Yes / Osteopontin (OPN) plays an important role in cancer progression, however its prognostic significance and its downstream factors are largely elusive. In this study, we have shown that expression of OPN was significantly higher in bladder cancer specimens with higher T-stage or tumor grades. In addition, a high level of OPN was significantly associated with poorer survival in two independent bladder cancer patient cohorts totaling 389 bladder cancer patients with available survival data. We further identified Matrix metallopeptidase 9 (MMP9) and S100 calcium-binding protein A8 (S100A8) were both downstream factors for OPN in bladder cancer specimens and bladder cancer cell lines. Expression of OPN was significantly positively associated with that of MMP9 and S100A8, while overexpression of OPN resulted in upregulation of MMP9 and S100A8, and knockdown of OPN showed consistent downregulation of MMP9 and S100A8 expression levels. Importantly, expression levels of both MMP9 and S100A8 were significantly associated with higher T-stage, higher tumor grade and a shorter survival time in the bladder cancer patients. Interestingly, OPN expression only predicted survival in MMP9-high, but not MMP9-low subgroups, and in S100A8-low but not S100A8-high subgroups. Our results suggest that OPN, MMP9 and S100A8 all play a significant role in bladder cancer progression and are potential prognostic markers and therapeutic targets in bladder cancer. The mechanistic link between these three genes and bladder cancer progression warrants further investigation. / University of Macau Multi-Year Research Grant (MYRG2015-00065-FHS)

The transcription factor NF-κB is a key regulator in many physiological processes, including innate and adaptative immune responses, inflammation and lymphoid organ development. NF-κB plays a crucial role in the development of B and T cells, by maintaining the cell death/survival equilibrium and therefore, constitutive activation of NF-κB is thought to contribute to the development and/or progression of B and T cell malignancies. The nfκb2 gene is frequently involved in chromosomal translocations associated with the development of various lymphomas and leukemias. These rearrangements all lead to the production of C-terminally truncated p100 proteins lacking variable portions of the ankyrin repeats domain, suggesting that this common feature may be involved in tumor development. However, whereas the oncogenic potential of such proteins is well established, the underlying mechanisms remain poorly understood. The aim of this work was to better understand these mechanisms by which such alterations contribute to lymphomagenesis. We then choose to investigate the functions of the protein Hut78 as representative of these p100ΔC mutants found in tumor cells. We identified mmp9 as a target gene of p52 and p52-producing NF-κB2 mutants and defined p52 as a key molecule for the invasive potential of lymphoma-derived cells harboring enhanced activity of the NF-κB alternative pathway. Moreover, we found that this p52/Hut78-mediated transcriptional induction of MMP9 involves the recruitment of MLL1 and MLL2 H3K4 histone methyltransferase complexes by p52 on the mmp9 gene promoter. Taken together, our results provide further insights into the oncogenic potential of the truncated p100 proteins, and by extension, will help to better understand how mutated IκB proteins contribute to deregulated NF-κB activities in haematological disorders.

Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-29T15:58:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO RAPHAELA PAULO DOS SANTOS.pdf: 1582818 bytes, checksum: 4215a83eff291ff2840c11ace4e2d890 (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-29T15:58:14Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO RAPHAELA PAULO DOS SANTOS.pdf: 1582818 bytes, checksum: 4215a83eff291ff2840c11ace4e2d890 (MD5) / Made available in DSpace on 2017-09-29T15:58:14Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO RAPHAELA PAULO DOS SANTOS.pdf: 1582818 bytes, checksum: 4215a83eff291ff2840c11ace4e2d890 (MD5) Previous issue date: 2013 / Universidade Federal Fluminense. Hospital Universitário Antonio Pedro. Centro de Ciências Médicas / A endometriose é definida pela presença de tecido endometrial (glândula e/ou estroma) fora da cavidade uterina, a patologia afeta 10-15% das mulheres em idade reprodutiva, além disso, os sintomas álgicos da doença implicam em impactos econômicos e sociais. A doença apresenta diagnóstico tardio e sua etiologia ainda não foi completamente elucidada, porém, sabe-se que a endometriose possui caráter poligênico e multifatorial. O objetivo do estudo foi avaliar se os polimorfismos no gene HSD17β1 (rs605059), envolvido na síntese de estrogênio, e nos genes MMP2 (rs243865) e MMP9 (rs17576), que atuam no remodelamento da matriz extracelular, estão associados com a endometriose quanto ao risco e o grau de severidade da doença. O estudo do tipo caso-controle foi composto por 231 mulheres, sendo 97 casos e 134 controles. Todas as pacientes do grupo caso possuíam diagnóstico histopatológico para a endometriose. O DNA genômico foi extraído a partir de saliva, e o polimorfismo no gene HSD17β1 foi detectado pela técnica de PCR- Nested seguido de digestão do produto de PCR pela enzima BstUI, os genes MMP2 e MMP9 foram genotipados pela técnica de PCR em Tempo Real. Não foi encontrada diferença estatisticamente significante entre as distribuições genotípicas e alélicas dos genes analisados entre os grupos estudados (p>0,05). Do mesmo modo não foi observada diferença significativa na frequência dos genótipos e alelos entre os diferentes estágios da doença (p>0,05). Os resultados do presente estudo sugerem que os polimorfismos nos genes HSD17β1 (rs605059), MMP2 (rs243865) e MMP9 (rs17576), não estão associado com a endometriose em pacientes brasileiras, mesmo quando avaliado as relações entre graus de severidade variados / Endometriosis is defined by the presence of endometrial tissue (glands and / or stroma) outside the uterine cavity, this condition affects 10-15% of women of reproductive age, in addition, the pain symptoms of the disease involves economic and social impacts. The disease presents late diagnosis and its etiology has not been fully elucidated, but it is known that endometriosis has multifactorial and polygenic character. The aim of the study was to assess whether the polymorphisms in the HSD17β1 (rs605059), involved in estrogen synthesis, and MMP2 genes (rs243865) and MMP9 (rs17576), which act in extracellular matrix remodeling are associated with endometriosis as the risk and severity of the disease. The case-control study consisted of 231 women, with 97 cases and 134 controls. All patients in the case group had histopathological diagnosis for endometriosis. Genomic DNA was extracted from saliva, and HSD17β1 gene polymorphism was detected by Nested-PCR followed by digestion of the PCR product by the enzyme BstUI, MMP2 and MMP9 genes were genotyped by Real Time-PCR. There was no statistically significant difference between the genotypic and allelic distributions of genes analyzed between groups (p> .05). Similarly there was no significant difference in the frequency of genotypes and alleles between different stages of the disease (p> .05). The results of this study suggest that polymorphisms in genes HSD17β1 (rs605059), MMP2 (rs243865) and MMP9 (rs17576), are not associated with endometriosis in Brazilian patients, even when the relations between different degrees of severity were evaluated

La pemphigoïde bulleuse (PB) est la plus fréquente et la plus grave des dermatoses bulleuses auto-immunes caractérisée par une cascade inflammatoire impliquant plusieurs cytokines et cellules inflammatoires, avec la libération de protéases (MMP-9, élastase) conduisant à la formation de la bulle. L’objectif de cette étude est de caractériser cette cascade inflammatoire en analysant la contribution de la chimiokine CXCL10 et des cellules inflammatoires comme les macrophages dans le mécanisme physiopathologique associé à la maladie. Nos résultats ont montré que CXCL10 est présent à des taux importants dans le sérum des patients et dans le liquide de bulle, de plus cette chimiokine reste fortement présente dans le sérum des patients qui rechutent. D’autre part nous avons montré pour la première fois que l’orientation des macrophages dans la PB est contrôlée dans un premier temps par les sérums de patients, qui préactivent les macrophages dérivés de monocytes, puis par le liquide de bulle qui différencient les macrophages vers une polarisation M2. Nous avons également montré que seulement les neutrophiles et les monocytes isolés de patients atteints de PB, mais pas leur lymphocytes, répondaient à une stimulation par CXCL10 par une augmentation de leur sécrétion en MMP-9 via l’activation des voies de signalisation ERK1/2, P38 et PI3K. Cette expression de la MMP-9 a été associée à une polarisation de type M2 dans les macrophages issus de monocytes de patients. Enfin, nous avons montré l’importance des activateurs sélectifs des récepteurs des glucocorticoïdes, tels que le composé A, qui était aussi efficace que le traitement avec la méthylprénisolone dans l'inhibition de la MMP-9, mais avec des effets distincts sur l'expression des marqueurs phénotypiques de macrophages. / Bullous pemphigoid (BP) is the most common and serious autoimmune bullous dermatosis characterized by inflammatory cascade involving many cytokines and inflammatory cells, with the release of proteases (MMP-9, HLE) leading to the formation of the blister. The objective of this study is to characterize the inflammatory cascade by analyzing the contribution of the CXCL10 chemokine and inflammatory cells such as macrophages in the pathophysiology associated with the disease. Our results showed that CXCL10 is present at significant levels in the serum of patients and in the blister fluid, this chemokine remains strongly present in the serum of patients who relapse. On the other hand we have shown for the first time that macrophages polarization in BP is controlled by the sera of patients, which activate macrophages derived from monocytes, and then blister fluid differentiate macrophages in an M2 polarization. We also showed that only neutrophils and monocytes isolated from patients, but not the lymphocytes responding to stimulation by CXCL10 by an increase in secretion of MMP-9 via the activation of signaling pathways ERK1 / 2, P38 and PI3K. This expression of MMP-9 has been associated with an M2 macrophages polarization derived from monocytes of patients. Finally, we have demonstrated the importance of selective glucocorticoid receptor activators, such as Compound A, which was as effective as treatment with methylprednisolone in inhibiting MMP-9, but with distinct effects on the expression of phenotypic markers of macrophages.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

L'insuffisance cardiaque consécutive à un infarctus du myocarde est une pathologie complexe qui apparaît suite au remodelage du ventricule gauche. Le renouvellement et la dégradation de la matrice extracellulaire cardiaque et l'inflammation sont impliqués dans la mise en place du remodelage post infarctus du myocarde. La métalloprotéinase matricielle 9 (MMP9) et le récepteur de l'immunité innée Toll-like 4 (TLR4) sont des médiateurs clés du remodelage ventriculaire. L'adénosine est un nucléoside cardioprotecteur et anti-inflammatoire dont les effets sur le remodelage sont encore mal caractérisés. Nous avons émis l'hypothèse que l'adénosine pourrait moduler les voies de la MMP9 et du TLR4 dans les macrophages, et ainsi réguler le remodelage ventriculaire. Nos résultats montrent que l'activation du récepteur A3 à l'adénosine augmente la production de MMP9 par les macrophages primaires humains. D'autre part, nous avons observé que l'adénosine réduit l'inflammation par une diminution de l'expression de surface du TLR4. Cet effet se traduit par une inhibition de la synthèse de cytokines pro-inflammatoires et est induit par l'activation du récepteur A2A. Ces résultats ont permis de caractériser certains mécanismes par lesquels l'adénosine agit sur le remodelage ventriculaire. Ils suggèrent de tester, dans un modèle animal, l'administration d'analogues de l'adénosine pour prévenir ou limiter le remodelage. La capacité à prédire la survenue du remodelage ventriculaire après un infarctus du myocarde est importante d'un point de vue clinique et bénéficierait de la découverte de nouveaux biomarqueurs. L'analyse par protéomique du plasma de 30 patients d'une cohorte test ayant développé un infarctus du myocarde a permis d'identifier l'haptoglobine comme biomarqueur pronostique potentiel. L'haptoglobine humaine possède 3 isoformes : [alpha]1-[alpha]1, [alpha]2-[alpha]1 et [alpha]2-[alpha]2. Nos résultats suggèrent que la présence du phénotype [alpha]2 et d'un taux plasmatique d'haptoglobine faible sont associés à l'apparition d'une insuffisance cardiaque. Cette étude preuve-du-concept suggère que l'haptoglobine pourrait être ajoutée à la liste existante des biomarqueurs de l'insuffisance cardiaque / Heart failure following myocardial infarction is a complex pathology that occurs as a result of left ventricular remodeling. Left ventricular remodeling is mediated in part by extracellular cardiac matrix turnover and inflammation. Matrix metalloproteinase 9 (MMP9) and innate immune receptor Toll-like 4 (TLR4) are key mediators of left ventricular remodeling. Cardioprotective and anti-inflammatory nucleoside adenosine acts on left ventricular remodeling through still poorly characterized mechanisms. We hypothesized that adenosine regulates left ventricular remodeling through modulation of MMP9 and TLR4 pathways in macrophages. Using human primary macrophages, we showed that adenosine increases MMP9 production through the A3 receptor. In a second set of experiments, we observed that adenosine dampens inflammation through decreased cell-surface expression of TLR4. This effect, which inhibits pro-inflammatory cytokines production, is induced by adenosine A2A receptor. All together, these results contribute to a better understanding of the mechanisms underlying left ventricular remodeling and suggest a potential therapeutic use of adenosine. Our data suggest testing a therapeutic strategy with different adenosine analogs to prevent or limit left ventricular remodeling. Prediction of left ventricular remodeling after myocardial infarction is clinically relevant and would benefit from the discovery of new biomarkers. Proteomic analysis of plasma samples from a test cohort of 30 myocardial infarction patients identified haptoglobin as a potential prognostic biomarker. Human haptoglobin has 3 distinct isoforms: [alpha]1-[alpha]1, [alpha]2-[alpha]1 and [alpha]2-[alpha]2. Our results suggest that the presence of [alpha]2 isoform, together with low plasma levels of total haptoglobin, is associated with the development of heart failure. This proof-of-concept study suggested that haptoglobin could be added to the panel of existing biomarkers of heart failure

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Matrix metalloproteinases (MMPs) are known for their role in extracellular matrix remodeling, but their role in regulating intracellular immune cell function is unknown. We reported that MMP inhibition down regulated T cell proliferation in response to alloantigens and autoantigens; but the direct role of MMP involvement in T cell activation has not been reported. Methods: MMP deficient or MMP sufficient wild-type CD4+ or CD8+ T cells from C57BL/6 mice were treated with SB-3CT, a specific inhibitor of MMP2 and MMP9, stimulated with anti-CD3 Ab, alone, or with IL-2 or CD28. Cellular activation and cytokine profiles were examined. A mouse model of antigen specific T cell mediated lung injury was used to examine MMP inhibition in antigen-specific T cell mediated lung injury. Results: SB-3CT (1-25μM) induced dose-dependent reductions in anti-CD3 Ab-induced proliferation (p<0.0001). Compared to wild-type, MMP9-/- CD4+ and CD8+ T cells proliferated 80-85% less (p<0.001) in response to anti-CD3 Ab. Compared to untreated or wild-type cells, anti-CD3 Ab-induced calcium flux was enhanced in SB-3CT-treated or MMP9-/- CD4+ and CD8+ T cells. Cytokine transcripts for IL-2, TNF-α and IFN-γ were reduced in both CD4+ and CD8+ MMP9-/- T cells, as well as in SB3CT treated CD4+ T cells. MMP inhibition dampened antigen-specific T cell mediated lung injury. Conclusions: Although known to be functional extracellularly, the current data suggest that MMPs function inside the cell to regulate intracellular signaling events involved in T cell activation. T cell targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases. - David S. Wilkes, M.D., Chair.