Collective cell migration is a defining feature of many morphogenetic processes. Diseases such as congenital heart diseases and cancer arise due to mis-regulation of collective migratory behaviour and animal models have played a pivotal role in dissecting the molecular mechanisms which underlie this process. During embryonic heart development, cardiac precursors undergo a stage of collective migration in both vertebrates and invertebrates. We developed a paradigm to quantitatively assess collective cell migration of cardiac precursors in live embryos of Drosophila, which is the simplest genetic model organism with a heart. Therefore, we studied processes which are commonly observed in most collective cell migration models such as guidance signalling and extracellular matrix remodelling. Our results demonstrate that leading edge of migrating cardioblasts is highly active and that this behaviour is regulated by guidance cues, Slit and Netrin and their respective receptors Robo/Robo2 and Frazzled/Uncoordinated5. These molecules cooperatively promote leading edge motility and epithelial characteristics of the cardioblasts. Next, we determined that matrix restructuring around the cardioblasts requires proteases Mmp1 and Mmp2, which are members of the highly conserved Matrix Metalloproteinase family. We demonstrate that Mmp1 and Mmp2 have distinct roles during lumen formation, however, both Mmp1 and Mmp2 are required for collective motility of the cardioblast leading edge. Hence, we propose that embryonic heart development in Drosophila is an effective and amenable model of collective cell migration which can be applied to discover unique mechanisms which coordinate cell movement in groups. / Thesis / Doctor of Philosophy (PhD)

No description available.

<p><strong> </strong>Fibrotic cataracts such as anterior subcapsular cataract (ASC) are induced by transforming growth factor beta (TGFβ). The mechanism which governs TGFβ-mediated ASC has not been elucidated. What is known is that TGFβ initiates the conversion of lens epithelial cells (LECs) to myofibroblast-like cells, through a process known as epithelial to mesenchymal transition (EMT). TGFβ-induced EMT leading to ASC has been associated with the upregulation of two matrix metalloproteinases (MMPs), MMP2 and MMP9. However, roles for either of these MMPs have yet to be established in ASC. To determine the involvement of MMP2 and MMP9 I used synthetic inhibitors in conjunction with an established <em>ex vivo </em>rat lens model initiated by TGFβ. The results demonstrated that co-culturing rat lenses with TGFβ and the matrix metalloproteinase inhibitor (MMPI), GM6001 or an MMPI specific for MMP2/9 suppressed ASC. Additionally, studies conducted on the conditioned media from these treatments revealed that TGFβ induces the cleavage of E-cadherin ectodomain which is suppressed by coculturing with either MMPI. To further delineate a role for MMP9 <em>in vivo</em>, ASC formation was examined in two models of lens specific TGFβ overexpression in the absence of functional MMP9. Adenoviral delivery of TGFβ to the anterior chamber of the eye in the absence of functional MMP9 resulted in complete suppression of ASC. Similarly, lens specific TGFβ overexpression in the absence of MMP9 suppressed ASC in 75% of mouse lenses. Additional studies determined that connective tissue growth factor is able to mediate ASC, albeit to a lesser degree than TGFβ.</p> / Doctor of Philosophy (PhD)

Indiana University-Purdue University Indianapolis (IUPUI) / The negative heath consequences of smoking are widely recognized, but there are still about 20% of the people in United States using tobacco products. Cigarette smoke condensate (CSC), the particulate matter of cigarette smoke, is comprised of thousands of chemicals (e.g., nicotine). Secondary only to bacterial plaque, cigarette smoking is a major risk factor for periodontal disease. Human gingival fibroblasts (HGFs) are the main cellular component of periodontal connective tissues. During the development of periodontal disease, collagen degradation occurs. Collagen is the major extracellular matrix component of the gingiva. The major extracellular matrix degrading enzymes produced by the HGFs are the matrix metalloproteinases (MMPs). The MMPs are mainly modulated by the tissue inhibitors of metalloproteinases (TIMPs). In this dissertation, three studies aimed at understanding the effects of tobacco on human gingival fibroblasts and their mechanisms have been conducted: the effects of CSC on HGF-mediated collagen degradation; comparison of the effects of CSC on HGFs with that of nicotine; and the combined effects of CSC and bacteria on HGFs. The cell proliferation of HGFs decreased and cytotoxicity increased in HGFs treated with increasing concentrations of CSC. CSC increased the collagen degrading ability of the HGFs by altering the production and localization of MMPs and TIMPs. Nicotine is one of the major components and the most pharmacologically active agent in tobacco. The percentage of nicotine in the CSC was 2.4%. CSC (100 µg/ml) increased the collagen degrading ability of the HGFs by affecting membrane associated MMP-2, MMP-14, and TIMP-2, but the level of nicotine in the CSC may only play a limited role in this process. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontal disease. The combined effects of CSC and P. gingivalis supernatant increased HGF-mediated collagen degradation by destroying the balance between the MMPs and TIMPs at the protein and mRNA levels. This project demonstrated that tobacco (with or without P. gingivalis) increased HGF mediated collagen degradation, as seen in the periodontal disease, through altering the MMPs and TIMPs.

Εισαγωγή: Ο καρκίνος του λάρυγγα, ιδιαιτέρως σε προχωρημένα στάδια, είναι μία καταστροφική νόσος η οποία χαρακτηρίζεται από αυξημένη διηθητικότητα και μεταστατικότητα. Η ανεύρεση ενός δείκτη πρώιμης διάγνωσης, παρακολούθησης και πρόγνωσης της νόσου θα ήταν ιδιαίτερα ευπρόσδεκτη. Συνεχώς αυξανόμενα δεδομένα στη βιβλιογραφία υποστηρίζουν την προγνωστική αξία των ζελατινασών και τον πιθανό ρόλο τους ως μοριακών δεικτών μεταξύ άλλων και στον καρκίνο του λάρυγγα. Σκοπός: Η διαπίστωση μεταβολών στα επίπεδα ορού των ζελατινασών Α και Β σε ασθενείς με καρκίνο του λάρυγγα μετά από εφαρμογή θεραπείας, καθώς και η πιθανή συσχέτιση με διάφορες κλινικοπαθολογικές παραμέτρους πριν και μετά τη θεραπευτική παρέμβαση. Υλικό και μέθοδος: Σαράντα εννέα ασθενείς και 8 υγιείς μάρτυρες συμπεριλήφθηκαν στη μελέτη. Ελήφθησαν προεγχειρητικά και μετεγχειρητικά δείγματα ορού τα οποία στη συνέχεια υποβλήθηκαν σε ζυμογραφία ζελατίνης. Η παρουσία ζελατινασών επιβεβαιώθηκε με την τεχνική western blotting. Οι ζώνες λύσης ποσοτικοποιήθηκαν με τη χρήση Scion Image PC. Η ανάλυση των αποτελεσμάτων πραγματοποιήθηκε με το πρόγραμμα SPSS 17 (SPSS Inc, Chicago, IL, USA). Αποτελέσματα: Στα ζυμογραφήματα αποτυπώθηκαν μόνο οι λανθάνουσες μορφές των ενζύμων (προένζυμα). Τα προ της θεραπείας επίπεδα και των δύο ζελατινασών στον ορό του αίματος των ασθενών με καρκίνο του λάρυγγα ήταν σημαντικά υψηλότερα σε σχέση με αυτά των υγιών μαρτύρων. Ασθενείς με υπεργλωττιδικό καρκίνωμα και ενεργοί καπνιστές είχαν σημαντικά υψηλότερα επίπεδα proMMP-2 σε σχέση με ασθενείς που έπασχαν από γλωττιδικό καρκίνωμα και με πρώην καπνιστές αντίστοιχα. Ασθενείς με πρωτοδιαγνωσμένη νόσο και ασθενείς με λεμφαδενικές μεταστάσεις είχαν σημαντικά χαμηλότερα προ της θεραπείας επίπεδα proMMP-9 σε σχέση με ασθενείς που προσήλθαν με υποτροπή και με ασθενείς στους οποίους δεν διαπιστώθηκε επιχώρια νόσος αντίστοιχα. Κατά τη διάρκεια της συστηματικής παρακολούθησης τα επίπεδα της proMMP-2 στον ορό παρουσίασαν σημαντική αύξηση τις πρώτες 10 με 15 ημέρες μετά την εφαρμογή θεραπείας, για να μειωθούν σταδιακά εντός των επόμενων μηνών. Οι ενεργοί καπνιστές παρουσίασαν σημαντική μείωση των επιπέδων της proMMP-2 κατά την περίοδο παρακολούθησης, σε αντίθεση με τους πρώην καπνιστές οι οποίοι εμφάνισαν σημαντική αύξηση κατά το ίδιο χρονικό διάστημα. Οι ασθενείς σταδίου ΙΙ είχαν σημαντικά χαμηλότερα επίπεδα proMMP-2 σε σχέση με ασθενείς προχωρημένων σταδίων πέντε με οκτώ μήνες μετά τη θεραπεία, όπως και οι ασθενείς οι οποίοι υποβλήθηκαν σε συντηρητική αντιμετώπιση σε σχέση με τους χειρουργημένους ασθενείς. Τα επίπεδα της proMMP-9 στον ορό επίσης παρουσίασαν σημαντική πτώση μετά την εφαρμογή θεραπείας. Διαφορές στο ρυθμό μείωσης των επιπέδων της proMMP-9 παρατηρήθηκαν μεταξύ των διαφόρων ομάδων ως προς το στάδιο, τη διαφοροποίηση, την εντόπιση, τον τύπο της νόσου (πρωτοδιαγνωσμένη ή υποτροπή), τις λεμφαδενικές μεταστάσεις, τον τρόπο αντιμετώπισης και την κατανάλωση αλκοόλ. Ωστόσο αυτή η διαφορά δεν διατηρήθηκε πέντε με οκτώ μήνες μετά την εφαρμογή θεραπείας, με εξαίρεση την ομάδα των χειρουργημένων ασθενών, οι οποίοι διατήρησαν σημαντικά υψηλότερα επίπεδα ενζύμου στον ορό. Αύξηση των ζελατινασών παρατηρήθηκε στον ορό ασθενών που εκδήλωσαν υποτροπή μετά από αντιμετώπιση πρωτοδιαγνωσμένης νόσου σε σχέση με αυτούς που δεν υποτροπίασαν. Ωστόσο εξαιτίας του μικρού δείγματος δεν είναι δυνατόν να εξαχθούν ασφαλή συμπεράσματα. Συμπεράσματα: Αν και δεν υφίστανται φυσιολογικές τιμές, το πρότυπο μεταβολής των επιπέδων της proMMP-9 στον ορό μετά από θεραπεία καταδεικνύει πιθανές ιδιότητες μοριακού δείκτη. Ωστόσο υπάρχουν ενδείξεις ότι και οι δύο ζελατινάσες θα μπορούσαν να χρησιμοποιηθούν για την εξατομικευμένη παρακολούθηση ασθενών με καρκίνο του λάρυγγα. Περαιτέρω έρευνα απαιτείται για την αποσαφήνιση του ζητήματος. / Introduction: Laryngeal cancer, especially in the advanced stages, is a highly devastating disease, characterized by increased invasiveness and high rates of metastasis. The identification of reliable tumour marker for prompt diagnosis, surveillance and prognosis would be highly desirable. There is a growing body of evidence with regard to the prognostic value of gelatinases and their possible role as tumour markers. Aim: To identify the pattern of alteration of serum gelatinases A and B in patients with laryngeal cancer following treatment, and a possible correlation with various clinicopathological parameters prior to and past treatment. Materials and methods: Forty nine patients and 8 healthy controls were included in the study. Pre-treatment and post-treatment serum samples were collected and processed by gelatin zymography. The presence of gelatinases was verified by western blotting. The zymograms were scanned by a digital scanner and the lysis bands were quantified by Scion Image PC. Analysis of the quantitative results was performed by using SPSS 17 (SPSS Inc, Chicago, IL, USA). Results: Only the latent forms of MMP-2 and -9 (proforms) were identified. Both gelatinases were increased in the serum of laryngeal cancer patients compared to healthy individuals. Patients with supraglottic tumours and active smokers had significantly higher pre-treatment levels of proMMP-2 than patients with glottic tumours and ex-smokers, respectively. Patients with primary disease and patients with lymph node involvement showed lower proMMP-9 pre-treatment levels than patients with recurrence and patients without neck disease, respectively. During the follow-up period the proMMP-2 serum levels increased significantly in the first ten to fifteen days after treatment, gradually decreasing over the following months. Smokers showed a very high decrease rate of proMMP-2 levels during the follow-up period, whereas in ex-smokers proMMP-2 levels significantly increased. Stage II patients showed significantly lower levels of circulating enzyme compared to patients with more advanced disease five to eight months past treatment. Similarly, conservative management was associated with lower levels of serum proMMP-2 compared to surgical management five to eight months following treatment. The proMMP-9 serum levels also showed a gradual decrease after treatment, which was statistically significant. Significant alterations in the rate of decrease developed among groups with regard to stage, grade, location, type of disease (primary or recurrence), regional disease, treatment modality and alcohol consumption. Nevertheless those differences were not maintained five to eight months past treatment, with the exception of patients who underwent surgery and who maintained higher levels of proMMP-9. An increase to the levels of both gelatinases were observed in patients with recurrent disease after having been treated for a primary compared to patients who did not develop a recurrence. However, the small sample of patients with recurrent disease during the follow-up period does not allow extrapolating sound conclusions. Conclusions: Although as yet normal values have not been established in the literature, the post-treatment alteration pattern of proMMP-9 serum levels indicates that this enzyme might play a role as a tumour marker. Nevertheless this study provides evidence that both gelatinases might be useful for surveillance on strictly individual basis in laryngeal cancer patients. Further research is necessary to clarify the contribution of both gelatinases to the disease progress and determine their role as prognostic factors and tumour markers.

Considerando-se a localização intra-óssea das lesões periapicais, o diagnostico clinico e dificultado e a avaliação radiográfica não fornece informações suficientes para o diagnostico de um periodonto apical sadio pós-tratamento. Dessa maneira, o objetivo deste estudo foi comparar os achados radiográficos e por tomografia computadorizada de feixe cônico com a avaliação microscópica após tratamento de canais radiculares em dentes de cães e avaliar a participação das metaloproteinases da matriz (MMPs) na lesão periapical e nos tecidos em processo de reparação, assim como em cistos e granulomas periapicais obtidos de humanos. A seguir, os mecanismos envolvidos na cementogênese apical foram investigados utilizando células do ligamento periodontal de humanos. Foram induzidas lesões periapicais em dentes de cães e o tratamento endodôntico foi realizado em sessão única ou apos utilização de um curativo de demora a base de hidróxido de cálcio [Ca(OH)2]. Avaliações radiográficas e tomográficas foram realizadas previamente, após a indução das lesões periapicais e 180 dias apos o tratamento endodôntico. Os tecidos periapicais foram avaliados por meio de microscopia de luz, imunofluorescência, imunoistoquímica e RT-PCR em tempo real. In vitro, células do ligamento periodontal foram utilizadas para avaliar os efeitos da estimulação com Ca(OH)2 nos processos de migração, proliferação, diferenciação celular e mineralização. As vias de sinalização envolvidas na diferenciação cementoblastica foram investigadas por meio de inibidores bioquímicos da via das proteínas quinases ativadoras de mitose (MAPK), bloqueadores de canais de cálcio e silenciadores de RNA para proteínas quinases reguladas por sinal extracelular (ERK-1 / ERK-2). De acordo com os resultados obtidos, a tomografia computadorizada permitiu a detecção de lesões periapicais com maior sensibilidade e acuracia que a radiografia periapical convencional, utilizando-se a avaliação microscópica como padrão-ouro. Histologicamente, as lesões periapicais experimentalmente induzidas apresentaram bactérias distribuídas pelo sistema de canais radiculares e lacunas de reabsorção do cemento e estavam associadas a desorganização das fibras colágenas e alta expressão de MMPs. A presença das MMPs em processos inflamatórios periapicais de humanos (cistos e granulomas) foi confirmada. Nos dentes com lesão periapical submetidos ao tratamento endodôntico em sessão única o desfecho do tratamento endodôntico foi caracterizado pela manutenção ou progressão da lesão periapical e alta expressão de MMPs. Por outro lado, nos dentes submetidos ao tratamento endodôntico utilizando o Ca(OH)2 como curativo de demora, maior numero de espécimes apresentaram regressão da lesão periapical e houve modulação da expressão de MMPs pelo tratamento. Neste grupo foi evidenciada neoformação de cemento no forame apical. Células do ligamento periodontal estimuladas com Ca(OH)2 expressaram proteínas especificas de cementoblastos (CEMP-1, CAP) e foram capazes de sintetizar nódulos de mineralização, mediados via ERK MAPK. A ação do Ca(OH)2 ocorreu via canais de cálcio, uma vez que o bloqueio destes canais inibiu a fosforização de ERK-1 e ERK-2 e, portanto, a expressão de CEMP-1 e CAP. CEMP-1 estimulou a migração, proliferação e mineralização mediada por células, exercendo um papel central na cementogenese, uma vez que o bloqueio de CEMP-1 inibiu a migração celular e mineralização. Juntos, estes resultados permitem concluir que a tomografia computadorizada e superior a radiografia periapical convencional para detecção de lesões periapicais refratarias ao tratamento de canais radiculares. Ainda, o tratamento endodôntico realizado utilizando o hidróxido de cálcio como curativo de demora propiciou um reparo apical e periapical mais favorável do que o tratamento endodôntico em sessão única, possivelmente devido a capacidade do Ca(OH)2 induzir a diferenciação de células do ligamento em células com um fenótipo cementoblastico e posterior mineralização. / Clinical diagnosis of apical periodontitis is difficult due to the intraosseous nature of the disease and radiographic evaluation does not provide sufficient information to determine a healthy apical periodontium following root canal therapy. Therefore, the aim of this study was to compare the radiographic and cone beam computed tomographic findings with microscopic evaluation following root canal treatment in dogs teeth. Then, the presence of matrix metalloproteinases (MMPs) in apical periodontitis and during the healing phase following treatment was evaluated and compared to the expression of MMPs in periapical cysts and granulomas obtained from human. Finally, the mechanisms involved in apical cementogenesis were investigated using human periodontal ligament cells. Apical periodontitis was induced in dogs teeth and then root canal treatment was performed in a single visit or using calcium hydroxide [Ca(OH)2] as the root canal dressing. Tomographic and radiographic evaluations were performed prior to and following induction of apical periodontitis, and 180 days following root canal therapy. Periapical tissues were evaluated by conventional light microscopy, immunofluorescence, immunohistochemistry, and real time RT-PCR. In vitro, periodontal ligament cells were used to evaluate the effects of Ca(OH)2 treatment on cell migration, proliferation, differentiation, and mineralization. The signaling pathways triggered by treatment with Ca(OH)2 were investigated using mitogen activated protein kinase (MAPK) biochemical inhibitors, calcium channel blockers, and extracellular regulated protein kinase (ERK-1 / ERK-2) silencing RNAs. Based on the results obtained, apical periodontitis was detected with higher sensitivity and accuracy by means of cone beam computed tomography compared to conventional periapical radiographs, using microscopic evaluation as the gold standard. Histological evaluation revealed that teeth with apical periodontitis presented microorganisms throughout the root canal system and in areas with resorption of cementum. Apical periodontitis was characterized by collagen fiber disorganization and high expression of MMPs. The presence and activity of MMPs was confirmed in periapical inflammatory diseases (cysts and granulomas) obtained from humans. Root canal treatment outcome was characterized by maintenance or progression of apical periodontitis and high expression of MMPs in teeth submitted to root canal treatment in a single visit, whereas root canal treatment outcome in teeth submitted to root canal treatment using Ca(OH)2 as the root canal dressing, higher number of teeth presented reduced apical periodontitis and lower expression of MMPs. In this group, cementum neogenesis was evident in the apical foramina. Periodontal ligament cells stimulated with Ca(OH)2 expressed cementoblastic specific proteins (CEMP-1, CAP) and were able to synthesize mineralized nodules via ERK MAPK. The effects of Ca(OH)2 occurred via calcium channels, since their blockade prevented ERK-1 and ERK-2 phosphorylation and therefore expression of CEMP-1 and CAP. CEMP-1 stimulated cell migration, proliferation, and mineralization and it was central to cementogenesis because blockade of activity of CEMP-1 prevented cell migration and mineralization. Taken together, these findings demonstrate that cone beam computed tomography is superior to conventional periapical radiography for detection of refractory apical periodontitis. Furthermore, the root canal treatment using Ca(OH)2 as the root canal dressing permitted a more favorable outcome than the root canal treatment performed in a single visit, probably due to the ability of Ca(OH)2 induce cementoblastic differentiation of periodontal ligament cells and mineralization.

O remodelamento vascular é uma resposta adaptativa a estímulos específicos, participando da fisiopatologia de diversas doenças cardiovasculares. Devido à intersecção de fatores de risco cardiovasculares relacionados tanto ao remodelamento vascular como à calcificação vascular (CV), propomos a investigação de mecanismos que inter-relacionam tais condições. Postulamos que camundongos ob/ob com obesidade e resistência à insulina têm resposta exacerbada de remodelamento vascular associado à CV quando comparado aos camundongos controles C57BL/6 (C57) após estímulo com vitamina D3 (VD) in vivo. Camundongos C57 e ob/ob (OB) machos foram injetados com 8x103 UI/kg de vitamina D3 intraperitoneal (IP) ou solução fisiológica (CT) durante 14 dias (n=6). Houve aumento da circunferência da lâmina elástica externa da aorta, determinando aumento da área circunferencial do vaso em camundongos OBVD. A hipervitaminose D aumentou o comprimento da lâmina elástica interna da aorta, aumentando o lúmen vascular em camundongos OBVD. Ocorreu também diminuição da espessura da parede do vaso em camundongos OBVD, caracterizando remodelamento vascular positivo hipotrófico. Observamos ainda maior deposição de colágeno na parede do vaso e elastólise em camundongos OBVD. O remodelamento vascular positivo em camundongos OBVD se correlacionou diretamente com o aumento da calcificação na aorta (R2=0,8; p < 0,003). Aortas de camundongos OBVD apresentaram aumento na expressão de espécies reativas de oxigênio (ERO), que foi associado a aumento da atividade de metaloproteinases de matriz (MMP). Estes resultados fornecem evidências que camundongos obesos, insulino-resistentes, e com diabetes tipo 2 desenvolveram remodelamento vascular positivo hipotrófico correlacionado diretamente com calcificação vascular em camundongos OBVD após estímulo com vitamina D3. O desenvolvimento de remodelamento vascular positivo hipotrófico neste modelo murino é possivelmente mediado pela ativação de MMP na parede da aorta e a geração de ERO pode ter contribuído para a ativação de MMP no nosso modelo / Vascular remodeling is a vessel response to mechanical and hemodynamic stimuli, which is a major determinant of changes in vessel lumen caliber. The mechanisms that influence arterial remodeling include calcification. We hypothesized that ob/ob mice develop positive vascular remodeling associated with calcification. We quantify and assess mechanisms of vascular remodeling and vascular calcification in ob/ob mice (OB) after vitamin D3 stimulation (VD) or phosphate buffered saline (CT), compared with (C57BL/6) mice. Both ob/ob (OBVD) and C57BL/6 (C57VD) mice received 8x103 IU/day of (IP) vitamin D3 for 14 days. Control ob/ob (OBCT) and C57BL/6 (C57CT) mice received IP phosphate buffered saline (PBS) for 14 days (n=6). Hypervitaminosis D increased the external and internal elastic length in aortas from OB mice, resulting in increased total vascular area and lumen vascular area respectively, which characterizes positive vascular remodeling. OBVD mice decreased the aortic wall thickness, resulting in hypotrophic vascular remodeling. We demonstrated increases in collagen deposition, elastolysis and calcification in the aortas of OBVD mice. These results showed a positive correlation between expansive vascular remodeling and vascular calcification in OBVD mice (R2=0,8; p < 0,003). Furthermore, aorta from OBVD increased oxidative stress, coincidently with augmented metalloproteinase activity. Our data provide evidence that obese type 2 diabetes mellitus and insulin-resistant mice (ob/ob) developed positive hypotrophic vascular remodeling correlated directly with increased vascular calcification in OBVD mice after chronic vitamin D3 stimulation. The development of positive hypotrophic vascular remodeling in this mouse model is possibly mediated by the activation in the aortic wall of MMP and ROS may have contributed to the activation of MMP in our model

A doença pulmonar obstrutiva crônica (DPOC) é uma das principais causas de morbidade crônica e mortalidade em todo o mundo e tem como principal manifestação o enfisema pulmonar, caracterizado pelo contínuo e progressivo remodelamento dos componentes da matriz extracelular (MEC). De acordo com estudos experimentais e clínicos esse remodelamento pode ser atribuído à ação proteolítica das metaloproteases de matriz (MMPs), especialmente da MMP-12, sobre os componentes da MEC, sendo a elastina considerada, até recentemente, a principal fibra alvo da proteólise. Dessa forma, só recentemente tem sido esclarecida a importância de outros componentes da MEC e outras MMPs, como as colagenases, no remodelamento da MEC. Sendo assim, o objetivo deste trabalho foi analisar a progressão do remodelamento das fibras no parênquima pulmonar e a expressão gênica das MMPs no início do desenvolvimento do enfisema. Para isso camundongos C57BL/6 receberam instilação intranasal de elastase pancreática suína (PPE 0,667 UI / Grupos ELA: n=40) ou o mesmo tratamento com solução salina (NaCl 0,9% / Grupos controle SAL: n=40). Os animais foram anestesiados, eutanasiados e separados em sub-grupos iguais (n=8) de acordo com o tempo após a instilação de PPE (1, 3, 6 horas, 3 e 21 dias) para remoção dos seus pulmões. Foram medidos o intercepto linear médio (Lm) e as proporções do volume de colágeno tipo I e tipo III, elastina e fibrilina. A expressão gênica para metaloproteinases (MMP-1a, MMP-1b, MMP-8, MMP-12 e MMP-13) no parênquima pulmonar foi avaliada por RT-PCR em tempo real. Os resultados demonstraram que o Lm aumentou desde 3 horas após a instilação de PPE, atingindo os maiores aumentos no 3º e 21º dias, sugerindo uma progressão no alargamento alveolar. Em comparação com o grupo SAL, os grupos ELA apresentaram uma diminuição no colágeno tipo I (no 3º dia) e colágeno tipo III (a partir de 6 horas até 3 dias) em tempos posteriores ao aumento da expressão gênica para MMP-8 (a partir de 3 até 6 horas) e - 13 (a partir de 1 até 6 horas). Após 21 dias, o colágeno tipo III apresentou aumento e o colágeno tipo I retornou a valores semelhantes aos do seu respectivo grupo controle. A expressão gênica para MMP-12 aumentou nos grupos ELA em tempos anteriores (3 e 6 horas) aos que constatamos a redução na proporção de elastina no pulmão (3º dia), reforçando a importância já estabelecida da MMP-12 na quebra deste componente da MEC. A proporção de volume de elastina aumentou no grupo ELA em relação ao respectivo grupo SAL no 21º dia. Não houve aumento na expressão gênica da MMP-1b em nenhum momento e a MMP-1a não teve expressão mensurável em nenhum dos grupos. Não foram observadas diferenças entre os grupos experimentais na avaliação da fibrilina. Nossos resultados serão úteis para melhor elucidar as alterações dinâmicas nos componentes da MEC e a importância, não só da metaloelastase, mas também das colagenases em estágios iniciais do enfisema, fornecendo novas ferramentas para futuros estudos sobre possíveis alvos terapêuticos / Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality worldwide and has as the major manifestation pulmonary emphysema in which the components of the extracellular matrix (ECM) are in a continuous process of remodeling. According to experimental and clinical studies that remodeling can be attributed to proteolytic action of matrix metalloproteinases (MMPs), particularly MMP-12, on the ECM components, with elastin being considered until recently, the primary target fiber of proteolysis. Therefore, only recently has been clarified the importance of other components of the ECM and other MMPs such as collagenases on ECM remodeling. Thus, the objective of this study was to analyze the progression of lung parenchymal fibers remodeling and the gene expression of MMPs in the early development of emphysema. For this C57BL/6 mice were given intranasal instillation of porcine pancreatic elastase (PPE 0,667 IU / ELA groups: n=40) or the same treatment with saline (0.9% NaCl / SAL Control groups: n=40). The animals were anesthetized and euthanized divided into equal sub-groups (n=8) according to time after PPE instillation (1, 3, 6 hours, 3 and 21 days) to remove their lungs. The mean linear intercept (Lm) and the volume proportions of type I and type III collagen, elastin and fibrillin were measured. Gene expression of metalloproteinases (MMP-1a, 1b-MMP, MMP-8, MMP-12 and MMP-13) in the lung parenchyma was evaluated by Real Time RT-PCR. The results demonstrated that the Lm has increased from 3 hours after PPE instillation, with the highest increases reached at 3rd and 21th day, suggesting a progression in the alveolar enlargement. Compared to SAL group, ELA group showed a decrease in type I collagen (3rd day) and collagen type III (from 6 hours to 3 days) on later times to the increase in MMP-8 gene expression (from 3 to 6 hours) and - 13 (from 1 to 6 hours). After 21 days, type III collagen showed an increase and collagen type I returned to values similar to those of their respective control group. Gene expression for MMP-12 increased on PPE groups in earlier times (3 and 6 hours) at which we detected reduction in elastin proportion in the lung (3rd day), reinforcing the importance already established of MMP-12 in the breakage of this ECM component. The elastin volume proportion increased in PPE group compared to respective SAL group at 21st day. There was no increase in MMP-1b gene expression at any time and MMP-1a shows no measurable expression in either group. There were no differences between the experimental groups in the evaluation of fibrillin. Our results will be useful to better elucidate the dynamic changes in ECM components and the importance not only of metalloelastase but also of collagenases in the early stages of emphysema, providing new tools for future studies of potential therapeutic targets

As metaloproteinases da matriz (MMPs) foram identificadas na saliva, na placa dental, na dentina e no cemento. Este trabalho teve como objetivos: Estudo I (I) - avaliar a expressão de MMP-2 e MMP-9 presentes na saliva total e parotidiana e no fluido gengival crevicular (FGC) de pacientes portadores e não portadores de lesões cervicais não cariosas (LCNC); Estudo II (II) - investigar se a presença de MMP-8 e -9/TIMPs poderia influenciar a remineralização de lesões artificialmente criadas na superfície radicular, com ou sem desgaste por abrasão. Os métodos utilizados foram: (I) Coleta de amostras de saliva e do FGC de 32 pacientes, com (n=16) e sem LCNC (n=16). A atividade gelatinolítica das MMPs foi avaliada através de análise zimográfica e Western Blot. (II): Espécimes de dentina humana radicular foram obtidos. O grupo controle G1(10) não sofreu nenhum tratamento. Os demais segmentos radiculares foram desmineralizados G2(60). O Grupo A não foi submetido à escovação e o Grupo B foi submetido à abrasão por escovação em uma máquina de escovação simulada. G2(10) foi apenas desmineralizado, G3(10) desmineralizado e remineralizado, e os Grupos G4(10), G5(10), G6(10), G7(10) foram desmineralizados e remineralizados em presença de tampão neutro, TIMP, MMP-8 e -9, MMP-8,-9 e TIMP, respectivamente. Para a análise elemental, as concentrações de Ca+2, P, Mg+2 assim como a relação molar Ca/P e Mg/Ca foram determinadas através de uma sonda eletrônica para microanálise (EPMA). A análise qualitativa por retrodispersão (BSE) foi realizada para demonstrar a distribuição global da densidade mineral. Os resultados (I) mostraram que a principal gelatinase presente, tanto na saliva total quanto no FGC, é a proMMP-9. Na saliva secretada pela glândula parótida, não foram detectadas bandas indicando a presença de gelatinases. Os resultados do estudo (II) indicaram que os espécimes escovados apresentaram maior conteúdo de Ca+2 a 20µm e maior conteúdo de Mg+2 a 30 e 50µm. Em presença de TIMPs, ocorreu uma redução do conteúdo de Ca+2 a 20µm. Para os espécimes não escovados, em todas as profundidades, as amostras incubadas com MMPs apresentaram maiores valores de Ca+2. Portanto, pode-se concluir que (I) a comparação entre pacientes com e sem LCNC mostrou não haver diferença estatisticamente significante quanto à atividade gelatinolítica; (II) quando não inibidas pelos TIMPs, as MMPs degradaram o colágeno completamente desmineralizado na superfície radicular, permitindo melhor recalcificação na superfície subjacente. Esse fenômeno foi também facilitado pela abrasão por escovação. / Matrix metalloproteinases (MMPs) have been identified in saliva, plaque, gingival crevicular fluid (GCF), dentin and cementum. Study (I) aimed at evaluating the presence and quantity of gelatinases MMP-2 and MMP-9 in total and parotid saliva and in GCF (GCF) of subjects with and without NCCL. Study (II) aimed at investigating whether the presence of matrix metalloproteinase (MMP)-8 and - 9/TIMPs would influence the remineralization of artificial root lesions with and without mechanical wear. (I) Total stimulated saliva, parotid saliva, and GCF from patients with (n=16) and without NCCL (n=16) were collected and assessed for gelatin zymography and for western immunoblot analysis. (II) Human root segments from Group A (n=35) were not brushed and from Group B (n=35) were subjected to machine-controlled brushing, simulating mechanical wear. Specimens from Group 1 (control, n=10) were left untreated. Group 2 (n=10), was just demineralized; Group 3 (n=10) was demineralized and remineralized. The other samples G4 (n=10), G5 (n=10), G6 (n=10), G7 (n=10) were subjected to remineralization with HEPES buffer, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), activated MMP-8 and MMP-9 and activated MMP-8, MMP-9 and TIMP-2, respectively. Ca+2, P, Mg+2 concentrations as well as Ca/P and Mg/Ca molar ratios were determined through an Electron Probe Microanalyser (EPMA). (I) Densitometric analysis revealed that the main gelatinase was proMMP-9. No statistically significant difference was observed for MMP-2 and MMP-9 levels, separately. In parotid saliva, gelatinolytic activity was very low or absent. Western immunoblots revealed that, while little immunoreactivity was detected for MMP-2, there was positive immunoreaction for MMP-9, both in total saliva and in GCF. Gelatinases do not seem to originate from parotid gland. (II) The results indicated that the brushed specimens presented higher Ca+2 levels at 20 µm and higher Mg+2 content at 30 and 50 µm. Ca+2 content at 20 µm decreased in the presence of TIMPs. For the non-brushed specimens, in all depths, samples incubated with MMPs showed highest Ca+2 values. It can be concluded that (I) the main gelatinase present in the oral cavity is MMP-9. No significant differences were found in total gelatinolytic activity among NCCL+ and NCCL- patients. (II) When not inhibited by TIMPs, MMPs degraded the completely demineralized collagen in the root surface, allowing for better recalcification in the deeper areas. This phenomenon was also facilitated by the brushing procedure.

Os tumores hipofisários mesmo sendo, em sua maioria, benignos, podem apresentar comportamento invasivo, com extensão para seio cavernoso, seio esfenoidal e clivo. As metaloproteinases de matriz tipo 2 (MMP-2) e 9 (MMP-9) e o inibidor tecidual de metaloproteinases-2 (TIMP-2) têm sido estudados em relação ao comportamento invasivo desses tumores, em especial quanto à invasão do seio cavernoso. Esse estudo teve como objetivo avaliar a expressão proteica das MMP-2, MMP-9 e do TIMP-2 nos tumores hipofisários e sua relação com invasão do seio cavernoso e, de maneira inédita, investigar a expressão dessas proteínas em nível plasmático. Adicionalmente, foram avaliadas a expressão dos RNAs mensageiros (RNAm) das MMP-2 e TIMP-2 com intuito de correlacioná-las com a expressão proteica no tecido tumoral, bem como a expressão do marcador de proliferação Ki67. Foram selecionados 77 casos, todos com amostras de tumor emblocado em parafina para análise imuno-histoquímica (IHQ). Destes, foram coletadas amostras de tumor fresco em 29 pacientes e de sangue periférico pré-operatório em outros 29 casos. A expressão proteica plasmática foi detectada de forma semi-quantitativa utilizando um arranjo de anticorpos em membrana comercial. A expressão dos RNAm das MMP-2 e TIMP-2 foi avaliada por reação em cadeia da polimerase quantitativo (qPCR) em tempo real. Do total de casos, 20 pacientes apresentavam tumores com invasão para o seio cavernoso. A expressão proteica tumoral das MMP-2, MMP-9 e TIMP-2 apresentou-se aumentada no grupo invasivo, contudo esta diferença não foi estatisticamente significante em relação ao grupo não- invasivo. A expressão plasmática das MMP-9 e TIMP-2 também não mostrou diferença entre os dois grupos e não se correlacionou com a expressão tumoral. A expressão plasmática da MMP-2 não foi detectada em nenhum caso. Quanto à expressão do RNAm das MMP-2 e TIMP-2, também não houve diferença significante entre os grupos e nem correlação com a expressão proteica tecidual ou plasmática. Foi observada uma diferença significante na dimensão tumoral [3.6 (2.5-5.2) x 2.0 (1.3-2.7); P < 0.001] e no índice do Ki67 [1.05 (0.27-25) x 0.5 (0.2-1.0); P < 0.001] entre os grupos invasivo e não-invasivo respectivamente. Em conclusão, em nossa coorte, não foi encontrada relação entre a expressão tecidual e plasmática das MMP-2, MMP-9 e TIMP-2 e a invasão para o seio cavernoso nos adenomas hipofisários / Pituitary tumors, although mostly benign, may present invasive behavior, with extension to the cavernous sinus, sphenoid sinus and clivus. Type 2 (MMP-2) and type 9 (MMP-9) matrix metalloproteinases and the metalloproteinase tissue inhibitor type 2 (TIMP-2) have been studied in relation to the invasive behavior of these tumors, especially regarding invasion of the cavernous sinus. The aim of this study was to evaluate the protein expression of MMP-2, MMP-9 and TIMP-2 in pituitary tumors and its relation with invasion of the cavernous sinus and, in an unprecedented way, to investigate the expression of these proteins at the plasma level. Additionally, expression of MMP-2 and TIMP-2 messenger RNAs (mRNAs) was evaluated in order to correlate with protein expression in tumor tissue, as well as Ki67 proliferation marker expression. A total of 77 cases were selected, all of them with paraffin embedded tumor samples for immunohistochemical analysis (IHC). Of these, fresh tumor samples were collected in 29 patients and preoperative peripheral blood in another 29 cases. Protein plasma expression was detected semi-quantitatively using a commercial membrane antibody array. Expression of MMP-2 and TIMP-2 mRNAs was evaluated by quantitative realtime polymerase chain reaction (qPCR). Of the total cases, 20 patients presented tumors invasive to the cavernous sinus. Tumor protein expression of MMP-2, MMP-9 and TIMP-2 was increased in the invasive group, not reaching, however, statistically significant difference as compared with the non-invasive group. Plasma expression of MMP-9 and TIMP-2 also did not differ between the two groups and did not correlate with tumor expression. Plasma expression of MMP-2 was not detected in any case. Concerning MMP-2 and TIMP-2 mRNA expression, there was also no significant difference between groups and no correlation with tissue or plasma protein expression was observed. A significant difference was observed in tumor size [3.6 (2.5-5.2) x 2.0 (1.3-2.7); P < 0.001] and in the Ki67 index [1.05 (0.27-25) x 0.5 (0.2-1.0); P < 0.001] between the invasive and non-invasive groups respectively. In conclusion, in our cohort, no relationship was found between the tissue and plasma expression of MMP-2, MMP-9 and TIMP-2 and the invasion of the cavernous sinus in pituitary adenomas