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1.
Int J Clin Pract ; 75(11): e14771, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34473881

RESUMO

BACKGROUND: Phosphatidylserine (PS) externalization out of the membrane facilitates the eryptotic erythrocytes (EE) binding to endothelial cells (EC), potentially leading to atherosclerosis. Thus, the levels of eryptosis and interactions of EE-EC in hypercholesterolemic patients, either non-medicated or medicated, compared with healthy subjects were studied. METHODS: A total of 56 subjects clustered into three groups: (control (n = 20), hypercholesterolemic non-treated (HCNT) (n = 15), and statin-treated (HCT) (n = 21)) were enrolled in this cross-sectional study. Biochemical parameters were determined with validated and standard methods. PS exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and GSH from CMFDA fluorescence by flow cytometry. The erythrocyte-EC adhesion assay was performed by using the parallel-plate flow chamber technique. RESULTS: Higher PS externalization and adhesion of erythrocytes to EC (P < .05) was found in hypercholesterolemic subjects, regardless of statin treatment, compared with the control group. Although no correlation between FSC and PS externalization with other parameters was found, GSH was inversely correlated with erythrocyte adhesion, which was significantly correlated with total cholesterol, LDL-c, and apolipoprotein B. CONCLUSION: The link between hypercholesterolemia and eryptosis suggests a possible detrimental impact of this binomial on endothelial function with possible further development of atherosclerosis and microcirculation problems in hypercholesterolemic patients, independently of statin therapy.


Assuntos
Eriptose , Inibidores de Hidroximetilglutaril-CoA Redutases , Cálcio , Estudos Transversais , Células Endoteliais , Endotélio , Eritrócitos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico
2.
Cell Physiol Biochem ; 55(4): 449-459, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34259420

RESUMO

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.


Assuntos
Eriptose , Eritrócitos/metabolismo , Oxigênio/sangue , Insuficiência Renal Crônica/sangue , Uremia/sangue , Adolescente , Adulto , Idoso , Anexina A5/sangue , Cálcio/sangue , Hipóxia Celular , Eritrócitos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/patologia , Uremia/patologia
3.
Turk J Med Sci ; 51(5): 2534-2542, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34174794

RESUMO

Background/aim: The present study aimed to assess erythrocyte morphology in newly diagnosed type 2 diabetes mellitus patients using scanning electron microscopy. Materials and methods: In total, 30 patients admitted to endocrine outpatient clinics were included in the study. The patients were divided into two groups according to their fasting blood glucose levels: type 2 diabetes mellitus (n = 15, fasting blood glucose levels ≥ 126 mg/dL) and control (n = 15, fasting blood glucose levels < 99 mg/dL). The patient's demographic characteristics, haemoglobin A1c levels, and scanning electron microscopy findings regarding erythrocyte morphology were recorded. Results: There was no significant difference between the control and type 2 diabetes mellitus group in terms of the participants' age (51.13 ± 8.53 vs. 50.33 ± 8.72 years, p = 0.8) and the male/female ratio (9/6 vs. 9/6). In the control group, discocytes were abundant, echinocytes were rare, and spherocytes were absent. On the other hand, discocytes were less common and echinocyte-shaped erythrocytes were more common in the type 2 diabetes mellitus group than in the control group. In addition, spherocytes were detected in the type 2 diabetes mellitus group. Moreover, the diameter of discocytes was significantly lower (p = 0.014), and blood glucose and haemoglobin A1c levels were significantly higher (p < 0.05 for both) in the type 2 diabetes mellitus group than in the control group. Conclusion: Our findings indicate that high glucose levels in type 2 diabetes mellitus patients lead to significant alterations in erythrocyte morphology, including decreased erythrocyte deformability and the formation of echinocytes and spherocytes due to eryptosis. The possibility of decreased erythrocyte deformability due to excessive eryptosis may disturb microcirculation in newly diagnosed, treatment-naïve type 2 diabetes mellitus patients who do not have any complications.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Eritrócitos/citologia , Hemoglobina A Glicada/análise , Microscopia Eletrônica de Varredura/métodos , Adulto , Idoso , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/diagnóstico , Eriptose , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto
4.
Methods Mol Biol ; 2326: 155-165, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097267

RESUMO

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.


Assuntos
Eriptose , Eritrócitos/citologia , Citometria de Fluxo/métodos , Hemólise , Necroptose , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Humanos , Necroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Testes de Toxicidade/métodos
5.
Front Cell Infect Microbiol ; 11: 630812, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777843

RESUMO

Erythrocytes possess an unusual programmed cell death mechanism termed eryptosis, and several compounds have been previously claimed to induce eryptosis in vitro. Malaria parasites (genus Plasmodium) reside in erythrocytes during the pathogenic part of their life cycle, and the potential of several eryptosis inducers to act as antimalarials has been tested in recent years. However, the eryptosis-inducing capacity of these compounds varies significantly between eryptosis-focused studies and malaria investigations. Here, we investigated the reasons for these discrepancies, we developed a protocol to investigate eryptosis in malaria cultures and we re-evaluated the potential of eryptosis inducers as antimalarials. First, we showed that eryptosis read-out in vitro is dependent on culture conditions. Indeed, conditions that have consistently been used to study eryptosis do not support P. falciparum growth and prime erythrocytes for eryptosis. Next, we defined culture conditions that allow the detection of eryptosis while supporting P. falciparum survival. Finally, we selected six eryptosis-inducers based on their clinical use, molecular target and antimalarial activities, and re-evaluated their eryptosis inducing capacities and their potential as antimalarials. We demonstrate that none of these compounds affect the viability of naïve or P. falciparum-infected erythrocytes in vitro. Nevertheless, three of these compounds impair parasite development, although through a mechanism unrelated to eryptosis and yet to be elucidated. We conclude that careful consideration of experimental set up is key for the accurate assessment of the eryptosis-inducing potential of compounds and their evaluation as potential antimalarials.


Assuntos
Antimaláricos , Eriptose , Malária Falciparum , Malária , Plasmodium , Eritrócitos , Humanos , Malária/tratamento farmacológico , Plasmodium falciparum
6.
Clin Exp Rheumatol ; 39(4): 838-843, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33124577

RESUMO

OBJECTIVES: Antiphospholipid syndrome (APS) is an autoimmune disease characterised by a hypercoagulable state and the presence of antiphospholipid antibodies (aPL). During the mechanism of red blood cells (RBCs) death, called eryptosis, RBCs can adhere to vascular wall participating in the development of a pro-thrombotic state. It is known that enhanced eryptosis contributes to several pathological conditions but the role of this process in APS has not been investigated yet. We analysed spontaneous eryptosis in a cohort of APS patients and aPL carriers (asymptomatic subjects with positive aPL tests). The effect on eryptosis of antibodies (Abs) purified from serum of APS patients and aPL carriers was also investigated. METHODS: In this study, 30 patients with primary APS (PAPS) and 17 aPL carriers were recruited. Twenty healthy donors (HD) and 13 patients affected by autoimmune haemolytic anaemia (AHIA) were also recruited. RBCs were incubated with PAPS and aPL carriers Abs, purified by ammonium sulfate precipitation. Levels of eryptosis were analysed by flow cytometry. RESULTS: In vitro Abs from APS patients induced eryptosis in RBCs isolated from HD after 4 h of culture. On the contrary, Abs from aPL carriers had no effect on the percentage of phosphatidylserine-exposing RBCs. Ex vivo, APS patients showed higher levels of spontaneous eryptosis compared to HD and aPL carriers. CONCLUSIONS: In this study, we demonstrated a potential new aspect of APS pathogenesis based on the ability of Abs isolated from APS patients, not identified in aPL carriers, to stimulate eryptosis suggesting a possible contribution of this process in the clinical manifestations of APS.


Assuntos
Síndrome Antifosfolipídica , Eriptose , Trombose , Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica/diagnóstico , Humanos , Fosfatidilserinas
7.
Chem Biol Interact ; 332: 109305, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33130048

RESUMO

Erythrocytes (RBCs) represent the main cell component in circulation and recently have become a topic of intensive scientific interest. The relevance of erythrocytes as a model for cytotoxicity screening of xenobiotics is under the spotlight of this review. Erythrocytes constitute a fundamental cellular model to study potential interactions with blood components of manifold novel polymer or biomaterials. Morphological changes, subsequent disruption of RBC membrane integrity, and hemolysis could be used to determine the cytotoxicity of various compounds. Erythrocytes undergo a programmed death (eryptosis) which could serve as a good model for evaluating certain mechanisms which correspond to apoptosis taking place in nucleated cells. Importantly, erythrocytes can be successfully used as a valuable cellular model in examination of oxidative stress generated by certain diseases or multiple xenobiotics since red cells are subjected to permanent oxidative stress. Additionally, the antioxidant capacity of erythrocytes, and the activity of anti-oxidative enzymes could reflect reactive oxygen species (ROS) generating properties of various substances and allow to determine their effects on tissues. The last part of this review presents the latest findings on the possible application of RBCs as drug delivery systems (DDS). In conclusion, all these findings make erythrocytes highly valuable cells for in vitro biocompatibility assessment, cytotoxicity screening of a wide variety of substances as well as drug delivery.


Assuntos
Materiais Biocompatíveis/farmacologia , Sistemas de Liberação de Medicamentos , Eritrócitos/metabolismo , Xenobióticos/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos
8.
Front Immunol ; 11: 551441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33250889

RESUMO

Chronic hemolysis, enhanced oxidative stress, and decreased nitric oxide (NO) bioavailability promote vasculopathy in sickle cell anemia (SCA). Oxidative stress and NO are known to modulate eryptosis in healthy red blood cells (RBCs); however, their role in SCA eryptosis and their impact on the genesis of RBC-derived microparticles (RBC-MPs) remains poorly described. RBC-MPs could play a role in vascular dysfunction in SCA. The aims of this study were to evaluate the roles of oxidative stress and NO in eryptosis and RBC-MPs release, and to determine whether RBC-MPs could be involved in vascular dysfunction in SCA. Markers of eryptosis and oxidative stress, plasma RBC-MPs concentration and arterial stiffness were compared between SCA and healthy (AA) individuals. In-vitro experiments were performed to test: 1) the effects of oxidative stress (antioxidant: n-acetylcysteine (NAC); pro-oxidant: cumene hydroperoxide) and NO (NO donor: sodium nitroprusside (SNP); NO-synthase inhibitor (L-NIO)) on eryptosis, RBC deformability and RBC-MP genesis; 2) the effects of SCA/AA-RBC-MPs on human aortic endothelial cell (HAEC) inflammatory phenotype and TLR4 pathway. Eryptosis, RBC-MPs, oxidative stress and arterial stiffness were increased in SCA. NAC increased RBC deformability and decreased eryptosis and RBC-MPs release, while cumene did the opposite. SNP increased RBC deformability and limited eryptosis, but had no effect on RBC-MPs. L-NIO did not affect these parameters. Arterial stiffness was correlated with RBC-MPs concentration in SCA. RBC-MPs isolated directly from SCA blood increased adhesion molecules expression and the production of cytokines by HAEC compared to those isolated from AA blood. TLR4 inhibition alleviated these effects. Our data show that oxidative stress could promote eryptosis and the release of RBC-MPs that are potentially involved in macrovascular dysfunction in SCA.


Assuntos
Anemia Falciforme/sangue , Micropartículas Derivadas de Células/metabolismo , Eriptose , Eritrócitos Anormais/metabolismo , Óxido Nítrico/sangue , Estresse Oxidativo , Rigidez Vascular , Adolescente , Adulto , Anemia Falciforme/patologia , Micropartículas Derivadas de Células/patologia , Criança , Pré-Escolar , Eritrócitos Anormais/patologia , Feminino , Humanos , Masculino
9.
ACS Chem Biol ; 15(10): 2673-2682, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32915529

RESUMO

Red blood cell death or erythrocyte apoptosis (eryptosis) is generally mediated by oxidative stress, energy depletion, heavy metals exposure, or xenobiotics. As erythrocytes are a major target for oxidative stress due to their primary function as O2-carrying cells, they possess an efficient antioxidant defense system consisting of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin 2 (Prx2). The oxidative stress-mediated activation of the Ca2+-permeable cation channel results in Ca2+ entry into the cells and subsequent cell death. Herein, we describe for the first time that selenium compounds having intramolecular diselenide or selenenyl sulfide moieties can prevent the oxidative stress-induced eryptosis by exhibiting an unusual Prx2-like redox activity under conditions when the cellular Prx2 and CAT enzymes are inhibited.


Assuntos
Antioxidantes/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Humanos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/química , Tiorredoxinas/metabolismo
11.
PLoS One ; 15(7): e0235335, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628695

RESUMO

Diabetes is associated with a dramatic mortality rate due to its vascular complications. Chronic hyperglycemia in diabetes leads to enhanced glycation of erythrocytes and oxidative stress. Even though erythrocytes play a determining role in vascular complications, very little is known about how erythrocyte structure and functionality can be affected by glycation. Our objective was to decipher the impact of glycation on erythrocyte structure, oxidative stress parameters and capacity to interact with cultured human endothelial cells. In vitro glycated erythrocytes were prepared following incubation in the presence of different concentrations of glucose. To get insight into the in vivo relevance of our results, we compared these data to those obtained using red blood cells purified from diabetics or non-diabetics. We measured erythrocyte deformability, susceptibility to hemolysis, reactive oxygen species production and oxidative damage accumulation. Altered structures, redox status and oxidative modifications were increased in glycated erythrocytes. These modifications were associated with reduced antioxidant defence mediated by enzymatic activity. Enhanced erythrocyte phagocytosis by endothelial cells was observed when cultured with glycated erythrocytes, which was associated with increased levels of phosphatidylserine-likely as a result of an eryptosis phenomenon triggered by the hyperglycemic treatment. Most types of oxidative damage identified in in vitro glycated erythrocytes were also observed in red blood cells isolated from diabetics. These results bring new insights into the impact of glycation on erythrocyte structure, oxidative damage and their capacity to interact with endothelial cells, with a possible relevance to diabetes.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Eritrócitos/patologia , Produtos Finais de Glicação Avançada/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Glicemia/metabolismo , Linhagem Celular , Técnicas de Cocultura , Diabetes Mellitus Tipo 2/patologia , Células Endoteliais , Eriptose , Deformação Eritrocítica , Eritrócitos/metabolismo , Hemoglobina A Glicada/análise , Voluntários Saudáveis , Hemólise , Humanos , Estresse Oxidativo , Cultura Primária de Células
12.
Biointerphases ; 15(4): 041001, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600052

RESUMO

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.


Assuntos
Membrana Celular/efeitos dos fármacos , Nanopartículas/toxicidade , Dióxido de Silício/química , Aminas/química , Membrana Celular/fisiologia , Microscopia Crioeletrônica , Eriptose/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Nanopartículas/química
13.
Apoptosis ; 25(9-10): 674-685, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32638182

RESUMO

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.


Assuntos
Benzofenantridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Eriptose/genética , Glucosefosfato Desidrogenase/genética , Proteína Quinase C-alfa/genética , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glutationa/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C-alfa/antagonistas & inibidores , Espécies Reativas de Oxigênio , Sesquiterpenos/farmacologia
14.
Cell Physiol Biochem ; 54(4): 605-614, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32543797

RESUMO

BACKGROUND/AIMS: Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. METHODS: Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. RESULTS: All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. CONCLUSION: Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.


Assuntos
Cálcio/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fosfatidilserinas/metabolismo , terc-Butil Hidroperóxido/farmacologia , Animais , Anexina A5/metabolismo , Benzofenantridinas/farmacologia , Caseína Quinase I/antagonistas & inibidores , Inibidores de Caspase , Caspases/metabolismo , Tamanho Celular/efeitos dos fármacos , Cães , Eriptose , Glucose/metabolismo , Ionomicina/farmacologia , Janus Quinase 3/antagonistas & inibidores , Pressão Osmótica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sacarose/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
15.
Mol Biol Rep ; 47(7): 5025-5032, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32524386

RESUMO

Pyrogallol, a polyphenolic component of Acacia nilotica has previously been reported to induce apoptosis of diverse cell types. Pyrogallol is in part effective by influencing gene expression and by interference with mitochondrial function. Despite lack of nuclei and mitochondria, erythrocytes may undergo eryptosis, a suicidal death apparent from phosphatidylserine translocation to the erythrocyte surface and cell shrinkage. Eryptosis is triggered by glucose depletion, by oxidation, by hyperosmotic cell shrinkage and by excessive Ca2+ entry. As enhanced eryptosis is a common cause of anemia, uncovering inhibitors and stimulators of eryptosis may, both, be of clinical interest. Here we tested, whether eryptosis of human erythrocytes is modified by pyrogallol. Utilizing flow cytometry, phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding and cell volume from forward scatter. Prior to determinations erythrocytes were incubated with or without glucose, without or with added oxidant tert-butyl-hydroperoxide (t-BOOH, 0.5 mM), without or with added hyperosmotic sucrose (550 mM) or without or with added Ca2+ ionophore ionomycin (1 µM). Treatment of erythrocytes with pyrogallol (2-8 µM) was without significant effect on annexin-V-binding and forward scatter. Glucose deprivation, t-BOOH, sucrose and ionomycin, each, triggered annexin-V-binding and decreased forward scatter. Pyrogallol significantly blunted the effects on annexin-V-binding but not on forward scatter. Pyrogallol thus blunts phosphatidylserine translocation in erythrocytes exposed to glucose depletion, oxidative stress, hyperosmotic shock and excessive Ca2+ entry.


Assuntos
Antioxidantes/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Pirogalol/farmacologia , Anexina A5/metabolismo , Cálcio/metabolismo , Tamanho Celular , Células Cultivadas , Eritrócitos/metabolismo , Glucose/deficiência , Humanos , Estresse Oxidativo
16.
Biomolecules ; 10(5)2020 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-32429353

RESUMO

BACKGROUND: Eryptosis is a physiological, apoptosis-like death of injured erythrocytes crucial to prevent premature haemolysis and the pathological sequalae generated by cell-free haemoglobin. When dysregulated, the process is associated to several inflammatory-based pathologies. 4-Hydroxy-trans-2-nonenal (HNE) is an endogenous signalling molecule at physiological levels and, at higher concentrations, is involved in the pathogenesis of several inflammatory-based diseases. This work evaluated whether HNE could induce eryptosis in human erythrocytes. METHODS: Measurements of phosphatidylserine, cell volume, intracellular oxidants, Ca++, glutathione, ICAM-1, and ceramide were assessed by flow cytometry. Scanning electron microscopy evaluated morphological alterations of erythrocytes. Western blotting assessed caspases. PGE2 was measured by ELISA. Adhesion of erythrocytes on endothelial cells was evaluated by gravity adherence assay. RESULTS: HNE in the concentration range between 10-100 µM induces eryptosis, morphological alterations correlated to caspase-3 activation, and increased Ca++ levels. The process is not mediated by redox-dependent mechanisms; rather, it strongly depends on PGE2 and ceramide. Interestingly, HNE induces significant increase of erythrocytes adhesion to endothelial cells (ECs) that are in turn dysfunctionated as evident by overexpression of ICAM-1. CONCLUSIONS: Our results unveil a new physiopathological role for HNE, provide mechanistic details of the HNE-induced eryptosis, and suggest a novel mechanism through which HNE could exert pro-inflammatory effects.


Assuntos
Aldeídos/farmacologia , Eriptose , Eritrócitos/efeitos dos fármacos , Peroxidação de Lipídeos , Adulto , Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/fisiologia , Eritrócitos/ultraestrutura , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo
17.
Semin Nephrol ; 40(2): 148-159, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32303278

RESUMO

Sepsis is a heterogeneous clinical syndrome that is complicated commonly by acute kidney injury (sepsis-AKI). Currently, no approved pharmacologic therapies exist to either prevent sepsis-AKI or to treat sepsis-AKI once it occurs. A growing body of evidence supports a connection between red blood cell biology and sepsis-AKI. Increased levels of circulating cell-free hemoglobin (CFH) released from red blood cells during hemolysis are common during sepsis and can contribute to sepsis-AKI through several mechanisms including tubular obstruction, nitric oxide depletion, oxidative injury, and proinflammatory signaling. A number of potential pharmacologic therapies targeting CFH in sepsis have been identified including haptoglobin, hemopexin, and acetaminophen, and early phase clinical trials have suggested that acetaminophen may have beneficial effects on lipid peroxidation and kidney function in patients with sepsis. Bedside measurement of CFH levels may facilitate predictive enrichment for future clinical trials of CFH-targeted therapeutics. However, rapid and reliable bedside tests for plasma CFH will be required for such trials to move forward.


Assuntos
Injúria Renal Aguda/metabolismo , Hemoglobinas/metabolismo , Sepse/metabolismo , Acetaminofen/uso terapêutico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/prevenção & controle , Anemia Falciforme/metabolismo , Animais , Ponte de Artéria Coronária , Coagulação Intravascular Disseminada/metabolismo , Eriptose , Deformação Eritrocítica , Haptoglobinas/metabolismo , Haptoglobinas/uso terapêutico , Heme/metabolismo , Hemoglobinas/imunologia , Hemólise , Hemopexina/metabolismo , Hemopexina/uso terapêutico , Humanos , Túbulos Renais , Malária/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sepse/complicações , Sepse/imunologia , Reação Transfusional/metabolismo
18.
Bioorg Med Chem ; 28(11): 115490, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32299660

RESUMO

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a chiral carbon at the C-2'' position of its furan ring and naturally occurs as a racemic mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-erypoegin K from genistein and separated the optical isomers by HPLC using a chiral column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and apoptosis-inducing activity, with an IC50 value of 90 nM, about 50% lower than that of its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity on HL-60 cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Erythrina/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Eriptose , Células HL-60 , Humanos , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
19.
J Vis Exp ; (155)2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-32065143

RESUMO

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.


Assuntos
Ionóforos de Cálcio/efeitos adversos , Eriptose/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Humanos
20.
Biochemistry (Mosc) ; 85(1): 119-129, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079523

RESUMO

Suicidal erythrocyte death, or eryptosis, is the key event in eliciting anemia in numerous pathological conditions, including diabetes, chronic kidney disease, cancer, sepsis, etc. Oxidative stress is an important trigger in the acceleration of erythrocyte loss via eryptosis and an underlying mechanism of anemia emergence in the above pathologies. Therefore, there is an increasing demand for identification of antioxidants and anti-eryptotic agents for the management of stress-related ailments. Here, we demonstrated the antioxidant and anti-eryptotic properties of the tamarind seed coat ethanol extract (TSCEE) against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress and eryptosis. The presence of probable secondary metabolites in the TSCEE extract was investigated by RP-HPLC. Active groups present in the TSCEE were studied by the Fourier-transform infrared spectroscopy. Cyclic voltammetric studies confirmed the antioxidant potential of TSCEE. The protective effect of TSCEE on red blood cells was confirmed by assessing various eryptotic markers, such as reactive oxygen species generation, intracellular calcium levels, and phosphatidylserine exposure. TSCEE reduced lipid peroxidation and protein carbonyl content and restored the levels of glutathione, antioxidant enzymes, and enzymes involved in glutathione replenishment. In conclusion, TSCEE was found to exhibit multiple therapeutic properties, which makes it a promising agent for treating oxidative stress-induced eryptosis and subsequent anemia in various pathologies.


Assuntos
Antioxidantes/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos , Extratos Vegetais/farmacologia , Tamarindus/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sementes/metabolismo
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