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.

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

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.

Matrix metalloproteinase2 (MMP2) was recently revealed to have targets and actions within the cardiac myocyte. In ischemia/reperfusion (I/R) injury, MMP2 is activated and degrades troponin I (TnI) and actinin. The regulation of intracellular MMP2 activity is relatively unknown and is thus the subject of this thesis. The localization of MMP2 in caveolae of endothelial cells suggests that caveolin1 (Cav1) may play a role in regulating MMP2. Whether Cav1 is responsible for regulating MMP2 in the heart is unknown. A Cav1 knockout mouse model was used to explore the role Cav1 may play in the regulation of MMP2 activity. The initial studies found that MMP2 and Cav1 were colocalized in cardiomyocytes and that MMP2 activity in Cav1/ hearts was markedly enhanced. Additionally, the caveolin scaffolding domain inhibited MMP2 activity in a concentrationdependent manner. To explore whether increased MMP2 in Cav1/ hearts translates to impaired cardiac function, Cav1+/+ and Cav1/ isolated working hearts were physiologically challenged with increasing increments of left atrial preload followed by increasing concentrations of isoproterenol. Cav1/ hearts show similar or better cardiac function compared to Cav1+/+ hearts following preload challenge or adrenergic stimulation in vitro, and this appears unrelated to changes in MMP2. Though the function of Cav1/ hearts appears similar to that of Cav1+/+ hearts during physiological situations, whether this is the case during I/R injury is not known. Cav1+/+ and Cav1/ isolated working mouse hearts exposed to global, noflow ischemia showed no functional differences. However, Cav1/ hearts had significantly higher levels of both TnI and actinin following I/R than Cav1+/+ hearts. Posttranslational modifications of the intracellular MMP2 substrates could alter susceptibility to MMP2 proteolysis. Isolated working mouse hearts were exposed to isoproterenol and/or I/R injury to examine the phosphorylation status of TnI. Isoproterenol and I/R both result in the phosphorylation of TnI, however, isoproterenol lead to a more highly phosphorylated form of TnI than that observed in hearts exposed I/R alone. These and subsequent studies will further reveal the molecular mechanisms that underlie the complex interactions between Cav1 and MMP2. This may eventually lead to a novel avenue of therapeutic intervention for heart diseases.

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 mechanical environment influences vascular smooth muscle cell (VSMC) functions related to the vascular remodeling. However, the relationships are not appropriately addressed by most mechanical models of arteries assuming homogeneity. Accounting for the effects of heterogeneity is expected to be important to our understanding of VSMC functions. We hypothesized that local stresses computed using a heterogeneous mechanical model of arteries positively correlate to the levels of matrix metalloproteinase (MMP)-2 and -9 in situ. We developed a mathematical model of an arterial wall accounting for nonlinearity, residual strain, anisotropy, and structural heterogeneity. The distributions of elastin and collagen fibers, quantified using their optical properties, showed significant structural heterogeneity. Anisotropy was represented by the direction of collagen fibers, which was measured by the helical angle of VSMC nuclei. The recruiting points of collagen fibers were computed assuming a uniform strain of collagen fibers under physiological loading conditions; an assumption motivated by the morphology. This was supported by observed uniform length and orientation of VSMC nuclei under physiological loading. The distributions of circumferential stresses computed using both heterogeneous and corresponding homogeneous models were correlated to the distributions of expression and activation of MMP-2 and -9 in porcine common carotid arteries, which were incubated in an ex vivo perfusion organ culture system under either normotensive or hypertensive conditions for 48 hours. While strains computed using incompressibility were identical in both models, the heterogeneous model, unlike the homogeneous model, predicted higher circumferential stresses in the outer layer. The tissue levels of MMP-2 and -9 were positively correlated to circumferential stresses computed using the heterogeneous model, which implies that areas of high stress are expected to be sites of localized remodeling and agrees with results from cell culture studies. The results support the role of mechanical stress in vascular remodeling and suggest the importance of structural heterogeneity in studying mechanobiological responses.

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs but principally attacks synovial joints. All the symptoms of RA are mainly caused by cell inflammation, which results in cellular infiltration and synovial hyperplasia, finally leading to severe bone erosion. Existing drugs (steroids, non-steroid antiinflammatory drugs, disease-modifying anti-rheumatic drugs, etc.) can attenuate the symptoms of RA; however, these drugs also have many side effects. Therefore, it is necessary to discover new drugs for RA. Excavatolide B (Exc-B) is derived from the gorgonian coral. In our preliminary observations, Exc-B strongly inhibited lipopolysaccharide (LPS)-induced proinflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW264.7 macrophages. The present study also showed that Exc-B significantly attenuates the expressions of osteoclast-like gene, cathepsin K, and matrix metalloproteinase (MMP)-9 in LPS-treated RAW 264.7 cells. Moreover, in the adjuvant-induced RA animal model, Exc-B effectively reduced the swelling and arthritic index from the morphological viewpoint as well as reduced bone erosion and synovial hyperplasia from the pathological viewpoint. Our data indicates that Exc-B can inhibit disease progression in RA. Hence, Exc-B may serve as a useful therapeutic agent for the treatment of RA.

No description available.

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.