suppressed production of prostanoids, 5) reduced COX-2 expression, and 6) reduced phosphoextracellular signal-regulated kinase (ERK) expression. Moreover, EPA treatment reduced both ERK2 and nuclear factor (NF)-κB activities in isolated OLETF aortas. We propose that EPA ameliorates endothelial dysfunction in OLETF rats by correcting the imbalance between endothelium-derived factors, at least partly, by inhibiting ERK, decreasing NF-κB activation, and reducing COX-2 expression.

The aim of this study was to examine the function of perivascular adiposa tissue (PVAT) on vascular relaxation response in spontaneously hypertensive rats (SHR) and the modulatory effects of the atorvastatin therapy on the PVAT functions. We investigated the mechanisms of the perivascular adipocyte-derived relaxation factor (PVRF) by using isolated rat's aortic rings and isometric contraction measurements. We found that contraction of the thoracic aorta induced by phenylephrine was significantly attenuated in the presence of PVAT from normotensive Wistar-Kyoto rats (WKY group) or the spontaneously hypertensive rats treated with atorvastatin (SHR-A group, atorvastatin 50mg/kg/day), whereas this effect was not observed in the thoracic aortic rings from the control SHR (SHR group). Transferring the solution incubated with PVAT-intact thoracic aorta to PVAT-free thoracic aorta, it induced a remarkable relaxation response in the WKY but not in the control SHR. Tetraethylammoniumchloride (TEA) could block the above relaxation. It was also shown that the PVRF function was likely, depending on the extracellular [Ca(2+)]; the anti-contractile effect of PVAT could be reduced by the inhibitor of the adenosine triphosphate (ATP)-dependent potassium channels, glibenclamide, and could be reduced by the inhibitor of cyclooxygenase by indomethacin. We thus infer that the PVAT function was distorted in hypertension rats, and the lipid-lowering treatment with atorvastatin could restore the PVAT function. The function of the PVRF may involve the Ca(2+)-activated potassium channels, the ATP-dependent potassium channels in vascular smooth muscle cell (SMC), and the release of PVRF from PVAT may involve prostaglandins (PGs) and the calcium metabolism. These results provide an insight into the pathological mechanisms of hypertension development, and indicate that the PVAT may be a potential new target for the hypertensive therapy.

Summary Since it was identified as the elusive endothelium-derived relaxing factor (EDRF) in the 1980s, nitric oxide (NO) has rapidly gained status as one of the most important signalling molecules in the cardiovascular system. Now, 20 years later, NO is regarded by most to be a ubiquitous mediator of cardio-protection. However, due to various complex underlying cellular mechanisms, the actions of NO often seem to be contradictory. This article sheds light on some of the mechanisms that may influence the variable actions of NO in the heart. Its role in conditions of oxygen deprivation (ischaemia and hypoxia) in particular is relevant to basic scientists and clinicians alike, since the prevalence of ischaemic heart disease is on the rise (in both the developed and the developing worlds) and novel therapeutic options are in constant demand. NO is a promising candidate molecule that could find therapeutic application. For this to be achieved, a sound understanding of this simple molecule and its complex actions is required.

Summary The purpose of this study was to examine the in vivo effects of Ekebergia capensis leaf ethanolic extract (EKE) on the blood pressure of anaesthetised normotensive male Wistar rats and conscious weanling Dahl salt-sensitive (DSS) rats, which develop hypertension as they age. To investigate possible mechanism(s) of the extract’s hypotensive effects, the contractile or relaxant responses to EKE in the absence or presence of reference drugs were evaluated in Wistar rat isolated aortic rings precontracted with methoxamine hydrochloride (ME, 10 μM). Acute intravenous administration of EKE elicited hypotensive responses in anaesthetised animals, while sub-chronic treatment with the extract averted the development of high blood pressure in weanling DSS rats. Isometric recordings of methoxamine hydrochloride (ME) pre-contracted, isolated, endothelium-intact and -denuded aortic rings revealed concentration-dependent relaxation responses to EKE (1–160 mg/ml). The potency was significantly less in the endothelium-denuded rings. Inhibitors of endothelium-derived relaxing factor (EDRF), L-NAME, methylene blue and indomethacin significantly reduced EKE-evoked vasorelaxations in endothelium-intact aortic rings. These results indicate that the vasorelaxant effect of EKE was in part mediated via EDRF-dependent or -independent pathways. These observations suggest that the hypotensive effect of EKE was in part mediated via modulation of total peripheral resistance of the vascular smooth muscles.

Aim: Endothelium-dependent relaxations to certain neurohumoral substances are mediated by pertussis toxin-sensitive Gi/o protein. Our experiments were designed to determine the role, if any, of pertussis toxin-sensitive G-proteins in relaxations attributed to endothelium-derived hyperpolarizing factor (EDHF). Methods: Pig coronary arterial rings with endothelia were suspended in organ chambers filled with Krebs-Ringer bicarbonate solution maintained at 37 °C and continuously aerated with 95%O 2 and 5% CO 2 . Isometric tension was measured during contractions to prostaglandin F 2α in the presence of indomethacin and N ω -nitro- L -arginine methyl ester ( L -NAME). Results: Thrombin, the thrombin receptor-activating peptide SFLLRN, bradykinin, substance P, and calcimycin produced dose-dependent relaxations. These relaxations were not inhibited by prior incubation with pertussis toxin, but were abolished upon the addition of charybdotoxin plus apamin. Relaxations to the α2-adrenergic agonist UK14304 and those to serotonin were abolished in the presence of indomethacin and L -NAME. Conclusion: Unlike nitric oxide-mediated relaxations, EDHF-mediated relaxations of pig coronary arteries do not involve pertussis toxin-sensitive pathways and are Gi/o protein independent.

Nitric oxide (NO) released by endothelial cells in response to hemodynamic shear stress is a key controller molecule of the vascular functions and antiatherogenic mechanisms. Endothelial dysfunction is associated with increased cardiovascular events. Therefore, several indirect techniques have been employed to evaluate endothelial function or NO bioavailability. However, a growing body of evidences suggests limitations of the indirect methods for evaluation of NO bioavailability. In years, it has been considered that NO is immediately oxidized or inactivated in blood stream. However, recent studies suggest that NO remain active in blood stream, causing remote biological response. Therefore, measuring plasma NO concentration directly in the circulation will contribute to clarify the kinetics and physiological roles of NO and to evaluate endothelial function. In this article, the measurement of plasma NO concentration using a newly developed catheter-type NO sensor will be described.

Background and Aims: Vasomotion consists in cyclic oscillations of the arterial diameter induced by the synchronized activity of the smooth muscle cells. So far, contradictory results have emerged in the literature about the role of the endothelium in the onset and maintenance of vasomotion. Here our aim is to understand how the endothelium may either abolish or promote vasomotion. Methods and Results: We investigate rat mesenteric arterial strips stimulated with phenylephrine (PE). Our results show that the endothelium is not necessary for vasomotion. However, when the endothelium is removed, the PE concentration needed to induce vasomotion is lower and the rhythmic contractions occur for a narrower range of PE concentrations. We demonstrate that endothelium-derived relaxing products may either induce or abolish vasomotion. On the one hand, when the strip is tonically contracted in a nonoscillating state, an endothelium-derived relaxation may induce vasomotion. On the other hand, if the strip displays vasomotion with a medium mean contraction, a relaxation may induce a transition to a nonoscillating state with a small contraction. Conclusion: Our findings clarify the role of the endothelium on vasomotion and reconcile the seemingly contradictory observations reported in the literature.

Asymmetric ( NG, NG)-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/− mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed.

The aim of this study was to investigate the vasorelaxant effect induced by diosgenin in superior mesenteric rings. In rings pre-contracted with phenylephrine (10 microM), diosgenin caused concentration-dependent relaxations [EC(50) = (3.3 +/- 1.2) x 10(- 4)M, E(max) = 94.2 +/- 2.6 %]. Vascular relaxation induced by diosgenin was significantly inhibited after removal of the endothelium (E(max) = 46 +/- 8.8%, p0.001) or after pre-treatment of the rings with N-nitro-L-arginine methyl esther (l-NAME) 100 or 300 microM (E(max) = 35.3 +/- 4%; 28.1 +/- 3.3%, respectively, p0.001), atropine 1 microM (E(max) = 24.6 +/- 3.4%, p0.001), hydroxocobalamin 30 microM (E(max) = 54.0 +/- 9.6%, p0.001), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) 10 microM (E(max) = 46.0 +/- 8.0%, p0.001) or indomethacin 1 microM (E(max) = 22.6 +/- 11.8%, p0.001). Vasorelaxation evoked by diosgenin was significantly inhibited after pre-treatment of preparations with both selective and non-selective inhibitors of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, iberiotoxin 100 nM or tetraethylammonium (TEA) 1mM, respectively (E(max) = 62.5 +/- 9.1%; 65.7 +/- 1.1%, p0.001). Conversely, in endothelium-denuded vessels, none of BK(Ca) channel blockers modified the relaxant effect induced by diosgenin. In mesenteric endothelial cells loaded with FURA-2 diosgenin was able to increase intracellular calcium concentrations, which were significantly decreased by atropine 1 microM. In addition, in isolated mesenteric rings, diosgenin induced marked increase in nitric oxide (NO) levels, which was completely abolished after functional endothelium removal. The results obtained here demonstrated that diosgenin-induced relaxation appears to involve endothelial muscarinic receptor activation with increase in intracellular calcium concentrations and consequent release of endothelium-derived relaxing factors (EDRFs), mainly NO and cyclooxygenase derivatives, which activate BK(Ca) channels. Nevertheless, further studies are necessary to clearly elucidate residual endothelium-independent relaxation induced by diosgenin.

The aim of the investigation was to study the possible effects of in vivo infusion of nitric oxide (NO) blockers upon the in vitro endothelium-dependent femoral reactivity. The experimental model tested herein was the inferior canine hindlimb global ischemia induced by infrarenal abdominal aortic cross-clamping followed by reperfusion. The NO blockers employed in the tests were N(G)-nitro-l-arginine methyl ester (L-NAME), aminoguanidine (AMG), and methylene blue (MB), which were infused immediately after the anesthesia induction. The research protocol was standardized in two main experimental groups, control and ischemia/reperfusion (I/R) injury, randomized in eight subgroups including controls and NO blockers. The femoral artery vascular reactivity was studied in vitro with the aid of a setup consisting of eight organ chambers, where segments of 4-5 mm were suspended and connected to force transducers in the presence of indomethacin to block the cyclooxygenase pathway. The NO-release pathway was evaluated by using specific pharmacological agonists in the in vitro experiments. The L-NAME in vivo infusion led to in vitro endothelium dysfunction in both groups and was associated with high mortality in the animals submitted to I/R. AMG and MB, two clinically used drugs, did not cause in vitro endothelium dysfunction in either of the two groups, which gives evidence that these drugs are not deleterious in the milieu of I/R injury. Nitrite/nitrate plasma levels were not significant except for the L-NAME groups, which presented significant NO decrease.

Objective : Sepsis-stimulated nitric oxide (NO) production impairs arteriolar responsiveness in skeletal muscle. Using wild type (WT), eNOS−/−, iNOS−/− and nNOS−/− mice, we aimed to determine the key nitric oxide synthase (NOS) isoenzyme(s) responsible for the arteriolar hyporesponsiveness to acetylcholine (ACh) in septic skeletal muscle. Methods : Sepsis was induced by the cecal ligation and perforation procedure (24 h model). We measured the post-ACh increase in red blood cell velocity ( V RBC) in a capillary fed by the stimulated arteriole as an index of vasodilation. NOS activity and protein expression in the muscle were measured by standard procedures. Results : In all non-septic mice, ACh increased V RBC by ∼150% from baseline. Sepsis impaired this response in WT, eNOS−/− and iNOS−/− mice, but not in nNOS−/− mice. Accordingly, pharmacological inhibition of nNOS with 7-nitroindazole reversed this impairment in WT mice. cNOS (eNOS+nNOS) activity was elevated in septic WT mice; Western blots indicated that this occurred through a post-translational mechanism. iNOS protein activity/expression was negligible. ACh caused dilation via endothelial-derived relaxing factor (EDRF) in WT mice and via endothelial-derived hyperpolarizing factor (EDHF) in eNOS−/− mice. Although exogenous NO reduced EDHF-mediated dilation in eNOS−/− mice, NOS inhibition did not reverse the sepsis-impaired dilation in these mice. Conclusions : In our 24-h mouse model of sepsis, NO in skeletal muscle is primarily derived from nNOS. Sepsis impairs both EDRF- and EDHF-mediated dilation in response to ACh. Both genetic deletion and inhibition of nNOS protect against this impairment when the dilation occurs via the EDRF but not EDHF pathway.

The reported EC 50 of vasorelaxation for authentic nitric oxide (NO) is from the nM to μM range. The cause of this large difference is undetermined. In this study, NO electrodes were used to monitor the actual NO concentration in the organ chamber during the recording of the relaxation of rat aortic rings. It was demonstrated that both the O 2 concentration in the solution and the rate of stirring the solution markedly affected the actual NO concentration, while the vasorelaxation response to NO was also changed with the composition of the buffer solution. It was observed that the apparent EC 50 of aortic relaxation for authentic NO is 340 ± 40 nM and 81 ± 4 nM in the conventional organ chamber containing Krebs–Ringer buffer bubbled with a high content (95% O 2 + 5% CO 2 ) and a low content (20% O 2 + 5% CO 2 + 75% N 2 ) of O 2 gas mixture, respectively. The apparent EC 50 was further reduced to 45 ± 2 nM after the Krebs–Ringer buffer, a bicarbonate buffer, was replaced by a phosphate buffer solution bubbled with a low content of O 2 gas mixture (20% O 2 + 80% N 2 ) or with air. By using an organ chamber, which makes low concentrations of NO more stable in the solution, it was determined that the apparent EC 50 was 9.7 ± 0.4 nM and the threshold of NO concentration in dilating the aortas was ∼0.3 nM. This modified organ chamber will be useful for quantitatively measuring EC 50 of vasorelaxation for authentic NO under physiological and pathological conditions.

Cardiovascular events are accelerated in chronic kidney disease (CKD). Although deranged nitric oxide (NO) pathways and asymmetric dimethylarginine (ADMA) cause endothelial dysfunction, no direct evidence for coronary artery endothelial dysfunction in CKD has been documented. CKD was induced in male dogs by heminephrectomy (1/2Nx) or five-sixths nephrectomy (5/6Nx). After 4 wk, renal ablation reduced GFR (control 76 [54 to 85]; 1/2Nx 38 [29 to 47]; 5/6Nx 15 [12 to 46] ml/min) and elevated plasma ADMA (control 1.88 [1.68 to 2.54]; 1/2Nx 2.51 [2.11 to 3.55]; 5/6Nx 3.84 [2.16 to 3.95] micromol/L). Coronary circulatory responses to acetylcholine revealed marked increases in coronary blood flow in control group (83 +/- 17% increment) but blunted responses in 1/2Nx (34 +/- 8% increment) and 5/6Nx (20 +/- 4% increment). The acetylcholine-induced changes in epicardial arteriolar diameter, using needle-lens probe charge-coupled device videomicroscopy, showed similar results. The responsiveness to sodium nitroprusside did not differ among three groups. Plasma nitrite/nitrate levels decreased in 1/2Nx and 5/6Nx, and the mRNA expressions of dimethylarginine dimethylaminohydrolase-II (DDAH-II), ADMA-degrading enzyme, and endothelial NO synthase (eNOS) in coronary arteries were downregulated in 1/2Nx and 5/6Nx. Finally, 4-wk treatment with all-trans retinoic acid restored the impaired endothelium-dependent vasodilation and reversed the expression of eNOS but not DDAH-II. Coronary endothelial function is impaired in the early stage of CKD. The dysfunction is attributed to the downregulation of eNOS and/or DDAH-II in coronary arteries. Furthermore, the manipulation of NO pathways may constitute a therapeutic strategy for the prevention of coronary dysfunction in CKD.

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.

Background: Metastasis, the dissemination of malignant cells to distant sites, remains one of the most significant factors responsible for death from cancer. Recent studies have shown some improvement in the rate of distant metastasis (DM) with the addition of chemotherapy to surgery and radiation for treatment of head and neck squamous cell carcinoma (HNSCC). However, diagnosis and treatment at an early stage ultimately leads to a better prognosis. The prediction of which patients will develop metastasis and the selection of treatment most effective at preventing and treating metastasis remains dependent on an incomplete understanding of prognostic factors and the biological and molecular basis for metastatic development. This study was undertaken using an in vivo model to investigate the possible role of nitric oxide (NO) in the development of metastasis from HNSCC. The findings will result in better understanding of the metastatic process for HNSCC, with the potential to develop and implement therapies that could prevent and treat metastasis in patients. Objectives/Hypothesis: 1) To analyze whether in vivo videomicroscopy (IVVM) is useful for the study of DM from squamous cell carcinoma of the head and neck; 2) with use of IVVM, investigate the effect of the biological mediators NO and interleukin (IL)-1 on the adhesion of circulating human HNSCC cells in the hepatic microcirculation. Study Design: Prospective study using an animal model. Methods: Phase 1: athymic nude rats and mice were used for IVVM experiments. The cremaster muscle and liver, used as arterial and venous flow models, were tested to determine whether IVVM was useful for the study of human HNSCC interactions with the microcirculation. A human squamous cell carcinoma cell line (FaDu) labeled with the intracytoplasmic fluorescent marker BCECF-AM. was used for all experiments. Videomicroscopic images of FaDu cells in the microcirculation were analyzed for cell adhesion, morphology, deformation, circulation, location of adhesion within the microcirculation, and alteration of microvascular circulation. Phase 2: the effect of IL-1, NO, and NO inhibitors on HNSCC cell adhesion in the hepatic microcirculation of nude mice was analyzed by IVVM. This was followed by histologic determination of the ratio of FaDu cells present for liver area analyzed. Nude mice were treated with 1) IL-1; 2) L-arginine (an NO substrate); or 3) L-N-monomethyl-L-arginine (an NO synthase inhibitor) alone or in combination. These data were analyzed statistically to determine the effect on cell adhesion in the liver. Results: IVVM provided a method for the study of circulating HNSCC with the microcirculation in both the cremaster and liver models. FaDu cells were arrested at the inflow side of the circulation, with maintenance of cell integrity. L-arginine and IL-1 both increased FaDu cell arrest in the liver above baseline (P = .00008 and P = .03), and the combination of these agents potentiated the effect (P = .000009). Conclusions: IVVM allows direct assessment of circulating HNSCC with the microcirculation and is a powerful model for the study of DM. NO and IL-1 play a role in increasing the arrest of HNSCC in the liver and are important in the generation of DM in patients with HNSCC.

Pulmonary arterial hypertension (PAH) is a rare and debilitating disease characterized by abnormal proliferation and contraction of pulmonary vascular smooth muscle cells. The resulting increase in pressure and pulmonary vascular resistance results in progressive right heart failure, low cardiac output, and ultimately death if left untreated. PAH is defined by a persistent elevation in pulmonary artery pressure with normal left-sided pressures, differentiating it from left-sided heart disease. Symptoms progress from shortness of breath and decreasing exercise tolerance to right heart failure, with peripheral edema and marked functional limitation. Exercise-induced syncope, worsening symptoms at rest, and intractable right heart failure indicate critical disease. PAH may be idiopathic with no identifiable cause or associated with collagen vascular diseases, drugs, HIV, liver disease, and/or congenital heart disease. Familial or genetically mediated PAH accounts for a small percentage of cases. Advances in the understanding of pathobiological pathways that contribute to vascular proliferation and remodeling have resulted in new therapies that improve quality of life and survival. Emerging therapies focus on the nitric oxide, prostacyclin, and endothelin pathways. Nursing interventions are critical to ensure patients' success with these expensive and complex treatments and their optimal adjustment to living with PAH.

Aims and objectives. This paper, therefore, aimed to review published literature in this area of pharmacological exploitation, to look at the therapeutic applications and clinical relevance and, by so doing, provide an accessible source for nurses to gain insight into the role of nitric oxide in the clinical setting. Background. Nitric oxide is a chemical mediator fundamental in the maintenance of adequate tissue perfusion and effective cardiovascular function; a major endogenous regulator of vascular tone. The use of nitrates are well established as pharmacological agents but it is only recently that it has been recognized that they act as a source of nitric oxide. Although widely addressed within the medical literature, there appears to be a paucity of nursing literature that explores either its physiological action, or its relevance to nursing practice. Conclusions. This literature review provides an overview of the use of nitric oxide and its implications for nursing practice and patient outcomes. Relevance to clinical practice. Knowledge of nitric oxide and its action is pertinent to nurses across diverse specialities. It helps in understanding the principles of many nitrogen-derived medications which nurses administer to their patients on a daily basis. In terms of oral medication, this is demonstrated by greater insights into the action of nitrates, the appreciation of surprising developments in medications such as sildenafil and the development of new drug opportunities such as nitric oxide–non-steroidal anti-inflammatory drugs. Equally, the use of inhaled nitric oxide therapy in adult and neonatal critical care units appears to be an increasingly valuable source of treatment. A particular research challenge is found in the attempt at nitric oxide inhibition in the management of septic shock. The authors argue that understanding such esoteric areas of therapeutic developments is increasingly to be part of the repertoire of knowledge and skills for nurses in the 21st century.

Treatment of children and adults with pulmonary hypertension (PH) with or without cardiac dysfunction has improved in the last two decades. The so-called pulmonary arterial hypertension (PAH)-specific medications currently approved for therapy of adults with PAH target three major pathways (endothelin, nitric oxide, prostacyclin). Moreover, some PH centres may use off-label drugs for compassionate use. Pulmonary hypertensive vascular disease (PHVD) in children is complex, and selection of appropriate therapies remains difficult. In addition, paediatric PAH/PHVD therapy is vastly based on experience and trial data from adult rather than paediatric studies; however, the first randomised paediatric PAH trials have been conducted recently. We present consensus recommendations for the treatment of children with PH. Class of recommendation and level of evidence were assigned based on paediatric data only or on adult studies that included >10% children. After a systematic literature search and analysis of the published data, we developed treatment strategies and algorithms that can guide goal-oriented PH therapy. We discuss early combination therapy (double, triple) in patients with PAH in functional class II-IV and in those with inadequate response to the initial pharmacotherapy. In those children with progressive, severe PAH and inadequate response, advances in drug development, and interventional and surgical approaches provide promising new strategies to avoid, reverse or ameliorate right heart failure and left ventricular compression. In particular, first follow-up data indicate that Potts shunt (left pulmonary artery to descending aorta anastomosis) may be an alternative destination therapy, or bridge to bilateral lung transplantation, in end-stage paediatric PAH.

Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction that results from an inflammatory process affecting the airways and lung parenchyma. Despite major abnormalities taking place in bronchial and alveolar structures, changes in pulmonary vessels also represent an important component of the disease. Alterations in vessel structure are highly prevalent and abnormalities in their function impair gas exchange and may result in pulmonary hypertension (PH), an important complication of the disease associated with reduced survival and worse clinical course. The prevalence of PH is high in COPD, particularly in advanced stages, although it remains of mild to moderate severity in the majority of cases. Endothelial dysfunction, with imbalance between vasodilator/vasoconstrictive mediators, is a key determinant of changes taking place in pulmonary vasculature in COPD. Cigarette smoke products may perturb endothelial cells and play a critical role in initiating vascular changes. The concurrence of inflammation, hypoxia and emphysema further contributes to vascular damage and to the development of PH. The use of drugs that target endothelium-dependent signalling pathways, currently employed in pulmonary arterial hypertension, is discouraged in COPD due to the lack of efficacy observed in randomized clinical trials and because there is compelling evidence indicating that these drugs may worsen pulmonary gas exchange. The subgroup of patients with severe PH should be ideally managed in centres with expertise in both PH and chronic lung diseases because alterations of pulmonary vasculature might resemble those observed in pulmonary arterial hypertension. Because this condition entails poor prognosis, it warrants specialist treatment.

Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders.Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba(2+) , ML-133) or in the arteries from EC-Kir2.1(-/-) mice. Potassium-induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba(2+) , did not affect currents through TRPV4, IK or SK channels. Endothelial cell-dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC-Kir2.1(-/-) . In angiotensin II-induced hypertension, the Kir channel function was not altered, although the endothelium-dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca(2+) -dependent activation of IK and SK channels.

Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.ConclusionsCombretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors. Objetivo Descrever e caracterizar os relaxamentos induzidos por um extrato das cascas de Combretum leprosum em anéis de artérias de diferentes espécies de animais.Métodos Anéis (3 a 4mm) de artérias de coelho, rato e porco foram montados em cubas para órgão isolado (Krebs, 37°C, 95%O2/5%CO2) para registro das contrações isométricas. Após um período de estabilização (2 a 3 horas), as contrações foram induzidas com fenilefrina (0,1 a 0,3µM) ou U46619 (10 a 100nM); no platô dessas contrações, adicionamos o extrato Combretum leprosum. Diferentes protocolos foram realizados para determinar potência, duração, reversibilidade e mecanismo dos relaxamentos induzidos pelo extrato.Resultados Em todas as preparações testadas, o extrato de Combretum leprosum (1,5µg/mL) provocou relaxamentos dependentes de endotélio. Em aorta torácica de coelho ou rato, os relaxamentos foram revertidos pela vitamina B12a ou L-NG-nitro-arginina. Em anéis de aorta abdominal de coelho e de artérias coronárias de porco, o extrato causou relaxamentos sensíveis e resistentes à L-NG-nitro-arginina. Em aorta torácica de coelho, o extrato foi relativamente muito potente (EC50=0,20μg/mL) e quando causou relaxamentos; intrigantemente o endotélio continuou a produzir fatores relaxantes por um longo período após remoção do extrato. A magnitude dos relaxamentos induzidos pelo extrato foi significativamente reduzida em ausência Ca2+ extracelular; ademais, o vermelho de rutênio (10μM), um bloqueador de canais TRPs, foi capaz de reverter os relaxamentos induzidos pelo extrato. Análises preliminares indicaram que o extrato continha compostos com reatividade química semelhante à polifenóis.Conclusão O extrato de Combretum leprosum contem compostos bioativos capazes de promover estimulação dependente de Ca2+ das células endoteliais a qual resulta numa produção prolongada de fatores relaxantes.

In pulmonary arterial hypertension (PAH), acute vasodilator response testing (AVT) is considered important to identify adult patients with favorable prognosis using calcium-channel blocker (CCB) therapy. However, in pediatric PAH, criteria used to identify acute responders and CCB use are insufficiently studied.This study sought to describe current clinical practice of AVT and subsequent treatment decisions in pediatric PAH.From January 2008 to May 2013, 529 consecutive children with confirmed pulmonary hypertension were enrolled in an international registry. We analyzed those children with evaluable AVT.Of 382 children with evaluable AVT, 212 had idiopathic/familial PAH (IPAH/FPAH) and 105 had PAH associated with congenital heart disease (PAH-CHD). In 70% of the patients, AVT was performed using inhaled nitric oxide; other agents were used in the remaining patients. In IPAH/FPAH patients, 78 (37%) patients were acute responders according to their physician, 62 (30%) according to REVEAL (Registry-to-Evaluate-Early-And-Long-term PAH disease management)-pediatric criteria, and 32 (15%) according to Sitbon criteria. For PAH-CHD patients, the numbers of AVT responders were 38 (36%), 14 (13%), and 7 (7%) respectively. Correlation between AVT responder status as judged by the treating physician and by published response criteria was poor. Moreover, of the IPAH/FPAH patients judged by the treating physician as acute responders, only 23% were treated with CCB without additional PAH-targeted therapy. The Sitbon criteria selected patients with better prognosis who had excellent outcome when treated with CCB.The current practice of identifying responders to AVT and subsequent treatment with CCB therapy demonstrated large discrepancies with current international guidelines. Also, in pediatric IPAH, the Sitbon criteria are the criteria of choice to identify patients with excellent survival when treated with CCB therapy.

Aging is a major risk factor for carotid artery disease that may lead to stroke and dementia. Vascular effects associated with aging include increased vasomotor tone, as well as enhanced contractility to endothelial vasoconstrictor prostanoids and reduced nitric oxide (NO) bioactivity partly due to increased oxidative stress. We hypothesized that vascular NADPH oxidase (Nox)-derived superoxide may be involved in prostanoid- and NO-related functional aging. NO-mediated relaxations and prostanoid-mediated contractions to acetylcholine as well as phenylephrine-dependent contractions were investigated in the carotid artery from young (4 months) and aged mice (24 months). Gene expression of Nox subunits and endothelial NO synthase (eNOS) was determined in the carotid artery and aorta. In young mice, the thromboxane-prostanoid receptor antagonist SQ 29,548 fully blocked acetylcholine-induced contractions while reducing responses to phenylephrine by 75 %. The Nox2-targeted inhibitor Nox2ds-tat and the superoxide scavenger tempol reduced acetylcholine-stimulated, prostanoid-mediated contractions by 85 and 75 %, respectively, and phenylephrine-dependent contractions by 45 %. Unexpectedly, in aged mice, the substantial Nox2-dependent component of acetylcholine- and phenylephrine-induced, prostanoid-mediated contractions was abolished. In addition, endothelium-dependent, NO-mediated relaxations were impaired with aging. The expression of Nox subunits was greater in the aorta compared with the carotid artery, in which Nox1 was undetectable. eNOS gene expression was reduced in the aorta of aged compared to young mice. In conclusion, aging decreases prostanoid-mediated contractility in the carotid artery involving a loss of Nox2 activity and is associated with impaired endothelium-dependent, NO-mediated relaxation. These findings may contribute to a better understanding of the pathophysiology of carotid artery disease and the aging process.

The present review provides an update of the biological actions of Pycnogenol® in the treatment of metabolic syndrome and related disorders such as obesity, dyslipidaemia, diabetes and hypertension. Pycnogenol® is a French maritime pine bark extract produced from the outer bark of Pinus pinaster Ait. Subsp. atlantica. Its strong antioxidant, antiinflammatory, endothelium-dependent vasodilator activity, and also its anti-thrombotic effects make it appropriate for targeting the multifaceted pathophysiology of metabolic syndrome. Clinical studies have shown that it can reduce blood glucose levels in people with diabetes, blood pressure in mild to moderate hypertensive patients, and waist circumference, and improve lipid profile, renal and endothelial functions in metabolic syndrome. This review highlights the pathophysiology of metabolic syndrome and related clinical research findings on the safety and efficacy of Pycnogenol®. The results of clinical research studies performed with Pycnogenol® are discussed using an evidence-based, target-oriented approach following the pathophysiology of individual components as well as in metabolic syndrome overall.

Hypertension is a major risk factor for cardiovascular disease, and reduction of elevated blood pressure significantly reduces the risk of cardiovascular events. Endothelial dysfunction, which is characterized by impairment of nitric oxide (NO) bioavailability, is an important risk factor for both hypertension and cardiovascular disease and may represent a major link between the conditions. Evidence suggests that NO plays a major role in regulating blood pressure and that impaired NO bioactivity is an important component of hypertension. Mice with disruption of the gene for endothelial NO synthase have elevated blood pressure levels compared with control animals, suggesting a genetic component to the link between impaired NO bioactivity and hypertension. Clinical studies have shown that patients with hypertension have a blunted arterial vasodilatory response to infusion of endothelium‐dependent vasodilators and that inhibition of NO raises blood pressure. Impaired NO bioactivity is also implicated in arterial stiffness, a major mechanism of systolic hypertension. Clarification of the mechanisms of impaired NO bioactivity in hypertension could have important implications for the treatment of hypertension.

Systemic lupus erythematosus is associated with accelerated atherosclerosis and increased cardiovascular morbidity and mortality. Objectives were to determine endothelial dysfunction with a non-invasive method in lupus patients and to analyse correlation with risk factors and atherosclerotic complications. Sixty-one SLE patients and 26 healthy age- and sex-matched control subjects were entered into the study. The diameters of brachial artery at rest, during reactive hyperaemia, and after glyceril trinitrate administration, as well as the intima-media thickness of the common carotid artery were measured using high-resolution B-mode ultrasonography. Demographic characteristics, lipid profile, paraoxonase activity, concentration of anti-phospholipid antibodies and anti-oxLDL were assessed together with atherosclerotic complications. The endothelium dependent vasodilation (FMD) was significantly impaired in SLE patients as compared to controls. The absolute difference of vessel diameter (Δd) was 0.25 ± 0.15 mm vs. 0.38 ± 0.16 mm ( p = 0.001), and Δd as in percent of the rest diameter was 7.31 ± 5.2% vs. 9.86 ± 3.87% ( p = 0.013) in lupus patients and controls, respectively. Nitrate mediated dilation (NMD) did not differ. FMD negatively correlated with age, systolic and diastolic blood pressure in SLE, but did not show significant correlation with the other examined parameters. However, FMD significantly differed between SLE patients with (5.54 ± 4.36%) and without (8.81 ± 5.28%) cardiovascular complications ( p = 0.01). The determination of flow-mediated vasodilation is a useful method to detect endothelial dysfunction in lupus patients, as reduced capacity of brachial artery may distinguish between SLE patients and healthy subjects, as well as lupus patients with and without atherosclerotic vascular complications.

Objectives This study examined the role of Na+/K+-ATPase in the vasodilator actions of nitric oxide (NO), S-nitrosothiols and the endothelium-dependent agonist, acetylcholine. Methods The vasodilator responses elicited by intravenous injections of (i) the NO-donors, sodium nitroprusside and MAHMA NONOate, (ii) the S-nitrosothiols, l -S-nitrosocysteine and S-nitrosocoenzyme A, and (iii) acetylcholine, in urethane-anesthetized rats. Results The NO-donors, S-nitrosothiols and acetylcholine elicited dose-dependent depressor responses and reductions in hindquarter (HQR) and mesenteric (MR) vascular resistances. The depressor responses and associated reductions in HQR elicited by NO-donors were markedly attenuated after injection of ouabain. In contrast, the depressor responses and reductions in HQR elicited by the S-nitrosothiols and acetylcholine were not affected. The reductions in MR elicited by all vasodilator agents were exaggerated after injection of ouabain. Finally, the decomposition of sodium nitroprusside, MAHMA NONOate, l -S-nitrosocysteine and S-nitrosocoenzyme A to NO upon addition to rat blood or vascular preparations was not affected by ouabain. Conclusion This study demonstrates that ouabain has opposing effects on NO-mediated vasodilation in resistance arteries in the hindquarter and mesenteric beds of the rat. The similarity of effects of ouabain on the vasodilator actions of acetylcholine, l -S-nitrosocysteine and S-nitrosocoenzyme A as opposed to the NO-donors supports the possibility that endothelium-derived relaxing factor released by acetylcholine in resistance arteries is an S-nitrosothiol.

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion ( P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.