RESUMO
Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.
Assuntos
Macrófagos/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Idoso , Animais , Apoptose , Autofagia , Feminino , Coração/fisiologia , Homeostase , Humanos , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina Quinase/metabolismoRESUMO
BACKGROUND: Senescence is a cellular aging-related process triggered by different stresses and characterized by the secretion of various inflammatory factors referred to as senescence-associated secretory phenotype (SASP), some of which are produced by the NLRP3 inflammasome. Here, we present evidence that the NLRP1 inflammasome is a DNA damage sensor and a key mediator of senescence. METHODS: Senescence was induced in fibroblasts in vitro and in mice. Cellular senescence was assessed by Western blot analysis of several proteins, including p16, p21, p53, and SASP factors, released in the culture media or serum. Inflammasome components, including NLRP1, NLRP3 and GSDMD were knocked out or silenced using siRNAs. RESULTS: In vitro and in vivo results suggest that the NLRP1 inflammasome promotes senescence by regulating the expression of p16, p21, p53, and SASP factors in a Gasdermin D (GSDMD)-dependent manner. Mechanistically, the NLRP1 inflammasome is activated in response to genomic damage detected by the cytosolic DNA sensor cGMP-AMP (cGAMP) synthase (cGAS). CONCLUSION: Our findings show that NLRP1 is a cGAS-dependent DNA damage sensor during senescence and a mediator of SASP release through GSDMD. This study advances the knowledge on the biology of the NLRP1 inflammasome and highlights this pathway as a potential pharmcological target to modulate senescence.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Senescência Celular , Dano ao DNA , Fibroblastos , Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato , Fenótipo Secretor Associado à Senescência , Animais , Inflamassomos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas NLR/metabolismo , Proteínas NLR/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Camundongos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Células Cultivadas , Camundongos Knockout , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , GasderminasRESUMO
BACKGROUND AND OBJECTIVE: Our aims were to improve the understanding of the pathogenic relationship between cardiovascular diseases and periodontitis and to generate new perspectives in the prevention and treatment of acute myocardial infarction (AMI) and periodontitis. The present study evaluates possible differences in inflammation, oxidative stress, and autophagy markers among subject suffering AMI, periodontitis, or both, to explore possible common pathogenic mechanisms. MATERIAL AND METHODS: A total of 260 subjects were enrolled in the study: 106 subjects that survived to a first AMI (AMI group) and 154 subjects had no cardiac events in their clinical record (control group). A questionnaire was used to assess age, height, weight, blood pressure, and heart rate. The clinical probing depth, clinical attachment loss, number of remaining teeth, and average number of sites with bleeding on probing were assessed. Lipid peroxidation and protein levels of phosphorylated AMP-activated protein kinase (p-AMPK) and microtubule-associated proteins 1A/1B-light chain 3-II (LC3-II) were determined in isolated peripheral blood mononuclear cells by thiobarbituric acid reactive substances (TBARS) assay and Western blot, respectively. Plasma levels of interleukin-1ß were determined using a commercial ELISA kit. All the obtained variables were compared between subjects suffering an AMI with or without periodontitis and control subject periodontal healthy or with periodontitis. RESULTS: A higher proportion of subjects suffering AMI + periodontitis than only AMI (without periodontitis) was found. Higher levels of TBARS were found in subjects with periodontitis than in subjects without periodontitis in both AMI and control subjects. Positive correlations between IL-1ß levels and TBARS and between IL-1ß levels and LC3-II were found only in control subjects. CONCLUSION: Results from the present study are consistent with the suggestion of periodontitis as a potential risk factor for AMI. Periodontitis association with circulating lipid peroxides in both AMI and control subjects were found. The absence of differences in IL-1ß levels between AMI subjects (only AMI vs AMI + periodontitis) suggests that oxidative stress could be the main pathogenic link between AMI and periodontitis.
Assuntos
Inflamação , Infarto do Miocárdio , Estresse Oxidativo , Periodontite , Índice de Placa Dentária , Humanos , Leucócitos Mononucleares , Infarto do Miocárdio/complicações , Perda da Inserção Periodontal , Índice Periodontal , Periodontite/complicaçõesRESUMO
BACKGROUND: Cathepsin C (CatC) is a lysosomal enzyme involved in activation of serine proteases from immune and inflammatory cells. Several loss-of-function mutations in the CatC gene have been shown to be the genetic mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis, and increased susceptibility to infections. Deficiencies or dysfunction in other cathepsin family proteins, such as cathepsin B or D, have been associated with autophagic and lysosomal disorders. OBJECTIVES: Here we characterized the basis for autophagic dysfunction in patients with PLS by analyzing skin fibroblasts derived from patients with several mutations in the CatC gene and reduced enzymatic activity. METHODS: Skin fibroblasts were isolated from patients with PLS assessed by using genetic analysis. Authophagic flux dysfunction was evaluated by examining accumulation of p62/SQSTM1 and a bafilomycin assay. Ultrastructural analysis further confirmed abnormal accumulation of autophagic vesicles in mutant cells. A recombinant CatC protein was produced by a baculovirus system in insect cell cultures. RESULTS: Mutant fibroblasts from patients with PLS showed alterations in oxidative/antioxidative status, reduced oxygen consumption, and a marked autophagic dysfunction associated with autophagosome accumulation. These alterations were accompanied by lysosomal permeabilization, cathepsin B release, and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Treatment of mutant fibroblasts with recombinant CatC improved cell growth and autophagic flux and partially restored lysosomal permeabilization. CONCLUSIONS: Our data provide a novel molecular mechanism underlying PLS. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for PLS.
Assuntos
Autofagia/efeitos dos fármacos , Catepsina C/farmacologia , Fibroblastos/efeitos dos fármacos , Adulto , Animais , Catepsina C/genética , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Insetos , Lisossomos/metabolismo , Masculino , Mutação , Doença de Papillon-Lefevre/tratamento farmacológico , Doença de Papillon-Lefevre/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Pele/citologia , Adulto JovemRESUMO
Activation of the NLRP3 inflammasome, a multiprotein complex, leading to caspase activation with production of proinflammatory IL-1ß represents a major pathway of inflammation. Recent, studies in mice and human patients uncovered several gain-of- function (GOF) mutations in inflammasome sensor proteins that allow inflammasome assembly in the absence of cognate ligands to trigger autoinflammatory syndromes. Cryopyrin-associated periodic syndromes (CAPS) are rare autoinflammatory diseases, comprising a broad disease spectrum with varying severity. CAPS are associated with GOF mutations in the NLRP3 inflammasome and activation of IL-1ß leading to episodes of fever, cutaneous, musculoskeletal, articular, ocular, and neurological symptoms. Here, we review current knowledge on different mutations leading to CAPS and related clinical syndromes. Homologous gene mutations in mice provide insights into the regulation and consequences of the activation of different inflammasomes in several autoinflammatory syndrome. In view of the critical role of IL-1ß in the pathogenesis of autoinflammatory GOF mutations such as CAPS, blockade of the action of IL-1ß is critical. Therapeutic administration of recombinant IL-1 receptor antagonists or monoclonal anti-IL-1ß antibody had a beneficial effect. Furthermore, novel inhibitors of inflammasome complex formation such as MCC950 and related compounds attenuate experimental and clinical disease. The discovery of new GOF mutants of inflammasomes leading to further insights in pathomechanisms and the development of novel inhibitors represent a great challenge.
Assuntos
Síndromes Periódicas Associadas à Criopirina/genética , Mutação com Ganho de Função/genética , Inflamassomos/genética , Multimerização Proteica/genética , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Autoimunidade , Síndromes Periódicas Associadas à Criopirina/imunologia , Síndromes Periódicas Associadas à Criopirina/terapia , Modelos Animais de Doenças , Humanos , Inflamassomos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologiaRESUMO
Cardiovascular diseases (CVD) are the leading cause of death worldwide, with high prevalence in industrialized countries. Cardiovascular risk factors are mainly influenced by diet, which like other lifestyle factors can be modified to either reduce or increase cardiovascular risk. Other metabolic diseases such as metabolic syndrome, type II diabetes mellitus, and obesity are associated to CVD and highly influenced by the diet. Inflammation has demonstrated to be a key factor in the biological progress of these diseases. Interestingly, IL-1ß which is associated to several steps in the development of atherosclerosis, heart disease, and the association of obesity and type II diabetes with CVD, is activated by the inflammasome complex, a multiprotein complex composed of an intracellular sensor, typically a Nod-like receptor (NLR), the precursor procaspase-1, and the adaptor ASC (apoptosis-associated speck-like protein containing a CARD. In the last years, inflammasome complex has been studied in depth and has been associated with the effect of unhealthy diets both from a clinical and experimental view point. We have reviewed the evidences supporting the role of the inflammasome complex in the development of cardiovascular pathology by unhealthy diets and the therapeutic perspectives.
Assuntos
Doenças Cardiovasculares/etiologia , Dieta Ocidental/efeitos adversos , Inflamassomos/imunologia , Inflamação/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Aterosclerose/etiologia , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/terapia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/terapia , Caspase 1/imunologia , Inibidores de Caspase/uso terapêutico , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidoresRESUMO
Cell cytoskeleton makes profound changes during apoptosis including the organization of an Apoptotic Microtubule Network (AMN). AMN forms a cortical structure which plays an important role in preserving plasma membrane integrity during apoptosis. Here, we examined the cytoskeleton rearrangements during apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. Using fixed and living cell imaging, we showed that CPT induced two dose- and cell cycle-dependent types of apoptosis characterized by different cytoskeleton reorganizations, time-dependent caspase activation and final apoptotic cell morphology. In the one referred as "slow" (~h) or round-shaped, apoptosis was characterized by a slow contraction of the actinomyosin ring and late caspase activation. In "slow" apoptosis the γ-tubulin complexes were not disorganized and microtubules were not depolymerized at early stages. In contrast, "fast" (~min) or irregular-shaped apoptosis was characterized by early caspase activation followed by full contraction of the actinomyosin ring. In fast apoptosis γ-tubulin complexes were disorganized and microtubules were initially depolymerized. However, after actinomyosin contraction, microtubules were reformed adopting a cortical but irregular disposition near plasma membrane. In addition to distinctive cytoskeleton reorganization kinetics, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocytes response. Our results suggest that the knowledge and modulation of the type of apoptosis promoted by genotoxic agents may be important for deciding a better therapeutic option and predicting the immune response in cancer treatment.
Assuntos
Apoptose/fisiologia , Camptotecina/farmacologia , Citoesqueleto/efeitos dos fármacos , Dano ao DNA , Inibidores da Topoisomerase I/farmacologia , Actomiosina/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Forma Celular , Citoesqueleto/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Células LLC-PK1 , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Fagocitose/efeitos dos fármacos , Suínos , Tubulina (Proteína)/efeitos dos fármacosRESUMO
Major Depressive Disorder (MDD, ICD-10: F-33) is a prevalent illness in which the pathogenic mechanism remains elusive. Recently an important role has been attributed to neuro-inflammation, and specifically the NLRP3-inflammasome complex, in the pathogenesis of MDD. This suggests a key role for immunomodulation as a key pathway in the treatment of this disorder. This study evaluates the involvement of nine common antidepressants in the NLRP3-inflammasome complex (fluoxetine, paroxetine, mianserin, mirtazapine, venlafaxine, desvenlafaxine, amitriptyline, imipramine and agomelatine), both in in vitro THP-1 cells stimulated by ATP, and in a stress-induced depressive animal or MDD patients. Antidepressant treatment induced inflammasome inhibition was observed by decreased serum levels of IL-1ß and IL-18 and decrease of NLRP3 and IL-1ß (p17) protein expression. This was also observed under stress-induced depressive behaviour and inflammasome activation in C57Bl/6 mice in vivo. Deletion of key autophagy mediator Atg5 in embryonic fibroblasts (MEF cells) showed an autophagy dependent-NLRP3-inflammasome inhibition by antidepressant treatment. These results suggest the NLRP3-inflammasome could be a biomarker for antidepressant treatment response in MDD patients, and therefore the monitoring of NLRP3 expression levels and/or IL-1ß/IL-18 release may have clinical value in drug selection. Existing evidence suggests an anti-inflammatory effect of some antidepressants shown by IL-1ß, IL-6 and TNF-α. Our data have shown that antidepressant-mediated autophagy may have a role in restoration of certain metabolic and immunological pathways in MDD patients.
Assuntos
Antidepressivos/uso terapêutico , Autofagia/efeitos dos fármacos , Transtorno Depressivo Maior/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Adulto , Animais , Antidepressivos/farmacologia , Linhagem Celular , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/imunologia , Feminino , Humanos , Inflamassomos/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
BACKGROUND: Fibromyalgia (FM) is a worldwide diffuse musculoskeletal chronic pain condition that affects up to 5% of the general population. Many symptoms associated with mitochondrial diseases are reported in patients with FM such as exercise intolerance, fatigue, myopathy and mitochondrial dysfunction. In this study, we report a mutation in cytochrome b gene of mitochondrial DNA (mtDNA) in a family with FM with inflammasome complex activation. METHODS: mtDNA from blood cells of five patients with FM were sequenced. We clinically and genetically characterised a patient with FM and family with a new mutation in mtCYB. Mitochondrial mutation phenotypes were determined in skin fibroblasts and transmitochondrial cybrids. RESULTS: After mtDNA sequence in patients with FM, we found a mitochondrial homoplasmic mutation m.15804T>C in the mtCYB gene in a patient and family, which was maternally transmitted. Mutation was observed in several tissues and skin fibroblasts showed a very significant mitochondrial dysfunction and oxidative stress. Increased NLRP3-inflammasome complex activation was observed in blood cells from patient and family. CONCLUSIONS: We propose further studies on mtDNA sequence analysis in patients with FM with evidences for maternal inheritance. The presence of similar symptoms in mitochondrial myopathies could unmask mitochondrial diseases among patients with FM. On the other hand, the inflammasome complex activation by mitochondrial dysfunction could be implicated in the pathophysiology of mitochondrial diseases.
Assuntos
Proteínas de Transporte/genética , Citocromos b/genética , Fibromialgia/genética , Inflamassomos/genética , Mutação , Adulto , Proteínas de Transporte/metabolismo , Citocromos b/química , Citocromos b/metabolismo , DNA Mitocondrial/genética , Feminino , Fibromialgia/patologia , Humanos , Inflamassomos/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , LinhagemRESUMO
Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/química , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Suplementos Nutricionais , Metabolismo Energético , Ativação Enzimática , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Subunidades Proteicas , Transdução de Sinais/efeitos dos fármacosRESUMO
Impaired AMPK is associated with a wide spectrum of clinical and pathological conditions, ranging from obesity, altered responses to exercise or metabolic syndrome, to inflammation, disturbed mitochondrial biogenesis and defective response to energy stress. Fibromyalgia (FM) is a world-wide diffused musculoskeletal chronic pain condition that affects up to 5% of the general population and comprises all the above mentioned pathophysiological states. Here, we tested the involvement of AMPK activation in fibroblasts derived from FM patients. AMPK was not phosphorylated in fibroblasts from FM patients and was associated with decreased mitochondrial biogenesis, reduced oxygen consumption, decreased antioxidant enzymes expression levels and mitochondrial dysfunction. However, mtDNA sequencing analysis did not show any important alterations which could justify the mitochondrial defects. AMPK activation in FM fibroblast was impaired in response to moderate oxidative stress. In contrast, AMPK activation by metformin or incubation with serum from caloric restricted mice improved the response to moderate oxidative stress and mitochondrial metabolism in FM fibroblasts. These results suggest that AMPK plays an essential role in FM pathophysiology and could represent the basis for a valuable new therapeutic target/strategy. Furthermore, both metformin and caloric restriction could be an interesting therapeutic approach in FM.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Restrição Calórica , Fibroblastos/metabolismo , Fibromialgia/metabolismo , Metformina/farmacologia , Mitocôndrias/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , DNA Mitocondrial/genética , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Estresse OxidativoRESUMO
Oxidative stress is implicated in several infectious diseases. In this regard, lipopolysaccharide (LPS), an endotoxic component, induces mitochondrial dysfunction and oxidative stress in several pathological events such as periodontal disease or sepsis. In our experiments, LPS-treated fibroblasts provoked increased oxidative stress, mitochondrial dysfunction, reduced oxygen consumption and mitochondrial biogenesis. After comparing coenzyme Q10 (CoQ10) and N-acetylcysteine (NAC), we observed a more significant protection of CoQ10 than of NAC, which was comparable with other lipophilic and hydrophilic antioxidants such as vitamin E or BHA respectively. CoQ10 improved mitochondrial biogenesis by activating PGC-1α and TFAM. This lipophilic antioxidant protection was observed in mice after LPS injection. These results show that mitochondria-targeted lipophilic antioxidants could be a possible specific therapeutic strategy in pharmacology in the treatment of infectious diseases and their complications.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Ubiquinona/análogos & derivados , Adulto , Animais , Células Cultivadas , Citrato (si)-Sintase/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/farmacologiaRESUMO
Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.
Assuntos
Apoptose , Temperatura Baixa , Temperatura Alta , Microtúbulos/ultraestrutura , Antineoplásicos Fitogênicos/farmacologia , Cálcio/metabolismo , Camptotecina/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/efeitos dos fármacos , Necrose , Oligopeptídeos/farmacologia , Paclitaxel/farmacologia , Fosfatidilserinas/metabolismoRESUMO
INTRODUCTION: Major depressive disorder (MDD) is a very prevalent disease which pathogenic mechanism remains elusive. There are some hypotheses and pilot studies suggesting that cytokines may play an important role in MDD. In this respect, we have investigated the role of NLRP3 inflammasome complex in the maturation of caspase-1 and the processing of its substrates, IL-1ß and IL-18, in blood cells from MDD patients. METHODS: Forty MDD patients were selected for this study, twenty without treatments and twenty treated with amitriptyline, a common tricyclic antidepressant. Blood samples from twenty healthy volunteers were included in the study. The inflammasome activation was studied by Western blot and real-time PCR of NLRP3 and caspase 1 and serum levels of IL-1ß and 18. RESULTS: We observed increased gene expression of NLRP3 and caspase-1 in blood cells, and increased serum levels of IL-1ß and IL-18 in non-treated patients. IL-1ß and IL-18 correlated with Beck Depression Inventory (BDI) scores of MDD patients. Interestingly, amitriptyline treatment reduced NLRP3 and caspase-1 gene expression, and IL-1ß and IL-18 serum levels. As it is well established that oxidative stress is associated with NLRP3 inflammasome activation, we next studied mitochondrial ROS and lipid peroxidation (LPO) levels in MDD patients. Increased levels of mitochondrial ROS and LPO were observed in MDD patients, however oxidative damage was higher in MDD patients treated with amitriptyline. CONCLUSIONS: These findings provide new insight into the pathogenesis of MDD and the effects of amitriptyline treatment on NLRP3 inflammasome activation and IL-1ß and IL-18 serum levels.
Assuntos
Proteínas de Transporte/metabolismo , Transtorno Depressivo Maior/sangue , Inflamassomos/metabolismo , Interleucina-18/sangue , Interleucina-1beta/sangue , Leucócitos Mononucleares/metabolismo , Caspase 1/metabolismo , Transtorno Depressivo Maior/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse OxidativoRESUMO
In order to analyze the association between body mass index (BMI), lipid profile and clinical symptoms in patients with fibromyalgia, we assessed BMI levels, lipid profile and its association with clinical symptoms in 183 patients with fibromyalgia. The patients were evaluated using tender points, FIQ and Visual Analogue Scales of pain (VAS). Serum lipid profile analysis (total cholesterol, triglyceride, HDL, LDL and VLDL), and biochemical parameters were measured in the biochemistry laboratory. The BMI distribution of the nonobese, overweight and obese patients' groups were relatively even with 37.7, 35.5 and 26.8%, respectively, with a mean BMI of 27.3 ± 4.9. The number of tender points showed significantly positive correlation with higher BMI (P < 0.05). A total of 57.9% of patients showed increased levels of total cholesterol, 63.4 % increased levels of LDL cholesterol and 19.9% high levels of triglycerides. BMI, total cholesterol and triglycerides showed high association with some clinical parameters. Overweight and lipid profile could be associated with fibromyalgia symptoms. A treatment program with weight loss strategies, and control in diet and increased physical activity is advised to patients.
Assuntos
Fibromialgia/sangue , Fibromialgia/fisiopatologia , Lipídeos/sangue , Obesidade/sangue , Obesidade/fisiopatologia , Sobrepeso/sangue , Sobrepeso/fisiopatologia , Adulto , Índice de Massa Corporal , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Dieta Redutora , Exercício Físico , Feminino , Fibromialgia/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Mialgia/fisiopatologia , Obesidade/terapia , Sobrepeso/terapia , Medição da Dor , Triglicerídeos/sangueRESUMO
An extensive number of pathologies are associated with mitochondrial dysfunction (MDF) and oxidative stress (OS). Thus, mitochondrial cofactors termed "mitochondrial nutrients" (MN), such as α-lipoic acid (ALA), Coenzyme Q10 (CoQ10), and l-carnitine (CARN) (or its derivatives) have been tested in a number of clinical trials, and this review is focused on the use of MN-based clinical trials. The papers reporting on MN-based clinical trials were retrieved in MedLine up to July 2014, and evaluated for the following endpoints: (a) treated diseases; (b) dosages, number of enrolled patients and duration of treatment; (c) trial success for each MN or MN combinations as reported by authors. The reports satisfying the above endpoints included total numbers of trials and frequencies of randomized, controlled studies, i.e., 81 trials testing ALA, 107 reports testing CoQ10, and 74 reports testing CARN, while only 7 reports were retrieved testing double MN associations, while no report was found testing a triple MN combination. A total of 28 reports tested MN associations with "classical" antioxidants, such as antioxidant nutrients or drugs. Combinations of MN showed better outcomes than individual MN, suggesting forthcoming clinical studies. The criteria in study design and monitoring MN-based clinical trials are discussed.
Assuntos
Quimioprevenção , Ensaios Clínicos como Assunto , Coenzimas/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/tratamento farmacológico , Estresse Oxidativo , Animais , HumanosRESUMO
Inflammation causes a wide range of health disorders. In this process, the formation of inflammasome complexes plays a key role. Although inflammasomes have been extensively studied during kidney disease, their role in kidney transplantation has not been fully elucidated. In this study, we evaluate the gene and protein expression of several components of the inflammasome pathway before and at several time points after kidney transplantation in a cohort of patients of different ages and receiving an organ from older or younger donors. Our findings indicate the activation of the NLRP1 inflammasome in several immune cell population, monocytes and CD4+ and CD8+ cells mainly, in renal transplant, and its level increases gradually in patients who receive an older organ, whereas it has the opposite effect on older patients who receive a younger organ. Despite treatment with immunosuppressants, inflammation persists in some patients. These results lead to the hypothesis that the donor's age is a critical factor in post-transplant inflammasome activation and that specific NLRP1 inflammasome inhibitors should be considered to increase the success of kidney transplantation long-term.
RESUMO
The role of the inflammasomes in aging and progeroid syndromes remain understudied. Recently, MCC950, a NLRP3 inhibitor, was used in Zmpste24-/- mice to ameliorate the phenotypes. However, the safety of MCC950 was questioned due to liver toxicity observed in humans. Nevertheless, inhibition of the inflammasomes would be a beneficial therapy for progeria. Here, we show that OLT1177 (dapansutrile), other NLRP3 inhibitor, improved cellular and animal phenotypes using progeroid fibroblasts and a LmnaG609G/G609G mouse model. In both cases dapansutrile reduced progerin accumulation, NLRP3-inflammasome activation and secretory phenotype of senescence, extended the lifespan of progeroid animals, preserved bodyweight, and reduced kyphosis, inflammation, and senescence. Interestingly, dapansutrile further improved the effect of lonafarnib, the only FDA-approved drug for the progeria. The combination of both drugs reduced the inflammation and senescence, extended survival and ameliorated various progeroid defects both in vitro and in vivo, compared with treatment using lonafarnib alone. These findings and the safety of dapansutrile demonstrated in several clinical trials proposes it as a possible co-adjuvant treatment with lonafarnid in HGPS.
RESUMO
Renal transplantation is an effective treatment for severe chronic kidney diseases. However, young patients often face a scarcity of kidneys from donors of similar age, resulting in the transplantation of older organs, which increase the risk of graft rejection and several complications compared with older individuals who receive kidneys from donors of similar age or younger. This article focuses on studying different senescence biomarkers in donors and patients who received kidneys from various age ranges complying with the STROBE requirements. We studied 61 patients subjected to renal transplant isolating blood samples 24 h before, and 24 h, 3 days, 7 days, 3 months, and 6 months after transplant. The patients were divided into three groups: older donor than the patient (Old Donor), younger donor than the patient (Young Donor), and similar age (Matched). We studied different senescence markers such as p16, p21, interleukin 6 (IL-6), and senescence-associated secretory phenotype (SASP) release. Young patients who receive older organs showed increased mRNA and protein expression of the senescence makers. Hence, increased SASP release was also observed in patients from older donor. In contrast, older patients who receive younger organs showed a slow but consistent improvement in their initial senescent phenotype. In addition, macrophage cell model treated with blood-derived serum from patients 6 months after the transplant showed a pro-senescence environment in macrophages proposed by the SASP from the patients. These results lead the hypothesis that senolytics could reduce the presence of senescent cells and mitigate the complications associated with the transplantation of older organs in young patients.
Assuntos
Biomarcadores , Senescência Celular , Transplante de Rim , Doadores de Tecidos , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Fatores Etários , Idoso , Fenótipo Secretor Associado à Senescência , Interleucina-6/metabolismo , Interleucina-6/sangueRESUMO
In the past, mitochondrial reactive oxygen species (mtROS) were considered a byproduct of cellular metabolism. Due to the capacity of mtROS to cause oxidative damage, they were proposed as the main drivers of ageing and age-related diseases. Today, we know that mtROS are cellular messengers instrumental in maintaining cellular homeostasis. As cellular messengers, they are produced in specific places at specific times, and the intensity and duration of the ROS signal determine the downstream effects of mitochondrial redox signalling. We do not know yet all the processes for which mtROS are important, but we have learnt that they are essential in decisions that affect cellular differentiation, proliferation and survival. On top of causing damage due to their capacity to oxidize cellular components, mtROS contribute to the onset of degenerative diseases when redox signalling becomes dysregulated. Here, we review the best-characterized signalling pathways in which mtROS participate and those pathological processes in which they are involved. We focus on how mtROS signalling is altered during ageing and discuss whether the accumulation of damaged mitochondria without signalling capacity is a cause or a consequence of ageing.