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1.
J Cell Physiol ; 239(3): e30958, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36748950

RESUMO

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.


Assuntos
Eritrócitos , Proteínas de Membrana , Humanos , Fosforilação , Eritrócitos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Hipóxia/metabolismo
2.
Int J Mol Sci ; 25(3)2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38338682

RESUMO

Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of abnormal extracellular matrix material in ocular and non-ocular tissues, including blood vessel walls. Clot-forming dysfunction might be responsible for venous thrombosis in PEX. We investigated global coagulation, the proteome, and functions of platelets in PEX patients and aimed to determine prognostic biomarkers for thrombosis risk in PEX. Peripheral blood was collected from PEX and retinal vein occlusion (RVO) patients, and age-sex matched controls. Viscoelastic hemostasis was evaluated by rotational thromboelastometry (ROTEM). Platelet markers (CD41, CD42, CD61, and CD62p) and endothelial markers (P-selectin, E-selectin, and von Willebrand factor) were investigated by flow cytometry and ELISA, respectively. The platelet proteome was analyzed by 2D fluorescence difference gel electrophoresis followed by mass spectrometry. Clot formation time (CFT) is significantly reduced in PEX patients compared to the controls (p < 0.05). P-selectin levels were higher in PEX patients than in controls (p < 0.05); E-selectin and von Willebrand factor remained unchanged. The monitorization of CFT by ROTEM, and soluble P-selectin, may help assess thrombotic risk in PEX patients. Proteomic analysis revealed differential expression of Profilin-1 in platelets. Profilin-1 regulates the stability of actin-cytoskeleton and may contribute to impaired platelet hemostatic functions. Increased P-selectin levels together with impaired coagulation dynamics might be responsible for the thrombotic events in PEX disease.


Assuntos
Síndrome de Exfoliação , Trombofilia , Humanos , Selectina-P , Profilinas , Proteoma , Fator de von Willebrand/metabolismo , Proteômica
3.
Int J Mol Sci ; 23(15)2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35955804

RESUMO

Vascular aging, characterized by structural and functional alterations of the vascular wall, is a hallmark of aging and is tightly related to the development of cardiovascular mortality and age-associated vascular pathologies. Over the last years, extensive and ongoing research has highlighted several sophisticated molecular mechanisms that are involved in the pathophysiology of vascular aging. A more thorough understanding of these mechanisms could help to provide a new insight into the complex biology of this non-reversible vascular process and direct future interventions to improve longevity. In this review, we discuss the role of the most important molecular pathways involved in vascular ageing including oxidative stress, vascular inflammation, extracellular matrix metalloproteinases activity, epigenetic regulation, telomere shortening, senescence and autophagy.


Assuntos
Sistema Cardiovascular , Epigênese Genética , Sistema Cardiovascular/metabolismo , Senescência Celular/fisiologia , Estresse Oxidativo/fisiologia , Encurtamento do Telômero
4.
Microvasc Res ; 135: 104124, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33359148

RESUMO

Red blood cell (RBC) deformability has vital importance for microcirculation in the body, as RBCs travel in narrow capillaries under shear stress. Deformability can be defined as a remarkable cell ability to change shape in response to an external force which allows the cell to pass through the narrowest blood capillaries. Previous studies showed that RBC deformability could be regulated by Ca2+/protein kinase C (PKC) signaling mechanisms due to the phosphorylative changes in RBC membrane proteins by kinases and phosphatases. We investigated the roles of Ca2+/PKC signaling pathway on RBC mechanical responses and impaired RBC deformability under continuous shear stress (SS). A protein kinase C inhibitor Chelerythrine, a tyrosine phosphatase inhibitor Calpeptin, and a calcium channel blocker Verapamil were applied into human blood samples in 1 micromolar concentration. Samples with drugs were treated with or without 3 mM Ca2+. A shear stress at 5 Pa level was applied to each sample continuously for 300 s. RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORRCA) and was calculated as the change in elongation index (EI) of RBC upon a range of shear stress (SS, 0.3-50 Pa). RBC mechanical stress responses were evaluated before and after continuous SS through the parameterization of EI-SS curves. The drug administrations did not produce any significant alterations in RBC mechanical responses when they were applied alone. However, the application of the drugs together with Ca2+ substantially increased RBC deformability compared to calcium alone. Verapamil significantly improved Ca2+-induced impairments of deformability both before and after 5 Pa SS exposure (p < 0.0001). Calpeptin and Chelerythrine significantly ameliorated impaired deformability only after continuous SS (p < 0.05). Shear-induced improvements of deformability were conserved by the drug administrations although shear-induced deformability was impaired when the drugs were applied with calcium. The blocking of Ca2+ channel by Verapamil improved impaired RBC mechanical responses independent of the SS effect. The inhibition of tyrosine phosphatase and protein kinase C by Calpeptin and Chelerythrine, respectively, exhibited ameliorating effects on calcium-impaired deformability with the contribution of shear stress. The modulation of Ca2+/PKC signaling pathway could regulate the mechanical stress responses of RBCs when cells are under continuous SS exposure. Shear-induced improvements in the mechanical properties of RBCs by this signaling mechanism could facilitate RBC flow in the microcirculation of pathophysiological disorders, wherein Ca2+ homeostasis is disturbed and RBC deformability is reduced.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Deformação Eritrocítica , Eritrócitos/enzimologia , Mecanotransdução Celular , Proteína Quinase C/metabolismo , Adulto , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Mecanotransdução Celular/efeitos dos fármacos , Pessoa de Meia-Idade , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Estresse Mecânico , Adulto Jovem
5.
Cytometry A ; 95(5): 488-498, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30980696

RESUMO

Red blood cells (RBCs) from sickle cell patients exposed to a low oxygen tension reveal highly heterogeneous cell morphologies due to the polymerization of sickle hemoglobin (HbS). We show that angle-resolved light scattering approach with the use of image-based flow cytometry provides reliable quantitative data to define the change in morphology of large populations of RBCs from sickle cell patients when the cells are exposed for different times to low oxygen. We characterize the RBC morphological profile by means of a set of morphological and physical parameters, which includes cell shape, size, and orientation. These parameters define the cell as discocyte, sickle, elongated, as well as irregularly or abnormal RBC shaped cells, including echinocytes, holly-leaf, and granular structures. In contrast to microscopy, quick assessment of large numbers of cells provides statistically relevant information of the dynamic process of RBC sickling in time. The use of this approach facilitates the understanding of the processes that define the propensity of sickle blood samples to change their shape, and the ensuing vaso-occlusive events in the circulation of the patients. Moreover, it assists in the evaluation of treatments that include the use of anti-sickling agents, gene therapy-based hemoglobin modifications, as well as other approaches to improve the quality of life of sickle cell patients. © 2019 International Society for Advancement of Cytometry.


Assuntos
Hemoglobina Falciforme/metabolismo , Citometria por Imagem/métodos , Luz , Espalhamento de Radiação , Forma Celular , Eritrócitos/metabolismo , Humanos , Oxigênio/metabolismo
6.
Mol Pharm ; 16(7): 2929-2934, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31038318

RESUMO

Anthracycline chemotherapy (e.g., doxorubicin or DOX) is associated with a cumulative dose-dependent cardiac dysfunction that may lead to congestive heart failure, which limits both its use and usefulness in the clinic. The cardiotoxicity may manifest acutely and/or months or years after treatment with doxorubicin has ended. Experimental and human data have demonstrated that angiotensin-converting enzyme/angiotensin-receptor antagonists mediate a cardioprotective effect against anthracycline toxicity. In this study, with the angiotensin receptor blocker, candesartan, as a positive control, we evaluated whether pretreatment with the hypoxic nitric oxide generating anticancer agent, RRx-001, could reduce acute DOX-induced cardiotoxicity. A total of 24 BALB/c mice were randomized for prophylactic treatment with vehicle, RRx-001, candesartan, or no-intervention control. Within each of the three intervention arms, mice received treatment with DOX. Murine pressure-volume analysis was performed with microconductance catheters to characterize the degree of cardiovascular dysfunction within each group. The following hemodynamic parameters were monitored: left ventricular systolic pressure (LVSP), heart rate, and maximal rate of increase of left ventricular pressure (±d P/d tmax). Five days after doxorubicin injection, untreated (with RRx-001) mice displayed significantly impaired systolic (LVSP, -27%; d P/d tmax, -25%; left ventricular developed pressure (LVDP), +33%; P < 0.05) and global (stroke volume (SV), -52%; ejection fraction (EF), -20%; stroke work (SW), -62.5%; heart rate (HR), -18%; cardiac output (CO), -57%; mean blood arterial pressure (MAP), -30%; systemic vascular resistance (SVR), +20%; P < 0.05) LV functions when compared with the untreated (with RRx-001) group. In contrast, RRx-001-treated mice showed improved variables of systolic (LVSP, +27%; d P/d tmax, +25%; LVDP, -33%; P < 0.05) and global (SV, +52%; EF, +20%; SW, +62.5%; HR, +18%; CO, +57%; MAP, +30%; SVR, -20%; P < 0.05) LV functions compared with untreated doxorubicin mice. Similar to the positive control, candesartan, the cardiotoxic effects of DOX in mice were partially attenuated by the prophylactic administration of RRx-001. These results suggest that RRx-001 as a multifunctional anticancer agent, which sensitizes cancer cells to the cytotoxic effects of chemotherapy and radiation, may also have beneficial cardioprotective effects.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Azetidinas/uso terapêutico , Cardiomiopatias/induzido quimicamente , Cardiotônicos/uso terapêutico , Doxorrubicina/toxicidade , Doxorrubicina/uso terapêutico , Neoplasias/tratamento farmacológico , Nitrocompostos/uso terapêutico , Doença Aguda , Animais , Antibióticos Antineoplásicos/administração & dosagem , Azetidinas/administração & dosagem , Benzimidazóis/uso terapêutico , Compostos de Bifenilo , Pressão Sanguínea/efeitos dos fármacos , Cardiotônicos/administração & dosagem , Cardiotoxicidade , Doxorrubicina/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nitrocompostos/administração & dosagem , Distribuição Aleatória , Volume Sistólico/efeitos dos fármacos , Tetrazóis/uso terapêutico
7.
J Pediatr ; 195: 228-235, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29449005

RESUMO

OBJECTIVES: To investigate the associations between several sickle cell disease genetic modifiers (beta-globin haplotypes, alpha-thalassemia, and glucose-6-phosphate dehydrogenase deficiency) and the level of oxidative stress and to evaluate the association between oxidative stress and the rates of vaso-occlusive events. STUDY DESIGN: Steady-state oxidative and nitrosative stress markers, biological variables, genetic modulators, and vaso-occlusive crisis events requiring emergency admissions were measured during a 2-year period in 62 children with sickle cell anemia (58 SS and 4 Sß0). Twelve ethnic-matched children without sickle cell anemia also participated as healthy controls (AA) for oxidative and nitrosative stress level measurement. RESULTS: Oxidative and nitrosative stress were greater in patients with sickle cell anemia compared with control patients, but the rate of vaso-occlusive crisis events in sickle cell anemia was not associated with the level of oxidative stress. The presence of alpha-thalassemia, but not glucose-6-phosphate dehydrogenase deficiency or beta-globin haplotype, modulated the level of oxidative stress in children with sickle cell anemia. CONCLUSION: Mild hemolysis in children with alpha-thalassemia may limit oxidative stress and could explain the protective role of alpha-thalassemia in hemolysis-related sickle cell complications.


Assuntos
Anemia Falciforme/genética , Anemia Falciforme/fisiopatologia , Predisposição Genética para Doença , Estresse Oxidativo , Índice de Gravidade de Doença , Adolescente , Anemia Falciforme/diagnóstico , Criança , Pré-Escolar , Feminino , Seguimentos , Marcadores Genéticos , Humanos , Modelos Lineares , Masculino , Estudos Retrospectivos , Fatores de Risco
8.
J Artif Organs ; 21(2): 142-149, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29478196

RESUMO

The aim of this work was to analyze a shrouded impeller pediatric ventricular assist device (SIP-VAD). This device has distinctive design characteristics and parameter optimizations for minimization of recirculation flow and reduction in high-stress regions that cause blood damage. Computational Fluid Dynamics (CFD) simulations were performed to analyze the optimized design. The bench-top prototype of SIP-VAD was manufactured with biocompatible stainless steel. A study on the hydrodynamic and hemodynamic performance of the SIP-VAD was conducted with predictions from CFD and actual experimentation values, and these results were compared. The CFD analysis yielded a pressure range of 29-90 mmHg corresponding to flow rates of 0.5-3 L/min over 9000-11000 rpm. The predicted value of the normalized index of hemolysis (NIH) was 0.0048 g/100 L. The experimental results with the bench-top prototype showed a pressure rise of 30-105 mmHg for the flow speed of 8000-12000 rpm and flow rate of 0.5-3.5 L/min. The maximum difference between CFD and experimental results was 4 mmHg pressure. In addition, the blood test showed the average NIH level of 0.00674 g/100 L. The results show the feasibility of shrouded impeller design of axial-flow pump for manufacturing the prototype for further animal trials.


Assuntos
Coração Auxiliar , Desenho de Prótese , Criança , Hemodinâmica , Hemólise , Humanos , Hidrodinâmica , Pressão
9.
Am J Physiol Heart Circ Physiol ; 313(2): H381-H391, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28526710

RESUMO

Adaptation to hypoxia requires compensatory mechanisms that affect O2 transport and utilization. Decreased hemoglobin (Hb) O2 affinity is considered part of the physiological adaptive process to chronic hypoxia. However, this study explores the hypothesis that increased Hb O2 affinity can complement acute physiological responses to hypoxia by increasing O2 uptake and delivery compared with normal Hb O2 affinity during acute severe hypoxia. To test this hypothesis, Hb O2 affinity in mice was increased by oral administration of 2-hydroxy-6-{[(2S)-1-(pyridine-3-carbonyl)piperidin-2yl] methoxy}benzaldehyde (GBT1118; 70 or 140 mg/kg). Systemic and microcirculatory hemodynamics and oxygenation parameters were studied during hypoxia in awake-instrumented mice. GBT1118 increased Hb O2 affinity and decreased the Po2 at which 50% of Hb is saturated with O2 (P50) from 43 ± 1.1 to 18.3 ± 0.9 mmHg (70 mg/kg) and 7.7 ± 0.2 mmHg (140 mg/kg). In a dose-dependent fashion, GBT1118 increased arterial O2 saturation by 16% (70 mg/kg) and 40% (140 mg/kg) relative to the control group during 5% O2 hypoxia. In addition, a GBT1118-induced increase in Hb O2 affinity reduced hypoxia-induced hypotension compared with the control group. Moreover, microvascular blood flow was higher during hypoxia in GBT1118-treated groups than the control group. The increased O2 saturation and improved blood flow in GBT1118-treated groups preserved higher interstitial tissue Po2 than in the control group during 5% O2 hypoxia. In conclusion, increased Hb O2 affinity enhanced physiological tolerance to hypoxia, as evidenced by improved hemodynamics and tissue oxygenation. Therefore, pharmacologically induced increases in Hb O2 affinity become a potential therapeutic approach to improve tissue oxygenation in pulmonary diseases characterized by severe hypoxemia.NEW & NOTEWORTHY This study establishes that pharmacological modification of hemoglobin O2 affinity can be a promising and novel therapeutic strategy for the treatment of hypoxic hypoxia and paves the way for the clinical development of molecules that prevent hypoxemia.


Assuntos
Benzaldeídos/farmacologia , Hipóxia/tratamento farmacológico , Niacinamida/análogos & derivados , Oxigênio/sangue , Oxiemoglobinas/metabolismo , Pele/irrigação sanguínea , Adaptação Fisiológica , Administração Oral , Animais , Benzaldeídos/administração & dosagem , Benzaldeídos/farmacocinética , Biomarcadores/sangue , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Modelos Animais de Doenças , Frequência Cardíaca , Hipóxia/sangue , Hipóxia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Niacinamida/administração & dosagem , Niacinamida/farmacocinética , Niacinamida/farmacologia , Fluxo Sanguíneo Regional , Índice de Gravidade de Doença
10.
Exp Physiol ; 100(8): 977-87, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26011432

RESUMO

NEW FINDINGS: What is the central question of this study? The aim was to evaluate the effect of perfusion pressure on blood flow in small arterioles. The hypothesis was that blood flow regulates the thickness of the red-cell-free layer and, therefore, blood flow determines blood apparent viscosity and local vascular resistance in vascular networks with limited myogenic or metabolic regulation of blood flow. What is the main finding and its importance? Reduced perfusion pressures lowered volumetric flow rates and increased local vascular resistance, due to increased blood apparent viscosity. Thus, the local vascular resistance of small arterioles with limited myogenic or metabolic regulation of blood flow, appeared to be determined by changes in blood rheology rather than blood vessel diameter. The study of blood flow regulation is important to understand and resolve pathological conditions. As blood is a complex non-Newtonian multiphase system, the foundations of blood rheological properties have been obtained mostly in viscometers. However, blood rheological behaviour in vivo depends on the concentration of red blood cells (RBCs), their mechanical properties and the RBC hydrodynamics, including RBC migration away from the vessel wall in shear flow. This migration promotes the formation of a RBC-depleted zone, or cell-free layer (CFL), which reduces the apparent viscosity of blood. We hypothesize that perfusion pressure determines blood apparent viscosity in microvessels, as shear rate affects axial migration of RBCs by influencing the CFL thickness. In this study, we analysed the effects of perfusion pressure on blood flow in individual arterioles within the rat cremaster muscle preparation. Perfusion pressures to this microvascular bed were controlled by occlusions of the iliac artery using a pressure cuff. Blood flow measurements were obtained from direct measurements of blood flow velocity profile, as well as determination of CFL thickness using intravital microscopy. Our results indicate that perfusion pressure determines shear rates and the CFL thickness and its variations. In addition, blood flow reduction increased local vascular resistance by augmenting blood apparent viscosity rather than vascular hindrance. In conclusion, blood rheology could act as an intrinsic mechanism to further limit blood flow to tissue with limited myogenic and metabolic responses at low perfusion pressures.


Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Pressão Sanguínea/fisiologia , Viscosidade Sanguínea/fisiologia , Microvasos/fisiologia , Resistência Vascular/fisiologia , Animais , Ratos , Ratos Sprague-Dawley
11.
Malar J ; 14: 218, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-26017006

RESUMO

BACKGROUND: The survival of malaria parasites, under substantial haem-induced oxidative stress in the red blood cells (RBCs) is dependent on the pentose phosphate pathway (PPP). The PPP is the only source of NADPH in the RBC, essential for the production of reduced glutathione (GSH) and for protection from oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, therefore, increases the vulnerability of erythrocytes to oxidative stress. In Plasmodium, G6PD is combined with the second enzyme of the PPP to create a unique bifunctional enzyme, named glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL). RRx-001 is a novel, systemically non-toxic, epigenetic anticancer agent currently in Phase 2 clinical development for multiple tumour types, with activity mediated through increased nitric oxide (NO) production and PPP inhibition. The inhibition of G6PD and NO overproduction induced by RRx-001 suggested its application in cerebral malaria (CM). METHODS: Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice is an experimental model of cerebral malaria (ECM) with several similar pathological features to human CM. This study uses intravital microscopy methods with a closed cranial window model to quantify cerebral haemodynamic changes and leukocyte adhesion to endothelial cells in ECM. RESULTS: RRx-001 had both single agent anti-parasitic activity and significantly increased the efficacy of artemether. In addition, RRx-001 preserved cerebral perfusion and reduced inflammation alone or combined with artemether. RRx-001's effects were associated with inhibition of PPP (G6PD and G6PD-6PGL) and by improvements in microcirculatory flow, which may be related to the NO donating properties of RRx-001. CONCLUSION: The results indicate that RRx-001 could be used to potentiate the anti-malarial action of artemisinin, particularly on resistant strains, and to prevent infection.


Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Azetidinas/uso terapêutico , Malária Cerebral/tratamento farmacológico , Nitrocompostos/uso terapêutico , Plasmodium berghei/efeitos dos fármacos , Animais , Artemeter , Hidrolases de Éster Carboxílico/metabolismo , Modelos Animais de Doenças , Combinação de Medicamentos , Glucosefosfato Desidrogenase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Parasitemia/tratamento farmacológico
12.
Transfusion ; 54(4): 1015-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23901933

RESUMO

BACKGROUND: Human red blood cells (RBCs) can be stored for up to 42 days under controlled conditions. Physical and chemical changes occur during RBC storage, altering their function. This study links stored cell mechanical changes with hemodynamic functional alterations upon transfusion. STUDY DESIGN AND METHODS: Mechanical properties of fresh and stored RBCs were evaluated in vitro. Their transfusion effects were evaluated in vivo using intravital microscopy of the rat's cremaster muscle preparation. Rats were hemodiluted to 30% hematocrit, to mimic an anemic state before transfusion, and then exchange-transfused with fresh or stored cells. RESULTS: In vitro studies on rheology and oxygen affinity of stored cells confirmed previously published results. Storage was found to modify static and dynamic RBC mechanic behavior. After transfusion, systemic hemodynamics were similar for fresh and stored cells; however, microvascular hemodynamics were drastically affected by stored cells. Stored cells reduced blood flow and oxygen delivery. Additionally, the presence of stored cells in circulation affected cell-to-cell and cell-to-wall interactions and affected cell hydrodynamics. Stored cells disrupted the RBC cell-free layer and wall shear stress signals. CONCLUSION: The reduced cell deformability due to RBC "storage lesions" caused pathologic changes in microvascular hemodynamics, endothelial cell mechanotransduction, and RBC dynamics. Thus, the mechanical changes of blood-banked cells can limit transfusion ability to achieve its intended goal.


Assuntos
Preservação de Sangue/efeitos adversos , Hemodinâmica/fisiologia , Microcirculação/fisiologia , Reação Transfusional , Doenças Vasculares/etiologia , Animais , Células Cultivadas , Módulo de Elasticidade , Deformação Eritrocítica , Humanos , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Resistência ao Cisalhamento
13.
Ther Apher Dial ; 28(4): 657-663, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38462734

RESUMO

INTRODUCTION: Peripheral blood stem cell (PBSC) collection via apheresis requires the administration of granulocyte colony-stimulating factor (filgrastim) to stem cell donors. Several reports have shown that filgrastim administration and apheresis procedure induce a hypercoagulable state across PBSC collection, which might predispose certain donors to thrombotic complications. METHODS: We evaluated the hemostatic functions of healthy allogeneic stem cell donors by rotational thromboelastometry (ROTEM). Blood samples from healthy donors (n = 30) were collected at defined time points: before filgrastim (baseline), on the day of apheresis before and after the procedure, and 1 week after apheresis. RESULTS: The results indicated that hemostatic changes are temporary since all parameters in both EXTEM and INTEM assays are restored to their initial values 1 week after the apheresis. CONCLUSION: We concluded that stem cell apheresis does not induce a hypercoagulable state in healthy donors. This is the first study evaluating the hemostatic functions of stem cell donors by ROTEM.


Assuntos
Remoção de Componentes Sanguíneos , Tromboelastografia , Humanos , Tromboelastografia/métodos , Remoção de Componentes Sanguíneos/métodos , Masculino , Feminino , Adulto , Filgrastim/farmacologia , Pessoa de Meia-Idade , Hemostasia/fisiologia , Transplante Homólogo/métodos , Doadores de Tecidos , Adulto Jovem
14.
Clin Hemorheol Microcirc ; 85(1): 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37482986

RESUMO

BACKGROUND: Thalassemia patients have reduced red cell deformability and decreased plasma zinc levels in their blood. OBJECTIVE: This study aimed to evaluate the effects of zinc (Zn) on the hemorheological parameters and antioxidant enzyme activities in ß-thalassemia major (TM) and healthy volunteers (HV). METHODS: Hemorheological parameters were measured using LORCA (laser-assisted optical rotational cell analyzer) after adjusting the hematocrit to 40%. Zinc sulfate (ZnSO4.7H2O) was used for Zn incubation with a concentration of 0.5µg/dl. Oxidative stress and antioxidant status were determined using commercial kits. RESULTS: Data showed that after Zn incubation, EImax, the area under the EI-osmolarity curve (Area), and Omax decreased in TM. However, no significant difference was observed in the osmotic deformability parameters of HV. The increased elongation index was obtained at different shear stresses for TM and HV, and SS1/2 decreased in both groups. The AMP and aggregation index (AI) decreased in TM, and the required time for half of the maximum aggregation (t1/2) increased in HV. However, Zn did not affect oxidative parameters in both groups. CONCLUSIONS: This study showed that Zn incubation increased deformability and decreased aggregation in thalassemic erythrocytes. It means that Zn supplementation will contribute to microcirculation in thalassemia patients.


Assuntos
Deformação Eritrocítica , Talassemia beta , Humanos , Talassemia beta/tratamento farmacológico , Antioxidantes/farmacologia , Zinco/farmacologia , Agregação Eritrocítica , Eritrócitos
15.
Front Physiol ; 14: 1215835, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37781231

RESUMO

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5 Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5 Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5 Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs.

16.
Am J Physiol Heart Circ Physiol ; 303(3): H271-81, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22636677

RESUMO

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.


Assuntos
Furaldeído/análogos & derivados , Hemodinâmica/efeitos dos fármacos , Hemoglobinas/metabolismo , Hipóxia/tratamento farmacológico , Oxigênio/sangue , Pele/irrigação sanguínea , Doença Aguda , Animais , Transporte Biológico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Capilares/efeitos dos fármacos , Capilares/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Coronária/efeitos dos fármacos , Cricetinae , Modelos Animais de Doenças , Furaldeído/administração & dosagem , Furaldeído/farmacologia , Hipóxia/sangue , Hipóxia/patologia , Hipóxia/fisiopatologia , Imuno-Histoquímica , Infusões Parenterais , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Microscopia de Vídeo , Miocárdio/metabolismo , Miocárdio/patologia , Pressão Parcial , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fatores de Tempo
17.
Am J Emerg Med ; 30(8): 1336-46, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22100469

RESUMO

Human serum albumin (HSA) is used as a plasma expander; however, albumin is readily eliminated from the intravascular space. The objective of this study was to establish the effects of various-sized polymerized HSAs (PolyHSAs) during small-volume resuscitation from hemorrhagic shock on systemic parameters, microvascular hemodynamics, and functional capillary density in the hamster window chamber model. Polymerized HSA size was controlled by varying the cross-link density (ie, molar ratio of glutaraldehyde to HSA). Hemorrhage was induced by controlled arterial bleeding of 50% of the animal's blood volume (BV), and hypovolemic shock was maintained for 1 hour. Resuscitation was implemented in 2 phases, first, by infusion of 3.5% of the BV of hypertonic saline (7.5% NaCl) then followed by infusion of 10% of the BV of each PolyHSA. Resuscitation provided rapid recovery of blood pressure, blood gas parameters, and microvascular perfusion. Polymerized HSA at a glutaraldehyde-to-HSA molar ratio of 60:1 (PolyHSA(60:1)) provided superior recovery of blood pressure, microvascular blood flow, and functional capillary density, and acid-base balance, with sustained volume expansion in relation to the volume infused. The high molecular weight of PolyHSA(60:1) increased the hydrodynamic radius and solution viscosity. Pharmacokinetic analysis of PolyHSA(60:1) indicates reduced clearance and increased circulatory half-life compared with monomeric HSA and other PolyHSA formulations. In conclusion, HSA molecular size and solution viscosity affect central hemodynamics, microvascular blood flow, volume expansion, and circulation persistence during small-volume resuscitation from hemorrhagic shock. In addition, PolyHSA can be an alternative to HSA in pathophysiological situations with compromised vascular permeability.


Assuntos
Ressuscitação/métodos , Albumina Sérica/uso terapêutico , Choque Hemorrágico/terapia , Animais , Pressão Sanguínea , Cricetinae , Frequência Cardíaca , Hemodinâmica , Humanos , Masculino , Mesocricetus , Albumina Sérica/administração & dosagem , Albumina Sérica/química , Albumina Sérica Humana , Viscosidade
18.
Cells ; 11(7)2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35406814

RESUMO

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs.


Assuntos
Adenilil Ciclases , Proteômica , Adenilil Ciclases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Deformação Eritrocítica/fisiologia , Eritrócitos/metabolismo , Diester Fosfórico Hidrolases/metabolismo
19.
Hum Mutat ; 32(10): E2283-93, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21913284

RESUMO

Benign familial neonatal seizures (BFNS) are a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K(V) 7.2 and K(V) 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K(V) 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-of-function with a dominant-negative effect of the mutant on WT K(V) 7.2 and K(V) 7.3 channels. Since single-channel recordings of K(V) 7.3-K(V) 7.2 and K(V) 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells-either at lower temperatures of <30°C or in presence of the agonist retigabine (RTG)-the N258S-derived currents increased fivefold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a K(V) 7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins.


Assuntos
Epilepsia Neonatal Benigna/genética , Canal de Potássio KCNQ2/genética , Mutação , Temperatura , Animais , Antracenos/farmacologia , Células CHO , Cricetinae , Expressão Gênica , Humanos , Canal de Potássio KCNQ2/antagonistas & inibidores , Canal de Potássio KCNQ2/química , Oócitos , Bloqueadores dos Canais de Potássio/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/genética
20.
Epilepsia ; 52(5): 975-83, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21320115

RESUMO

PURPOSE: To further evaluate the previously shown linkage of absence epilepsy (AE) to 2q36, both in human and WAG/Rij absence rat models, a 160-kb region at 2q36 containing eight genes with expressions in the brain was targeted in a case-control association study involving 205 Turkish patients with AE and 219 controls. METHODS: Haplotype block and case-control association analysis was carried out using HAPLOVIEW 4.0 and inhibin alpha subunit (INHA) gene analysis by DNA sequencing. KEY FINDINGS: An association was found between the G allele of rs7588807 located in the INHA gene and juvenile absence epilepsy (JAE) syndrome and patients having generalized tonic-clonic seizures (GTCS) with p-values of 0.003 and 0.0002, respectively (uncorrected for multiple comparisons). DNA sequence analysis of the INHA gene in 110 JAE/GTCS patients revealed three point mutations with possible damaging effects on inhibin function in three patients and the presence of a common ACTC haplotype (H1) with a possible dominant protective role conferred by the T allele of rs7588807 with respective p-values of 0.0005 and 0.0014. SIGNIFICANCE: The preceding findings suggest that INHA could be a novel candidate susceptibility gene involved in the pathogenesis of JAE or AE associated with GTCS.


Assuntos
Epilepsia Tipo Ausência/genética , Epilepsia Tônico-Clônica/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Haplótipos/genética , Inibinas/genética , Convulsões/genética , Animais , Estudos de Casos e Controles , Cromossomos Humanos Par 2/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Ratos
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