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1.
Artigo em Inglês | MEDLINE | ID: mdl-33748918

RESUMO

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.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33079828

RESUMO

Involvement of the vascular endothelium in the complications of COVID-19 is now recognized. Chief among these are pulmonary endotheliitis, cytokine storm, endotoxic shock and cardiovascular collapse. This Perspectives paper 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 COVID-19.

3.
Basic Res Cardiol ; 115(4): 43, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533377

RESUMO

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.

4.
Neuropharmacology ; 143: 327-338, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30219501

RESUMO

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.


Assuntos
Dopamina/análogos & derivados , Histamina/metabolismo , Região Hipotalâmica Lateral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Animais , Dopamina/farmacologia , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/crescimento & desenvolvimento , Região Hipotalâmica Lateral/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Técnicas de Cultura de Tecidos
5.
Oxid Med Cell Longev ; 2018: 8309698, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29854098

RESUMO

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.


Assuntos
Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase Tipo II/genética , Animais , Aorta/metabolismo , Cardiomegalia/diagnóstico por imagem , Glutationa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Fator 2 Relacionado a NF-E2/deficiência , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Ultrassonografia , Função Ventricular Esquerda/fisiologia
6.
Front Physiol ; 9: 332, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867516

RESUMO

The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.

7.
Curr Med Chem ; 25(34): 4457-4474, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29521199

RESUMO

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.


Assuntos
Eritrócitos/metabolismo , Exercício Físico , Óxido Nítrico Sintase Tipo III/metabolismo , Conexinas/metabolismo , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/prevenção & controle , Eritrócitos/citologia , Humanos , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico/metabolismo , Resistência ao Cisalhamento , Transdução de Sinais
8.
Br J Pharmacol ; 175(10): 1607-1620, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29465763

RESUMO

BACKGROUND AND PURPOSE: Non-allergic angio-oedema is a life-threatening disease mediated by activation of bradykinin type 2 receptors (B2 receptors). The aim of this study was to investigate whether activation of B2 receptors by endogenous bradykinin contributes to physiological extravasation. This may shed new light on the assumption that treatment with an angiotensin converting enzyme inhibitor (ACEi) results in an alteration in the vascular barrier function predisposing to non-allergic angio-oedema. EXPERIMENTAL APPROACH: We generated a new transgenic mouse model characterized by endothelium-specific overexpression of the B2 receptor (B2tg ) and established a non-invasive two-photon laser microscopy approach to measure the kinetics of spontaneous extravasation in vivo. The B2tg mice showed normal morphology and litter size as compared with their transgene-negative littermates (B2n ). KEY RESULTS: Overexpression of B2 receptors was functional in conductance vessels and resistance vessels as evidenced by B2 receptor-mediated aortic dilation to bradykinin in presence of non-specific COX inhibitor diclofenac and by significant hypotension in B2tg respectively. Measurement of dermal extravasation by Miles assay showed that bradykinin induced extravasation was significantly increased in B2tg as compared with B2n . However, neither endothelial overexpression of B2 receptors nor treatment with the ACEi moexipril or B2 antagonist icatibant had any effect on spontaneous extravasation measured by two-photon laser microscopy. CONCLUSIONS AND IMPLICATIONS: Activation of B2 receptors does not appear to be involved in spontaneous extravasation. Therefore, the assumption that treatment with an ACEi results in an alteration in the physiological vascular barrier function predisposing to non-allergic angio-oedema is not supported by our findings.


Assuntos
Edema/metabolismo , Receptor B2 da Bradicinina/metabolismo , Pele/metabolismo , Animais , Bradicinina/sangue , Bradicinina/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor B2 da Bradicinina/genética
9.
J Hypertens ; 35(1): 76-88, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27861245

RESUMO

OBJECTIVES: Endothelial dysfunction and oxidative stress are associated with hypertension but whether endothelial superoxide may play a role in the early development of essential hypertension remains uncertain. We investigated whether endothelial nitric oxide synthase (eNOS)-derived endothelial oxidative stress is involved in the regulation of SBP. METHODS: Wild-type eNOS [mice with endothelium-specific overexpression of bovine endothelial NO-synthase (eNOS-Tg)] or a novel dimer-destabilized eNOS-mutant harboring a partially disrupted zinc-finger [mice with endothelium-specific overexpression of destabilized bovine eNOS destabilized by replacement of Cys 101 to Ala (C101A-eNOS-Tg)] was introduced in C57BL/6 in an endothelial-specific manner. Mice were monitored for aortic endothelium-dependent relaxation, SBP, levels of superoxide and several posttranslational modifications indicating activity and/or increased vascular oxidative stress. Some groups of mice underwent voluntary exercise training for 4 weeks or treatment with the superoxide dismutase mimetic Tempol. RESULTS: C101A-eNOS-Tg showed significantly increased superoxide generation, protein-tyrosine-nitration and eNOS-tyrosine-nitration, eNOS-S-glutathionylation, eNOS phosphorylation and AMP kinase-α phosphorylation at Thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared with eNOS-Tg and wild-type controls. Exercise training increased phosphorylation of eNOS at Ser and AMP kinase-α in wild-type. These physiologic adaptations were absent in C101A-eNOS-Tg. Maximal aortic endothelium-dependent relaxation was similar in all strains. C101A-eNOS-Tg displayed normal SBP despite higher levels of eNOS, whereas eNOS-Tg showed significant hypotension. Tempol completely reversed the occurring protein modifications and significantly reduced SBP in C101A-eNOS-Tg but not in wild-type. CONCLUSION: Oxidative stress generated by endothelial-specific expression of genetically destabilized C101A-eNOS selectively prevents SBP-reducing activity of vascular eNOS, while having no effect on aortic endothelium-dependent relaxation. These data suggest that oxidative stress in microvascular endothelium may play a role for the development of essential hypertension.


Assuntos
Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/fisiologia , Animais , Bovinos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Mutação/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fosforilação , Estabilidade Proteica
10.
Antioxid Redox Signal ; 26(16): 917-935, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27927026

RESUMO

SIGNIFICANCE: In this review, we discuss the role of nitric oxide (NO) as a key physiological mechanotransducer modulating both local and systemic heterocellular communication and contributing to the integrated (patho)physiology of the cardiovascular system. A deeper understanding of mechanotransduction-mediated local and systemic nodes controlling heterocellular communication between the endothelium, blood cells, and other cell types (e.g., cardiomyocytes) may suggest novel therapeutic strategies for endothelial dysfunction and cardiovascular disease. Recent Advances: Mechanical forces acting on mechanoreceptors on endothelial cells activate the endothelial NO synthase (eNOS) to produce NO. NO participates in (i) abluminal heterocellular communication, inducing vasorelaxation, and thereby regulating vascular tone and blood pressure; (ii) luminal heterocellular communication, inhibiting platelet aggregation, and controlling hemostasis; and (iii) systemic heterocellular communication, contributing to adaptive physiological processes in response to exercise and remote ischemic preconditioning. Interestingly, shear-induced eNOS-dependent activation of vascular heterocellular communication constitutes the molecular basis of all methods applied in the clinical routine for evaluation of endothelial function. Critical Issues and Future Directions: The integrated physiology of heterocellular communication is still not fully understood. Dedicated experimental models are needed to analyze messengers and mechanisms underpinning heterocellular communication in response to physical forces in the cardiovascular system (and elsewhere). Antioxid. Redox Signal. 26, 917-935.


Assuntos
Células Endoteliais/citologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animais , Comunicação Celular , Células Endoteliais/enzimologia , Humanos , Mecanotransdução Celular , Estresse Mecânico
11.
Antioxid Redox Signal ; 26(13): 718-742, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27889956

RESUMO

SIGNIFICANCE: Recent clinical evidence identified anemia to be correlated with severe complications of cardiovascular disease (CVD) such as bleeding, thromboembolic events, stroke, hypertension, arrhythmias, and inflammation, particularly in elderly patients. The underlying mechanisms of these complications are largely unidentified. Recent Advances: Previously, red blood cells (RBCs) were considered exclusively as transporters of oxygen and nutrients to the tissues. More recent experimental evidence indicates that RBCs are important interorgan communication systems with additional functions, including participation in control of systemic nitric oxide metabolism, redox regulation, blood rheology, and viscosity. In this article, we aim to revise and discuss the potential impact of these noncanonical functions of RBCs and their dysfunction in the cardiovascular system and in anemia. CRITICAL ISSUES: The mechanistic links between changes of RBC functional properties and cardiovascular complications related to anemia have not been untangled so far. FUTURE DIRECTIONS: To allow a better understanding of the complications associated with anemia in CVD, basic and translational science studies should be focused on identifying the role of noncanonical functions of RBCs in the cardiovascular system and on defining intrinsic and/or systemic dysfunction of RBCs in anemia and its relationship to CVD both in animal models and clinical settings. Antioxid. Redox Signal. 26, 718-742.


Assuntos
Anemia/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico/metabolismo , Animais , Humanos , Oxirredução
12.
Biochem Pharmacol ; 112: 24-36, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27235748

RESUMO

Increasing vascular NO levels following up-regulation of endothelial nitric oxide synthase (eNOS) is considered beneficial in cardiovascular disease. Whether such beneficial effects exerted by increased NO-levels include the vascular renin-angiotensin system remains elucidated. Exposure of endothelial cells originated from porcine aorta, mouse brain and human umbilical veins to different NO-donors showed that expression of the angiotensin-II-type-2-receptor (AT2) mRNA and protein is up-regulated by activation of soluble guanylyl cyclase, protein kinase G and p38 mitogen-activated protein kinase without changing AT2 mRNA stability. In mice, endothelial-specific overexpression of eNOS stimulated, while chronic treatment with the NOS-blocker l-nitroarginine inhibited AT2 expression. The NO-induced AT2 up-regulation was associated with a profound inhibition of angiotensin-converting enzyme (ACE)-activity. In endothelial cells this reduction of ACE-activity was reversed by either the AT2 antagonist PD 123119 or by inhibition of transcription with actinomycin D. Furthermore, in C57Bl/6 mice an acute i.v. bolus of l-nitroarginine did not change AT2-expression and ACE-activity suggesting that inhibition of ACE-activity by endogenous NO is crucially dependent on AT2 protein level. Likewise, three weeks of either voluntary or forced exercise training increased AT2 expression and reduced ACE-activity in C57Bl/6 but not in mice lacking eNOS suggesting significance of this signaling interaction for vascular physiology. Finally, aortic AT2 expression is about 5 times greater in female as compared to male C57Bl/6 and at the same time aortic ACE activity is reduced in females by more than 50%. Together these findings imply that endothelial NO regulates AT2 expression and that AT2 may regulate ACE-activity.


Assuntos
Células Endoteliais/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Animais , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Sistema Renina-Angiotensina/efeitos dos fármacos , Suínos , Regulação para Cima
13.
Neuropharmacology ; 107: 100-110, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27012889

RESUMO

Nicotinic acetylcholine receptor (nAChR) subtypes containing the α4 subunit, particularly α4ß2 nAChRs, play an important role in cognitive functioning. The impact of the smoking cessation aid varenicline, a selective partial α4ß2 nAChR agonist, on (1) changes of central protein and mRNA expression of this receptor and (2) on memory deficits in a mouse model of cognitive impairment was investigated. Protein and mRNA expression of both the α4 and ß2 receptor subunits in mouse brain endothelial and hippocampal cells as well as hippocampus and neocortex tissues were determined by western blot and realtime PCR, respectively. The ß2 antibody showed low specificity, though. Tissues were examined following a 2-week oral treatment with various doses of varenicline (0.01, 0.1, 1, 3 mg/kg/day) or vehicle. In addition, episodic memory of mice was assessed following this treatment with an object recognition task using (1) normal mice and (2) animals with anticholinergic-induced memory impairment (i.p. injection of 0.5 mg/kg scopolamine). Varenicline dose-dependently increased protein expression of both the α4 and ß2 subunit in cell cultures and brain tissues, respectively, but had no effect on mRNA expression of both subunits. Scopolamine injection induced a significant reduction of object memory in vehicle-treated mice. By contrast, cognitive performance was not altered by scopolamine in varenicline-treated mice. In conclusion, a 2-week oral treatment with varenicline prevented memory impairment in the scopolamine mouse model. In parallel, protein, but not mRNA expression was upregulated, suggesting a posttranscriptional mechanism. Our findings suggest a beneficial effect of varenicline on cognitive dysfunction.


Assuntos
Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Nootrópicos/farmacologia , Receptores Nicotínicos/metabolismo , Vareniclina/farmacologia , Administração Oral , Animais , Encéfalo/metabolismo , Linhagem Celular , Cognição/fisiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Escopolamina
14.
Exp Clin Transplant ; 14(1): 86-92, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862826

RESUMO

OBJECTIVES: Methods for conservation and preservation of vascular grafts are often controversially discussed. Furthermore, immunologic monitoring or immunotherapy for allogeneic graft is not considered necessary in many cases. The present study was initiated to examine the cellular vitality and functional efficiency of vein transplant during preservation. MATERIALS AND METHODS: Twenty-seven human vein segments (vena saphena magna) were stored after explant in University of Wisconsin solution or histidine-tryptophan-ketoglutarate solution at 4 °C. After 3, 24, 48, 72, and 96 hours, vein functionality was tested. Ring segments were fixed by triangles in Krebs-Henseleit buffer. Contractile function was measured after addition of potassium chloride solution (80 mM) and phenylephrine (0.2, 2, or 20 µM). To investigate endothelium-dependent vasorelaxation, 1 µM acetylcholine was added. RESULTS: Of 27 segments, 5 showed endothelium-dependent relaxation. Vasorelaxation continued for up to 48 hours after administration of acetylcholine in University of Wisconsin solution and for up to 24 hours in histidine-tryptophane-ketoglutarate solution. At 48 hours, potassium chloride solution-induced vasocontraction was 17% more effective than phenylephrine in University of Wisconsin solution. University of Wisconsin solution was significantly more effective than histidine-tryptophane-ketoglutarate solution in terms of preservation of phenylephrine (0.2, 2 µM)-induced vasocontraction. Phenylephrine (2 µM)-induced contraction was retained in University of Wisconsin solution after 24 hours by 81% and after 48 hours by 55%, with comparable results in histidine-tryptophane-ketoglutarate solution of only 62% and 34% after 24 and 48 hours. CONCLUSIONS: At 48 hours, human saphenous vein transplants had better endothelium and smooth muscle function when preserved in University of Wisconsin solution versus histidine-tryptophane-ketoglutarate solution.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Veia Safena/efeitos dos fármacos , Preservação de Tecido/métodos , Adenosina/farmacologia , Alopurinol/farmacologia , Temperatura Baixa , Relação Dose-Resposta a Droga , Endotélio Vascular/transplante , Endotélio Vascular/ultraestrutura , Glucose/farmacologia , Glutationa/farmacologia , Humanos , Insulina/farmacologia , Manitol/farmacologia , Músculo Liso Vascular/transplante , Músculo Liso Vascular/ultraestrutura , Cloreto de Potássio/farmacologia , Procaína/farmacologia , Rafinose/farmacologia , Veia Safena/transplante , Veia Safena/ultraestrutura , Fatores de Tempo , Coleta de Tecidos e Órgãos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
15.
Neuropharmacology ; 106: 102-15, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26297536

RESUMO

Using a reporter mouse model with expression of the tomato fluorescent protein under the dopamine transporter promoter (Tmt-DAT) we discovered a new group of neurons in the histaminergic tuberomamillary nucleus (TMN), which, in contrast to tuberoinfundibular dopaminergic neurons of the dorsomedial arcuate nucleus, do not express tyrosine hydroxylase but can synthesize and store dopamine. Tmt-DAT neurons located within TMN share electrophysiological properties with histaminergic neurons: spontaneous firing at a membrane potential around -50 mV and presence of hyperpolarization-activated cyclic nucleotide-gated ion channels. Histamine (30 µM) depolarizes and excites Tmt-DAT neurons through H1R activation but inhibits histaminergic neurons through H3R activation thus allowing a pharmacological identification of the different neurons. Single-cell RT-PCR revealed that all histaminergic neurons expressing histidine decarboxylase (HDC) also express H3R. This includes neurons retrogradely traced from the striatum whose inhibition by a selective H3R agonist was indistinguishable from the whole population. Prolonged depolarization reduces the autoinhibition. The potency of histamine at H3R depends on membrane potential and on extracellular and intracellular calcium. Autoinhibition can be impaired by preincubation with capsaicin, a ligand of the calcium-permeable TRPV1 channel or by blockade of Ca(2+)-ATPase with thapsigargin. The pharmacology of autoinhibition is revisited and physiological conditions for its functionality are determined. Usage of reporter mouse models for the safe identification of aminergic neurons under pathophysiological conditions is recommended. This article is part of the Special Issue entitled 'Histamine Receptors'.


Assuntos
Histamina/metabolismo , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptores Histamínicos H3/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Histidina Descarboxilase/metabolismo , Região Hipotalâmica Lateral/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Canais de Cátion TRPV/metabolismo , Técnicas de Cultura de Tecidos
16.
Arterioscler Thromb Vasc Biol ; 36(2): e9-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26586662

RESUMO

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.


Assuntos
Doenças das Artérias Carótidas/enzimologia , Glucuronosiltransferase/deficiência , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Neointima , Animais , Becaplermina , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/enzimologia , Artéria Carótida Primitiva/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Genótipo , Glucuronosiltransferase/genética , Hialuronan Sintases , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Transdução de Sinais , Transcrição Genética , Transfecção
17.
Free Radic Biol Med ; 89: 906-17, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26475037

RESUMO

Increased production of reactive oxygen species and failure of the antioxidant defense system are considered to play a central role in the pathogenesis of cardiovascular disease. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch controlling the expression of antioxidant and protective enzymes, and was proposed to participate in protection of vascular and cardiac function. This study was undertaken to analyze cardiac and vascular phenotype of mice lacking Nrf2. We found that Nrf2 knock out (Nrf2 KO) mice have a left ventricular (LV) diastolic dysfunction, characterized by prolonged E wave deceleration time, relaxation time and total diastolic time, increased E/A ratio and myocardial performance index, as assessed by echocardiography. LV dysfunction in Nrf2 KO mice was associated with cardiac hypertrophy, and a downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in the myocardium. Accordingly, cardiac relaxation was impaired, as demonstrated by decreased responses to ß-adrenergic stimulation by isoproterenol ex vivo, and to the cardiac glycoside ouabain in vivo. Surprisingly, we found that vascular endothelial function and endothelial nitric oxide synthase (eNOS)-mediated vascular responses were fully preserved, blood pressure was decreased, and eNOS was upregulated in the aorta and the heart of Nrf2 KO mice. Taken together, these results show that LV dysfunction in Nrf2 KO mice is mainly associated with cardiac hypertrophy and downregulation of SERCA2a, and is independent from changes in coronary vascular function or systemic hemodynamics, which are preserved by a compensatory upregulation of eNOS. These data provide new insights into how Nrf2 expression/function impacts the cardiovascular system.


Assuntos
Cardiomegalia/patologia , Cardiomiopatias/patologia , Endotélio Vascular/patologia , Fator 2 Relacionado a NF-E2/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Disfunção Ventricular Esquerda/patologia , Animais , Western Blotting , Cardiomegalia/etiologia , Cardiomiopatias/etiologia , Proliferação de Células , Células Cultivadas , Ecocardiografia , Endotélio Vascular/metabolismo , Feminino , Hemodinâmica , Células Endoteliais da Veia Umbilical Humana , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Disfunção Ventricular Esquerda/etiologia , Remodelação Ventricular
18.
Antioxid Redox Signal ; 23(9): 711-23, 2015 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-25764009

RESUMO

AIMS: Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia. RESULTS: A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only. INNOVATION: Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease. CONCLUSION: eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation.


Assuntos
Endotélio Vascular/metabolismo , Neointima/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Alanina/metabolismo , Animais , Aorta/metabolismo , Bovinos , Citrulina/metabolismo , Cisteína/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Superóxidos/metabolismo , Vasodilatação
19.
Biochem Biophys Res Commun ; 458(3): 576-583, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25680465

RESUMO

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.


Assuntos
Pressão Sanguínea , Endotélio Vascular/fisiopatologia , Hipertensão/genética , Hipertensão/fisiopatologia , Óxido Nítrico Sintase Tipo III/genética , Animais , Bradicardia/complicações , Bovinos , Endotélio Vascular/metabolismo , Hipertensão/complicações , Hipertensão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo
20.
Hypertension ; 63(2): 265-72, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24191281

RESUMO

Apolipoprotein E-deficient (apoE(-/-)) mice fed on Western diet are characterized by increased vascular resistance and atherosclerosis. Previously, we have shown that chronic angiotensin (Ang)-(1-7) treatment ameliorates endothelial dysfunction in apoE(-/-) mice. However, the mechanism of Ang-(1-7) on vasoconstrictor response to Ang II is unknown. To examine Ang-(1-7) function, we used apoE(-/-) and wild-type mice fed on Western diet that were treated via osmotic minipumps either with Ang-(1-7) (82 µg/kg per hour) or saline for 6 weeks. We show that Ang II-induced renal pressor response was significantly increased in apoE(-/-) compared with wild-type mice. This apoE(-/-)-specific response is attributed to reactive oxygen species-mediated p38 mitogen-activated protein kinase activation and subsequent phosphorylation of myosin light chain (MLC(20)), causing renal vasoconstriction. Here, we provide evidence that chronic Ang-(1-7) treatment attenuated the renal pressor response to Ang II in apoE(-/-) mice to wild-type levels. Ang-(1-7) treatment significantly decreased renal inducible nicotinamide adenine dinucleotide phosphate subunit p47phox levels and, thus, reactive oxygen species production that in turn causes decreased p38 mitogen-activated protein kinase activity. The latter has been confirmed by administration of a specific p38 mitogen-activated protein kinase inhibitor SB203580 (5 µmol/L), causing a reduced renal pressor response to Ang II in apoE(-/-) but not in apoE(-/-) mice treated with Ang-(1-7). Moreover, Ang-(1-7) treatment had no effect in Mas(-/-)/apoE(-/-) double-knockout mice confirming the specificity of Ang-(1-7) action through the Mas-receptor. In summary, Ang-(1-7) modulates vascular function via Mas-receptor activation that attenuates pressor response to Ang II in apoE(-/-) mice by reducing reactive oxygen species-mediated p38 mitogen-activated protein kinase activity.


Assuntos
Angiotensina I/farmacologia , Hipertensão Renal/tratamento farmacológico , Naftalenos/farmacologia , Fragmentos de Peptídeos/farmacologia , Pirazóis/farmacologia , Circulação Renal/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Feminino , Hipertensão Renal/genética , Hipertensão Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas-G/genética , Circulação Renal/fisiologia , Resistência Vascular/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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