<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)

Στην παρούσα διατριβή μελετήθηκε η έκφραση του παράγοντα αναστολής της μετανάστευσης των μακροφάγων (MIF) στον ιστό από ρινικό πολύποδα αλλά και στον φυσιολογικό ρινικό βλεννογόνο, καθώς και η ικανότητα αυτού να εξουδετερώνει την ανασταλτική δράση των γλυκοκορτικοειδών (ΓΚ) στην επαγόμενη από διάφορους αυξητικούς παράγοντες παραγωγή διαμεσολαβητών, όπως η IL-6 η MMP-1, η MMP-3 το κολλαγόνο τύπου-Ι και ο TIMP-1, που εμπλέκονται στη παθογένεια του ρινικού πολύποδα (ΡΠ). Ο MIF ανιχνεύθηκε στο μέσο καλλιέργειας όλων των ιστών και σε όλα τα εκχυλίσματα. Η έκφρασή του ήταν αυξημένη στον ρινικό πολύποδα σε σχέση με τον φυσιολογικό ρινικό βλεννογόνο. O TGF-β1 προκάλεσε δοσο- και χρονο-εξαρτώμενη αύξηση των επιπέδων της IL-6 του TIMP-1 και του κολλαγόνου τύπου-Ι, και παράλληλα ο TNF-α προκάλεσε δοσο- αλλά και χρονο-εξαρτώμενη διέγερση στην παραγωγή της IL-6 του TIMP-1 και των μεταλλοπρωτεασών MMP-1 και MMP-3. Η δεξαμεθαζόνη προκάλεσε στατιστικά σημαντική και δοσοεξαρτώμενη μείωση της επαγόμενης από τον TGF-β1 και TNF-α, παραγωγής της IL-6 του TIMP-1 του κολλαγόνου τύπου-Ι και των μεταλλοπρωτεασών MMP-1 και MMP-3. Διερευνώντας τον μηχανισμό μέσω του οποίου η δεξαμεθαζόνη ασκεί την κατασταλτική της δράση στην επαγόμενη τόσο από τον TGF-β1 όσο και από τον TNF-α, παραγωγή της IL-6, φάνηκε πως αυτή εκδηλώνεται κυρίως μέσω της επαγωγής αλλά και της προστασίας της ΜΚΡ-1 και κατά συνέπεια της καταστολής του μονοπατιού των ΜΑΡΚ και της ενεργοποίησης του ΑΡ-1, και λιγότερο μέσω της καταστολής της ενεργοποίησης του NF-κB. Ο ISO-1, ένας αναστολέας της δράσης του MIF, ενίσχυσε σημαντικά την κατασταλτική επίδραση της δεξαμεθαζόνης στα επίπεδα της IL-6 και του TIMP-1 στο μέσο καλλιέργειας ιστού από ΡΠ, ενώ αντίθετα προκάλεσε αναστροφή της κατασταλτικής δράσης της δεξαμεθαζόνης, η οποία ήταν στατιστικά σημαντική για την ΜΜΡ-1 όχι όμως και για την ΜΜΡ-3. Η ενίσχυση της κατασταλτικής δράσης της δεξαμεθαζόνης παρουσία του ISO-1, που κυμάνθηκε από 15.0% έως 20.5% θα πρέπει μάλλον να οφείλεται στην αναστολή του ενδογενούς MIF από τον ISO-1. Συμπερασματικά, η παρουσία του MIF στον ιστό του ρινικού πολύποδα, φαίνεται να εξασθενίζει το κατασταλτικό αποτέλεσμα της δεξαμεθαζόνης στην παραγωγή IL-6 και TIMP-1 από αυτόν τον ιστό, ενώ η ταυτόχρονη χρήση του αναστολέα του MIF, ISO-1 οδηγεί σε μια περαιτέρω ενίσχυση της κατασταλτικής δράσης της δεξαμεθαζόνης. Έτσι, είναι λογικό κατ΄αρχήν, να προταθεί πως η δημιουργία ενός φαρμακευτικού σχήματος που περιέχει κορτιζόλη και ένα αναστολέα του MIF, θα μπορούσε να είναι πιο αποτελεσματικό στην θεραπεία της ΡΠ. Απαιτούνται περαιτέρω πειράματα με συνδυασμό ΓΚ και αναστολέων του MIF για να μελετηθεί η επίδρασή τους στη παραγωγή και άλλων παραγόντων που εμπλέκονται στη παθογένεια της ΡΠ προκειμένου να εξαχθούν ασφαλέστερα συμπεράσματα. / In the present study we investigated the expression of macrophage migration inhibitory factor (MIF) in nasal polyp tissues and also in normal nasal mucosa. The ability of MIF to neutralize the inhibitory effect of glucocorticoids on various growth factors induced expression of IL-6, TIMP-1, collagen type-I and matrix metalloproteinases MMP-1 and MMP-3, involved in the pathogenesis of nasal polyps, was studied. MIF was detected in all polyp tissue extracts and tissue culture conditioned media and its expression was increased in nasal polyps compared with normal nasal mucosa. TGF-b1 caused a dose-and time-dependent increase in levels of IL-6 of TIMP-1 and collagen type-I, while the TNF-a induced a dose-and time-dependent stimulation in the production of IL-6 of TIMP-1 and metalloproteinases MMP-1 and MMP-3. Dexamethasone caused a statistically significant and dose-dependent reduction induced by TGF-b1 and TNF-a, production of IL-6 of TIMP-1 of collagen type-I and the metalloproteinases MMP-1 and MMP-3. Investigating the mechanism by which dexamethasone exercises the suppressive action on both induced by TGF-b1 and by TNF-a, production of IL-6, showed that this occurs mainly through the induction and protection of MKP-1 and hence the suppression of the MAPK pathway and activation of AP-1, and less through the suppression of the activation of NF-kB. The ISO-1, an inhibitor of the action of MIF, significantly enhanced the suppressive effect of dexamethasone on the levels of IL-6 and TIMP-1 in tissue culture medium from nasal polyps. In contrary, ISO-1 induced inversion of the suppresive action of dexamethasone, which was statistically significant for MMP-1 but not for MMP-3. Enhancing of the suppresive action of dexamethasone in the presence of ISO-1, which ranged from 15.0% to 20.5% would probably be due to inhibition of endogenous MIF by ISO-1. In conclusion, the presence of MIF in nasal polyp tissue, appears to attenuate the suppressor effect of dexamethasone on the production of IL-6 and TIMP-1by this tissue, while simultaneously using the inhibitor of MIF, ISO-1 leads to an enhancement of dexamethasone activity. Therefore, it is reasonable to propose that the creation of a pharmaceutical regimen containing cortisol and an inhibitor of MIF, might be more effective in the treatment of nasal polyposis. Of course, requires further experiments with a combination of glucocorticoids and MIF inhibitors to study their effect on production of other factors involved in the pathogenesis of nasal polyposis in order to draw safer conclusions.

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.

Thèse réalisée en cotutelle avec Dre Christine Des Rosiers / Le processus de l’athérosclérose est associé à des changements vasculaires structuraux et mécaniques dont la rigidification carotidienne et aortique. Ce phénomème est bien connu et contraste avec l’augmentation paradoxale de la distensibilité cérébrovasculaire observée dans les artères cérébrales exposées aux facteurs de risque cardiovasculaire, tels que l’hypertension. L’impact de l’athérosclérose sur le remodelage, la compliance et la fonction des artères cérébrales est inconnu. En ciblant l’endothélium, l’athérosclérose induit une dysfonction endothéliale cérébrale sévère qui interfère avec le contrôle du débit sanguin cérébral et ultimement avec les fonctions cognitives. Dans les artères cérébrales, le remodelage de la paroi artérielle est toujours accompagné d’une perte des fonctions vasodilatatrices, ce qui suggère que ces deux évènements sont au cœur d’un cercle vicieux. Nos études visent à vérifier l’hypothèse selon laquelle le remodelage de la paroi est déterminé par la fonction endothéliale au niveau cérébrovasculaire alors qu’au niveau de la carotide, le stress mécanique du pouls sanguin régule les propriétés structurales et biomécaniques. Afin de vérifier cette hypothèse, dans une première étude, nous avons sélectionné trois interventions thérapeutiques aux mécanismes d’action différents qui modulent la fonction endothéliale indirectement en diminuant le stress mécanique exercé sur la paroi via une diminution de la fréquence cardiaque. Suite à un traitement chronique de trois mois chez la souris athérosclérotique, LDLr-/-; hApoB-100+/+, l’efficacité de l’ivabradine, du métoprolol et de l’exercice physique volontaire dans la prévention de l’augmentation de la compliance cérébrovasculaire s’est avérée proportionnelle à l’étendue des bénéfices sur la fonction endothéliale. La rigidification carotidienne n’a été prévenue que par les interventions qui réduisent vraiment la fréquence cardiaque, c’est-à-dire l’ivabradine et le métoprolol. Dans une deuxième étude, nous avons confirmé nos résultats en utilisant un traitement antioxydant dans le but de cibler plus directement l’endothélium. La catéchine ne réduit pas la fréquence cardiaque, mais elle est reconnue pour protéger l’endothélium cérébral en neutralisant le stress oxydant. Ainsi, la carotide est restée rigide alors que le remodelage cérébral a été prévenu. Une technique d’imagerie novatrice, la tomographie par cohérence optique, nous a permis de valider nos observations in vivo et de proposer que la catéchine prévient l’hypoperfusion du cerveau en protégeant la fonction endothéliale et l’intégrité de la paroi vasculaire cérébrale. Finalement, les deux études identifient la métalloprotéinase de type 9 comme un joueur potentiellement impliqué dans l’augmentation de la compliance cérébrovasculaire. Nos études démontrent que les changements structuraux et biomécaniques affectant la paroi des artères cérébrales sont indubitablement dépendants de l’endothélium alors que dans la carotide, le stress mécanique est le paramètre le plus déterminant. Somme toute, en protégeant indirectement l’endothélium cérébral on empêche les processus de remodelage, telle que l’activation de la métalloprotéinase de type 9. De nombreuses études ont suggéré l’implication des dysfonctions cérébrovasculaires dans la maladie d’Alzheimer. En effet, les affections vasculaires qui compromettent chroniquement le débit sanguin cérébral, telles la dysfonction endothéliale et la réduction de la lumière artérielle, vont entraîner un déficit métabolique des neurones à l’origine de la neurodégénérescence. Les traitements préventifs cardioprotecteurs, tels que l’ivabradine, l’exercice physique et la catéchine améliorent la fonction endothéliale, la structure et la biomécanique des artères cérébrales, et pourraient donc prévenir l’hypoperfusion chronique du cerveau et le déclin cognitif dans l’athérosclérose. / Large artery stiffness and endothelial dysfunction are markers of atherosclerosis. Stiffening of the carotid arteries contrast with the paradoxical increase in distensibility of cerebral arteries that was reported in the presence of risk factors for cardiovascular diseases, such as hypertension. However, our knowledge concerning the influence of atherosclerosis on cerebrovascular compliance and structure remains incomplete. By targeting the endothelium, atherosclerosis induces a severe cerebral endothelial dysfunction affecting chronically the cerebral blood flow and potentially leading to cognitive dysfunctions. Few studies have shown that the paradoxical increase in cerebrovascular distensibility is consistently reported in animal model of risk factors for cardiovascular diseases exhibiting a cerebral endothelial dysfunction. That being said, we hypothesized that the compliance and structure of cerebral arteries is essentially controlled by the endothelium. To validate our hypothesis, in a first study, we selected three distinct therapeutic approaches that modulated the cerebral endothelial function and the mechanical stress imposed to the vascular wall by lowering heart rate in a mouse model of atherosclerosis, LDLr-/-; hApoB-100+/+ during three months. Ivabradine, metroprolol and voluntary physical training protected, with different efficiencies, the cerebral flow-mediated dilation and this was reflected by a prevention, or not, of the increase in compliance. A 13.5 % heart rate reduction with ivabradine and metoprolol limited carotid artery stiffening. Voluntary physical training did not induce an overall reduction of heart rate explaining the lack of effect on carotid mechanics and suggesting that carotids compliance is more influenced by the mechanical stress imposed to the vascular wall by the cardiac cycle. In a second study, we confirmed our previous findings using a diatery approach that targeted more directly the endothelium, the polyphenol antioxidant catechin. Catechin was previously proven, by us and others, to reverse endothelial dysfunction, reduce inflammation and neutralize reactive oxygen species in diverse vascular beds from animal models of atherosclerosis. Accordingly, we found that catechin prevents adverse cerebral wall remodeling but, again, without a significant heart rate reduction, carotids remained stiff. We also integrated a new live imaging technology allowing us to confirm our findings in vivo and to demonstrate that endothelial, structural and mechanical protection by catechin can result in an improvement of basal cerebral blood flow. Finally, both studies identified metalloproteinase -9 as a potential player in the process leading the weakening of the cerebral artery walls. Taken together, our studies highlight that structural and biomechanical alterations are genuinely triggered by endothelial dysfunction. In carotids, mechanicals stress seems to be the main factor controlling remodeling. In essence, indirect protection of the endothelium impedes in cerebral vessels the remodeling processes, such as the activation of metalloproteinase -9. Numerous studies have revealed that vascular, especially cerebral endothelial dysfunction is implicated in the pathogenesis of Alzheimer’s disease. When brain perfusion is compromised, the suboptimal energy delivery causes neuronal death. Deleterious cerebrovascular outcomes that promote the impairment of vasodilation and the encroachment of the lumen will limit cerebral blood flow in a chronic manner. Chronic treatment with ivabradine, voluntary physical training and catechin preserved the endothelial function, the structure and the mechanics of cerebral arteries, which guarantees a closer management of cerebral blow flow in atherosclerotic mice and a reduce propensity to develop cognitive deficiency.

Les métalloproteases matricielles (MMPs) appartiennent à une famille d'endopéptidases dépendantes du zinc, présentent sous forme secrétée ou membranaire (MT-MMP) et qui jouent un rôle fondamental dans la signalisation cellulaire. L'activité des MMPs est régulée par leur inhibiteurs endogènes, les inhibiteurs tissulaires des MMPs (TIMPs). Le système MMP/TIMP régule les interactions cellule-cellule et cellule-matrice extra cellulaire et module la motilité cellulaire par clivage protéolytique des composants de la matrice extra cellulaire aussi bien lors de processus physiologiques que dans des situations pathologiques.Dans un premier temps, nous avons mis en évidence le rôle de TIMP-1 dans la modulation de la croissance neuritique et la morphologie neuronale, via l'inhibition de MMP-2 et non de MMP-9. souches de la muqueuse olfactive (OE-MSCs). Nous montrons dans cette étude que les gélatinases MMP-2 et MMP-9 ainsi que la MMP membranaire MT1-MMP, sont impliquées dans la migration des OE-MSCs. Nous montrons également que les gélatinases sont probablement impliquées dans les propriétés neurotrophiques des OE-MSCs et des cellules engainantes olfactives.L'ensemble de ces résultats apporte de nouveaux éléments fondamentaux, dans la compréhension du rôle du système MMP/TIMP dans les processus post-lésionnels qui ont lieu au sein du système nerveux central. / The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), which play a fundamental role in the cell signalling. The activity of the MMPs is regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). The MMP / TIMP system regulates the cell-cell and cell-extracellular matrix interactions and modulates the cellular motility through the cleavage of protein components of the extracellular matrix, as well during physiological and pathological conditions.Our results suggest that TIMP-1 is implicated in the modulation of the neurite outgrowth and morphology of cortical neurons through the inhibition at least in part, of MMP-2 and not MMP-9. Afterward, we study of the system MMP / TIMP in the migration of the stem cells of olfactory ectomesenchymal stem cells (OE-MSCs). We show that gelatinases MMP-2 and MMP-9 as well as MT1-MMP, are involved in OE-MSCs migration. We also show that gelatinases are probably involved in neurotrophic properties of the OE-MSCs and olfactory ensheathing cells.Altogether, these results provide new evidences on the role of MMP/TIMP system in central nervous system post-lesional processes.

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.

Introdução e Objetivo: A doença mixomatosa da valva mitral leva ao comprometimento de sua matriz devido à alteração em sua composição tecidual provocada pelo desequilíbrio na quantidade de ácidos mucopolissacarídeos ou glicosaminoglicanos. Sua etiologia ainda não está totalmente esclarecida, podendo ocorrer em formas familiares com transmissão autossômica dominante de penetrância variável, que pode ser dependente do tempo ou de prováveis fatores ambientais, situações em que a interação de agentes infecciosos necessita de maiores esclarecimentos. O objetivo deste estudo é a análise dos produtos dos patógenos da Chlamydophila pneumoniae, Mycoplasma pneumoniae e Borrelia burgdorferi em segmentos de cúspide retirados da valva mitral com degeneração mixomatosa, comparada ao grupo controle e a relação dos produtos bacterianos com aumento de marcadores inflamatórios (CD20, CD48, CD68) e de metaloproteinase (MMP9) na etiopatogenia da degeneração mixomatosa da valva mitral. Método: Estudo observacional, analítico, tipo caso-controle, que analisou 2 grupos contendo 20 pacientes cada e divididos em grupo 1, composto por fragmentos de tecido valvar mitral com diagnóstico de degeneração mixomatosa extraídos em procedimentos de troca ou plásticas valvares mitrais; e grupo 2, formado por segmentos de valvas mitrais sem valvopatia retirados no serviço de verificação de óbito. Foram realizadas colorações de hematoxilina e eosina e Movat para diagnóstico histológico da degeneração mixomatosa e técnica de imunohistoquímica para detecção de antígenos da Borrelia burgdorferi, Mycoplasma pneumoniae, mediadores inflamatórios (CD20, CD45, CD68) e marcadores de metaloproteinase (MMP9). A presença de antígenos da Chlamydophila pneumonia e foi pesquisada pela técnica de hibridização in situ. A análise quantitativa dos aspectos microscópicos foi realizada com o analisador de imagens Aperio. A pesquisa de elementos bacterianos foi feita através de microscopia eletrônica de transmissão. Resultados: No grupo 1, 14 (70%) pacientes são do gênero masculino e 6 (30%) do gênero feminino. A idade média é de 67,4 anos (51 a 79 anos, dp = 9,2). No grupo 2, 11 (55%) pacientes são do gênero masculino e 9 (45%) do gênero feminino. A idade média é de 67,6 anos (42 a 84 anos, dp= 12,0). Na análise da porcentagem de degeneração mixomatosa pela coloração Movat, houve diferença com significância estatística entre os grupos DM (G1), com média de 54,6 % ± 23,7 e grupo controle (G2) com média de 35,5 % ± 22,5 com valor de p = 0,013. Houve um maior número de células CD20 positivas/mm2 no grupo com DM com mediana igual a 17,8 (6,7 - 27,9) x 4,6 (3,6 - 9,8) com p = 0,007 para a área 1. Houve maior número de células CD45 positivas/mm2 no grupo com DM com mediana igual a 17,3 (3,4 - 92,5) x 2,8 (1,4 - 10,1) com p = 0,008 para a área 1. Houve maior número de células CD68 positivas/mm2 no grupo controle (G2), porém sem significância estatística com mediana igual a 38,7 (26,6 - 81,8) x 70 (42,7 - 120,4) com p = 0,098 para a área 1. Em relação à presença de antígenos de Mycoplasma pneumoniae, houve uma maior área (?m2) de antígenos detectados no grupo 1, quando comparadas com o grupo 2 com diferença estatisticamente significante para as duas áreas. Na área 1, mediana de 180.993 (24.856 - 387.477) x 7.970 (2.736 - 15.992) com p < 0,001 e na área 2, mediana igual a 105.968 (2.503 - 416.585) x 7.190 (3.314 - 17.833) com p = 0,02 A análise da presença de antígenos de Chlamydophila pneumoniae demonstrou que em ambas as áreas, houve uma maior área (?m2) de antígenos detectados no grupo de valvas com degeneração mixomatosa, quando comparadas com o grupo controle, porém sem diferença estatística com mediana para o G1 de 9.905 (4.716 - 16.912) x 5.864 (2.382 - 8.692) com p = 0,2 e para o G2, mediana de 3.199 (1.791 - 10.746) x 2.536 (683 - 6.125) com p = 0,3. Em relação à presença de antígenos de Borrelia burgdorferi, houve uma maior área (um2) de antígenos detectados no grupo 2 em relação ao grupo 1, em ambas as áreas. Na área 1, mediana de 7.596 (3.203 - 13.519) x 10.584 (7.223 - 15.974) com p = 0,14 e na área 2, mediana igual a 5.991 (3.009 - 8.475) x 8.403 (1.626 - 27.887) com p = 0,47. Em relação à presença da metaloproteinase MMP9, observamos maior área (um2) de antígeno marcado de MMP9 no grupo com degeneração mixomatosa tanto na área 1 quanto na área 2, com diferença estatística significante. Na área 1, mediana de 503.894 (202.428 - 938.072) x 269.244 (111.953 - 354.022) com p = 0,03. Na área 2, houve diferença estatística representada pela mediana de 389.844 (214.459 - 679.711) x 144.397 (29.894 - 247.453) com p < 0,001. No grupo DM houve correlação positiva entre Borrelia burgdorferi e porcentagem de DM com R = 0,52 e p = 0,018. Em relação às células inflamatórias, houve correlação positiva entre CD45 e Mycoplasma pneumoniae com R = 0,51 e p = 0,02. A presença de MMP9 se correlacionou positivamente com a presença de Mycoplasma pneumoniae com R = 0,45 e p = 0,04. Estas correlações estiveram ausentes no grupo controle. Conclusões: Houve associação de agentes infecciosos Mycoplasma pneumoniae e Borrelia burgdorferi na etiopatiopatogenia da degeneração mixomatosa da valva mitral. Na análise da relação dos produtos bacterianos com os marcadores inflamatórios e com a metaloproteinase, houve relação positiva entre o marcador inflamatório CD45 e a metaloproteinase (MMP9) apenas com a Mycoplasma pneumoniae, nas valvas com degeneração mixomatosa. O marcador inflamatório CD68 foi encontrado em maior número no grupo controle / Background: The myxomatous mitral valve disease leads to impairment due to changes in their tissue composition caused by the imbalance in the amount of acid mucopolysaccharides or glycosaminoglycans. Its etiology is not yet fully understood and may occur in familial forms of autosomal dominant trait with variable penetrance that can be time-dependent or probable environmental factors, where the interaction of infectious agents requires further elucidation. The purpose of this study is the analysis of the pathogens products of Chlamydophila pneumoniae, Mycoplasma pneumoniae and Borrelia burgdorferi in removed cusp segments of the mitral valve with myxoid degeneration, compared to the control group and the ratio of bacterial products with increased inflammatory markers (CD20, CD48, CD68) and metalloproteinase (MMP9) in the pathogenesis of myxomatous degeneration of the mitral valve. Method: Observational, analytical, case-control study which analyzed 2 groups of 20 patients each and divided in group 1, consisting of fragments of mitral valve tissue with diagnosis of myxomatous degeneration extracted in replacement procedures or mitral valve repair; group 2, formed by segments of mitral valves without valvolpaty clinial disease removed in the coroner service. Hematoxylin and eosin and Movat stains were done for histological diagnosis of myxoid degeneration and immunohistochemical technique for the detection of Borrelia burgdorferi, Mycoplasma pneumonia antigens, inflammatory mediators (CD20, CD45, CD68) and markers of metalloproteinase (MMP9). The presence of Chlamydophila pneumonia antigens was verified through an in situ hybridization technique. The quantitative analysis of the microscopic aspects was performed with the Aperio image analyzer. The research of bacterial elements was performed by a transmission electron microscopy. Results: In group 1, 14 (70%) patients were male and 6 (30%) were female. The mean age was (51 to 79 years, sd = 9.2). In group 2, 11 (55%) patients were male gender and 9 (45%) were female. The mean age was 67,6 years (42 to 84 years, sd= 12). In the analysis percentage of myxomatous tissue by Movat staining, there was a significant difference between the DM (G1) groups, with a media of 54.6 % ± 23,7 and control group (G2) with a media of 35.5 % ± 22.5 with p = 0.013. There was an increased number of CD20 cells/mm2 in myxomatous degeneration group (G1) with a median of 17.8 (6.7 - 27.9) x 4.6 (3.6 - 9.8) with p = 0.007 for the area 1. There was a higher number of CD45 cells/mm2 in myxomatous degeneration group (G1) with a median of 17.3 (3.4 - 92.5) x 2.8 (1.4 - 10.1) with p = 0.008 for the area 1. There was a higher number of CD68 cells/mm2 in control group (G2) without a statistically significant difference, with a median of 38.7 (26.6 - 81.8) x 70 (42.7 - 120.4) with p = 0,098 for the area 1. In quantifying Mycoplasma pneumoniae we observed a higher area (um2) antigen marked by, there was a higher amount of antigen detected in myxomatous degeneration group. In area 1, a median of 180,993 (24,856 - 387,477) x 7,970 (2,736 - 15,992) with p < 0.001 and in area 2, a median of 105,968 (2,503 - 416,585) x 7,190 (3,314 - 17,833) with p = 0.02. The analysis of the presence of Chlamydophila pneumoniae antigens showed that in both area, there was a larger area (?m2) antigens detected in the group of valves with MD when compared with the control group, but without significant differences with median for the G1 of 9,905 (4,716 - 16,912) x 5,864 (2,382 - 8,692), with p = 0.2 and the G2, median 3,199 (1,791 - 10,746) x 2,536 (683 - 6,125) with p = 0.3. Regarding the presence of Borrelia burgdorferi antigens, there was a greater area (?m2) antigens detected in group 2 than in group 1, in both areas. In one area, median 7,596 (3,203 - 13,519) x 10,584 (7,223 - 15,974), with p = 0.14 and in area 2, a median of 5,991 (3,009 - 8,475) x 8,403 (1,626 - 27,887) with p = 0.47. Regarding the presence of metalloproteinase MMP9, we observed a higher area (um2) antigen marked by MMP9 in the group with MD both in area 1and area 2, with statistically significant difference. In area 1, median of 503,894 (202,428 - 938,072) x 269,244 (111,953 - 354,022), p = 0.03. In area 2, median 389,844 (214,459 - 679,711) x 144,397 (29,894 - 247,453) with p < 0.001. In the DM group there was a positive correlation between Borrelia burgdorferi and the percentage of MD with R = 0.52 and p = 0.018. Regarding inflammatory cells, there was a positive correlation between CD45 and Mycoplasma pneumoniae with R = 0.51 and p = 0.02. The presence of MMP9 was positively correlated with the presence of Mycoplasma pneumoniae with R = 0.45 and p = 0.04. These correlations were absent in the control group. Conclusions: There was an association of infectious agents Mycoplasma pneumoniae and Borrelia burgdorferi in etiopathogeny of myxoid degeneration of the mitral valve. In the analysis of the relationship of bacterial products with the inflammatory markers and the metalloproteinase, there was a positive relationship between the inflammatory marker CD45 and metalloproteinase (MMP9) only with Mycoplasma pneumoniae. The inflammatory marker CD68 was found in greater numbers in the control group

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 sintomatologia dolorosa é um dos principais estímulos capaz de caracterizar ações nocivas e, portanto, é um importante mecanismo de defesa. No estudo da dor orofacial, as alterações musculoesqueléticas de cabeça, pescoço e face caracterizam um grupo de sintomatologia dolorosa, denominadas de Disfunções Temporomandibulares (DTMs). As DTMs possuem etiologia multifatorial, incluindo alterações estruturais, sensoriais e influências emocionais-motivacionais, atingindo cada vez mais indivíduos na sociedade atual. Para o tratamento desta patologia é necessário o conhecimento detalhado dos mecanismos moleculares e fisiológicos envolvidos, os quais têm sido objetivo de alguns pesquisadores nos últimos anos. Entretanto, ainda se faz necessário a continuidade deste estudo para caracterização minuciosa das DTMs, com novas possibilidades de intervenção e tratamento da sintomatologia dolorosa. Como principais fatores associados à DTM, a inflamação e as alterações estruturais e moleculares da articulação temporomandibular (ATM) desempenham importante papel nesta patologia. Ainda, sabe-se que a dor, em particular a dor inflamatória, promove a ativação de mecanismos locais e sistêmicos, os quais liberam mensageiros fundamentais para a modulação da resposta do organismo, como proteínas e citocinas inflamatórias gerando a inflamação propriamente dita. Segundo a literatura, durante o estabelecimento da resposta inflamatória, muitas proteínas, principalmente as denominadas, metaloproteinases da matriz extracelular (MMPs), são responsáveis pela reabsorção de colágeno, e de outras macromoléculas estão intimamente envolvidas neste processo. Além disso, as MMPs também participam da reorganização no sistema nervoso que ocorre juntamente com os processos inflamatórios persistentes. Desde meados da década de 90, tem sido utilizado com considerável sucesso a laserterapia de baixa intensidade para o tratamento das DTMs na clínica odontológica. Dentro desta perspectiva, estudos demonstraram que este método de tratamento possui atividade antiinflamatória e colaboram no reparo tecidual. Estudo recente demonstrou que o desenvolvimento da inflamação bilateral das ATMs promoveu alteração das MMPs no gânglio trigeminal, bem como desencadeou um processo de alodinia mecânica e hiperalgesia orofacial. Desta forma, o presente projeto teve como objetivo, verificar se o uso da laserterapia de baixa intensidade (LLLT) altera a expressão das MMPs no gânglio trigeminal, nas diferentes fases de desenvolvimento da inflamação intraarticular, (1, 3, 5, 7 e 10 dias) bem como na alodinia mecânica e da hiperalgesia orofacial em ratos. Os resultados obtidos neste trabalho mostraram que a LLLT em ratos inflamação persistente das ATMs, induzida por administração de Adjuvante Completo de Freund, reduziu a alodinia mecânica, avaliada pelo teste de von Frey, bem como a hiperalgesia orofacial no teste da formalina. Adicionalmente, observou-se redução do aumento da MMP-9 e da MMP-2, nos ratos tratados com LLLT. / Painful symptoms is one of the main stimuli that characterizing threatening situation and represent an important defense mechanism. In the study of orofacial pain, musculoskeletal abnormalities of the head, neck and face featuring a group of painful symptoms, which are known as Temporomandibular Disorders (TMD). The TMDs have a multifactorial etiology, including structural changes, sensory and emotional-motivational influences. For the treatment of this pathology is necessary detailed knowledge of the physiological and molecular mechanisms involved, which have been the goal of several researchers in the last few years. However, it is still necessary to continue this study for detailed characterization of TMDs, with new possibilities for intervention and treatment of painful symptom. It is noteworthy that inflammation, as well as molecular and structural changes of the temporomandibular joint (TMJ) play an important role in TMD. Also, it is known that pain, particularly inflammatory pain, promotes the activation of local and systemic mechanisms which release essential mediators, such as proteins and inflammatory cytokines generating inflammation itself. Furthermore, during the establishment of the inflammatory response, many proteins, in particular, the extracelular matrix metalloproteinases (MMPs) are responsible for the resorption of collagen and other macromolecules. Moreover, MMPs also participate in the reorganization that occurs in the nervous system during persistent inflammatory processes. Since the 90s, has been used with considerable success to low intensity laser therapy for the TMD treatment in clinical dentistry. In addtion, studies have shown that this therapy has antiinflammatory activity and collaborate in tissue repair. A recent study has demonstrated that the development of bilateral inflammation of TMJs promote alteration of MMPs expression in the trigeminal ganglion, and triggered a process of orofacial mechanical allodynia and hyperalgesia. Thus, this study aimed to determine whether the use of low intensity laser therapy (LLLT) alters the expression and activity of MMPs in the trigeminal ganglion, at different stages of development of intraarticular inflammation (1,3, 5, 7 and 10 days), as well as mechanical allodynia and hyperalgesia orofacial in rats. So, the results showed that LLLT, reduced mechanical allodynia (as measured by the von Frey test), and orofacial hyperalgesia (formalin test) in rats submmitted to persistent inflammation of TMJs induced by administration of Freund\'s Complete Adjuvant (CFA). Additionally, whereas CFA administration induced an increase of MMP-9 and MMP-2 expression in the trigeminal ganglion, LLLT reduce these effects.