Tissue and sex especifities in the Ca2+ handling by isolated mitochondria : evaluations under conditions avoiding the permeability transition

Orientadores: Tiago Rezende Figueira, Roger Frigério CastilhoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Algumas das características das mitocôndrias, incluindo as suas funções de transporte de Ca2+, podem apresentar dimorfismo sexual e especificidades teciduais. No entanto, as mensurações do transporte de Ca2+ em mitocôndrias isoladas estão sujeitas a artefatos secundários a abertura do poro de transição de permeabilidade mitocondrial (PTP) induzido pelo acúmulo excessivo de Ca2+ nesta organela. Neste estudo, o objetivo inicial foi avaliar se a inibição do PTP pela ciclosporina A (CsA) afeta a mensuração de diversas variáveis que descrevem o transporte de Ca2+por mitocôndrias isoladas de fígado de rato. Os resultados obtidos indicam que as concentrações de estado estável do Ca2+ externo a mitocôndria e as taxas deefluxo mitocondrial de Ca2+através de trocadores seletivos foram superestimados em até 4 vezes quando o PTP não foi inibido farmacologicamente pela CsA. O objetivo subsequente foi analisar o transporte de Ca2+ em mitocôndrias isoladas de fígado, de músculo esquelético, de coração e de cérebro de ratos machos e fêmeas sob condições experimentais específicas (i.e. meio de incubação contendo inibidores TPM, substratos energéticos ligados a NAD e níveis relevantes de Ca2+, Mg2+e Na+). Os dados indicaram que a taxa de influxo de Ca2+em mitocôndrias de fígado foi ~4 vezes superior a dos outros tecidos, as quais foram semelhantes entre si. Em contrapartida, as taxas de efluxo de Ca2+ apresentaram uma maior diversidade entre tecidos, especialmente na presença de Na+. Curiosamente, o efluxo de Ca2+na ausência de Na+foi significativamente mais elevado nas mitocôndrias cardíacas (~4nmol/mg/min) em relação às taxas observadas nos outros tecidos, contrariando a concepção de que o efluxo de Ca2+de mitocôndrias de coração é dependente, quase que exclusivamente, de um trocador que requer Na+. A especificidade em relação ao sexo só foi observada em dois índices relacionados a homeostase mitocondrial de Ca2+(i.e. cinética geral normalizada da captação de Ca2+ e a concentração de estado estável do Ca2+ externo a mitocôndria) em mitocôndrias isoladas de coração (mais lentos ou maiores na fêmea) e na respiração estimulada por ADP em mitocôndrias de fígado (~20% maior na fêmea). O presente estudo demonstrou a importância metodológica de se prevenir a abertura do PTP para a análise das propriedades e da variabilidade fisiológica do transporte de Ca2+por mitocôndrias isoladas. Adicionalmente, concluímos que sob as condições experimentais aqui utilizadas, o efluxo de Ca2+ mitocondrial apresenta grandes especificidades teciduais e que alguns achados desafiam conceitos estabelecidos em estudos anteriores sob condições arguivelmente menos controladasAbstract: The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sex dimorphism. Because the measurements of the Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluates the extent to which MPT inhibition by cyclosporine-A affects the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to 4-fold if MPT pore opening is not prevented. Then, we analyzed the Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats under incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. Except for the liver mitochondria displaying values4-fold higher, the Ca2+ influx rates were similar among the other tissues. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (~4 nmol/mg/min), thus challenging the view that heart mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was only observed in two kinetic indexes (i.e. the normalized overall kinetics of Ca2+ uptake and the steady-state levels of external Ca2+) of heart mitochondrial Ca2+ homeostasis (slower or higher in female)and in the ADP-stimulated respiration of liver mitochondria (~20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria. Moreover, we conclude that mitochondrial Ca2+ efflux exhibits great tissue specificity under our conditions, which may challenge some concepts raised in previous studies that employed experimental conditions that are arguably not well controlledMestradoFisiopatologia MédicaMestra em Ciência

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Evaluation of the role of platelets in sickle cell anemia

Orientador: Nicola Amanda Conran ZorzettoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A anemia falciforme (AF) resulta da homozigose de uma mutação de ponta no gene HBB, resultando na produção da cadeia ß-globina alterada (ßS). A doença é caracterizada por anemia hemolítica crônica, crises dolorosas de vaso-oclusão, lesões de órgãos-alvo e um estado inflamatório crônico. Pacientes com AF apresentam contagens elevadas de leucócitos, e evidências indicam a participação dos leucócitos no início e propagação dos processos vaso-oclusivos, pois estas células são relativamente grandes e rígidas, fazendo com que seu recrutamento para a microcirculação reduza o fluxo sanguíneo nos vasos. O papel das plaquetas no processo vaso-oclusivo é pouco conhecido, apesar de sabermos que há ativação das plaquetas na AF. Sendo assim, este projeto teve como principal objetivo avaliar o papel das plaquetas no processo vaso-oclusivo, estimulado pela citocina TNF, em camundongos quimeras para AF, visualizando e diferenciando as células participantes deste processo inflamatório. Adicionalmente, o efeito da depleção ou diminuição da atividade das plaquetas na resposta inflamatória causada por TNF em camundongos com AF foi verificado. No decorrer deste projeto, realizamos as técnicas de transplante de medula óssea de camundongos transgênicos Berkeley para camundongos C56BL/6 (com o intuito de produzir camundongos quimeras por AF e aumentar a colônia de animais com AF), além de microscopia convencional intravital e microscopia intravital confocal da microcirculação do musculo cremaster com marcação fluorescente para plaquetas e leucócitos. Nós verificamos que, após estímulo dos animais AF com TNF, a plaqueta atua como primeiro sinal da resposta inflamatória, possuindo a função de recobrir o endotélio inflamado da microcirculação e então recrutando leucócitos (com ambos, alta e baixa expressão da molécula Ly6G), tanto em animais controles como em animais AF. Além disso, em associação ao aumento da adesão de plaquetas a parede vascular, houve uma redução da velocidade do fluxo sanguíneo destes animais e da sua sobrevida. Verificamos que tratamentos capazes de diminuir a quantidade de plaquetas ou a ativação plaquetária (uso de anticorpo anti-CD42b e prasugrel, respectivamente) foram capazes de reduzir, ou até reverter, o recrutamento de plaquetas e leucócitos na microcirculação e, portanto, os processos vaso-oclusivos em camundongos AF. A administração de hidroxiureia em animais AF reduziu o recrutamento das plaquetas e dos leucócitos e a formação de agregados plaquetas/ leucócitos na microcirculação, melhorando a velocidade de fluxo nos mesmos. Finalmente, comparamos o mecanismo de vaso-oclusão, estimulado por processos de hipóxia e reperfusão, com a vaso-oclusão induzida por TNF; apesar de um maior recrutamento de leucócitos na microcirculação destes camundongos AF, verificamos um menor recrutamento das plaquetas com menor diminuição do fluxo sanguíneo. Esses dados nos levam a concluir que a plaqueta possui um papel importante nos processos inflamatórios induzidos por TNF, iniciando e propagando os processos vaso-oclusivos em animais com AF. Apesar dos dados decepcionantes do primeiro ensaio clinico multicêntrico com prasugrel na AF, agentes antiplaquetários ainda devem ser estudados como forma de terapia em combinação, ou não, com a hidroxiureia para diminuir a frequência das crises vaso-oclusivas ou até para uso em pacientes hospitalizados por este motivoAbstract: Sickle cell anemia (SCA) results from a homozygotic mutation in the HBB gene, resulting in the production of the altered ß-globin (ßS) chain. The disease is characterized by chronic hemolytic anemia, painful vaso-occlusive crises, target organ damage and a chronic inflammatory state. Patients with SCA have high leukocyte counts, and evidence indicates the participation of leukocytes in the triggering and propagation of vaso-occlusive processes, as these cells are relatively large and rigid, causing their recruitment to blood vessel walls in the microcirculation and reducing blood flow. The role of platelets in the vaso-occlusive process is poorly understood, although we know that platelet activation occurs in SCA. Thus, this project aimed to evaluate the role of platelets in vaso-occlusive processes, stimulated by TNF cytokine, in chimeric mice with SCA, visualizing and differentiating the cells participating in this inflammatory process. Additionally, the effect of the depletion of platelets or the inhibition of their activity on the inflammatory response caused by TNF in mice with SCA was evaluated. For this project, we produced chimeric SCA mice by transplanting the bone marrow of SCA Berkeley transgenic mice into C56BL/6 mice (in order to increase the SCA mouse colony size), in addition to conventional intravital microscopy and intravital confocal microscopy of the cremaster muscle microcirculation, with fluorescent labeling for platelets and leukocytes. We found that, after stimulation of SCA animals with TNF, the platelets act as a primary response signal, covering the inflamed endothelium of microcirculation and subsequently recruiting leukocytes (with both high and low expressions of the Ly6G adhesion molecule), both in Control (CON) and SCA animals. Moreover, in association with increased platelet adhesion to the vascular wall, there was a reduction in blood flow velocity and animal survival. We found that treatments capable of decreasing platelet count or platelet activation (use of anti-CD42b antibody or prasugrel, respectively) were able to reduce, or even reverse, the recruitment of platelets and leukocytes in the microcirculation, and thus vaso-occlusive processes in SCA mice. Administration of hydroxyurea in SCA animals reduced platelet and leukocyte recruitment and the formation of platelet / leukocyte aggregates in the microcirculation, therefore improving blood flow rate. Finally, we compared the mechanism of vaso-occlusion, when stimulated by hypoxia and reperfusion processes, with TNF-induced vaso-occlusion in SCA mice; despite an increased recruitment of leukocytes in the microcirculation of the SCA mice subjected to hypoxia/reperfusion, we observed less platelet recruitment with a less significant decrease in blood flow. These data lead us to conclude that the platelet plays an important role in TNF-induced inflammatory processes, initiating and propagating vaso-occlusive mechanisms in animals with SCA. Despite the disappointing data from the first multicenter clinical trial of prasugrel in SCA, antiplatelet agents should still be studied as a form of therapy for the disease, in combination or not with hydroxyurea, to decrease the frequency of vaso-occlusive episodes or even for use in patients hospitalized for acute vaso-occlusive crisisDoutoradoFisiopatologia MédicaDoutora em Ciências2015/18369-6001FAPESPCAPE

Tissue And Sex Specificities In Ca2+ Handling By Isolated Mitochondria In Conditions Avoiding The Permeability Transition

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)New Findings What is the central question of this study? The assessment of Ca2+ handling by isolated mitochondria can be biased by dysfunctions secondary to Ca2+-induced mitochondrial permeability transition (MPT). As a result of this uncertainty and the differing experimental conditions between studies, the tissue and sex diversities in mitochondrial Ca2+ transport are still unsettled questions. What is the main finding and its importance? If MPT is not prevented during Ca2+ transport assays, some measured variables are biased. Accounting for the implied importance of preventing MPT, we observed substantial tissue specificities in the mitochondrial Ca2+ handling, particularly in the Ca2+ efflux pathways. The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sexual dimorphism. Given that measurements of Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluated the extent to which MPT inhibition by ciclosporin affected the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to fourfold if MPT pore opening is not prevented. We analysed Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats in incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. The Ca2+ influx rates were similar among the samples, except that the liver mitochondria displayed values fourfold higher. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (approximate to 4nmolmg(-1)min(-1)), thus challenging the view that cardiac mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was observed in only two kinetic indexes of heart mitochondrial Ca2+ homeostasis and in the ADP-stimulated respiration of liver mitochondria (approximate to 20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria.100910731092Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [11/50400-0]FAPESP [2011/51800-1

TGF-β1 Reduces Neutrophil Adhesion and Prevents Acute Vaso-Occlusive Processes in Sickle Cell Disease Mice

Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-β1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-β1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-β1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-β, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-β1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-β1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-β1 can reduce the adhesive properties of these cells; however, direct effects of TGF-β1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed

Didox (3,4-dihydroxybenzohydroxamic acid) reduces the vascular inflammation induced by acute intravascular hemolysis

Sem informação81FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/00984-

A kidney resident macrophage subset is a candidate biomarker for renal cystic disease in preclinical models

Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206(+) resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206(+) macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206(+) resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206(+) resident macrophages and cyst formation, and reveal that the CD206(+) resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper