Made available in DSpace on 2015-01-26T13:21:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-07Bitstream added on 2015-01-26T13:30:22Z : No. of bitstreams: 1 000797249_20150903.pdf: 551884 bytes, checksum: 6f249a087df5eb16de47f2472dd301e1 (MD5) Bitstreams deleted on 2015-09-03T13:12:49Z: 000797249_20150903.pdf,. Added 1 bitstream(s) on 2015-09-03T13:13:21Z : No. of bitstreams: 1 000797249.pdf: 3130829 bytes, checksum: b6ad1f9fa1251f1ff059a82f758f9992 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidases são complexos multienzimáticos associados à membrana celular cuja principal função é catalisar a redução de oxigênio molecular a ânion superóxido (O2 •-). Em leucócitos, este é o mecanismo primário pelo qual estas células produzem espécies reativas de oxigênio (EROs), as quais estão envolvidas tanto nos mecanismos de defesa imune inato, quanto em processos oxidativos deletérios característicos de muitas patologias de cunho crônico inflamatório. Neste trabalho apresentamos os resultados obtidos e proposta de mecanismo de inibição do complexo NADPH oxidase por um conjunto de ésteres alquílicos sintéticos derivados do ácido protocatecúico, sendo este último um ácido fenólico presente em diversas plantas e com destacada capacidade antiradicalar. Nossa hipótese foi de que o aumento da hidrofobicidade provocado pela esterificação do ácido protocatecúico poderia facilitar o seu acesso à membrana celular e assim alterar seus efeitos biológicos. Esta hipótese se confirmou, pois muito mais do que melhorar a sua capacidade anti-radicalar (modelos in vitro), a esterificação provocou uma melhora significativa na capacidade de inibição do complexo NADPH oxidase em leucócitos (modelos ex vivo). Este efeito se propagou às EROs decorrentes de ânion superóxido e produzidas por leucócitos como peróxido de hidrogênio e ácido hipocloroso, sem entretanto alterar a sua capacidade fagocítica. Cabe frisar que não se trata de ação supressora sobre estas EROs, mas sim efetiva inibição de sua formação, o que foi demonstrado pelos diversos controles empregados. A esterificação do ácido protocatecúico também causou efetiva melhora na capacidade deste como inibidor das citocinas TNF-a e IL-10 produzidas por leucócitos mononucleares de sangue periférico. Considerando a baixa toxicidade e baixo custo de síntese desses ésteres, propomos que os mesmos ... / Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are multienzymatic complexes associated to the cell membranes whose main function is to catalyze the reduction of molecular oxygen to superoxide (O2 •-). In leukocytes, this is the primary mechanism through which these cells produce reactive oxygen species (ROS), which are involved in both the innate immune defense mechanisms and deleterious oxidative processes, which characterizes many chronic inflammatory diseases. In this thesis we present the results and proposed mechanism of inhibition of the NADPH oxidase complex by a group of synthetic alkyl esters derived from protocatechuic acid, a phenolic acid present in many plants with detached antiradical capacity. Our hypothesis was that the increase in hydrophobicity caused by esterification of protocatechuic acid could facilitate their access to the cell membrane and thereby alter their biological effects. This hypothesis was confirmed, since not only its anti-radical activity was increased (in vitro models), but also caused a significant improvement in their ability to inhibit NADPH oxidase complex in leukocytes (ex vivo models). This effect has spread to ROS derived from superoxide anion and produced by leukocytes such as hydrogen peroxide and hypochlorous acid, without altering their phagocytic capacity. It must be emphasize that the observed cellular effects were not due to simple suppressive action on ROS, but effective inhibition of its formation, which was demonstrated by the various control experiments. The esterification of protocatechuic acid also caused improvement in their capacity as inhibitor of TNF-a and IL-10 production by peripheral blood mononuclear leukocytes. Considering the low toxicity and low cost of synthesis of these esters, we suggest that they could be used in in vivo models as promising anti-inflammatory ...

Made available in DSpace on 2016-06-07T17:12:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2016-06-07T17:16:41Z : No. of bitstreams: 1 000865881.pdf: 980130 bytes, checksum: 18fb986e61f4ae49274bf00edf7afc5d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As doenças cardiovasculares (DCV) são a maior causa de invalidez e mortalidade em todo o mundo. Entre os principais fatores de risco para o desenvolvimento das DCV estão o diabetes mellitus (DM) e a hipertensão arterial sistêmica (HAS). Frequentemente, há co-existência de DM e HAS e isso ocasiona aumento do risco de eventos cardiovasculares. Os danos causados tanto pela HAS como pelo DM têm sido associados com o aumento do estresse oxidativo. A família de enzimas nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidase constitui uma das principais fontes de produção de espécies reativas de oxigênio no sistema cardiovascular. A apocinina (APO) tem sido caracterizada como um inibidor da NADPH oxidase desde a década de 1980. Apesar de evidências promissoras do uso da APO no tratamento de inúmeras doenças, há estudos questionando seu poder inibidor da NADPH oxidase. Além de controvérsias do uso da APO no bloqueio da NADPH oxidase em células não fagocíticas, poucos estudos avaliaram os efeitos desse bloqueio sobre o remodelamento cardíaco. Além disso, a escassez de informações é maior quando se associa HAS e DM. Portanto, o objetivo foi analisar a influência da inibição da NADPH oxidase por apocinina sobre o remodelamento cardíaco em ratos espontaneamente hipertensos (SHR) com diabetes mellitus. Métodos: SHR, machos, com 7 meses de idade, foram divididos em quatro grupos: controle (CTL, n=18), controle+apocinina (CTL+APO, n=18); diabético (DM, n=20) e diabético+apocinina (DM+APO, n=20). DM foi induzido por estreptozotocina (40 mg/kg, ip, dose única). Os grupos CTL+APO e DM+APO receberam APO (16 mg/kg/dia, diluída na água dos animais) durante 8 semanas. A avaliação estrutural e funcional in vivo do coração foi realizada por meio do ecocardiograma. O estudo funcional in vitro foi realizado pela técnica do músculo papilar do ventrículo esquerdo (VE). Para análise estrutural in vitro, foram medidos os... / Cardiovascular diseases (CVD) are the major cause of morbidity and mortality worldwide. Diabetes mellitus (DM) and arterial hypertension (AH) are the main risk factors for the development of CVD. The coexistence of diabetes and hypertension is common and leads to an increased risk of cardiovascular events. Organs damage caused by hypertension and DM have been associated with increased oxidative stress. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes family is a major source of reactive oxygen species in the cardiovascular system. Apocynin (APO) has been characterized as an inhibitor of NADPH oxidase since the 1980's decade. Despite promising evidence of APO in the treatment of many diseases, there are studies questioning its power as an inhibitor of NADPH oxidase. Besides controversies on the use of apocynin in blocking NADPH oxidase in non-phagocytic cells, few studies have evaluated the effects of its blockade on cardiac remodeling. In addition, there is a lack of information when AH and DM are associated. Therefore, the aim was to analyze the influence of NADPH oxidase inhibition by apocynin on cardiac remodeling in spontaneously hypertensive rats (SHR) with diabetes mellitus. Methods: Seven-month-old male SHR were divided into four groups: control (CTL, n=18); CTL+APO (n=18); DM (n=20); DM+APO (n=20). DM was induced by streptozotocin (40 mg/kg, i.p., single dose). CTL+APO and DM+APO groups received APO (16 mg/kg/day, diluted in the water) for 8 weeks. In vivo cardiac structures and functions were assessed by echocardiogram. In vitro functional study was performed by left ventricular papillary muscle study. In vitro left ventricle (LV), right ventricle and atria weights were measured. Samples of these structures, liver and lung were used to calculate the wet/dry weight ratio. LV tissue samples were obtained to measure myocyte diameters, interstitial collagen fraction, and hydroxyproline concentration. Left ...

Submitted by MURILO EDUARDO GRATON null (murilo.graton@yahoo.com.br) on 2017-05-30T04:15:45Z No. of bitstreams: 1 Dissertação de Mestrado - Murilo Eduardo Graton - Oficial.pdf: 2455052 bytes, checksum: 9fdb49043c056012a6f5db47564916b4 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-31T17:10:09Z (GMT) No. of bitstreams: 1 graton_me_me_araca.pdf: 2455052 bytes, checksum: 9fdb49043c056012a6f5db47564916b4 (MD5) / Made available in DSpace on 2017-05-31T17:10:09Z (GMT). No. of bitstreams: 1 graton_me_me_araca.pdf: 2455052 bytes, checksum: 9fdb49043c056012a6f5db47564916b4 (MD5) Previous issue date: 2017-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A enzima NAD(P)H oxidase (NOX) é a principal fonte de espécies reativas de oxigênio (ERO) no sistema cardiovascular e sua atividade e expressão podem ser regulada pela angiotensina (Ang) II. Demonstramos previamente que o tratamento crônico com apocinina, um inibidor de NOX, reduziu a pressão arterial e preveniu o desenvolvimento da disfunção endotelial em SHR. Estes efeitos da apocinina foram associados a redução de geração de ERO e ao aumento da biodisponibilidade de óxido nítrico em células endoteliais de SHR. Dados de nosso laboratório mostraram que o tratamento com apocinina, também reduziu o efeito pressor da Ang II em SHR. A associação entre Ang II, via receptores AT1, e o estresse oxidativo tem sido implicada na patogênese da hipertensão. Levantamos a hipótese que a apocinina, ao alterar a sinalização redox, reduz a expressão de receptores AT1 e a resposta vasoconstritora da Ang II em SHR. Neste estudo, avaliamos o efeito do tratamento crônico com apocinina sobre as respostas contráteis à Ang II em vasos sanguíneos de SHR e os mecanismos envolvidos nestes efeitos, utilizando ensaios bioquímicos, biomoleculares e funcionais. SHR foram tratados com apocinina (30 mg/Kg, v.o.) da 4ª a 10ª semana de vida e ratos Wistar foram utilizados como controle normotenso. Analisamos os efeitos da apocinina na capacidade antioxidante plasmática, expressão de NOX, geração de ERO, níveis de nitrato/ nitrito, expressão de receptores AT1 e AT2, e respostas vasoconstritoras à Ang II em artéria mesentérica e aorta. O tratamento de SHR com apocinina aumentou a capacidade antioxidante plasmática, os níveis de nitrato/nitrito, não alterou a expressão de receptores AT1 ou AT2 em artérias mesentéricas, mas aumentou a expressão de AT2, mas não de AT1, em aorta de SHR. Além disto, o tratamento com apocinina diminuiu a expressão de NOX2 e p47phox e a produção de ERO. O tratamento com apocinina aumentou a modulação do endotélio e/ou da atividade da NOS sobre as respostas vasoconstritoras à Ang II em artérias mesentéricas de SHR, mas não alterou a reatividade de aortas de SHR à Ang II. A menor reatividade de artérias de resistência à Ang II levaria a menor resistência vascular periférica e consequentemente a redução da pressão arterial média e do efeito pressor da Ang II em SHR tratados com apocinina, como observado previamente. O mecanismo de ação da apocinina envolvido neste efeito está associado a importantes alterações redox que determinam uma maior modulação endotelial dependente de NOS das respostas vasoconstritoras da Ang II, mas não envolve alterações na expressão de receptores AT1 em vasos de SHR. / NAD(P)H oxidase (NOX) is the major source of reactive oxygen species (ROS) in the cardiovascular system and its activity and expression could be regulated by angiotensin (Ang) II. We previously demonstrated that chronic treatment with apocynin, a NOX inhibitor, reduced blood pressure and prevented the development of endothelial dysfunction in SHR. These effects of apocynin have been associated with reduced generation of ROS and increased bioavailability of nitric oxide in endothelial cells of SHR. Data from our laboratory showed that treatment with apocynin also reduced the pressor effect of Ang II on SHR. The association between Ang II, via AT1 receptors, and oxidative stress has been implicated in the pathogenesis of hypertension. We hypothesized that apocynin, altering redox signaling, could reduce expression of AT1 receptors and Ang II vasoconstrictor response in SHR. In this study, we evaluated the effect of chronic treatment with apocynin on the contractile responses to Ang II in blood vessels of SHR and the mechanisms involved in the effects of these on biochemical, biomolecular and functional assays. SHR were treated with apocynin (30 mg/kg, p.o.) from the 4 th to the 10th week of life and Wistar rats were used as normotensive control. Analysis of the effects of apocynin on plasma antioxidant capacity, NOX expression, ROS generation, nitrate/ nitrite levels, expression of AT1 and AT2 receptors, and vasoconstrictor responses to Ang II on mesenteric and aortic arteries. Treatment of SHR with apocynin increased plasma antioxidant capacity, nitrate/ nitrite levels, did not alter AT1 or AT2 receptor expression in mesenteric arteries, but increased expression of AT2, but not AT1, in SHR aorta. In addition, treatment with apocynin decreased expression of NOX2 and p47phox and ROS generation. Treatment with apocynin increased endothelium modulation and/or NOS activity on Ang II vasoconstrictor responses in mesenteric arteries of SHR, but did not alter the reactivity to Ang II in aortas of SHR. The lower reactivity of Ang II in resistance arteries would lead to lower peripheral vascular resistance and consequently the reduction of mean arterial pressure and Ang II pressor effect in SHR treated with apocynin, as previously observed. The mechanism of action of the apostate involved in this effect is associated with important redox changes that determine a greater NOS-dependent endothelial modulation of the vasoconstrictor responses of Ang II, but does not involve expression in the expression of AT1 receptors in SHR vessels.

Orientador : Valdecir Farias Ximenes / Coorientador: Luiz Marcos da Fonseca / Banca: Patricia Palmeira Daenekas Jorge / Banca: Alexander Batista Duharte / Banca: Maria Luiza Zeraik / Banca: Alexandra Ivo de Medeiros / Resumo: Nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidases são complexos multienzimáticos associados à membrana celular cuja principal função é catalisar a redução de oxigênio molecular a ânion superóxido (O2 •-). Em leucócitos, este é o mecanismo primário pelo qual estas células produzem espécies reativas de oxigênio (EROs), as quais estão envolvidas tanto nos mecanismos de defesa imune inato, quanto em processos oxidativos deletérios característicos de muitas patologias de cunho crônico inflamatório. Neste trabalho apresentamos os resultados obtidos e proposta de mecanismo de inibição do complexo NADPH oxidase por um conjunto de ésteres alquílicos sintéticos derivados do ácido protocatecúico, sendo este último um ácido fenólico presente em diversas plantas e com destacada capacidade antiradicalar. Nossa hipótese foi de que o aumento da hidrofobicidade provocado pela esterificação do ácido protocatecúico poderia facilitar o seu acesso à membrana celular e assim alterar seus efeitos biológicos. Esta hipótese se confirmou, pois muito mais do que melhorar a sua capacidade anti-radicalar (modelos in vitro), a esterificação provocou uma melhora significativa na capacidade de inibição do complexo NADPH oxidase em leucócitos (modelos ex vivo). Este efeito se propagou às EROs decorrentes de ânion superóxido e produzidas por leucócitos como peróxido de hidrogênio e ácido hipocloroso, sem entretanto alterar a sua capacidade fagocítica. Cabe frisar que não se trata de ação supressora sobre estas EROs, mas sim efetiva inibição de sua formação, o que foi demonstrado pelos diversos controles empregados. A esterificação do ácido protocatecúico também causou efetiva melhora na capacidade deste como inibidor das citocinas TNF-a e IL-10 produzidas por leucócitos mononucleares de sangue periférico. Considerando a baixa toxicidade e baixo custo de síntese desses ésteres, propomos que os mesmos ... / Abstract: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are multienzymatic complexes associated to the cell membranes whose main function is to catalyze the reduction of molecular oxygen to superoxide (O2 •-). In leukocytes, this is the primary mechanism through which these cells produce reactive oxygen species (ROS), which are involved in both the innate immune defense mechanisms and deleterious oxidative processes, which characterizes many chronic inflammatory diseases. In this thesis we present the results and proposed mechanism of inhibition of the NADPH oxidase complex by a group of synthetic alkyl esters derived from protocatechuic acid, a phenolic acid present in many plants with detached antiradical capacity. Our hypothesis was that the increase in hydrophobicity caused by esterification of protocatechuic acid could facilitate their access to the cell membrane and thereby alter their biological effects. This hypothesis was confirmed, since not only its anti-radical activity was increased (in vitro models), but also caused a significant improvement in their ability to inhibit NADPH oxidase complex in leukocytes (ex vivo models). This effect has spread to ROS derived from superoxide anion and produced by leukocytes such as hydrogen peroxide and hypochlorous acid, without altering their phagocytic capacity. It must be emphasize that the observed cellular effects were not due to simple suppressive action on ROS, but effective inhibition of its formation, which was demonstrated by the various control experiments. The esterification of protocatechuic acid also caused improvement in their capacity as inhibitor of TNF-a and IL-10 production by peripheral blood mononuclear leukocytes. Considering the low toxicity and low cost of synthesis of these esters, we suggest that they could be used in in vivo models as promising anti-inflammatory ... / Doutor

Orientador: Katashi Okoshi / Banca: Silméia Garcia Zanati Bazan / Banca: Marcos Ferreira Minicuci / Banca: Fábio Fernandes / Banca: Francis Lopes Pacagnelli / Resumo: As doenças cardiovasculares (DCV) são a maior causa de invalidez e mortalidade em todo o mundo. Entre os principais fatores de risco para o desenvolvimento das DCV estão o diabetes mellitus (DM) e a hipertensão arterial sistêmica (HAS). Frequentemente, há co-existência de DM e HAS e isso ocasiona aumento do risco de eventos cardiovasculares. Os danos causados tanto pela HAS como pelo DM têm sido associados com o aumento do estresse oxidativo. A família de enzimas nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidase constitui uma das principais fontes de produção de espécies reativas de oxigênio no sistema cardiovascular. A apocinina (APO) tem sido caracterizada como um inibidor da NADPH oxidase desde a década de 1980. Apesar de evidências promissoras do uso da APO no tratamento de inúmeras doenças, há estudos questionando seu poder inibidor da NADPH oxidase. Além de controvérsias do uso da APO no bloqueio da NADPH oxidase em células não fagocíticas, poucos estudos avaliaram os efeitos desse bloqueio sobre o remodelamento cardíaco. Além disso, a escassez de informações é maior quando se associa HAS e DM. Portanto, o objetivo foi analisar a influência da inibição da NADPH oxidase por apocinina sobre o remodelamento cardíaco em ratos espontaneamente hipertensos (SHR) com diabetes mellitus. Métodos: SHR, machos, com 7 meses de idade, foram divididos em quatro grupos: controle (CTL, n=18), controle+apocinina (CTL+APO, n=18); diabético (DM, n=20) e diabético+apocinina (DM+APO, n=20). DM foi induzido por estreptozotocina (40 mg/kg, ip, dose única). Os grupos CTL+APO e DM+APO receberam APO (16 mg/kg/dia, diluída na água dos animais) durante 8 semanas. A avaliação estrutural e funcional in vivo do coração foi realizada por meio do ecocardiograma. O estudo funcional in vitro foi realizado pela técnica do músculo papilar do ventrículo esquerdo (VE). Para análise estrutural in vitro, foram medidos os... / Abstract: Cardiovascular diseases (CVD) are the major cause of morbidity and mortality worldwide. Diabetes mellitus (DM) and arterial hypertension (AH) are the main risk factors for the development of CVD. The coexistence of diabetes and hypertension is common and leads to an increased risk of cardiovascular events. Organs damage caused by hypertension and DM have been associated with increased oxidative stress. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes family is a major source of reactive oxygen species in the cardiovascular system. Apocynin (APO) has been characterized as an inhibitor of NADPH oxidase since the 1980's decade. Despite promising evidence of APO in the treatment of many diseases, there are studies questioning its power as an inhibitor of NADPH oxidase. Besides controversies on the use of apocynin in blocking NADPH oxidase in non-phagocytic cells, few studies have evaluated the effects of its blockade on cardiac remodeling. In addition, there is a lack of information when AH and DM are associated. Therefore, the aim was to analyze the influence of NADPH oxidase inhibition by apocynin on cardiac remodeling in spontaneously hypertensive rats (SHR) with diabetes mellitus. Methods: Seven-month-old male SHR were divided into four groups: control (CTL, n=18); CTL+APO (n=18); DM (n=20); DM+APO (n=20). DM was induced by streptozotocin (40 mg/kg, i.p., single dose). CTL+APO and DM+APO groups received APO (16 mg/kg/day, diluted in the water) for 8 weeks. In vivo cardiac structures and functions were assessed by echocardiogram. In vitro functional study was performed by left ventricular papillary muscle study. In vitro left ventricle (LV), right ventricle and atria weights were measured. Samples of these structures, liver and lung were used to calculate the wet/dry weight ratio. LV tissue samples were obtained to measure myocyte diameters, interstitial collagen fraction, and hydroxyproline concentration. Left ... / Doutor

Note: Portions of this abstract have been previously published in the journal Blood, Basiorka et al. Blood. 2016 Oct 13, and has been reproduced in this manuscript with permission from the publisher. Myelodysplastic syndromes (MDS) are genetically diverse hematopoietic stem cell malignancies that share a common phenotype of cytological dysplasia, ineffective hematopoiesis and aberrant myeloid lineage maturation. Apoptotic cell death potentiated by inflammatory cytokines has been considered a fundamental feature of MDS for over two decades. However, this non-inflammatory form of cell death cannot account for the inflammatory nature of these disorders. We report that a hallmark of lower-risk (LR) MDS is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptosis, a caspase-1-dependent programmed cell death induced by danger-associated molecular pattern (DAMP) signals. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress pyroptosis-related transcripts, inflammasome proteins and manifest activated NLRP3 inflammasome complexes that direct caspase-1 activation, IL-1β and IL-18 maturation and pyroptotic cell death. Using the S100A9 transgenic (S100A9Tg) mouse model that phenocopies human MDS, we demonstrated that forced expression of S100A9 was sufficient to drive pyroptosis in vivo, implicating pyroptosis as the principal mechanism of HSPC cell death in S100A9Tg mice. The lytic cell death releases intraceullar contents that include alarmins and catalytically active ASC specks, which can propagate bystander inflammation. Notably, MDS mesenchymal stromal cells (MSC) and stromal-derived linages were found to predominantly undergo pyroptosis, with marked activation of caspase-1 and NLRP3 inflammasome complexes. These findings may account for the clusters of both HSPC and stromal cell death previously described in the bone marrows of patients with MDS. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPC and bone marrow (BM) plasma. Further, both somatic gene mutations and S100A9-induced signaling activate NADPH oxidase (NOX), generating reactive oxygen species (ROS) that initiate cation influx, cell swelling and β-catenin activation. Accordingly, ROS and active β-catenin were significantly increased in MDS BM mononuclear cells (BM-MNC) and S100A9Tg mice compared to normal controls, as well as in human cell lines harboring gene mutations and in murine models of gene mutation knock-in or gene loss. ROS and β-catenin nuclear translocation were significantly reduced by NLRP3 or NOX inhibition, indicating that S100A9 and somatic gene mutations prime cells to undergo NOX1/4-dependent NLRP3 inflammasome assembly, pyroptosis and β-catenin activation. Together, these data explain the concurrent proliferation and inflammatory cell death characteristic of LR-MDS. Given that loss of a gene-rich area in del(5q) disease results in derepression of innate immune signaling, we hypothesized that this genetic deficit would trigger assembly of the NLRP3 inflammasome complex, akin to the pathobiological mechanism characteristic of non-del(5q) MDS. To this end, we utilized two distinct murine models of del(5q) disease, namely in the context of Rps14 haploinsufficiency and concurrent loss of mDia1 and microRNA (miR)-146a. In both models, pyroptosis was not evident in the HSPC compartment; however, early erythroid progenitors displayed high fractions of pyroptotic cells. This was associated with significant increases in caspase-1 and NLRP3 inflammasome activation, ROS and nuclear localization of β-catenin, which was extinguished by inflammasome or NOX complex inhibition. These data suggest that early activation of the inflammasome drives cell death and prevents terminal maturation of erythroid precursors, accounting for the progressive anemia characteristic of del(5q) disease, whereby hematopoietic defects are primarily restricted to the erythroid compartment. Importantly, these data implicate a similar pathobiological mechanism in del(5q) MDS as is observed in non-del(5q) patients. The identification of the NLRP3 inflammasome as a pathobiological driver of the LR non-del(5q) and del(5q) MDS phenotype allows for novel therapeutic agent development. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS, and are sufficient to restore effective hematopoiesis. In del(5q) murine models, inhibition of the NLRP3 inflammasome significantly improved erythroid colony forming capacity by a mechanism distinct from that of lenalidomide, highlighting the translational potential for targeting this innate immune complex in this subset of MDS. Thus, alarmins and founder gene mutations in MDS license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. Furthermore, aggregated clusters of the NLRP3 adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] are referred to as ASC specks. During pyroptosis, ASC specks are released from dying cells and function as DAMP signals that propagate inflammation. In this way, specks are a surrogate marker for NLRP3 inflammasome activation and pyroptotic cell death. Given that pyroptosis is the predominant mechanism of cell death in MDS and ASC specks are readily quantified by flow cytometry, we hypothesized that BM or peripheral blood (PB) plasma-derived ASC specks may be a biologically rational biomarker for the diagnosis of MDS. The percentage of ASC specks were significantly increased in MDS BM plasma compared to normal, healthy donors, which was validated by confocal microscopy. PB plasma-derived ASC specks were significantly greater in LR- versus HR-MDS, consistent with the greater extent of cell death and myeloid-derived suppressor cell (MDSC) expansion in LR disease. As hyperglycemia induces NLRP3 inflammasome activation, plasma glucose levels were measured to adjust for this confounding variable. Subsequently, the percentage of glucose-adjusted, PB plasma-derived ASC specks was measured in a panel of specimens of varied hematologic malignancies. The corrected percentage of ASC specks was significantly increased in MDS compared to normal donors and to each other malignancy investigated, including other myeloid and lymphoid leukemias, myeloproliferative neoplasms and overlap syndromes, like chronic myelomonocytic leukemia (CMML). These data indicate that the glucose-adjusted ASC speck percentage is MDS-specific and may be a valuable diagnostic biomarker. At a cutoff of 0.039, the biomarker minimizes misclassification error and achieves 95% sensitivity and 82% specificity in classifying MDS from normal donors, other hematologic malignancies and T2D. Lastly, the biomarker declined with treatment response to lenalidomide in LR-MDS patients, but not to erythropoietin stimulating agent (ESA) or hypomethylating agent (HMA) therapy. As such, the percentage of ASC specks represents the first biologically rational, diagnostic biomarker for MDS that can be implemented with current diagnostic practices to reduce diagnostic error.

Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

Yes / Quercetin is reported to reduce blood pressure in hypertensive but not normotensive humans, but the role of endothelial redox signaling in this phenomenon has not been assessed. This study investigated the effects of physiologically obtainable quercetin concentrations in a human primary cell model of endothelial dysfunction in order to elucidate the mechanism of action of its antihypertensive effects. Angiotensin II (100 nM, 8 h) induced dysfunction, characterized by suppressed nitric oxide availability (85 ± 4% p<0.05) and increased superoxide production (136 ± 5 %, p<0.001). These effects were ablated by an NADPH oxidase inhibitor. Quercetin (3 μM, 8 h) prevented angiotensin II induced changes in nitric oxide and superoxide levels, but no effect upon nitric oxide or superoxide in control cells. The NADPH oxidase subunit p47(phox) was increased at the mRNA and protein levels in angiotensin II-treated cells (130 ± 14% of control, p<0.05), which was ablated by quercetin co-treatment. Protein kinase C activity was increased after angiotensin II treatment (136 ± 51%), however this was unaffected by quercetin co-treatment. Physiologically obtainable quercetin concentrations are capable of ameliorating angiotensin II-induced endothelial nitric oxide and superoxide imbalance via protein kinase C-independent restoration of p47(phox) gene and protein expression. / Innovate UK and Boots Pharmaceuticals

No description available.

It is known that African American ethnicity is an independent risk factor for exaggerated oxidative stress which is intricately intertwined with inflammation, hypertension (HT), and cardiovascular disease (CVD). The purpose of this dissertation study was to examine the racial differences that exist between African Americans and Caucasians in oxidative stress levels at the molecular level using an in vitro model of Human Umbilical Vein Endothelial Cells (HUVECs). African American HUVECs were found to have significantly higher baseline levels of oxidative stress in vitro compared to Caucasian HUVECs. In order to establish proof of concept, three preliminary studies were conducted. The first preliminary study, an acute exercise protocol was conducted in young healthy adults in order to measure plasma oxidative stress markers in response to a single moderate intensity treadmill exercise bout. In this study, it was found that the treadmill exercise did not elicit a race-dependent responses, but that African American adults had higher level of oxidative stress at all sample times when compared to the Caucasians. A second preliminary study was conducted using a parallel cell culture design to measure basal oxidative stress levels in African American and Caucasian HUVECs without stimulation. These data were shown in relation to the plasma levels of oxidative stress in resting African American and Caucasian adults. This was done in order to show that the common oxidative stress markers measured in human plasma can also be measured in cell culture supernatant and lysate. It was found that both African American adults and HUVECs had heightened oxidative stress and inflammatory markers when compared to their Caucasian counterparts. The third preliminary study was conducted using tumor Necrosis Factor-#945; (TNF-#945;) as an inflammatory stimulant and measuring the oxidative stress response in both African American and Caucasian HUVECs. This was done in order to show that cells of different race respond differently to stimuli. It was found that the response to TNF-α was blunted in African American HUVECs. The final step was to use laminar shear stress (LSS) as an exercise mimetic in order to examine whether HUVECs from different race respond differently. HUVECs from both race were harvested under static condition (no LSS), with low LSS at 5 dyne/cm2, and with a moderate level of LSS at 20 dyne/cm2. It was found that despite the fact that African American HUVECs had higher levels of oxidative stress under static conditions, when LSS was applied, protein expressions and oxidative stress biomarkers adjusted to levels that were similar to the Caucasian HUVEC adaptations to LSS. From this, it appears that African American HUVECs have a larger response to LSS stimulus indicating that aerobic exercise prescriptions may be valuable for this population since the potential exists for large improvements in oxidative stress levels for this population. / Kinesiology