Papel do estresse oxidativo na fisiopatologia da esquizofrenia / Role of oxidative stress in the pathophysiology of schizophrenia

Introdução: a fisiopatologia da esquizofrenia é ainda pouco esclarecida. Vários estudos descrevem o importante papel da inflamação e do estresse oxidativo nessa explicação. Esquizofrênicos parecem ter seus mecanismos de defesa alterados e resposta distinta dos controles ao estresse oxidativo, que é lesivo a várias estruturas celulares, entre elas a matriz extracelular (MEC). Uma MEC íntegra e estruturada é essencialpara a boa condução sináptica. Objetivo: avaliar a resposta de genes que codificam proteínas da MEC ao estresse oxidativo em esquizofrênicos crônicos e controles. Metodologia: foi feita cultura de fibroblastos a partir de biópsias de pele de esquizofrênicos e controles. Estas foram tratadas com TBHQ, um pró-oxidante, ou DMSO, o veículo, e então se quantificou a expressão dos genes MMP16, GALNT6, SULF1, ADAMTS1 e ACSL1pelo método de PCR em tempo real e dos seus produtos por western blot. Resultados: existe resposta de ambos os grupos ao estresse oxidativo, no entanto, essa resposta é distinta entre pacientes e controles, com esquizofrênicos expressando menos o gene GALNT6 e sua proteína. Conclusão: o gene GALNT6 codifica uma enzima responsável por glicosilar componentes da MEC. Pode-se hipotetizar que a resposta de esquizofrê-nicos ao estresse oxidativo torna essa MEC mais suscetível aos seus efeitos deletérios, colaborando com a fisiopatologia da doença.

Introduction: The pathophysiology of schizophrenia is still poorly understood. Several studies suggest an important role of inflammation and oxidative stress in this explanation. Schizophrenics seem to have altered defense mechanisms and a distinct response to oxidative stress than that of controls. Oxidative stress is harmful to various cellular structures,among them the extracellular matrix (ECM). A intact and structured ECM is essential for proper synaptic signaling. Objective: To evaluate the response of genes encoding ECM proteins to oxidative stress in chronic schizophrenics and controls. Methodology: Fibroblasts were cultivated from schizophrenic and controls? skin biopsies. They were treatedwith TBHQ, a pro-oxidant, or DMSO (the vehicle), and then had the expression of MMP16, GALNT6, SULF1, ADAMTS1 and ACSL1 genes quantified by the method of real time PCR and their products by the method of Western blot. Results: There was response from both groups to oxidative stress, however, this response was different between patients and controls, with schizophrenics expressing less the GALNT6 gene and its protein. Conclusion: GALNT6 gene encodes an enzyme responsible for glycosylating ECM components. We can hypothesize that the schizophrenic?s response to oxidative stress renders the ECM moresusceptible to its harmful effects, contributing to the pathophysiology of the disease.

Inmunodetección de metaloproteinasas de matriz extracelular (MMPs)-2, -9, -13 y -14 en lesiones apicales asociadas con periodontitis apical asintomática / Immunodetection of matrix metalloproteinases (MMPs)-2, -9, -13 and -14 in periapical lesions associated with asymptomatic apical periodontitis

La periodontitis apical asintomática (PAa) es una patología infecciosa caracterizada por destrucción ósea perirradicular asociada a un proceso inflamatorio crónico y producción de mediadores inflamatorios, entre los cuales se encuentran las metaloproteinasas de matriz extracelular (MMPs). Entre éstas, las MMPs-13, -14, -2 y -9, son producidas por el tejido óseo y degradan sinérgicamente el colágeno tipo I, principal componente de los tejidos periodontales, y gelatina, producto de la degradación y desnaturación del colágeno. El objetivo de este estudio fue determinar el patrón de expresión de las MMPs-2, -9, -13 y -14 en granulomas periapicales (GPAs), quistes radiculares inflamatorios (QRIs) y ligamento periodontal sano (LS). Materiales y Métodos: Se seleccionaron 12 pacientes con diagnóstico clínico de PAa e indicación de exodoncia a partir de los cuales se obtuvieron biopsias de lesiones periapicales (LPAs). Como controles, se seleccionaron 7 individuos con indicación de exodoncia de premolares por ortodoncia, obteniéndose biopsias de LS. Se efectuó el diagnóstico anátomo-patológico de los especímenes y se caracterizó la expresión de las MMPs en estudio mediante inmunohistoquímica. Resultados: Las MMPs en estudio sólo se detectaron en GPAs y QRIs, y se inmunolocalizaron principalmente en el infiltrado inflamatorio de éstos. Adicionalmente, la MMP-2 se identificó en fibroblastos del tejido conectivo. Conclusiones: MMPs-2, -9, -13 y -14 se expresan predominantemente en el infiltrado inflamatorio de las LPAs y no en LS, y por tanto se sugiere la participación de estos mediadores en la patogénesis de la PAa.

Asymptomatic apical periodontitis (aAP) is an infectious disease characterized by perirradicular bone destruction associated with chronic inflammation and release of inflammatory mediators, such as matrix metalloproteinases (MMPs). MMPs-13, -14 and -2, -9 are bone-expressed enzymes that can synergistically degrade collagen I, the main component of periodontal extracellular matrix, and gelatin, the product of degradation and denaturation of collagen. The aim of this study was to characterize the expression pattern of MMPs-2, -9, -13, and -14 in periapical granulomas (PGs), radicular cysts (RCs) and healthy periodontal ligament (PDL). Materials and Methods: Individuals with clinical diagnosis of aAP and indication of extraction were selected (N=12), and biopsies of periapical lesions (PLs) were obtained. For controls, 7 subjects with indication of premolar extraction for orthodontic reasons were selected, and PDL biopsies were obtained. Samples were diagnosed by anatomopathological examination and immunohistochemical staining was carried out to characterize MMPs expression. Results: MMPs-2, -9, -13 and -14 detection was limited to PLs and were localized mainly to inflammatory infiltrate on both, PGs and RCs. Additionally, MMP-2 was immunolocalized to fibroblasts from the connective tissue. Conclusions: Whereas MMPs-2, -9, -13 and -14 were not detected in healthy periodontal ligament, they were highly expressed on inflammatory infiltrate from PGs and RCs, suggesting a role of these mediators in aAP pathogenesis.

Efeitos da terapêutica estrogênica sobre a remodelação da parede vascular em pacientes pós-menopáusicas / Effects of the estrogen therapy on vascular wall remodeling in postmenopausal women

O processo de aterogênese e o risco das doenças cardiovasculares sofrem influências significativas do hipoestrogenismo pós-menopáusico. Entretanto, os possíveis mecanismos anti-aterogênicos de proteção da parede vascular exercidos pelos estrogênios não são completamente conhecidos. Por sua vez, a matriz extracelular dos vasos tem importância reconhecida no controle da sua homeostase, especialmente nos processos de remodelação vascular. Entre as enzimas secretadas pela matriz extracelular envolvidas na remodelação da parede arterial estão as metaloproteinases. Os estrogênios exercem influência sobre a secreção, ativação e inibição da ação das metaloproteinases. Estas ações variam consoante o estágio de integridade da parede vascular. Quando a parede arterial é saudável ou quando o processo aterosclerótico encontra-se em fases iniciais, a ação estrogênica propicia elevação nos níveis das metaloproteinases 2 e 9 que conseguem degradar o acúmulo de proteína da matriz prevenindo a progressão da lesão. Entretanto, nos estágios ateroscleróticos mais avançados, a administração terapêutica de estrogênios ao elevar os níveis das metaloproteinases, especialmente da metaloproteinase 9, pode levar a um enfraquecimento e desestabilização da placa aterosclerótica através da sua digestão enzimática. Pelas razões consideradas e do ponto de vista das influências sobre a saúde vascular parece ser de crucial importância o momento de início da terapêutica estrogênica no período climatérico.

Extracellular breakdown of collagen by mice decidual cells. A cytochemical and ultrastructural study

The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates (disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

In vitro anti-proliferation/cytotoxic activity of cantharidin (Spanish Fly) and related derivatives

The anti-cancer therapeutic promise of cantharidin is limited because of its high mammalian toxicity. In order to find new anti-cancer lead compounds with reduced toxicity of the cantharidin prototype, the following seven derivatives were screened against the human SH-SY5Y neuroblastoma and MCF-7 breast cancer cells in vitro: 2,3-dimethyl-7-oxabicylo-[2.2.1]heptane-2,3-dicarboxylic anhydride (cantharidin) [1], 1-cyclohexen-1,2-dicarboxylic anhydride [2], cis-4-cyclohexen-1,2-dicarboxylic anhydride [3], cis-1, 2-cyclohexanedicarboxylic anhydride [4], exo-7-oxabicyclo[2.2.1]hept-5-ene-2-3 dicarboxylic anhydride [5], exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) [6], and (S)-(-)-O-acetylmalic anhydride [7]. Cantharidin, was found to be the most effective anti-proliferative compound on both cell lines. However, on the human neuroblastoma cells cantharidin was of equal toxicity to compound [6]. Mode of action studies revealed that cantharidin inhibited growth factor-mediated activation of mitogen activated protein kinase (MAPkinase) and attenuated the de-phosphorylation of the extracellular regulated kinases 1 and 2 (erk1 and erk2)

Upregulation of extracellular matrix metalloproteinase inducer (EMMPRIN) and gelatinases in human atherosclerosis infected with Chlamydia pneumoniae: The potential role of Chlamydia pneumoniae infection in the progression of atherosclerosis

Chlamydia pneumoniae infection implicated as an important etiologic factor of atherosclerosis, especially in coronary artery disease (CAD), was found in vitro to be associated with the induction of matrix metalloproteinases (MMPs). An extracellular matrix metalloproteinase inducer (EMMPRIN)/membrane-type 1 matrix metalloproteinase (MT1-MMP) system which induces and activates MMPs,is suggested to be functional and were upregulated in the failing myocardium. However, the upstream regulation of MMPs by C. pneumoniae within atheroma itself remains unclear. We evaluated the seroepidemiologic study of C. pneumoniae infection in CAD patients (n = 391) and controls (n = 97) and performed histopathological and in vitro analysis in atherosclerotic vascular tissues obtained from patients with seropositive to C. pneumoniae (n = 20), by using immunochemistry for C. pneumoniae, EMMPRIN/MT1-MMP, MMP-2, and MMP-9. The seropositive rates of both anti-C. pneumoniae IgG and IgA were 56.7% in CAD group and 43.3% in control group (P =0.033). Seropositive rate was increased in subgroups of CAD patients without conventional coronary risk factors compared to those with conventional risk factors. Immunoreactivities of EMMPRIN, MT1-MMP, MMP-2, and MMP-9 were increased in the atheromatous plaque itself, predominantly in immunoreactive macrophages/mononuclear cells to C. pneumoniae. Furthermore, Western blot analysis showed that EMMPRIN and MMP-2 were detected more prominently in atherosclerotic tissues infected with C. pneumoniae compared to control tissues. Zymographic analysis revealed that activities of MMP-2 and MMP-9 were more increased in atherosclerotic tissues infected with C. pneumoniae compared to control tissues. The present study demonstrated upstream regulation of MMPs can be induced by C. pneumoniae within atheromatous plaque itself. These findings help to understand the potential role of C. pneumoniae in the progression of atherosclerosis.

Matrix metalloproteinases and lung injury

The dynamic equilibrium of extracellular matrix (ECM) under different physiological conditions is a consequence of the balance between the regulation of synthesis and degradation of ECM components. Matrix metalloproteinases (MMPs), a family of structurally related zinc-dependent endopeptidases, are the physiological mediators of matrix remodeling. The expression and activity of these enzymes are highly regulated at several intra- and extracellular levels, so that in vivo enzymatic activity is the final result of a complex series of events including gene expression, zymogen activation, matrix binding, and enzymatic inhibition. MMPs are expressed at low levels in normal adult tissues, and their upregulation appears to play an important role in the development of a number of pathological processes. In acute lung injury, a disorder characterized by a severe disruption of the gas exchange alveolo-capillary structures, the upregulation of interstitial collagenase and gelatinases A and B strongly suggests that MMPs contribute to acute lung damage by facilitating the migration of inflammatory cells, as well as to the disruption of basement membrane components and extracellular matrix remodeling.

Transición de la forma enzimática latente a activa como un modelo de regulación de la degradación de matriz extracelular en el corioamnios durante el trabajo de parto humano / Transition from the laten to active enzymatic form asa regulation model for the degradation of choriomniotic extracelular matrix during human labor

Las metaloprotesas de matriz extracelular (MMP) son las mediadoras fisiológicas de la degradación de la colágena y su participación en la fisiopatogenia de la ruptura prematura de membranas ha sido sugerida por nuestro grupo. Con objeto de definir si algunas MMP se activan de manera coordinada con el trabajo de parto en las membranas fetales, analizamos la actividad enzimática y proteína inmunorreactiva presente en extractos de membranas obtenidas durante cesáreas, sin trabajo de parto, aunque su actividad/cantidad fue apenas detectable. En cambio los extractos de membranas fetales obtenidas durante el trabajo de parto activo, mostraron gran actividad/cantidad de ésta MMP. Con ayuda de un anticuerpo monoclonal, fue posible demostrar que la forma activa de la MMP-9 se podía encontrar sólo en las muestras con trabajo de parto. La MMP-9 y su ARN mensajero correspondiente fueron localizados por inmunohistoquímica e hibridación in situ en el epitelio amniótico, en algunos fibroblastos de la capa compacta y en células con características de trofoblasto en el corion. Se concluye que: 1. La actividad y la cantidad de la MMP-9 se incrementa de manera selectiva asociada al trabajo de parto y 2. que esta enzima es expresada por diferentes poblaciones celulares de la membrana fetal

Regulation of extracellular matrix-degrading proteases

Extracellular matrix-degrading enzymes have a very important role in many normal and pathological processes. Members of the matrix metalloproteinase and plasminogen activator families are the major modulators of extracellular matrix degradation. Here, we discuss some topics about these enzymes giving special attention to the transcriptional and extracellular regulation of their expression.