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1.
Br J Pharmacol ; 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956895

RESUMO

BACKGROUND AND PURPOSE: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline-rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. EXPERIMENTAL APPROACH: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. KEY RESULTS: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. CONCLUSION AND IMPLICATIONS: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes.

2.
Circulation ; 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38836358

RESUMO

BACKGROUND: Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. METHODS: We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and NO consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. RESULTS: In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb and plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. CONCLUSIONS: AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05603520. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01805739.

3.
Br J Pharmacol ; 181(11): 1553-1575, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38519837

RESUMO

In 2023, seventy novel drugs received market authorization for the first time in either Europe (by the EMA and the MHRA) or in the United States (by the FDA). Confirming a steady recent trend, more than half of these drugs target rare diseases or intractable forms of cancer. Thirty drugs are categorized as "first-in-class" (FIC), illustrating the quality of research and innovation that drives new chemical entity discovery and development. We succinctly describe the mechanism of action of most of these FIC drugs and discuss the therapeutic areas covered, as well as the chemical category to which these drugs belong. The 2023 novel drug list also demonstrates an unabated emphasis on polypeptides (recombinant proteins and antibodies), Advanced Therapy Medicinal Products (gene and cell therapies) and RNA therapeutics, including the first-ever approval of a CRISPR-Cas9-based gene-editing cell therapy.


Assuntos
Aprovação de Drogas , United States Food and Drug Administration , Humanos , Europa (Continente) , Estados Unidos
4.
Antioxidants (Basel) ; 13(2)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38397777

RESUMO

Ozone-based chemiluminescence detection (CLD) has been widely applied for determining nitric oxide (•NO) and its derived species in many different fields, such as environmental monitoring and biomedical research. In humans and animals, CLD has been applied to determine exhaled •NO and •NO metabolites in plasma and tissues. The main advantages of CLD are high sensitivity and selectivity for quantitative analysis in a wide dynamic range. Combining CLD with analytical separation techniques like chromatography allows for the analytes to be quantified with less disturbance from matrix components or impurities. Sampling techniques like microdialysis and flow injection analysis may be coupled to CLD with the possibility of real-time monitoring of •NO. However, details and precautions in experimental practice need to be addressed and clarified to avoid wrong estimations. Therefore, using CLD as a detection tool requires a deep understanding of the sample preparation procedure and chemical reactions used for liberating •NO from its derived species. In this review, we discuss the advantages and pitfalls of CLD for determining •NO species, list the different applications and combinations with other analytical techniques, and provide general practical notes for sample preparation. These guidelines are designed to assist researchers in comprehending CLD data and in selecting the most appropriate method for measuring •NO species.

5.
Physiol Genomics ; 56(2): 113-127, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37982169

RESUMO

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.


Assuntos
Endotélio Vascular , Genes Mitocondriais , Humanos , Camundongos , Animais , Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Artérias , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo/metabolismo
6.
J Clin Invest ; 133(17)2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37655658

RESUMO

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.


Assuntos
Cardiotônicos , GMP Cíclico , Eritrócitos , Guanilil Ciclase Solúvel , Animais , Camundongos , Hipóxia , Miócitos Cardíacos , Nitratos , Óxido Nítrico , Ratos , Humanos
7.
Br J Pharmacol ; 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37658519

RESUMO

Red blood cells (RBCs) have traditionally been seen as simple carriers of gases and nutrients in the body. One important non-canonical function of RBCs in the cardiovascular system is the regulation of nitric oxide (NO) metabolism. It has been shown that RBCs can scavenge NO, transport NO metabolites and produce NO in hypoxic conditions, thereby inducing hypoxic vasodilation. RBCs also express endothelial nitric oxide synthase (eNOS). However, the physiological significance of RBC eNOS has been controversial for many years. This review article provides a comprehensive overview of the experimental research on RBC eNOS signalling in vivo. The data show that RBC eNOS signalling modulates intracellular NO production and NO-haem levels, as well as participating in extracellular paracrine NO metabolite signalling, which contributes to regulating peripheral vascular resistance, blood pressure and cardioprotection. Additionally, this article explores the molecular mechanisms of sytemic regulation mediated by RBC eNOS and the implications of RBC eNOS in cardiovascular health and disease.

8.
Antioxidants (Basel) ; 12(9)2023 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-37760039

RESUMO

Beyond their established role as oxygen carriers, red blood cells have recently been found to contribute to systemic NO and sulfide metabolism and act as potent circulating antioxidant cells. Emerging evidence indicates that reactive species derived from the metabolism of O2, NO, and H2S can interact with each other, potentially influencing common biological targets. These interactions have been encompassed in the concept of the reactive species interactome. This review explores the potential application of the concept of reactive species interactome to understand the redox physiology of RBCs. It specifically examines how reactive species are generated and detoxified, their interactions with each other, and their targets. Hemoglobin is a key player in the reactive species interactome within RBCs, given its abundance and fundamental role in O2/CO2 exchange, NO transport/metabolism, and sulfur species binding/production. Future research should focus on understanding how modulation of the reactive species interactome may regulate RBC biology, physiology, and their systemic effects.

10.
Redox Biol ; 60: 102612, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36681048

RESUMO

BACKGROUND & AIMS: Nitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. METHODS & RESULTS: RBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. CONCLUSIONS: RBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia.


Assuntos
Doenças Cardiovasculares , Pré-Eclâmpsia , Doenças Vasculares , Camundongos , Feminino , Humanos , Gravidez , Animais , Endotélio Vascular/metabolismo , Doenças Cardiovasculares/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Arginase/genética , Arginase/metabolismo , Pré-Eclâmpsia/metabolismo , Estresse Oxidativo , Óxido Nítrico/metabolismo , Eritrócitos/metabolismo
11.
Nat Commun ; 13(1): 6405, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302779

RESUMO

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.


Assuntos
Óxido Nítrico , Nitrito Redutases , Camundongos , Animais , Nitrito Redutases/genética , Nitrito Redutases/farmacologia , Óxido Nítrico/farmacologia , Nitritos , alfa-Globinas/genética , Hipóxia , Endotélio Vascular , Hemoglobinas/genética , Vasodilatação/fisiologia
12.
Redox Biol ; 54: 102370, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35759945

RESUMO

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.


Assuntos
Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Eritrócitos , Coração , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Traumatismo por Reperfusão Miocárdica/genética , Óxido Nítrico , Óxido Nítrico Sintase Tipo III/genética , Vasodilatadores
13.
EXCLI J ; 21: 623-646, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35721574

RESUMO

Living organisms need to be able to cope with environmental challenges and other stressors and mount adequate responses that are as varied as the spectrum of those challenges. Understanding how the multi-layered biological stress responses become integrated across and between different levels of organization within an organism can provide a different perspective on the nature and inter-relationship of complex systems in health and disease. We here compare two concepts which have been very influential in stress research: Selye's 'General Adaptation Syndrome' and Sies's 'Oxidative Stress' paradigm. We show that both can be embraced within a more general framework of 'change and response'. The 'Reactive Species Interactome' allows each of these to be considered as distinct but complementary aspects of the same system, representative of roles at different levels of organization within a functional hierarchy. The versatile chemistry of sulfur - exemplified by hydrogen sulfide, glutathione and proteinous cysteine thiols - enriched by its interactions with reactive oxygen, nitrogen and sulfur species, would seem to sit at the heart of the 'Redox Code' and underpin the ability of complex organisms to cope with stress.

14.
Nitric Oxide ; 125-126: 69-77, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35752264

RESUMO

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.


Assuntos
Arginase , Óxido Nítrico Sintase Tipo III , Animais , Arginase/genética , Arginase/metabolismo , Arginina/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ornitina , Tamoxifeno/metabolismo , Ureia/metabolismo
15.
Cells ; 11(3)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35159317

RESUMO

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.


Assuntos
Arginase , Células Estreladas do Fígado , Proteína Oncogênica p21(ras) , Animais , Arginase/metabolismo , Cromatografia Líquida , Células-Tronco Embrionárias/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Proteína Oncogênica p21(ras)/metabolismo , Ratos , Espectrometria de Massas em Tandem
16.
Physiol Rev ; 102(2): 859-892, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34486392

RESUMO

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.


Assuntos
Fenômenos Fisiológicos Cardiovasculares , Citoglobina/metabolismo , Células Endoteliais/metabolismo , Globinas/metabolismo , Animais , Humanos , Mioglobina/metabolismo , Neuroglobina/metabolismo
17.
Circulation ; 144(11): 870-889, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34229449

RESUMO

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.


Assuntos
Pressão Sanguínea/fisiologia , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Acetilcolina/farmacologia , Animais , Doenças da Aorta/tratamento farmacológico , Arginina/análogos & derivados , Arginina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Contagem de Eritrócitos/métodos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Camundongos
18.
Nitric Oxide ; 115: 44-54, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34325012

RESUMO

Diaminofluoresceins (DAFs) are fluorescent probes widely applied to measure nitric oxide (NO) formation in cells and tissues. The main advantages of these compounds are their availability and low cost, and the general availability of instruments able to detect green fluorescence in all laboratories; these include fluorimeters, flow cytometers, and fluorescent microscopes. What made these molecules particularly interesting for many scientists approaching the NO field is that they are apparently very easy to use, as compared with other techniques requiring specific instrumentation and knowledge like chemiluminescence and electron paramagnetic resonance. However, the reactivity and biological chemistry of these probes in the cellular environment is rather complex and still not fully understood. Moreover, secondary reactions with ascorbate, or interference with thiols occur in cells. Therefore, the use of DAFs requires specific experimental planning and a careful interpretation of the results obtained. In this methodological review, we described in detail what is known about the reactivity of DAFs, their application in biological assays, list some principles to help experimental planning, including the necessary controls, and list the caveats concerning result interpretation. These guiding principles will help to understand the "Method behind our DAF-madness".


Assuntos
Fluoresceína/química , Corantes Fluorescentes/química , Óxido Nítrico/análise , Estrutura Molecular
19.
Free Radic Biol Med ; 168: 95-109, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33789125

RESUMO

The fundamental physiology of circulating red blood cells (RBCs) and platelets involving regulation of oxygen transport and hemostasis, respectively, are well-described in the literature. Their abundance in the circulation and their interaction with the vascular wall and each other have attracted the attention of other putative physiological and pathophysiological effects of these cells. RBCs and platelets are both important regulators of redox balance harboring powerful pro-oxidant and anti-oxidant (enzymatic and non-enzymatic) capacities. They are also involved in the regulation of vascular tone mainly via export of nitric oxide bioactivity and adenosine triphosphate. Of further importance are emerging observations that these cells undergo functional alterations when exposed to risk factors for cardiovascular disease and during developed cardiometabolic diseases. Under these conditions, the RBCs and platelets contribute to increased oxidative stress by their formation of reactive species including superoxide anion radical, hydrogen peroxide and peroxynitrite. These alterations trigger key changes in the vascular wall characterized by enhanced oxidative stress, reduced nitric oxide bioavailability and endothelial dysfunction. Additional pathophysiological effects are triggered in the heart resulting in increased susceptibility to ischemia-reperfusion injury with impairment in cardiac function. Pharmacological interventions aiming at restoring circulating cell function has been shown to exert marked beneficial effects on cardiovascular function. In this review, we summarize the current knowledge of RBC and platelet biology with special focus on redox biology, their roles in the development of cardiovascular disease and potential therapeutic strategies targeting RBC and platelet dysfunction. Finally, the complex and scarcely understood interaction between RBCs and platelets is discussed.


Assuntos
Plaquetas , Doenças Cardiovasculares , Eritrócitos , Humanos , Óxido Nítrico , Oxirredução , Estresse Oxidativo
20.
Biol Chem ; 402(3): 317-331, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33544503

RESUMO

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.


Assuntos
Eritrócitos/metabolismo , Cisteína/metabolismo , Humanos , Oxirredução , Espectrina/metabolismo
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