ROCHA, MG. Mediadores inflamatórios na dor pélvica crônica Identificação de possíveis marcadores séricos da doença. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2010. Introdução: Dor pélvica crônica é uma doença de elevada prevalência e fisiopatologia complexa. Os métodos diagnósticos muitas vezes são insuficientes e, em decorrência, o tratamento e seguimento das mulheres é difícil. Inúmeras doenças que se apresentam com dor crônica tem um perfil inflamatório, que ainda não foi investigado para o tema em questão. Objetivos: Quantificar os níveis de óxido nítrico (NO) e das metaloproteinases 2 (MMP-2) e 9 (MMP-9) no plasma de mulheres com dor pélvica crônica. Pacientes e métodos: Foram incluídas 64 mulheres, subdivididas em 02 grupos: dor pélvica crônica e grupo controle, com 37 pacientes no primeiro grupo e 27 pacientes no segundo grupo. As pacientes do grupo de estudo eram seguidas no Ambulatório de Dor Pélvica e Endoscopia e as pacientes do grupo controle eram seguidas no Ambulatório de Anticoncepção do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto USP. Foi realizada a mensuração clínica da dor através de uma escala unidimensional (VAS) e uma escala multidimensional (McGill). Também foram preenchidas as escalas de ansiedade e depressão (HAD). Indivíduos com qualquer evidência de processos inflamatórios, hipertensão, tabagismo ou uso de contraceptivos hormonais foram excluídos. Indivíduos tomando medicação para a dor, como analgésicos ou antiinflamatórios foram solicitados a pará-los 72 horas antes de participar do estudo. Foi coletada uma amostra sanguínea de 10 ml, no ato da consulta. Esse material foi armazenado em frasco próprio com anti-coagulante, processado imediatamente no local para separação do plasma e armazenado em freezer, a -70C para mensuração subseqüente. As concentrações de espécies relacionadas ao NO (nitrato) em líquidos foram medidas, sempre em triplicata, pelo método da quimioluminescência, que é um dos métodos mais simples, sensíveis e precisos disponíveis para medir NO. Foi utilizado um analisador de NO (Sievers Model 280 NO Analyzer - Boulder, CO, EUA), o qual permite medir NO em quantidades tão pequenas quanto 1 pmol. A atividade das MMP-2 e MMP-9 no plasma serão determinadas pelo método da zimografia, que consiste em uma eletroforese das amostras em um sistema SDS/PAGE que inclui o substrato da enzima (gelatina) no gel de separação, de modo a permitir a evidenciação e quantificação da atividade da enzima. Resultados: Os níveis plasmáticos de NO foram maiores nas pacientes com DPC quando comparadas às pacientes do grupo controle (16.8 ± 7.9 versus 12.2 ± 2.4, respectivamente (P = 0.0016). Especificamente, os níveis plasmáticos de NO foram maiores nas pacientes com DPC de origem visceral quando comparadas às pacientes com dor exclusivamente somática ou aos controles saudáveis (19.2 ± 8.9 versus 12.4 ± 1.8 versus 12.2 ± 2.4, respectivamente) (P=0.0001). Não observamos uma correlação entre os níveis plasmáticos de NO e a duração dos sintomas (em meses) (Spearman r = 0.04, 95%CI:-0.34 to 0.40, P = 0.84) ou com a intensidade dos sintomas dolorosos: EAV (Spearman r =:-0.18, 95%CI:-0.52 to 0.20, P=0.34), ou McGill (Spearman r = -0.06, 95%CI:-0.41 to 0.30, P =0.72). Com relação às MMP´s, não houve diferença estatística entre os dois grupos. Conclusões: Os níveis plasmáticos de NO encontram-se elevados em mulheres com DPC, especialmente naquelas com dor de origem visceral. Este fato pode ser considerado uma possibilidade no seguimento de pacientes com DPC, visto que pode ser usado como um marcador sérico para a doença. Já a dosagem das MMP-s não se mostrou útil como marcador plasmático para mulheres com DPC. / Background: Chronic pelvic pain is a disease of high prevalence and a complex pathophysiology. The diagnostic methods are often inadequate and, consequently, treatment and follow-up of women is quite difficult. Several diseases that present with chronic pain has an inflammatory profile, which has not yet been investigated for the topic. Aim: to quantify levels of nitric oxide (NO) and metalloproteinases 2 (MMP-2) and 9 (MMP-9) in plasma of women with chronic pelvic pain. Methods: 64 women were included in the sudy and divided into 02 groups: chronic pelvic pain and control group with 37 patients in the first group and 27 patients in the second group. Patients in the study group were followed at the Endoscopy and Pelvic Pain Unit and the control group patients were followed in the Contraception Unit of the Hospital of the Medical School of Ribeirão Preto University of São Paulo. We performed the measurement of clinical pain by a unidimensional scale (VAS) and a multidimensional scale (McGill). Anxiety and depression scales were also filled. Individuals with any evidence of inflammation, hypertension, smoking or use of hormonal contraceptives were excluded. Individuals taking medication for pain, such as painkillers or antiinflammatory drugs were asked to stop them 72 hours before entering the study. A blood sample was collected from 10 ml during the appointment. This material was stored in bottle itself with anti-coagulant (EDTA and / or heparin), processed immediately on site for plasma separation and stored in a -70 ºC freezer. The concentrations of species related to NO were measured in liquid, always in triplicate by the method of chemiluminescence, which is one of the most simple, sensitive and accurate available to measure NO. We used a NO analyzer (Sievers Model 280 NO Analyzer - Boulder, CO, USA), which allows the measurement of NO in quantities as small as 1 pmol. The activity of MMP-2 and MMP-9 in plasma was determined by the zymography method, which consists of an electrophoresis of the samples in an SDS / PAGE system, which includes the enzyme substrate (gelatin) in the gel separation, allowing the disclosure and quantification of enzyme activity. Results: Plasma NO levels were higher in CPP women than in controls (16.8 ± 7.9 versus 12.2 ± 2.4, respectively) (P=0.0016). Furthermore, plasma nitrate levels were higher in CPP women with evidence of pain of visceral origin than in CPP women with evidence of an exclusive somatic component or controls (19.2 ± 8.9 versus 12.4 ± 1.8 versus 12.2 ± 2.4, respectively) (P=0.0001). No correlation was detected between NO levels and duration of symptoms or intensity of pain. Regarding the MMP\'s, there was no statistical difference between the two groups. Conclusion: Plasma levels of NO are elevated in women with CPP, especially those with visceral pain. This fact can be considered an option in treating patients with CPP as it can be used as a serum marker for the disease. On the other hand, MMP´s did not turn out to be a good serum marker for women with CPP.

Orientador: José Eduardo Tanus dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T04:17:50Z (GMT). No. of bitstreams: 1 Marson_BernardoPavinato_D.pdf: 29131187 bytes, checksum: 6a16c5b929f162ad17cdee05fa1926ce (MD5) Previous issue date: 2012 / Resumo: A insuficiência renal crônica é uma complicação grave de diferentes doenças, como diabetes, hipertensão e glomerulopatias. Quando os rins entram em falência, é preciso substituir a função renal, terapia geralmente feita através de hemodiálise. A população de pacientes dependentes de hemodiálise, cujo número cresce de forma geométrica, está exposta a taxas extremamente elevadas de eventos cardiovasculares fatais e não fatais. Fatores de risco clássicos e específicos da uremia se somam conferindo alterações patológicas severas na parede dos vasos. A própria sessão de hemodiálise ativa a inflamação e induz aterogênese. A degradação da elastina e a apoptose das células musculares lisas evoluem para a mudança do fenótipo da camada média, que se expande reduzindo o lúmem em um processo de acentuada calcificação arterial. Alterações na atividade das metaloproteinases de matriz extracelular (MMPs) 2 e 9 e desequilíbrios com seus inibidores endógenos, os TIMPs, ajudam a compor este cenário ao estimular o remodelamento cardiovascular e reorganizar a matriz extracelular, permitindo a expansão tecidual e o depósito de cálcio. Os níveis circulantes de MMP-2 e -9 estão relacionados com maior severidade de doenças cardiovasculares na hemodiálise. Diversos polimorfismos genéticos foram associados com alterações na concentração e/ou atividade destas enzimas, e é possível que diferenças nas distribuições dos polimorfismos ajudem a discriminar indivíduos expostos a níveis plasmáticos mais elevados de MMP-2 e -9 tanto antes como após a sessão de hemodiálise. Os principais polimorfismos são: um polimorfismo de nucleotídeo único (SNP) (C-1562T) e um microssatélite (-90 CA14-24) na região promotora, e um SNP no exon 6 (A855T, Q279R) da MMP-9, e dois SNPs (C-1306T e C-735T) no promotor da MMP-2. Como estes polimorfismos também foram associados com diversas doenças, o propósito deste estudo foi avaliar se eles influem na concentração plasmática de MMP-2 e MMP-9 em pacientes submetidos à hemodiálise crônica, e se afetam o efeito que a sessão de hemodiálise tem sobre os seus níveis circulantes. Para atingir nosso objetivo, estudamos 98 pacientes com idades entre 18 e 65 anos e submetidos à hemodiálise há mais de 3 meses. Amostras de sangue venoso foram coletadas em dois momentos, antes do início e após o término da sessão de hemodiálise. As concentrações de MMP-2 foram determinadas por zimografia e as de MMP-9, TIMP-1 e TIMP-2 foram analisadas por ELISA. O DNA genômico foi extraído a partir do sangue total e amostras foram então genotipadas para os polimorfismos da MMP-2 e MMP-9. As frequências dos haplótipos da MMP-2 e -9 foram estimadas pelo programa PHASE. Nossos resultados mostraram que a sessão de hemodiálise reduz os níveis circulantes de MMP-2 e não altera o TIMP-2, ao passo que os níveis de MMP-9 e TIMP-1 se encontram aumentados ao final da sessão. Encontramos uma associação entre os genótipos que envolvem o alelo de análise T do polimorfismo C-735T e o haplótipo CT demonstrando níveis pré hemodiálise significativamente aumentados de MMP-2 (P= 0,0077 e P= 0,01, respectivamente), mas não de TIMP-2. Os genótipos da MMP-2 não alteram o efeito da sessão da hemodiálise, que reduziu a MMP-2 e o TIMP-2 independente de marcadores genéticos. Marcadores genéticos da MMP-9 mostraram estar associados a níveis maiores de MMP-9 após a hemodiálise: os genótipos CC e QQ (P= 0,0081 e P= 0,0415, respectivamente) e o haplótipo CLQ (P= 0,0012). As concentrações de TIMP-1 aumentaram significativamente após a hemodiálise nos genótipos HH e QR (P= 0,0375 e P= 0,0113, respectivamente) e no haplótipo CHR (P= 0,0008). Adicionalmente, marcadores genéticos da MMP-9 não alteraram os níveis basais de MMP-9 e TIMP-1. Estes achados sugerem que marcadores genéticos da MMP-2 e -9 interferem nos níveis circulantes destas proteases no contexto da hemodiálise / Abstract: Chronic renal disease is a serious complication which may occur in patients who suffer from a vast range of diseases, such as diabetes, hypertension and glomerulonephritis, among others. When the kidneys fail, it becomes necessary to substitute the renal function, which is usually made through hemodialysis. The population of patients that are dependent of hemodialysis are rapidly growing in number. These patients are exposed to extremely high rates of cardiovascular events. Both traditional and uremic specific factors account for severe pathologic alterations on the walls of the vessels. The session of hemodialysis itself stimulates inflammation and induces atherogenesis. The degradation of elastin and the apoptosis of smooth muscle cells eventually progresses to a change in the phenotype of the media layer, which expands, thus reducing the arterial lumen in a process of accelerated calcification. Alteration in the activity of the matrix metalloproteinases (MMPs) 2 and 9 and imbalancements with its endogenous inhibitors - the TIMPs - help to set the scenario for cardiovascular diseases by stimulating cardiovascular remodelling and reorganizing the extracellular matrix, allowing tissue expansion and calcium deposits. The circulating levels of MMP-2 and -9 are associated with greater severity of cardiovascular diseases on patients undergoing hemodialysis. Diverse genetic polymorphisms were associated with alterations on the concentration and with the activity of these enzymes, and it is possible that differences on the distribution of these polymorphisms may help to discriminate individuals exposed to increased plasmatic levels of MMP-2 and -9, both before and after hemodialysis. The main polymorphisms known are: one single nucleotide polymorphism (SNP) (C-1562T) and one microsatellite (-90 CA14-24) on the promoter region, and one SNP on exon 6 (A855G, Q279R) of MMP-9, and two SNPs (C-1306T and C-735T) on the promoter of MMP-2. These polymorphisms were also associated with a number of diseases. Our purpose was to study whether they influence the circulating levels of MMP-2 and -9 in patients undergoing hemodialysis, and whether they affect the levels of these proteases after hemodialysis. In order to reach our aim, we have studied 98 patients whose ages ranged between 18 and 65 years of age and who were undergoing chronic hemodialysis for at least 3 months. Venous samples were collected in two moments, before and after hemodialysis. The concentrations of MMP-2 were assayed with gelatin zymography, and MMP-9, TIMP-1, and TIMP-2 were analyzed with ELISA. Genomic DNA were extracted and samples were genotypied for MMP-2 and -9 polymorphisms. The haplotypic frequencies were analyzed by the PHASE software. Our results show that sessions of hemodialysis reduce the levels of MMP-2, however, it does not alter TIMP-2, while MMP-9 and TIMP-1 suffer an increase after the hemodialysis session. We found an association amidst the genotypes with the variant allele T on the SNP C-735T and on the haplotype CT showing elevated pre hemodialysis levels of MMP-2 (P= 0,0077 and P= 0,01, respectively), but not on TIMP-2. The MMP-2 genotypes do not modify the effect of a hemodialysis session. Genetic markers of the MMP-9 were associated with enhanced levels of MMP-9 after hemodialysis: the CC and QQ genotypes (P= 0,0081 and P= 0,0415, respectively) and the haplotype CLQ (P= 0,0012). The concentrations of TIMP- 1 increased significantly after hemodialysis on the genotypes HH and QR (P= 0,0375 and P= 0,0113, respectively) and on the haplotype CHR (P= 0,0008). Furthermore, genetic markers of the MMP-9 have not altered the basal levels of MMP-9 and TIMP-1. These findings suggest that the genetic markers of MMP-2 and -9 interfere on circulating levels of these proteases on the hemodialysis setting / Doutorado / Farmacologia / Doutor em Farmacologia

Made available in DSpace on 2014-08-20T14:30:05Z (GMT). No. of bitstreams: 1 dissertacao_Rodrigo_Varella_de_Carvalho.pdf: 598355 bytes, checksum: 322409e41faaa4fcdb65cd8f7b7e200d (MD5) Previous issue date: 2006-05-29 / The aim of the present study was to evaluate the gelatinase activity (MMP-2 and 9), and proliferation of human pulp fibroblasts in vitro, after irradiation with a low level diode laser (780nm). The activity was evaluated with zymography and the proliferation rate was obtained through growth curves made in SigmaPlot (8.0), after the counting in Neubauer chamber.in light microscope. Cells used to zimography were cultived in DMEM, 10% fetal bovine serum and incubated at 37ºC in atmosphere of 5% CO2 and 95% air, in 100% humidity. Cells used to proliferation rate were cultivated in nutritional deficit (5% SFB). Groups to zymography and cell proliferation were determined: zymography - Z1 = not irradiated, Z2 = 3J/cm2 e Z3 = 6J/cm2, cell proliferation: L1 = not irradiated, L2 = 3J/cm2 e L3 = 6J/cm2. The counting was made after 2, 4 and 6 days. Assays were performed in triplicate. The zymography showed that the MMP-2 activity was major with MMP-9, and, Z2 e Z3 produced a higher activity than MMP-2, when compared to Z1. Results of the zymography and proliferation test were compared by either ANOVA complemented by Tukey´s test. The level of significance was 5% (p<0,05). Statistical analysis demonstrate difference between the groups in different days. The L3 obtained higher proliferation in the finish of the six days. L2 demonstrated higher growth than L1. Irradiated fibroblasts with the low power laser expressed a higher amount of MMP-2 activity and 6J/cm2 produced a higher proliferation rate in the initial days / O objetivo do presente estudo foi avaliar a atividade das gelatinases (MMP-2 e MMP-9), e a proliferação de fibroblastos pulpares humanos in vitro, após a irradiação com um diodo laser de baixa de baixa potência (780nm). A atividade foi avaliada com zimografia e as taxas de proliferação e contagem celular foram obtidas através de curvas de crescimento feitas com o software SigmaPlot (8.0), após a contagem em câmara de Neubauer ao microscópio óptico. As células usadas para a zimografia foram cultivvadas em DMEM, com SFB a 10% e incubadas a 37ºC em atmosfera de 5% CO2 e 95% de ar, em 100% de umidade. Já, as células usadas para a taxa de proliferação e contagem celular foram cultivadas em déficit nutricional (5% SFB). Os grupos para zimmografia e para a contagem e taxa de proliferação foram determinados: a) zimografia - Z1 = sem irradiação, Z2 = 3J/cm2 e Z3 = 6J/cm2 e b) proliferação e contagem celular - L1 = sem irradiação, L2 = 3J/cm2 e L3 = 6J/cm2. A contagem celular foi realizada em após 2, 4 e 6 dias. Os ensaios foram realizados em triplicata. A zimografia mostrou que a ativiidade da MMP-2 foi maior que a MMP-9, e Z2 e Z3 produziram uma grande atividade, quando comparados ao rupo Z1. Os resultados da zimografia e dos testes de contagem e proliferação celular foram comparados por análise de variância (ANOVA) e complementado pelo teste Tukey. O nível de significância utilizado foi de % (p<0,05). A análise estatística demonstrou diferença entre os grupos nos diferentes dias para a proliferação e contagem celular. O grupo L3 obteve a maior contagem celular ao final dos seis dias avaliados. O grupo L2 demonstrou maior proliferação que o grupo L1. Os fibroblastos irradiados com o laser de baixa potência expressaram uma alta atividade de MMP-2 e a dose 6J/cm2 produziu uma alta taxa de proliferação nos primeiros dias

La cicatrisation cornéenne représente un processus de réparation complexe qui vise à restaurer l'intégrité, la structure et la transparence de la cornée. Cependant, dans un certain nombre de cas, ce processus peut évoluer de façon anormale et se stabiliser en entraînant la formation d'une opacité cornéenne installée. Les mécanismes impliqués dans la formation de ces cicatrices persistantes ne sont pas encore complètement élucidés, mais il est établi que la composition et l'organisation de la matrice extracellulaire du stroma jouent un rôle majeur dans la restauration de la transparence de la cornée. Ce projet s’est concentré sur la métalloprotéase extracellulaire BMP-1 (Bone Morphogenetic Protein 1), déjà connue pour son rôle dans l'assemblage de la matrice extracellulaire et l'activation du TGF-bêta. Afin d’identifier les processus contrôlés par BMP-1 dans la cornée, nous avons d’abord effectué une comparaison systématique des inhibiteurs de BMP-1 connus ou potentiels, de différentes origines, pour caractériser leurs propriétés à la fois in vitro et dans des cultures cellulaires. Ensuite, nous avons mené une étude approfondie du sécrétome des cellules stromales de la cornée humaine (kératocytes), et des conséquences de la différenciation de ces cellules en myofibroblastes. Enfin, nous avons analysé les événements protéolytiques médiés par BMP-1 dans le sécrétome des kératocytes en utilisant principalement une approche de protéomique quantitative basée sur le marquage iTRAQ des protéines entières (technique TAILS). La comparaison des inhibiteurs disponibles de BMP-1 a permis de mettre en évidence différents profils d’efficacité, de spécificité et de toxicité et a conduit à l’identification d’un inhibiteur hydroxamate et d’un inhibiteur protéique efficaces, peu toxiques et très spécifiques de BMP-1. Le sécrétome des kératocytes s’est avéré être un modèle adéquat pour l’étude des activités de BMP-1 dans le contexte cornéen. Plus de 2022 protéines ont été identifiées, dont la métalloprotéase BMP-1 et 16 de ses 33 substrats connus jusqu’à présent. Enfin, 76 protéines modifiées par l’activité de BMP-1 ont été identifiées dans le sécrétome des kératocytes. Ces résultats confirment les liens forts entre BMP-1, l'assemblage de la matrice extracellulaire et le TGF-bêta, mais suggèrent également de nouveaux rôles pour la protéase dans l'inflammation. Certains des substrats nouvellement identifiés (TGFBI, HSP47 et collagène VI) sont très pertinents dans le contexte de la cicatrisation de la cornée et ont été validés d’un point de vue biochimique. En conclusion, BMP-1 est confirmée comme une cible potentielle intéressante pour traiter ou prévenir la formation des opacités cornéennes et la caractérisation des inhibiteurs disponibles ouvre des perspectives importantes pour des études précliniques chez l’animal / When the cornea is injured, a complex multi-step healing process is triggered which aims at restoring corneal integrity, structure and transparency. However, in some cases, corneal healing results in the formation of a stable scar associated with a prolonged loss of corneal transparency and with functional blindness. The mechanisms involved in the formation of these persistent scars are still not fully understood but it is known that the composition and organization of the extracellular matrix significantly contributes to the maintenance of corneal transparency. This work focused on the extracellular metalloproteinase called BMP-1 (Bone Morphogenetic Protein 1), a major player in the control of extracellular matrix assembly and TGF-beta activation, which was previously shown to be up-regulated in corneal healing and scarring. In order to further probe BMP-1 functions in corneal healing, we first performed a systematic comparison of known or potential BMP-1 inhibitors from different origins to characterize their properties both in vitro and in cell cultures. We then carried out an in-depth study of the secretome of human corneal stromal cells (keratocytes) and of the consequences of the differentiation of these cells into myofibroblasts. Finally, we analyzed BMP-1-mediated proteolytic events in keratocyte secretomes, mainly using a quantitative proteomic approach based on iTRAQ labeling of proteins (TAILS technique). The comparison of BMP-1 available inhibitors revealed different profiles of efficacy, specificity and toxicity and led to the identification of one hydroxamate inhibitor and one protein inhibitor, which were very efficient, non-toxic and very specific of BMP-1. The keratocyte secretome was shown to be a suitable model for the study of BMP-1 activities in the corneal context. More than 2022 proteins were identified, including the BMP-1 metalloprotease and 16 of its 33 already known substrates. Finally, 76 proteins modified by BMP-1 activity were identified in the keratocyte secretome. These results confirm the strong links between BMP-1, extracellular matrix assembly and TGF-beta, but also suggest new roles for this protease in cell proliferation and inflammation. Some of the newly identified substrates (TGFBI, HSP47 and collagen VI) are highly relevant in the context of corneal healing and were validated at the biochemical standpoint. In conclusion, BMP-1 is confirmed as a potential target to treat or prevent the formation of corneal opacities and the characterization of available inhibitors opens up important perspectives for preclinical studies in animals

Abstract Tissue repair and cancer progression involve similar mechanisms, including degradation of extracellular matrix in which matrix metalloproteinases (MMPs) play essential roles. The action of MMPs is important in normal physiological processes but MMPs also contribute to various pathological conditions. MMP-8 belongs to a family of collagenases with a diverse set of substrates. MMP-8 action is involved in skin wound healing and in various human cancers. The function of MMP-8 in cancer appears to be highly complex and varies depending on the cancer type and location. Little is known about the involvement of MMP-8 in oral physiology and pathology. The aim of this study was to clarify the role of MMP-8 in oral tissue repair and oral tongue squamous cell carcinoma (OTSCC). Studies with MMP-8 deficient mice revealed that the function of MMP-8 in tissue repair is highly dependent on the spatial aspects. In alveolar bone, MMP-8 increased inflammation and affected collagen metabolism. In tongue wounds, MMP-8 impaired early healing and reduced transforming growth factor (TGF) -β1 levels. This study also revealed the protective role of MMP-8 in OTSCC patients, in agreement with previous studies indicating positive features of MMP-8 in cancer. Low MMP-8 level and high vascular endothelial growth factor (VEGF) -C levels in tumors correlated with worse prognosis in these patients. In mouse tongue fibroblast cell cultures, MMP-8 reduced TGF-β1 signaling molecule phosphorylated Smad2 levels and impaired the collagen contraction ability. TGF-β1, apoptosis factor Fas-ligand (Fas-L) and estrogen receptors (ERs) were identified as novel MMP-8 substrates. In OTSCC cell cultures, MMP-8 impaired cell migration and invasion. Diminished TGF-β1 levels were involved in the defective migration of MMP-8 overexpressing cells. Moreover, MMP-8 affected the expression of MMP-9, MMP-1, cathepsin-K, VEGF-C and TGF-β1. In mouse models of OTSCC, MMP-8 protected against tumor development but was not able to prevent metastasis formation. The main findings of this study were that 1) MMP-8 action in tissue repair depends on the site of the injury and 2) in OTSCC, MMP-8 has tumor suppressive effects, but in mouse, MMP-8 does not inhibit metastasis formation. In addition, 3) four novel MMP-8 substrates (TGF-β1, Fas-L, ER-α and -β) were identified that may explain the spatial and diverse roles of MMP-8. / Tiivistelmä Kudosvaurioiden paranemiseen ja syövän etenemiseen liittyy useita samankaltaisia mekanismeja. Molemmissa toimivat soluvälitilan muokkaamiseen osallistuvat proteaasit, joista matriksin metalloproteinaaseilla (MMP) on tärkeä merkitys; ne osallistuvat lukuisiin elimistön keskeisiin fysiopatologisiin prosesseihin. Kollagenaasi MMP-8 muokkaa monentyyppisiä molekyylejä. Se on mukana ihohaavan paranemisessa ja useissa syövissä. MMP-8:n toiminta syöpätiloissa on hyvin moninainen riippuen syöpätyypistä ja sijainnista. Väitöstutkimuksessa selvitettiin MMP-8:n merkitystä suun kova- ja pehmytkudosvaurioprosesseissa sekä kielisyövässä, joissa se on ollut tuntematon. MMP-8 poistogeenisillä hiirillä tehdyissä pehmyt- ja kovakudoshaavoissa MMP-8:n vaikutusmekanismit riippuivat kohdekudoksesta. Alveoliluun paranemisen yhteydessä MMP-8 lisäsi tulehdusta ja osallistui kollageenin muokkaamiseen. Akuutin kielihaavan paranemisessa MMP-8 hidasti haavan umpeutumista ja vähensi transformoivan kasvutekijä-β1:n (TGF-β1) määrää. Kuten useissa muissakin syövissä, myös kielisyövässä todettiin MMP-8:lla olevan suojaava vaikutus. Potilaan ennuste huononi, jos kasvainsolukon matala MMP-8-taso yhdistyi korkeaan verisuonten kasvutekijä-C:n (VEGF-C) määrään. Hiiren kielen normaaleissa fibroblastiviljelmissä MMP-8 vähensi TGF-β1:n solunsisäistä signalointia välittävän fosforyloidun Smad2:n määrää sekä solujen kykyä supistaa kollageenikiekkoja. Koeputkessa MMP-8 pilkkoi TGF-β1:tä, estrogeenireseptoreja (ER) ja apoptoositekijä Fas-ligandia (Fas-L). Ihmisen kielikarsinoomasoluviljelmissä korkea MMP-8:n määrä vähensi solujen migraatiota ja invaasiota sekä muutti MMP-1:n, MMP-9:n, katepsiini-K:n, TGF-β1:n ja VEGF-C:n ilmentymistä. Migraation heikentyminen MMP-8:aa tuottavissa soluissa johtui osin vähentyneestä TGF-β1:n määrästä. Hiiren kokeellisissa kielisyövissä MMP-8 hidasti syövän muodostumista mutta ei estänyt etäpesäkkeiden muodostusta. Tässä väitöskirjatutkimuksessa on kolme päälöydöstä: 1) MMP-8:n vaikutus kudoksen paranemisprosessiin riippuu vauriokohdasta, 2) MMP-8 on kielisyövän kehittymisessä puolustuksellinen molekyyli, mutta sen lisääntynyt tuotto ei hiirikokeissa estänyt etäpesäkkeiden muodostusta, 3) MMP-8:lle löydettiin neljä uutta kohdemolekyyliä (TGF-β1, Fas-L, ER-α ja -β), joiden muokkaus saattaa osin selittää MMP-8:n monipuoliset kudos- ja prosessispesifit vaikutukset.

Abstract Collagen XVII (BP180) is a transmembrane component of hemidesmosomes, which connect basal keratinocytes to the basement membrane. The extracellular domain of collagen XVII is proteolytically shed from the cell surface and released to the extracellular matrix. Apart from its function in epithelial cell adhesion, collagen XVII has been suggested to participate in keratinocyte motility. The collagen XVII expression pattern was studied in wounds of oral mucosa and in epithelial tumors. During re-epithelialization, collagen XVII was expressed in the keratinocytes distal to the wound edge, but not in the leading cells of the epithelial tip. Collagen XVII upregulation was observed in moderate/severe dysplasias of oral mucosa. In follicular ameloblastomas and basal cell carcinomas, collagen XVII expression was reduced in peripheral cells, whereas cytoplasmic staining was detected in central tumor cells. Tongue squamous cell carcinomas showed increased collagen XVII expression in grade II/III tumors, particularly in areas of invasive growth. The results suggest a correlation between overexpression of collagen XVII and the invasive potential of the tumor. For the first time, the role of collagen XVII in the regulation of malignant migration was explored. The presence of COL15, the cell adhesion domain of collagen XVII, induced migration of tongue squamous cell carcinoma cells in transmigration assays. Experiments with specific function-blocking integrin antibodies revealed that the promigratory function of COL15 is mediated by αv and α5 integrins. The role of the matrix metalloproteinase (MMP) family of proteolytic enzymes in wound re-epithelialization was studied in a transgenic mouse model. In these mice, a specific inhibitor of MMPs, TIMP-1, was overexpressed in cells that normally produce MMP-9. The healing of cutaneous wounds was found to be significantly delayed, but not prevented, due to the impaired ability of keratinocytes to migrate to the wound area. These results suggest that collagen XVII may participate in epithelial tumor progression and invasion by promoting migration of tumor cells. Based on the present study, epithelial cell-derived MMPs play a significant role in the migration of wound keratinocytes during re-epithelialization.

O câncer de mama é no Brasil o segundo maior causador de morte entre mulheres. Os hormônios sexuais estão dentre os vários fatores indutores ou promotores da carcinogênese. ADAMTS (uma desintegrina e metaloproteinase com domínios trombospondina) é uma enzima Zn2+/Ca2+dependente. Avaliamos a influência dos hormônios na expressão e localização da ADAMTS-1 em 3 linhagens de mama. qPCR demonstrou que a progesterona estimulou o aumento de mRNA de ADAMTS-1 na MCF-10A e na MDA-MB-231, o tratamento com estrógeno e progesterona estimulou a diminuição. No Western blot observamos nas linhagens MCF-10A e MCF-7 que os hormônios tendem a aumentar os níveis de ADAMTS-1, e o estrógeno estimulou uma acentuada secreção em MCF-7. Na linhagem MDA-MB-231, os tratamentos demonstraram uma tendência a diminuir os níveis de ADAMTS-1 intracelular. Na imunofluorescência e Western blot observamos a predominância de ADAMTS-1 nuclear. Os resultados sugerem que os hormônios regulam a expressão de ADAMTS-1 nas células normais e tumorais, e que está predominantemente presente no núcleo celular. / Breast cancer is the second largest cause of death among women in Brazil. Sex hormones are one of the several inducers factors or cancer promoters. ADAMTS (a disintegrin and metaloproteinase with thrombospondin motifs) are dependent on Zn2+/Ca2+ enzymes. We evaluated influence of hormones on ADAMTS-1 levels and localization in three different breast cell lines. qPCR showed that progesterone stimulated an increase of ADAMTS-1 mRNA in MCF-10A, however, in MDA-MB-231, the treatment by estrogen and progesterone decreased levels. Western blot analysis showed a tendency to increased ADAMTS-1 levels in MCF-10A and MCF-7 due to the hormone treatment. Treatment by estrogen led to a marked secretion in MCF-7. In MDA-MB-231 the treatment showed a tendency to decreased intracellular ADAMTS-1 protein levels. Immunofluorescence and Western Blot we observed ADAMTS-1 predominant in the nucleus. Results suggest that sex hormones regulate the ADAMTS-1 expression in normal and tumoral breast cells, ADAMTS-1 is predominant in the cellular nucleus.

The extracellular matrix is a multi-functional environment that cells inhabit to form living tissue. To maintain the tissue, cells require constant telemetry with the matrix and respond to a variety of cues by remodeling matrix architecture. In this study the physical and biochemical manipulation of the matrix by resident cells is explored to better understand how these are used to remodel tissue. Cell-populated collagen hydrogels are used as a controllable in vitro tissue model. To directly measure mechanical forces involved with gel contraction, a culture force monitor was designed and built. Measuring dimensional changes together with contractile forces presents a method of separating mechanisms that influence tissue remodeling. Together, these techniques revealed a correlation between contractile force and gel deformation, suggesting a novel method for examining the material properties of the matrix. Limiting matrix metalloproteinase (MMP) activity altered the correlation as predicted, indicating a stiffer matrix. Contractile force was found to be regulated independent of MMP activity. In contrast, contractile force was found to be dependent on α2β1 integrin function. Collagen gel contraction correlated with both α2β1 function and MMP activity, and was significantly enhanced when combined. The results of this study indicate cells have the capacity to use multiple mechanisms for remodeling the extracellular matrix and may alternately use them together or independently to vary the rate of matrix contraction.

No description available.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure. / Shaukat Khanam Memorial Cancer Hospital and Research Centre, Pakistan and University of Bradford