Melanoma é o câncer mais agressivo e a mutação BRAF V600E é a mais frequente entre os pacientes. O vemurafenibe foi o primeiro inibidor específico desta mutação aprovado pela Food and Drug Administration. Entretanto, após cerca de seis meses há recidiva e superar os mecanismos de resistência responsáveis por este fenômeno ainda é um desafio. A curcumina é um tumérico com características antitumorais e anti-inflamatórias, entretanto sua baixa biodisponibilidade e estabilidade limitam seu uso e por isso impulsionaram a busca por análogos capazes de serem eficientes e comercializados. O DM-1, é um análogo monocetônico que apresentou efeitos antiumorais in vitro e in vivo em estudos anteriores. O objetivo deste trabalho foi avaliar os efeitos citotóxicos do DM-1 em células de melanoma sensíveis (naive) e resistentes ao vemurafenibe, bem como na modulação de metaloproteinases. As células de melanoma foram tratadas com diferentes concentrações de DM-1, e este composto foi citotóxico para linhagens sensíveis e resistentes ao vemurafenibe, além de induzir parada de ciclo celular em G1/G0 e diminuir o número de colônias, entretanto ele não foi seletivo em ensaios de citotoxicidade realizados com melanócitos e fibroblastos. O tratamento dessas células em doses subtóxicas resultou na modulação de metaloproteinases importantes no processo de invasão celular. O DM-1 reduziu as concentrações das metaloproteinases -1, -2 e -9 (MMP-1, -2 e -9) em um ensaio de quantificação de MMPs e a atividade das MMP-2 e -9 em um ensaio de zimografia de maneira célula dependente. As modulações negativas do inibidor de MMP TIMP-2 e MMP-14 para SKMEL-28 naive foram associadas a diminuição das atividades de MMP-2 e -9, enquanto que as modulações positivas para SKMEL-19 naive foram relacionadas ao aumento de MMP-2. Este composto ainda inibiu a migração das células e a formação de tubos por células endoteliais. / Malignant melanoma is the most aggressive cancer and the BRAF V600E mutation is the most frequent among patients. Vemurafenib was the first specific inhibitor for this mutation approved by Food and Drug Administration. Therefore around six months later there is relapse and overcoming it is still a challenge. Curcumin is a turmeric and it has been deeply researched because of its anti-inflammatory and antitumoral effects. However the low stability limits its use, therefore, encouraged the investigation of analogues capable to be efficient and commercialized. DM-1 is a monoketone curcumin analog and it showed antitumoral effects in vitro and in vivo in previous studies The aim of this project was to evaluate the cytotoxical effects of DM-1 for vemurafenib responsive (naïve) and resistant melanoma cells, as well as metalloproteinases modulation. Melanoma cells were treated with different DM-1 concentrations, and this compound was cytotoxic for responsive and resistant cell lines, besides inducing G1/G0 cell cycle arrest and reducing the number of colonies, nonetheless it was not selective in assays performed with melanocytes and fibroblasts. Subtoxic treatment of those cells modulated important MMPs in the cell invasion process. DM-1 reduced metalloproteinases -1, -2 and -9 (MMP-1,-2 and -9) in a quantification assay, and MMP-2 and -9 activities by zymography in a cell-dependent way. Negative modulations of MMP inhibitor TIMP-2 and MMP-14 for SKMEL-28 naïve were associated with MMP-2 and -9 reduced activities, whereas positive modulations for SKMEL-19 naïve were correlated to MMP-2 increase. Furthermore, this compound reduced migration of those cells and endothelial cell tube formation.

Matrix metalloproteinase-2 (MMP-2, gelatinase A, type IV collagenase) is a member of a family of zinc-dependent metalloendopeptidases that functions in the degradation of elastin, collagens, and other components of extracellular matrix (ECM). Both secretion and activation of MMP-2 are elevated in human atherosclerotic lesions and abdominal aortic aneurysms (AAA). In this dissertation project, we sought to test the hypothesis that MMP-2 plays a critical role in both atherosclerosis and AAA. We also sought to determine the detailed mechanism. We first examined the atherosclerosis and AngII-induced AAAs development in MMP-2-/- x apolipoprotein (apoE)-/- mice in vivo. It was surprising that MMP-2 deficiency did not reduce the incidence of AngII-induced AAAs or the size of atherosclerosis in apoE-/- mice. However, the cellular and ECM content of atherosclerotic plaques were modified in MMP-2-/- x apoE-/- mice as compared to MMP-2+/+ x apoE-/- control mice. To explain the apparent paradox between this result and the hypothesis, we investigated the morphological characteristics of the aortic wall of MMP-2-/- mice. We detected an enhanced MMP-9 level in the aortic wall of MMP-2-/- x apoE-/- mice compared with MMP-2+/+ x apoE-/- mice. Interestingly, we also observed more branching of the elastin fibers in aortic wall of MMP-2-/- mice as compared with aorta of wild type mice. We also examined the behavior of macrophages from MMP-2-/- mice. Reduced adhesion, migration, and expression of integrin beta 3 were detected in MMP-2 deficient macrophages compared with wild type macrophages. Lastly, we examined whether MMP-2 deficiency in bone marrow-derived cells may influence AAAs and atherosclerosis using bone marrow transplantation technique. There was a significant reduction of both atherosclerosis development and AAAs formation in mice that were reconstituted MMP-2-/- bone marrow cells. In conclusion, the findings in this dissertation suggest that MMP-2 might play an important role in atherosclerosis and aneurysm through influencing inflammatory cell infiltration.

Kawasaki disease (KD) is a multisystem vasculitis that results in localized coronary artery elastin breakdown and aneurysm formation. It is the leading cause of acquired heart disease of children in North America. Despite conventional treatment, a significant proportion of patients continue to develop coronary sequelae. The mechanisms of arterial aneurysm formation in KD are not known. Using a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis, the processes leading to coronary aneurysm formation were examined. Vessel damage occurred as a result of the increased enzymatic activity of the elastase, matrix metalloproteinase (MMP)-9. MMP-9 protein and activity levels were elevated in the heart post-disease induction. Expression and activity were specific for and localized to inflamed coronary arteries. The pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, was required for increasing local MMP-9 expression. Importantly, MMP-9-deficient animals had a significantly reduced incidence of elastin breakdown. Furthermore, in a cohort of KD patients, serum MMP-9 did not correlate with coronary outcome, highlighting the importance of local expression of this elastase. Intravenous immunoglobulin (IVIG) and aspirin/salicylate are therapeutic agents in current use for the treatment of KD, though their exact mechanisms of action in KD are not known. The biologic effects of IVIG and salicylate on critical stages of disease development were examined. IVIG and salicylate had differential effects on TNF-α expression, with therapeutic concentrations of IVIG inhibiting, and salicylate inducing, TNF-α expression leading to an indirect modulation of MMP-9 expression. Interestingly, TNF-α expression and MMP-9 activity were both directly inhibited by the metal-chelating drug doxycycline. Treatment of affected mice with doxycycline significantly improved coronary outcome. Inhibiting both the inflammatory response as well as the downstream effects of inflammation were of therapeutic value in this model of KD. These results taken together demonstrate the importance of MMP-9 in the pathogenesis of coronary artery aneurysms in KD. Targeting MMP activity holds the promise of transforming KD from the leading cause of acquired heart disease to a self-limited febrile illness.

Age-related degeneration of the equine endometrium is an established and important cause of fertility problems in thoroughbred mares, causing great loss to the industry. As a part of the age-related endometrial degeneration complex, an excessive deposition of collagen leading to endometrial fibrosis is particularly important due to the limitations it causes to uterine function. The consequences include reduced efficacy of uterine defence mechanisms and a decrease in the uterine capacity for foetal nutrition. Extensive research into the process of fibrosis in other organs has shown that this condition results from the malfunction of physiological tissue repair mechanisms. These mechanisms revolve around tissue fibroblasts that due to continuous stimulation secrete excessive amounts of collagen and inhibit the activation of factors essential to the normal collagen degradation occurring in scar resolution. Among these factors are the MMPs, an enzyme family with the ability to degrade extracellular matrix components such as collagen during the normal repair mechanisms following tissue injury. The malfunction in the regulation of these enzymes is important in the development of fibrosis in the liver and other organs. In this study it was demonstrated that MMPs are involved in the acute uterine inflammatory response and that they were secreted by infiltrating inflammatory cells. The cellular mechanisms observed during endometritis in normal mares were comparable to the normal repair mechanisms known to be altered in the fibrosis of other organs. These enzymes were present in equine foetal fluids, and their regulation may be important in the process of abortion and stillbirth. It was demonstrated that inbreeding may be correlated with increased deposition of endometrial collagen in a study population of the Icelandic horse breed even though this breed appears to exhibit less severe endometrial degeneration than what is known in lighter breeds. It is likely that genetic predisposition leads to the disruption of normally self-limiting inflammatory and repair mechanisms in the endometrium, resulting in constant activation of collagen synthesis by local and infiltrating cells. This thesis has shown that tissue repair mechanisms involving MMPs are likely to be involved in endometrial fibrosis in the mare. An inherent alteration in these mechanisms may play a role in the pathogenesis of this condition, and might arise due to genetic predisposition. Further understanding of the pathways leading to excess collagen amounts in the endometrium may produce preventative measures, and even therapeutic targets.

Proteolytic processing of transmembrane receptors and ligands can have dramatic effects on cell signaling and subsequent cellular responses. Previous studies demonstrated that a disintegrin and metalloproteinase 10 (ADAM10) may cleave numerous B cell-expressed receptors, including the low affinity IgE receptor (CD23). However, lethality of ADAM10-deficient embryos has limited examination of these cleavage events in lymphocytes. To investigate their role in B cell development and function, we generated B cell-specific ADAM10 knockout mice. Intriguingly, deletion prevented development of the entire marginal zone B cell (MZB) lineage. Further analysis revealed that ADAM10 is required for S2 cleavage of the Notch2 receptor and initiation of Notch2 signaling, which is required for MZB development. Additionally, cleavage of CD23 was dramatically impaired in ADAM10-deficient B cells. This finding and results of ex vivo cleavage assays demonstrated that ADAM10 is the principal in vivo sheddase of CD23. Previous studies have demonstrated that Notch signaling and CD23 cleavage regulate antibody production. Accordingly, deletion of ADAM10 profoundly inhibited germinal center formation, and T-dependent and T-independent antibody responses to immunization, implicating ADAM10 as a novel regulator of adaptive immunity. Additionally, to determine the role of ADAM10 activity in hematopoiesis, we generated transgenic mice (A10Tg) that overexpress the protease on lymphoid and myeloid progenitors. Surprisingly, this markedly suppressed B2 cell development and promoted dramatic expansion of myeloid-derived suppressor cells (MDSCs) via a cell intrinsic mechanism. A10Tg MDSCs inhibited T cell proliferation and adoptive immunotherapy of B16 melanoma, resulting in exacerbated metastatic progression that was prevented by MDSC depletion. Thus, A10Tg mice represent a novel model for the examination of MDSC development and MDSC-mediated immune suppression in a tumor-free environment. Finally, hematopoietic stem cell cultures revealed that ADAM10 overexpression directs myeloid development by dysregulating Notch signaling via uncoupling the highly regulated proteolysis of Notch receptors. Collectively, these findings demonstrate that ADAM10 is a critical regulator of Notch signaling, B cell development, and MDSC expansion. Moreover, they have important implications for the treatment of numerous CD23 and Notch mediated pathologies, ranging from allergy to cancer.

O objetivo desse estudo foi sintetizar e caracterizar um primer experimental contendo Nanopartículas (NLS) de Quitosana e avaliar algumas de suas propriedades. Foram utilizados 50 molares humanos os quais foram seccionados no sentido mesio-distal e oclusal. Esses foram polidos com lixas de diferentes granulações. Após foi realizada a síntese e caracterização de primers experimentais contendo nanopartículas de Quitosana. Os grupos experimentais foram determinados por um ensaio de atividade antimicrobiana e alocados da seguinte forma: 1) Sem aplicação de primer experimental, 2) Aplicação de um primer experimental contendo Clorexidina (CHX) 2% 3) Aplicação de um primer experimental de NLS Quitosana 2% 4) Aplicação de um primer experimental de NLS Quitosana 0,6% 5) Aplicação de um primer experimental de NLS Quitosana 0,4%. Os primers experimentais foram aplicados sobre a dentina sadia. Para a análise de Microtração, foi confeccionado um platô de resina composta Z350 (3M Dental Products, St, Paul, MN, EUA), onde foram realizadas secções para a confecção dos palitos. Foi realizada a caracterização dos primers experimentais por meio de espalhamento de luz dinâmica e o ensaio da atividade de MMPs por meio de Zimografia in situ. Para a analise de Microtração foi utilizada a análise de variância (ANOVA) \"one way\" (p=0,9054). Para a análise de Zimografia in situ foi utilizada ANOVA seguido do teste de Holm-Sidak (p<0,05). A caracterização das NLS de Quitosana demonstrou sua estabilidade em todos os tempos analisados. No ensaio de atividade antimicobriana foi observado concentração de 0,4% para CIM e 0,6% para CBM. Na Resistência a Microtração, não foi observada diferença estatística entre os grupos experimentais (p=0,9054). O ensaio de Zimografia in situ mostrou que o primer experimental contendo Quitosana 2% apresentou maior atividade de inativação de MMPs comparado com os demais grupos experimentais (p<0,05). O primer experimental contendo NLS de Quitosana possui atividade antimicrobiana, não altera a resistência adesiva e possui efeito de inativação de MMPs presentes na dentina / The objective of this study was to synthesize and characterize an experimental primer containing Chitosan Nanoparticles (NLS) and to evaluate some of its properties. Fifity human molars were used which were sectioned mesio-distally and occlusally. These were polished with sandpaper of different granulations. After the synthesis and characterization of experimental primers containing chitosan nanoparticles were performed. The experimental groups were determined by an antimicrobial activity assay and were allocated as follows: 1) Without application of experimental primer 2) Application of an experimental primer containing Chlorhexidine (CHX) 2% 3) Application of an experimental primer of NLS Chitosan 2 % 4) Application of an experimental primer of NLS Chitosan 0.6% 5) Application of an experimental primer of NLS Chitosan 0.4%. The experimental primers were applied on the dentin sound. A composite resin plateau Z350 (3M Dental Products, St, Paul, MN, USA) was used for the analysis of Microtration, where sections were made for making the toothpicks. Characterization of the experimental primers was carried out by means of dynamic light scattering and the assay of MMP activity by means of Zimography in situ. The analysis of variance (ANOVA) \"one way\" (p = 0.9054) was used for the analysis of Microtraction. For the analysis of Zimography in situ was used ANOVA followed by the Holm-Sidak test (p <0.05). The characterization of the NLS of Chitosan demonstrated its stability at all times analyzed. In the antimycobrian activity assay, a concentration of 0.4% for MIC and 0.6% for CBM was observed. In the Microtensile Strength, no statistical difference was observed between the experimental groups (p = 0.9054). The in situ Zimography assay showed that the experimental primer containing Chitosan 2% presented higher inactivation activity of MMPs compared to the other experimental groups (p <0.05). The experimental primer containing Chitosan NLS has antimicrobial activity, does not alter the adhesive resistance and has an inactivation effect of MMPs present in dentin

The 23 matrix metalloproteinases (MMPs) in humans catalyze the turnover of all protein components of the extracellular matrix (ECM) and have important roles in tissue remodeling, wound healing, embryo implantation, cell migration and shedding of cell surface proteins. Excess MMP activities are associated with many diseases including arthritis, heart disease and cancer. The activities of MMPs are regulated by a family of four protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), that are endogenous inhibitors of matrix metalloproteinases (MMPs), ADAMs (A Disintegrin And Metalloproteinase) and ADAMTS (disintegrin-metalloproteinase with thrombospmdin motifs) .... The balance between TIMPs and active metzinicins is very important and imbalances are linked to human diseases such as arthritis, cancer, and atherosclerosis. The engineering of TIMPs to produce specific inhibitors of individual MPs could provide new therapeutic principles for disease treatment, but this requires a detailed understanding of the biophysical and structural basis of the interactions of TIMPs and MMPs and ADAMs. / by Wu Ying. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Peçonhas de serpentes são ricas em proteínas, peptídeos, compostos inorgânicos, lipídeos e carboidratos. Entre os principais constituintes protéicos podemos destacar as metaloproteases (SVMPs), responsáveis pelos processos hemorrágicos e inflamatórios, induzindo a formação de edema e infiltração de leucócitos. Da peçonha de Bothrops atrox, foi isolada uma metaloprotease denominada Batroxase. Esta SVMP foi isolada a partir de três passos cromatográficos, sendo duas exclusões moleculares, uma clássica e outra em FPLC, e uma troca aniônica utilizando CLAE. As SVMPs podem ser classificadas de acordo com os domínios existentes em sua estrutura. Entre as diversas classes, existem as SVMPs da classe PI, que possuem fraca atividade hemorrágica e baixo peso molecular. A Batroxase apresentou massa molecular de 27 kDa (dados não mostrados) e DHM de 10µg, podendo ser classificada como uma metaloprotease de classe PI. A atividade proteolítica da Batroxase foi avaliada sobre diferentes substratos presentes na matriz extracelular e em algumas proteínas presentes no plasma, responsáveis pela ativação da coagulação. Dos constituintes da matriz extracelular (MEC) avaliadas, a Batroxase apresentou atividade sobre o colágeno tipo IV e sobre a fibronectina, esta atividade facilita o processo hemorrágico, desde que estes substratos são encontrados na MEC de tecidos epiteliais e vasos sanguíneos. A atividade sobre proteínas presentes no plasma foi avaliada, evidenciando a capacidade de degradar o fibrinogênio e a fibrina, principalmente a cadeia . Esta atividade pode ser inibida na presença de inibidores de metaloproteases EDTA e EGTA, e na presença do redutor de proteínas -mercaptoetanol. A atividade sobre a fibrina e sobre o fibrinogênio interfere no processo de coagulação, dificultando a interrupção de processos hemorrágicos. Após a formação de um coágulo, a Batroxase foi capaz de dissolvê-lo completamente, capacidade esta, relacionada à sua atividade fibrinolítica. As plaquetas quando na presença da Batroxase não foram induzidas a se agregarem, e quando, na adição de ADP, não foi capaz de inibir a agregação. A atividade inflamatória foi elucidada, evidenciando a participação na formação de edema e hiperalgesia, bem como infiltração de leucócitos. Na avaliação farmacológica foi observada a participação de mediadores pró-inflamatórios como a histamina via receptores H1, a serotonina via receptores 5-HT1, e a formação de leucotrienos no edema e a histamina via receptores H1 e formação de leucotrienos na hiperalgesia. A infiltração de leucócitos observada foi mediada principalmente por neutrófilos nas primeiras horas, seguida do aumento de células mononucleares. A Batroxase possui citotoxicidade ausente ou baixa sobre as linhagens tumorais testadas, com análise significativa apenas para JURKAT, B16-F10, SK-BR3 em altas concentrações. Em células como EL-4 e PBMC sugerem que a Batroxase apresenta atividade imunorregulatória, induzindo proliferação celular em baixas concentrações e inibindo este crescimento em concentrações mais altas como observado em células JURKAT, A-20 e PBMC. É capaz de estimular a produção de citocinas próinflamatórias e antiinflamatórias de maneira distinta induzindo preferencialmente a produção de citocinas do padrão Th1 como IFN-, que, após adição de estímulo policlonal anti-CD3, apresentou atividade supressora na capacidade proliferativa de células T, inibindo a produção de citocinas do padrão Th1 como a IL-2 e IFN-, citocinas do padrão Th2 como a IL-4 e citocinas do padrão Th17 como a IL-17A. / Snake venoms are rich in components like proteins, peptides, lipids, carbohydrates and inorganic compounds. Among their main proteins components, the metalloproteinases (SVMPs) are responsible for hemorrhagic and inflammatory effects, edema formation and leucocytes recruitment. Batroxase, a new metalloproteinase isolated from Bothrops atrox snake venom, was purified using three chromatographic steps, namely: two molecular exclusion steps, one as classic chromatography and another as FPLC, and an anionic exchange in HPLC. SVMPs can be classified by specific domains in their structure. Among the various classes, there are SVMP PI, which has weak hemorrhagic activity and low molecular weight. Batroxase showed a molecular mass of 27 kDa (data not shown) and DHM of 10 µg. The proteolytic activity was evaluated on different substrates in the extracellular matrix and some plasma proteins, responsible for activation of coagulation. Batroxase was able to degrade extracellular matrix (ECM) components like, type IV collagen and fibronectin, inducing bleeding process, since these components are found in the ECM of epithelial tissues and blood vessels. The activity of proteins present in plasma was evaluated, demonstrating the ability to degrade fibrinogen and fibrin, especially the chain, which can be inhibited in the presence of metalloproteinase inhibitors as EDTA and EGTA, as well as with a reducing protein agent -mercaptoethanol. Activity on fibrin and fibrinogen interfere with the clotting process, making difficult to interrupt processes like bleeding. After clot formation, Batroxase was able to dissolve it completely, which is related to their fibrinolytic activity. The platelets in the presence of Batroxase were not induced to aggregate, and when, in addition to ADP, was not able to inhibit aggregation. Inflammatory activity has been elucidated, suggesting the participation in edema and hyperalgesia, as well as infiltration of leukocytes. Pharmacological evaluation was observed with participation of pro-inflammatory mediators such as histamine via H1 receptors, serotonin 5-HT1 receptor via, and the synthesis of leukotrienes and histamine on edema via H1 receptors and synthesis of leukotrienes in hyperalgesia. Infiltration of leukocytes was observed mainly mediated by neutrophils in the early hours, followed by the increase of mononuclear cells. Batroxase have absent or very low cytotoxicity on tumor cell lines tested, with significant only for Jurkat analysis, B16-F10, SK-BR3 in high concentrations. In EL-4 cells and PBMC, inducing cell proliferation at low concentrations and inhibiting this growth in higher concentrations as observed in Jurkat cells, A-20 and PBMC. It can stimulate production of proinflammatory and antiinflammatory cytokines, preferentially induces the production of Th1 cytokines such as IFN-, which, after addition of polyclonal anti-CD3 stimulation showed activity in suppressing proliferative capacity of T cells, inhibiting the production of Th1 cytokines such as IL-2 and IFN-, Th2 cytokines such as IL-4 and Th17 cytokine pattern as IL-17A.

As metaloproteinases são abundantes em venenos de serpentes. Estas enzimas são homólogas às de mamíferos, encontradas em níveis elevados em inflamações articulares. Neste estudo avaliou-se a capacidade da BaP1, induzir: i) a inflamação e incapacitação articulares e a participação do TNF-<font face=\"symbol\">a e PGE2 nesses efeitos e ii) a ativação de macrófagos em cultura e a natureza de sua interação com estas células. A BaP1 induziu aumento da permeabilidade vascular, liberação de TNF-<font face=\"symbol\">a, MMP-9 e PGE2 e acúmulo de leucócitos na cavidade articular e tecido sinovial. Ainda, induziu dor articular. O pré-tratamento dos animais com indometacina ou anti-TNF-<font face=\"symbol\">a reduziu a dor e o influxo leucocitário, induzidos pela BaP1. A BaP1 induziu a expressão de COX-2 e de TNF-<font face=\"symbol\">a e a liberação desta citocina, em macrófagos isolados. Nestas células detectou-se a internalização da BaP1. Em conclusão, a BaP1 induz inflamação e nocicepção articulares, dependentes de TNF-<font face=\"symbol\">a e PGE2. A COX-2 deve estar envolvida na liberação de PGE2 e os macrófagos são alvos importantes para as ações dessa metaloproteinase. / Metalloproteinases are major enzymes in snake venoms showing high grade of homology with mammal matrix metalloproteinases, present in high levels in inflamed joints. In this study we examined the ability of BaP1, to induce: i) inflammation and hypernociception in rat articular joints and participation of TNF-<font face=\"symbol\">a and PGE2 in these effects, and ii) activation of cultured macrophages. BaP1 increased vascular permeability, induced release of TNF-<font face=\"symbol\">a, PGE2 and pro-MMP-9 in joint cavities, and leucocyte influx into joint cavities and synovial tissues. Moreover, BaP1 induced articular hypernociception. Treatment of animals with indomethacin or antiserum anti-TNF-<font face=\"symbol\">a significantly reduced hypernociception and leukocyte influx induced by BaP1. Incubation of macrophages with BaP1 caused expression of TNF-<font face=\"symbol\">a and COX-2 as well as TNF-<font face=\"symbol\">a release. In conclusion, BaP1 induces inflammation and hypernociception in articular joints. These effects are dependent on PGE2 and TNF-<font face=\"symbol\">a. COX-2 may contribute for BaP1-induced PGE2 release and macrophages are key targets for BaP1 induced effects.

Atheromas contain hypoxic areas which upregulate HIF1α expression, promoting angiogenesis and unstable lesion formation. Simvastatin stabilises atheromas through preventing rupture and neovascularisation. Atheromas express matrix metalloproteinase 14 (MMP14) which degrades matrix proteins and promotes neovascularisation. MMP14 is upregulated by hypoxia and contains Hypoxic-Inducible Factor (HIF) recognition sequences (5’-RCGTG-3’). My project sought to investigate if HIF1α interacts with the MMP14 promoter to enhance MMP14 expression, and whether simvastatin attenuates this effect, inhibiting angiogenesis. Immunostaining of atheromas identified MMP14 and HIF1α localisation. Protein-DNA binding assays were performed on human umbilical vein endothelial cells (HUVECs) and showed HIF1α bound to the MMP14 promoter in hypoxia, which was significantly decreased by simvastatin. To assess gene regulation, a human MMP14 promoter-firefly luciferase reporter construct was transfected into C166 endothelial cells alongside HIF-overexpression plasmids and mutations of the MMP14 promoter region at HIF recognition sequences. Overexpression of HIF1α and HIF1β increased MMP14 activity which was abolished by introducing the mutations and diminished by simvastatin in a HIF-dependent manner. Immunoblots, flow cytometry, scratch assays and bromodeoxyuridine incorporation showed HIF1α knockdown and simvastatin significantly attenuated hypoxia upregulated MMP14 expression, migration and proliferation in a HIF1α-dependent manner. Angiogenesis was assessed using in vivo sponge angiogenesis assays and ex vivo aortic ring assays cultured in hypoxia or normoxia, with or without 0.1μM simvastatin, and MMP14 inhibitor, utilising HIF1αfl/flTie1Cre+ and wildtype littermates. Simvastatin perturbed angiogenesis through decreasing MMP14 expression in a HIF1α-dependent manner. The results show hypoxia upregulates MMP14 through HIF1α interaction with the MMP14 promoter. Simvastatin attenuates MMP14 upregulation which reduces HIF1α:MMP14 promoter interaction. HIF1α knockdown and simvastatin treated HUVECs show less migration and proliferation, equivalent to that of MMP14 inhibition. Simvastatin inhibits neovascularisation in a HIF1α-dependent manner. These results suggest simvastatin may stabilise atheromas through inhibiting MMP14 driven angiogenesis which may have further implications in the treatment of atherosclerosis.