Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice.

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

Endothelial Hyaluronan Synthase 3 Augments Postischemic Arteriogenesis Through CD44/eNOS Signaling.

Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 HAS isoenzymes (hyaluronan synthases 1-3) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3-deficient (Has3-KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3-KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT (protein kinase B)-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3-KO thigh muscles. Endothelial-specific Has3-KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3-deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.

Targeting the Vascular Endothelium in the Treatment of COVID-19.

ABSTRACT: The involvement of the vascular endothelium in the complications of coronavirus disease 2019 is now recognized. Chief among these are pulmonary endotheliitis, cytokine storm, endotoxic shock, and cardiovascular collapse. This Perspectives article is focused on therapeutical strategies to reduce the risk of these complications by targeting the vascular endothelium as a part of the overall treatment of coronavirus disease 2019.

Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia.

Anaemia is frequently present in patients with acute myocardial infarction (AMI) and contributes to an adverse prognosis. We hypothesised that, besides reduced oxygen carrying capacity, anaemia is associated with (1) red blood cell (RBC) dysfunction and a reduced circulating nitric oxide (NO) pool, (2) compensatory enhancement of vascular and cardiac endothelial nitric oxide synthase (eNOS) activity, and (3) contribution of both, RBC dysfunction and reduced circulatory NO pool to left ventricular (LV) dysfunction and fatal outcome in AMI. In mouse models of subacute and chronic anaemia from repeated mild blood loss the circulating NO pool, RBC, cardiac and vascular function were analysed at baseline and in reperfused AMI. In anaemia, RBC function resulted in profound changes in membrane properties, enhanced turnover, haemolysis, dysregulation of intra-erythrocytotic redox state, and RBC-eNOS. RBC from anaemic mice and from anaemic patients with acute coronary syndrome impaired the recovery of contractile function of isolated mouse hearts following ischaemia/reperfusion. In anaemia, the circulating NO pool was reduced. The cardiac and vascular adaptation to anaemia was characterised by increased arterial eNOS expression and activity and an eNOS-dependent increase of end-diastolic left ventricular volume. Endothelial dysfunction induced through genetic or pharmacologic reduction of eNOS-activity abrogated the anaemia-induced cardio-circulatory compensation. Superimposed AMI was associated with decreased survival. In summary, moderate blood loss anaemia is associated with severe RBC dysfunction and reduced circulating NO pool. Vascular and cardiac eNOS are crucial for the cardio-circulatory adaptation to anaemia. RBC dysfunction together with eNOS dysfunction may contribute to adverse outcomes in AMI.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice.

OBJECTIVE: Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. APPROACH AND RESULTS: Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. CONCLUSIONS: HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.

Sustained hypertension despite endothelial-specific eNOS rescue in eNOS-deficient mice.

The aim of the study was to evaluate the possible contribution of non-endothelial eNOS to the regulation of blood pressure (BP). To accomplish this, a double transgenic strain expressing eNOS exclusively in the vascular endothelium (eNOS-Tg/KO) has been generated by endothelial-specific targeting of bovine eNOS in eNOS-deficient mice (eNOS-KO). Expression of eNOS was evaluated in aorta, myocardium, kidney, brain stem and skeletal muscle. Organ bath studies revealed a complete normalization of aortic reactivity to acetylcholine, phenylephrine and the NO-donors in eNOS-Tg/KO. Function of eNOS in resistance arteries was demonstrated by acute i.v. infusion of acetylcholine and the NOS-inhibitor L-NAME. Acetylcholine decreased mean arterial pressure in all strains but eNOS-KO responded significantly less sensitive as compared eNOS-Tg/KO and C57BL/6. Likewise, acute i.v. L-NAME application elevated mean arterial pressure in C57BL/6 and eNOS-Tg/KO, but not in eNOS-KO. In striking contrast to these findings, mean, systolic and diastolic BP in eNOS-Tg/KO remained significantly elevated and was similar to values of eNOS-KO. Chronic oral treatment with L-NAME increased BP to the level of eNOS-KO only in C57BL/6, but had no effect on hypertension in eNOS-KO and eNOS-Tg/KO. Taken together, functional reconstitution of eNOS in the vasculature of eNOS-KO not even partially lowered BP. These data suggest that the activity of eNOS expressed in non-vascular tissue might play a role in physiologic BP regulation.

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro.

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu(2+)) can dissociate from their physiological carrier proteins. Reduced by the body's own antioxidants, the resultant Cu(1+) might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu(1+)-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu(2+) ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe(2+)) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu(1+)-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Nitric Oxide Is the Cause of Nitroglycerin Tolerance: Providing an Old Dog New Tricks for Acute Heart Failure.

Our paper highlights the past 50 years of research focusing solely on tolerance involving nitroglycerin (glyceryl trinitrate, GTN). It also identifies and discusses inconsistencies in previous mechanistic explanations that have failed to provide a way to administer GTN continuously, free of limitations from tolerance and without the requirement of a nitrate-free interval. We illustrate, for the first time in 135 years, a mechanism whereby nitric oxide, the mediator of vasodilation by GTN, may also be the cause of tolerance. Based on targeting superoxide from mitochondrial complex I, uncoupled by glutathione depletion in response to nitric oxide from GTN, a novel unit dose GTN formulation in glutathione for use as a continuous i.v. infusion has been proposed. We hypothesize that this will reduce or eliminate tolerance seen currently with i.v. GTN. Finally, to evaluate the new formulation we suggest future studies of this new formulation for the treatment of acute decompensated heart failure.

Alterations in endothelial nitric oxide synthase activity and their relevance to blood pressure.

Among all physiologic functions of nitric oxide (NO) known so far, NO-dependent regulation of vascular tone is one of the most important. Under physiological conditions vascular NO is mainly generated by endothelial NO-synthase (eNOS), the major isoform of NOS in the cardiovascular system. NO produced in vascular endothelial cells displays complex physiologic activities considered to be vasoprotective. Of those, the initially detected vasodilation was most rigorously investigated. Increasing the activity of eNOS by genetic approaches in mouse models, non-pharmacologic interventions such as exercise training and treatment with a variety of drugs, for example ACE-inhibitors, reduces blood pressure. Conversely, several experimental and clinical conditions reducing the activity of eNOS and/or initiating the development of endothelial dysfunction show the opposite effect. While robust evidence suggest that endothelial dysfunction occurs in overt hypertension, it is still a matter of debate whether endothelial dysfunction might be an underlying cause of essential hypertension. Therefore, investigations using transgenic mice expressing mutant eNOS enzymes as well as clinical studies demonstrating an association of hypertension with some loss-of-function alleles in the promoter and in exon 7 of the eNOS gene were highlighted in this review. It is concluded that present experimental and clinical data strongly support the view that endothelial dysfunction contributes to the well-known genetic causes of hypertension and should be considered as a pre-hypertensive treatment option.

Red blood cell eNOS is cardioprotective in acute myocardial infarction.

Red blood cells (RBCs) were shown to transport and release nitric oxide (NO) bioactivity and carry an endothelial NO synthase (eNOS). However, the pathophysiological significance of RBC eNOS for cardioprotection in vivo is unknown. Here we aimed to analyze the role of RBC eNOS in the regulation of coronary blood flow, cardiac performance, and acute myocardial infarction (AMI) in vivo. To specifically distinguish the role of RBC eNOS from the endothelial cell (EC) eNOS, we generated RBC- and EC-specific knock-out (KO) and knock-in (KI) mice by Cre-induced inactivation or reactivation of eNOS. We found that RBC eNOS KO mice had fully preserved coronary dilatory responses and LV function. Instead, EC eNOS KO mice had a decreased coronary flow response in isolated perfused hearts and an increased LV developed pressure in response to elevated arterial pressure, while stroke volume was preserved. Interestingly, RBC eNOS KO showed a significantly increased infarct size and aggravated LV dysfunction with decreased stroke volume and cardiac output. This is consistent with reduced NO bioavailability and oxygen delivery capacity in RBC eNOS KOs. Crucially, RBC eNOS KI mice had decreased infarct size and preserved LV function after AMI. In contrast, EC eNOS KO and EC eNOS KI had no differences in infarct size or LV dysfunction after AMI, as compared to the controls. These data demonstrate that EC eNOS controls coronary vasodilator function, but does not directly affect infarct size, while RBC eNOS limits infarct size in AMI. Therefore, RBC eNOS signaling may represent a novel target for interventions in ischemia/reperfusion after myocardial infarction.

Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue.

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.

Inhibition of hyaluronan synthesis accelerates murine atherosclerosis: novel insights into the role of hyaluronan synthesis.

BACKGROUND: Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. METHODS AND RESULTS: Four-week-old apolipoprotein E-deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. CONCLUSIONS: The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.

Regulation of vascular guanylyl cyclase by endothelial nitric oxide-dependent posttranslational modification.

In isolated cells, soluble guanylyl cyclase (sGC) activity is regulated by exogenous nitric oxide (NO) via downregulation of expression and posttranslational S-nitrosylation. The aim of this study was to investigate whether such regulatory mechanism impact on endothelium-dependent vasodilation in a newly developed mouse strain carrying an endothelial-specific overexpression of eNOS (eNOS(++)). When compared with transgene negative controls (eNOS(n)), eNOS(++)-mice showed a 3.3-fold higher endothelial-specific aortic eNOS expression, increased vascular cGMP and VASP phosphorylation, a L-nitroarginine (L-NA)-inhibitable decrease in systolic blood pressure, but normal levels of peroxynitrite and nitrotyrosine formation, endothelium-dependent aortic vasodilation and vasodilation to NO donors. Western blot analysis for sGC showed similar protein levels of sGC-α1 and sGC-ß1 subunits in eNOS(n) and eNOS(++). In striking contrast, the activity of isolated sGC was strongly decreased in lungs of eNOS(++). Semiquantitative evaluation of sGC-ß1-S-nitrosylation demonstrated that this loss of sGC activity is associated with increased nitrosylation of the enzyme in eNOS(++), a difference that disappeared after L-NA-treatment. Our data suggest the existence of a physiologic NO-dependent posttranslational regulation of vascular sGC in mammals involving S-nitrosylation as a key mechanism. Because this mechanism can compensate for reduction in vascular NO bioavailability, it may mask the development of endothelial dysfunction.

Treating Acute Decompensated Heart Failure in Patients with COVID-19 Using Intravenous Nitroglycerin in 5% Glutathione.

The purpose of this current opinion article is to illustrate a novel approach to the treatment of acute decompensated heart failure (ADHF) in coronavirus disease 2019 (COVID-19) patients. The approach described herein relies on a reformulation of intravenous nitroglycerin in 5% glutathione, itself novel, and is felt to have the potential to not only improve the rate of resolution of ADHF, but also reduce the risk of complications of heart failure seen in patients with COVID-19.

Reactive oxygen species as cardiovascular mediators: lessons from endothelial-specific protein overexpression mouse models.

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.

Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo.

Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS(++)) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS(++) mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

Potential role of vasomotor effects of fibrinogen in bradykinin-induced angioedema.

BACKGROUND: Although bradykinin is known to play a major role in the pathophysiology of hereditary and angiotensin-converting enzyme inhibitor (ACEi)-induced angioedema, other factors acting as triggers or enhancers are likely important as well. OBJECTIVE: We hypothesized that fibrinogen might contribute to ACEi-induced angioedema (eg, through direct actions on vascular tone). METHODS: Plasma levels of fibrinogen were determined in 59 patients with acute angioedema. Vascular activity of human and bovine fibrinogen and its effects on bradykinin-induced vasodilation and phosphorylation of vasodilator-stimulated phosphoprotein were investigated in small (0.8-1.4 mm in diameter) porcine coronary artery and human internal thoracic artery (ITA) segments. RESULTS: In patients with ACEi-induced angioedema, fibrinogen levels (481 +/- 22 mg/dL, n = 39) were significantly higher than in patients with idiopathic angioedema (302 +/- 15 mg/dL, P < .001). Fibrinogen (1-15 mumol/L) induced a concentration-dependent vasodilation in preconstricted small porcine coronary arteries (n = 13), reaching a maximum vasodilator effect of 70% +/- 4.7%. Likewise, fibrinogen induced a 52.1% +/- 9.1% (n = 7) vasodilation in ITA rings. Fibrinogen vasorelaxations were completely inhibited by abciximab and diminished by endothelial denudation and treatment with the nitric oxide synthase inhibitor L-nitroargininemethylester and glibenclamide (P < .01). Importantly, fibrinogen increased the vasodilator potency of bradykinin by 10-fold (P < .0001) and increased bradykinin-induced vasodilator-stimulated phosphoprotein phosphorylation (P < .01). CONCLUSION: The increase of plasma fibrinogen levels, its vasodilator activity in human ITAs, and the potentiation of bradykinin-induced vasodilation suggest that fibrinogen might contribute to the pathophysiology of ACEi-induced angioedema. Thus acute-phase proteins, such as fibrinogen, might be viewed as risk factors for bradykinin-induced angioedema.

Exercise-Induced Cardioprotection via eNOS: A Putative Role of Red Blood Cell Signaling.

Moderate exercise training is a key aspect of primary and secondary prevention strategies. Shear-induced upregulation of eNOS activity and function in the vascular endothelium is considered as one of the main molecular mechanisms of exercise-induced protection against myocardial ischemia/ reperfusion (I/R) injury. It has been reported that levels of plasma nitrite, which are largely dependent on eNOS activity, were increased in healthy subjects after acute exercise, while this increase was abolished in coronary artery disease (CAD) patients. Our group and others demonstrated that RBCs contain a functional eNOS, which contributes to systemic nitrite homeostasis and to cardioprotection; moreover, expression and activity of red cell eNOS are decreased in CAD patients and significantly correlated with flow-mediated dilation, a diagnostic marker of endothelial function. Therefore, in addition to vascular eNOS, also red cell eNOS (or in more general terms NO metabolic activity of RBCs) may play a role in exercise-dependent changes of NO-bioavailability. In this review, we will focus on what is known and what is unknown about the role of RBCs in exercise-dependent cardioprotection with emphasis on RBC signaling and red cell eNOS. In detail, we will discuss the effects and molecular mechanisms of shear stress and exercise training on RBC signaling and function, review how these changes may influence blood rheology and systemic hemodynamics and highlight the potential role of red cell eNOS-mediated cardiovascular protection induced by physical activity against myocardial injury in animal and human studies and in clinical settings.

Mechanisms of N-oleoyldopamine activation of central histaminergic neurons.

Histaminergic (HA) neurons located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus fire exclusively during waking and support many physiological functions. We investigated the role of the endovanilloid N-oleoyldopamine (OLDA) in TMN, where dopamine synthesis and its conjugation with oleic acid likely occur. We show that several known targets of OLDA including TRPV1 and cannabinoid receptors are expressed in HA neurons. In contrast to capsaicin, which failed to increase firing of HA neurons in TRPV1 knockout mice (TRPVI KO), OLDA was still able to induce excitation. This excitation was not sensitive to the blockade of cannabinoid receptors 1 and 2 and could result from OLDA interaction with GPR119, as the ligand of GPR119, oleoylethanolamide (OEA), also increased the firing of HA neurons. However, we ruled out this possibility as OEA- (but not OLDA-) excitation was abolished by the PPAR (peroxisome proliferator activated receptor) alpha antagonist MK886. The dopamine uptake blocker nomifensine blanked OLDA-excitation and dopamine receptor antagonists abolished the OLDA action in TRPV1 KO mice. Therefore OLDA excites HA neurons through multiple targets suggesting a central role of the histaminergic system in the behavioral stimulation seen after systemic OLDA application.