Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.

Neutrofilai ir makrofagai – vienos svarbiausių uždegiminiame procese dalyvaujančių ląstelių. Lėtinės obstrukcinės plaučių ligos (LOPL) metu aktyvintos šios ląstelės sintetina biologiškai aktyvias medžiagas, skatinančias intensyvesnį sisteminį ir kvėpavimo takuose vykstantį uždegimą. Iki šiol nėra aiškūs neutrofilų ir makrofagų sintetinamų žymenų (citokinų, proteinazių) ypatumai sergant LOPL ir atsižvelgiant į rūkymo įprotį. Nėra žinoma, kaip kraujo neutrofilų funkcines savybes tiesiogiai įtakoja pats sergančiųjų LOPL kvėpavimo takų sekretas. Šio tyrimo tikslas buvo įvertinti neutrofilų ir makrofagų funkcinių savybių ypatumus sergant lėtine obstrukcine plaučių liga ir galimus jų skirtumus priklausomai nuo rūkymo įpročio. Ištirti sergančių LOPL rūkančiųjų ir neberūkančiųjų matrikso metaloproteinazės-12 raiškos kvėpavimo takų sekreto makrofaguose ypatumai. Įvertintos sergančiųjų LOPL biologiniais ir cheminiais veiksniais aktyvintų kraujo neutrofilų funkcinės savybės (chemotaksis, fagocitozė, reaktyviųjų deguonies formų susidarymas), analizuotas potencialus moduliacinis kvėpavimo takų sekreto poveikis šioms funkcinėms savybėms. Nustatyta, jog sergančiųjų lėtine obstrukcine plaučių liga neutrofilai ir makrofagai buvo labiau aktyvūs nei sveikų asmenų ląstelės; rūkymo įprotis įtakojo kai kurias uždegimines neutrofilų ir makrofagų savybes kvėpavimo takų sekrete ir kraujyje. / Neutrophils and macrophages play an important role in inflammatory process. Activated during chronic obstructive pulmonary disease (COPD), these cells synthesize a variety of biologically active compounds, which may further amplify both systemic and local airway inflammation. The features of biologically active compounds (cytokines, proteinases) released by neutrophils and macrophages during COPD and according to smoking status are unknown. Still, a direct influence of airway mucous from COPD patients on functional neutrophil features is unknown. The aim of this study was to evaluate the functional features of neutrophils and macrophages during chronic obstructive pulmonary disease and possible differences according to smoking status. An expression of matrix metalloproteinase-12 in airway mucous of COPD smokers and ex-smokers was evaluated. The functional features (chemotaxis, phagocytosis, production of reactive oxygen species) of biologically and chemically activated blood neutrophils and a modulator effect of airway mucous were also analysed. The neutrophils and macrophages of patients with chronic obstructive pulmonary disease were more activated than the cells of healthy individuals; smoking status has influenced some inflammatory features of neutrophils and macrophages in airway mucous and blood.

Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.

Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.

Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.

Perinatal hypoxic ischemic (HI) injury is a leading cause of long-term neurological complications in newborn babies. Matrix metalloproteinases (MMPs) are a family of endopeptidases that are capable of degrading the extracellular matrix (ECM) components. They are considered to be integral in many physiological processes. However, recently it has been demonstrated that the inappropriate activity of these proteases, particularly MMP-2 and 9, contribute to the pathogenesis of cerebral ischemia in the adult brain. Given that ECM disruption is frequently observed following injury to the developing brain, it is possible that MMPs play an important role in HI injury processes in the developing brain. Therefore, this thesis evaluated the hypothesis that MMP-2 and 9 participate in the pathophysiology of HI injury to the developing brain. Since ECM remodelling is a fundamental process during brain development it was important to first characterise the MMP-2 and 9 profiles in the normal developing forebrain. We demonstrated that MMP-2, which mainly was observed in cortical plate neurons, declined with age, thus indicating a potential role in the development and differentiation of the cortical plate. Conversely, MMP-9 was increased with age, particularly during active myelination, indicating that it may contribute in myelination. Secondly, we showed an upregulation of MMP-9 within the ischemic core during the early hours following HI injury, suggesting that MMP-9 may be involved in the development of delayed injury processes following hypoxic ischemia. On the contrary, MMP-2 was strongly upregulated during a later stage following injury surrounding the ischemic core possibly suggesting that it plays a role in wound repair processes. Thirdly, the profiles of tissue (tPA) and urokinase (uPA) plasminogen activators were characterised following HI injury since they are known to be major upstream activators of MMPs. uPA upregulation paralleled that of MMP-2 suggesting a function for uPA in wound repair processes following HI injury to the developing brain through activation of MMP-2. In contrast with uPA, tPA activity remained unaffected following injury at both ages. Finally, MMP-9 activity was inhibited using a very specific MMP-2/9 inhibitor, SB-3CT, to determine if the MMP-9 deficiency protects the developing brain from HI injury. The elevated MMP-9 activity following HI injury was attenuated by the SB-3CT treatment. Although SB-3CT failed to confer any significant neuroprotection, we recommend that further investigations are needed before discounting the role of MMP-9 during HI injury to the developing brain. In conclusion, we suggest that MMP-9 is induced following an insult to the developing brain potentially contributing to the delayed neuronal death whilst MMP-2 is involved in essential developmental, differentiation and wound repair processes.

Η HARP (Heparin Affin Regulatory Peptide) είναι ένας αυξητικός παράγοντας με Μ.Β. 18 kDa που ανήκει στην οικογένεια των αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Eμπλέκεται στην ανάπτυξη των νευριτών, την επούλωση πληγών και φαίνεται να παίζει σημαντικό επαγωγικό ρόλο στις διαδικασίες της ογκογένεσης, καθώς επάγει την αγγειογένεση και εμφανίζεται σε υψηλές συγκεντρώσεις τόσο σε καρκινικούς ιστούς, όσο και σε κυτταρικές σειρές καρκινικών κυττάρων. Στο πλαίσιο μελέτης της σχέσης δομής/δράσης του αυξητικού αυτού παράγοντα, χρησιμοποιούνται τόσο συνθετικά πεπτίδια, όσο και ανασυνδυασμένες τροποποιημένες μορφές του αυξητικού αυτού παράγοντα. Σε φυσιολογικές συνθήκες, η εκκρινόμμενη HARP πέπτεται από ένζυμα του κυτταρικού μικροπεριβάλλοντος και προκύπτουν πεπτίδια που παρουσιάζουν βιολογικές δράσεις παρόμοιες ή και αντίθετες από αυτές της HARP. Φαίνεται λοιπόν πως η δράση του αυξητικού αυτού παράγοντα ρυθμίζεται τόσο στο επίπεδο βιοσύνθεσης και έκκρισης, όσο και από τη δράση ενζύμων του εξωκυττάριου χώρου. Στην παρούσα εργασία μελετήθηκε η δράση ενός συνθετικού πεπτιδίου το οποίο αντιστοιχεί στα αμινοξέα 65-97 που εντοπίζονται στην ΤSR περιοχή προς το καρβοξυτελικό άκρο της HARP. Με δεδομένο ότι τι πεπτίδιο αυτό εμφανίζει αντιαγγειογενετική δράση, πραγματοποιήθηκαν χρονοεξαρτώμενα και δοσοεξαρτώμενα πειράματα, με σκοπό τη μελέτη της δράσης του στον πολλαπλασιασμό, τη μετανάστευση και την επούλωση πληγών. Στο πλαίσιο αυτών των μελετών, ελέγξαμε τη δράση του στην έκφραση των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9, των αναστολέων τους ΤΙMP-1 και ΤΙMP-2 καθώς και του κολλαγόνου και της ελαστίνης σε πρωτογενείς καλλιέργειες ενδοθηλιακών κυττάρων από ομφάλιο λώρο (HUVEC cells). Τα αποτελέσματα έδειξαν πως το συνθετικό αυτό πεπτίδιο καταστέλλει τον πολλαπλασιασμό, την μετανάστευση αλλά και την επούλωση πλήγών των κυττάρων HUVEC με δοδοεξαρτώμενο και στατιστικώς σημαντικό τρόπο. Επιπλέον από τα πειράματά μας δεν παρατηρήθηκε μεταβολή στα πρωτεϊνικά επίπεδα έκφρασης των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9 καθώς και των αναστολέων τους ΤΙMP-1 και ΤΙMP-2. Ωστόσο, παρατηρήθηκε στατιστικώς σημαντική μεταβολή στα επίπεδα γονιδιακής έκφρασης των αναστολέων ΤΙMP-1 και ΤΙMP-2 όπως επίσης και της ελαστίνης και του κολλαγόνου IV. / Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. HARP was originally described as a neurite outgrowth promoting molecule, which appears to increases during recovery from injury and is thought to be involved in angiogenesis expression, playing a major role in the cell growth and differentiation that are associated with regeneration in several tissues. HARP is expressed in several human tumors and tumor cell lines and is also indicated in high serum levels of patients with different types of cancer. HARP contains two random coiled clusters of basic residues (N- and C-terminal) and two b-sheet domain. Each b-sheet domain contains a thrombospondin repeat I (TSR-I) motif, which have been suggested to be responsible for the interaction of HARP with heparin. Our project is based on C-TSR-I domain, corresponding to amino acids 65–97 of HARP peptide, respectively, required for the neurite outgrowth activity of HARP. In this study, we investigate the impact of C-TSR on basic biological functions of endothelial cells (HUVEC) such as proliferation, migration, the expression of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2) that contribute to the ECM remodeling. Time course and dose-response experiments revealed that CTSR reduces proliferation, migration and wound healing, without affecting the protein levels of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2). Moreover, CTSR inhibits the expression of TIMP-1 and TIMP-2 contributing to the ECM remodeling. Concluding, HARP could act as pro- or anti-angiogenic factor, depending on the system used and the cell microenvironment.

Nox4 est une source de ROS constitutive et ubiquitaire intervenant dans la signalisation intracellulaire. Une dérégulation de son activité a été associée à l'athérosclérose, la fibrose pulmonaire, le diabète et nous suspectons son rôle dans l'arthrose. L'arthrose est la résultante de phénomènes mécaniques et biologiques conduisant à la dégradation du cartilage et de l'os sous-chondral. Le chondrocyte, synthétisant et libérant des métalloprotéases matricielles (MMP), principales effectrices de la protéolyse matricielle, en est l'acteur principal. Dans la lignée de chondrocytes C-20/A4, l'IL-1β induit la synthèse de la collagénase MMP-1 de manière dépendante de l'activité de Nox4.Ces données suggèrent que Nox4 pourrait jouer un rôle dans la physiopathologie de l'arthrose. De manière à éclaircir le rôle de Nox4 dans le chondrocyte mais également pour proposer des pistes permettant de moduler cette activité, ce projet à été découpé en trois parties étroitement liées.1- Une meilleure connaissance de la topologie de Nox4 permettrait de mieux comprendre son fonctionnement pour mieux appréhender le développement d'inhibiteurs de son activité. L'étude de la topologie membranaire de Nox4 a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode (ToDUFA) a permis pour la première fois de proposer expérimentalement une topologie de Nox4 avec 6 passages transmembranaires. 2- L'indentification de nouveaux partenaires modulateurs de l'activité de Nox4 pourrait être une première étape vers le traitement de l'arthrose. L'hème oxygénase (HO-1) est l'enzyme limitante dans le catabolisme de l'hème, molécule indispensable à l'activité catalytique des Nox. Les résultats montrent que HO-1 est capable de réprimer l'activité de Nox4 et suggèrent un mécanisme dépendant de la libération de monoxyde de carbone (CO) produit au cours de l'activité de HO-1. Ces résultats soulèvent également le rôle de l'hème dans le processus de maturation du complexe Nox4/p22phox. 3- Le rôle de Nox4 dans la physiopathologie de l'arthrose doit être validé sur des cultures primaires. Les résultats montrent que Nox4 est la seule Nox exprimée dans les chondrocytes primaires humains. Nox4, stabilisée par une augmentation protéique de p22phox après stimulation des chondrocytes par IL-1β, conduirait à la synthèse des MMP-1, MMP-13 et de l'ADAMTS4. Ces travaux montrent enfin que l'activité de Nox4 est impliquée dans le rétrocontrôle positif conduisant à la néosynthèse de l'IL-1β et à la persistance du catabolisme.En conclusion, les résultats suggèrent un rôle significatif de Nox4/p22phox dans la dégénérescence cartilagineuse. Nous décrivons un nouveau mécanisme de régulation de l'activité de Nox4 par HO-1 dans la lignée C-20/A4 et dans les chondrocytes primaires humains, préfigurant d'un rôle thérapeutique potentiel de HO-1. Enfin, le premier modèle topologique de Nox4 basé sur nos données expérimentales pourrait constituer une base pour le développement d'inhibiteurs ou de peptides compétiteurs. / Nox4 is a ubiquitous and constitutive source of ROS involved in intracellular signaling. However, a dysregulation of its activity has been shown to be involved in atherosclerosis, pulmonary fibrosis, diabetes, and we suspect its role in osteoarthritis (OA). OA is the consequence of mechanical and biological processes leading to the breakdown of cartilage and subchondral bone. Chondrocytes by synthesizing and releasing matrix metalloproteinases (MMP) mediates proteolysis of the extracellular matrix and is therefore the main actor of OA. In the C-20/A4 chondrocytes cell line, IL-1β leads to the synthesis of the MMP-1 collagenase in a Nox4 dependent manner suggesting that Nox4 might be a good therapeutic candidate for osteoarthristis treatment.In order to shed light on the role of Nox4 in the chondrocyte and to propose new strategies to modulate its activity, 3 mains objectives have been developed in this work.1 - A better understanding of the topology of Nox4 could allow a better comprehension of its function and the development of inhibitors: Membrane topology of Nox4 was assessed by preparing fusion proteins with ubiquitin fused with GFP's tag. This method (ToDUFA) allowed us for the first time to determine experimentally a topology organization for Nox4 with 6 transmembrane domains.2 - The identification of novel partners of modulation of Nox4 activity might be a first step toward the treatment of osteoarthritis: Heme oxygenase (HO-1) is the rate limiting enzyme in heme catabolism, an essential molecule for the catalytic activity of Nox. Our results showed that HO-1 was able to inhibit Nox4 activity and suggest a mechanism involving the release of carbon monoxide (CO) that was produced during the activity of HO-1. These findings also highlight the role of heme in the maturation process of Nox4/p22phox hetero-complex.3 - The role of Nox4 in the pathophysiology of osteoarthritis must be validated in primary cultures: Our results showed that Nox4 is the sole Nox isoform expressed in human primary chondrocytes. Nox4 activity, stabilized by the induced expression of p22phox in the IL-1β-stimulated chondrocytes, might mediate the synthesis of MMP- 1, MMP-13 and ADAMTS4. Finally, our data demonstrated that the activity of Nox4 is involved in the positive feedback leading to the neo-synthesis of IL-1β, mechanism that sustains the catabolism.In conclusion, our data suggest a significant role of Nox4/p22phox in cartilage degeneration. We describe a new mechanism by which HO-1 regulates the activity of Nox4 in C-20/A4 cell line and in primary human chondrocytes, predicting a potential therapeutic effect of HO-1. Finally, the first topological model of Nox4 based on our experimental data might provide a basis for the development of inhibitors or competitor peptides.

A degradação da dentina pelas metaloproteinases da matriz (MMPs) pode facilitar a progressão de lesões erosivas. Objetivos: Avaliar, por meio de uma série de 5 subprojetos: 1) A atividade de MMPs em dentina bovina e humana; 2) O efeito do chá verde contra erosão/abrasão de dentina; 3 e 4) Géis contendo inibidores de MMPs (epigallocatechin-3-galatte-EGCG, clorexidina-CHX e sulfato ferroso-FeSO4) sobre a prevenção de erosão de dentina sozinha ou associada à abrasão, respectivamente, e 5) Sobre a degradação de colágeno e desgaste. Material e Métodos: 1) Extração proteica de dentina de coroa e raiz bovina e humana foi realizada (ácido cítrico a 0,87 M, pH 2,3) e testada por zimografia e atividades gelatinolíticas; 2) Voluntários (n=10) bochecharam chá verde ou água (1 min, 10 mL) entre os desafios erosivos (Coca-Cola, pH 2,6, 4x/dia/5 min, extraoralmente) e abrasivos. A abrasão (escova elétrica + dentifrício não fluoretado) foi realizada imediatamente ou 30 min depois da erosão por 30 s. O desgaste da dentina foi analisado por perfilometria (µm); 3 e 4) Voluntários (n=10-13) utilizaram dispositivos palatinos contendo 12 blocos de dentina e distribuídos aleatoriamente para 6 grupos, de acordo com o tipo de gel aplicado ou não (não tratado-NT). Os géis tinham composição idêntica, exceto pela presença de EGCG (400 µM), CHX (0,012%), FeSO4 (1 mM), flúor (NaF-1,23%) ou sem ativo (Placebo-P). Os géis foram ou não aplicados sobre os espécimes em fina camada e removidos depois de 1 min. A erosão (Coca-Cola, pH 2,6, 4x5min/dia, extraoralmente) foi realizada por 5 ou 10 dias, respectivamente. A cada dia, depois do primeiro e último desafios erosivos, os blocos eram (ERO) ou não escovados (ERO+ABR) por 15 s (escova elétrica + solução de dentifrício não fluoretado). O desgaste da dentina foi avaliado por perfilometria (µm) depois de 5 e 10 dias. 5) Dentina (n=45/grupo) foi desmineralizada com ácido cítrico (0,87 M, pH 2,3) por 36 h. Na sequência, foi ou não tratada (NT) com os mesmos géis descritos acima e estocada em saliva artificial (5 dias, 37ºC), contendo inibidores de protease EDTA-free e colagenase (Clostridium histolyticum, 238 U/mL). A degradação de colágeno foi analisada pelo conteúdo de hidroxiprolina (µg/mL) e o desgaste dentinário avaliado por perfilometria (µm, n=12/grupo,). Os dados foram analisados por ANOVA (p<0,05). Resultados: A dentina bovina mostrou ser um substrato confiável para estudos envolvendo atividades de MMPs. O chá verde reduziu o desgaste dentinário para todas as condições. A média de desgaste e de degradação de colágeno foi significativamente reduzida para os géis experimentais quando comparados aos géis NaF e P ou NT, em todos os protocolos testados. Para ERO, o desgaste não aumentou significativamente com tempo, enquanto que para ERO+ABR, foi observado um aumento significativo com o tempo para os gel de NaF e NT. Conclusão: O uso de bochecho com chá verde, bem como de um gel como veículo de aplicação de inibidores de MMP demonstrou reduzir consistentemente a degradação de colágeno e o desgaste e promoveu prevenção duradoura contra erosão e erosão mais abrasão em dentina. / The dentin degradation by matrix metalloproteinases (MMPs) can increase the progression of erosive lesions. Objectives: Five studies were conducted to evaluate: 1) The activity of MMPs in bovine and human dentin; 2) The effect of green tea rinse against dentin erosion/abrasion; 3 and 4) Gels containing MMP inhibitors (epigallocatechin-3-galatte-EGCG, chlorexidine-CHX and ferrous sulphate-FeSO4) to prevent dentin erosion alone or associated with abrasion, respectively, and 5) On collagen degradation and wear. Material and Methods: 1) Protein extraction from crown and root of bovine and human dentin was performed (0.87 M citric acid, pH 2.3) and tested by zymography and gelatinolytic activities; 2) Volunteers (n=10) rinsed with green tea or water (1 min, 10 mL) between the erosive (Coke, pH 2.6, 4x5min/day, extraorally) and abrasive challenges. The abrasion (electric toothbrush + fluoride-free toothpaste) was performed immediately or 30 min after erosion for 30 s. Dentin wear was analyzed by profilometry (µm); 3 and 4) Volunteers (n=10-13) wore palatal devices containing 12 bovine dentin blocks randomly allocated to 6 groups, according to the type of gels applied or not (not treated-NT). The gels had identical composition, except for the presence of EGCG (400 µM), CHX (0.012%), FeSO4 (1 mM), fluoride (NaF-1.23%) or not (Placebo-P). Gels were applied or not on specimens once in a thin layer and removed after 1 min. Erosion (Coke, pH 2.6, 4x5 min/day, extraorally) was performed for 5 or 10 days, respectively. Each day, after first and last erosive challenges, blocks were (ERO) or not brushed (ERO+ABR) for 15 s (electric toothbrush + fluoride-free toothpaste slurry). Dentin wear was evaluated by profilometry (µm) after days 5 and 10. 5) Demineralization of dentin (n=45/group) was performed with 0.87 M citric acid, pH 2.3, for 36 h. In sequence, dentin was or not (NT) treated with the same gels described above. Specimens were stored in artificial saliva (5 days, 37ºC), containing EDTA-free protease inhibitors and collagenase (Clostridium histolyticum, 238 U/mL). Collagen degradation was analyzed by hydroxyproline content (µg/mL) and dentin wear was evaluated by profilometry (µm, n=12/group). Data were analyzed by ANOVA (p<0.05). Results: Bovine dentin showed to be a reliable substrate for studies involving the activity of MMPs. Green tea significantly reduced the dentin wear for all conditions. The mean wear and collagen degradation was significantly reduced for the experimental gels when compared to NaF and P gels or NT in all protocols tested. For ERO, wear did not significantly increase with time, while for ERO+ABR, significant increase along time was observed for NaF, P and NT. Association between ERO and ABR was only able to significantly increase the wear at 10 days for NaF and NT. Conclusion: The use of green tea rinse and a gel as a vehicle to deliver MMP inhibitors to dentin was shown to consistently reduce collagen degradation and provide long-lasting prevention against dentin erosion and erosion plus abrasion.

A evolução das resinas compostas fez com que esses materiais passassem a ter uma durabilidade maior e características estéticas muito boas, mas o risco de cárie recorrente é ainda um problema a ser resolvido. Na tentativa de solucionar esse problema, estudos vêm sendo conduzidos na tentativa de se formularem resinas compostas contendo agentes antibacterianos, como é o caso da incorporação de clorexidina (CHX). Outro fato que impede a longevidade deste material é a degradação de matriz de colágeno por proteases ativadas por pH ácido. Para tentar contornar esse problema, a adição de clorexidina, assim como Epigallocatechin gallate (EGCg), clássicos antibacterianos e inibidores de proteases da matriz , como as metaloproteinases da matriz (MMP) a resinas, poderia melhorar a eficácia destes materiais como substitutos de dentina em procedimentos restauradores, aumentando a longevidade do tratamento restaurador, mediante preservação das propriedades mecânicas do material. Assim, o objetivo desse estudo é avaliar o poder de inibição de resinas experimentais contendo inibidores conhecidos de proteases da matriz sobre gelatinases e colagenase. Para isso, copolímeros experimentais foram preparados combinando Bis-GMA com o diluente TEGDMA (70/30 mol%). Com exceção do copolímero placebo (sem drogas), EGCg ou CHX foram incorporados a 1% em peso isoladamente ou em combinação, a 0,5% em peso cada. Amostras contendo EGCg, CHX ou EGCg e CHX concentradas 10X foram obtidas do armazenamento de espécimes polimerizados da resina experimental em água deionizada (1 mL) após o período de 24h a 37°C e sua posterior concentração. O efeito da ação dos inibidores foi checado por zimografia e confirmado por um ensaio enzimático específico para colagenases e gelatinases. Os dados passaram por teste de homogeneidade (Bartlett) e normalidade (Kolmogorov-Smirnov) e foram avaliados por ANOVA a 2 critérios, seguido pelo teste de Bonferroni para comparações individuais (p<0,05). Os resultados do presente estudo, mostraram que, in vitro, a liberação de EGCg e CHX incorporados em resinas é capaz de reduzir a atividade gelatinolítica das MMPs -2 e -9, bem como a atividade da colagenase bacteriana, sugerindo um efeito potencial no aumento da longevidade de restaurações de resinas. Com isso, podemos afirmar que a liberação de ativos de resinas experimentais é possível e que esses ativos são capazes de inibir as MMPs, assim sugerindo um novo substituto para dentina em procedimentos restauradores. / The evolution of composite resins made these materials to have a greater durability and very good esthetics characteristics, but the risk of recurrent caries is still a problem to be solved. In the attempt to solve this problem, studies are being conducted with the purpose to formulate composite resins containing antibacterial agents, such as chlorhexidine (CHX). Another fact that prevents the longevity of this material is the degradation of the collagen matrix by the proteases activated by acidic pH. In order to solve this problem, the addition of chlorhexidine and/or Epigallocatechin gallate (EGCg), classical antibacterial agents and inhibitors of matrix proteases, such as matrix metalloproteinases (MMP) in resins, could improve the efficacy of these materials as dentin substitutes in restorative procedures, increasing the longevity of the restorative treatment, while preserving the mechanical properties of the material. Thus, the aim of this study is to evaluate the ability of experimental resins containing known matrix protease inhibitors on the inhibition of gelatinases and collagenase. For this purpose, experimental copolymers were prepared combining Bis-GMA with the diluent TEGDMA (70/30 mol%). Except for the placebo copolymer (drug free), EGCg or CHX were incorporated at 1% in weight, isolated or in combination (0.5% in weight each). Samples containing EGCg, CHX or EGCg and CHX concentrated 10X were obtained after storage of polymerized specimens of the experimental resin in deionized water (1 mL) after the period of 24 h, at 37°C and after that were concentra. The effect of the action of the inhibitors was checked by zymography and confirmed by an enzymatic test specific for collagenases and gelatinases. The data passed in the tests of homogeneity (Bartlett test) and normality (Kolmogorov-Smirnov test), and were evaluated by 2-way ANOVA, followed by Bonferroni test for individual comparisons (p<0.05). The results of this study showed that the in vitro release of EGCG and CHX incorporated in resins was able to reduce the gelatinolytic activity of MMPs-2 and -9 and bacterial collagenase activity, suggesting a potential effect in increasing the longevity of resin restorations. It can be concluded that the release of drugs from experimental resins is possible and that these drugs are able to inhibit MMPs, thereby suggesting a new substitute for dentin in restorative procedures.