Le rôle important joué par les protéases dans plusieurs processus physiologiques (reproduction, cicatrisation et régénération tissulaire) nexclut pas leurs contributions parfois primordiales dans divers pathologies, telles que la croissance tumorale et les métrorragies dysfonctionnelles. Lors de ce travail, nous nous sommes intéressés à létude des protéases et de langiogenèse au niveau de deux aspects distincts de la reproduction : 1) Une situation physiologique quest la placentation par le biais dun modèle murin ; 2) Une situation pathologique chez les femmes souffrant de métrorragies dysfonctionnelles suite à lutilisation dun système intra-utérin à libération de progestatif comme moyen de contraception

Abstract The assay for the carboxyterminal telopeptide of type I collagen, ICTP, has been shown to be a reliable marker in many pathological conditions but insensitive to changes in physiological bone turnover. This has induced uncertainty and confusion regarding the role of ICTP assay in the study of collagen metabolism in bones. Especially, since another assay for the carboxyterminal telopeptide of type I collagen, serum CrossLaps ELISA, sensitively follows the changes in physiological bone turnover. To find out the reasons for the discrepancy we characterized the antigenic determinant of the ICTP assay by comparing human and bovine antigens after trypsin and chymotrypsin treatments. An assay for bovine ICTP was developed contemporarily with the present study. The epitope lies on the phenylalanine rich region of two telopeptide chains. We were able to show that the region is destroyed by cathepsin K, an osteoclastic enzyme responsible for physiological bone turnover, but not by several matrix metalloproteinases (MMPs), which are important collagen degrading enzymes in pathological conditions. Cathepsin K treatment had no effect on the CrossLaps assay. The CrossLaps assay is also able to measure the MMP-derived fragments, but usually their amount is so low in serum that it is masked by the cathepsin K-derived collagen degradation. The results explain the apparent discrepancy regarding the different behaviour of ICTP and CrossLaps assays in various conditions as also verified in our study with rheumatoid arthritis patients. The ICTP assay was also found to measure only trivalently cross-linked forms of the carboxyterminal telopeptide which contains two telopeptide chains, and is therefore unable to react with divalently or histidinohydroxylysinonorleucine (HHL)-cross-linked forms of the carboxyterminal telopeptide. These forms can be measured with the SP4 (synthetic peptide 4) assay. We utilized this property in analyzing the skin samples of 18 breast cancer patients on both the irradiated and unirradiated side. The content of HHL was increased on the irradiated side, as were type I collagen synthesis and degradation. In conclusion, there are two assays for two different degradation products of the trivalently cross-linked carboxyterminal telopeptide of type I collagen, ICTP and CrossLaps, the former measuring the MMP-derived and the latter the cathepsin K-derived collagen degradation.

No description available.

Ανάμεσα στα διάφορα είδη του γένους Crocus, ο C. sativus έχει μελετηθεί εκτενώς ως προς τη σύσταση και τις βιολογικές ιδιότητες των στύλων του, οι οποίοι αποτελούν το γνωστό άρτυμα (κρόκος ή ζαφορά) το οποίο χρησιμοποιείται ευρέως στη Μεσογειακή, την Ινδική και την Κινέζικη μαγειρική. Ανά τους αιώνες, στον κρόκο έχουν αποδοθεί πολλές θεραπευτικές ιδιότητες. Παρά την ύπαρξη πολλών μελετών, οι οποίες αναφέρουν ότι η κροκετίνη και οι κροκίνες από τον κρόκο έχουν ποικίλες βιολογικές δράσεις, θέματα που αφορούν στον τρόπο χορήγησης του κρόκου καθώς και στην απορρόφηση και στο μεταβολισμό των καροτενοειδών του σε ανθρώπους δεν έχουν ακόμα απαντηθεί. Στην παρούσα μελέτη, ισοκρατική μέθοδος υγρής χρωματογραφίας ανάστροφης φάσης αναπτύχθηκε και επικυρώθηκε για την ανίχνευση κροκετίνης σε πλάσμα. Τα αποτελέσματα έδειξαν υψηλή συγκέντρωση της κροκετίνης μετά από 2 ώρες (1.24 -3.67 μΜ) και παρουσία της ακόμα και στις 24 ώρες (0.10-0.24). Ενδιαφέρον προκαλεί η παρουσία του cis-ισομερούς σε ποσοστά 25 με 50%, προτείνοντας in vivo ισομερείωση. Με χρήση υγρής χρωματογραφίας (HPLC) και UV/vis φασματοσκοπίας δείχνουμε για πρώτη φορά την παρουσία υδρόφιλων καροτενοειδών στους στύλους τριών ενδημικών ειδών Crocus: C. boryi ssp. tournefortii, C. boryi ssp. boryi και C. niveus και ότι στα ενδημικά είδη η αλκαλική υδρόλυση οδηγεί σε ένα άγλυκο trans-καροτενοειδές. Με σκοπό την περαιτέρω έρευνα ως προς την επίδραση των μεθανολικών εκχυλισμάτων αλλά και συστατικών του saffron στον πολλαπλασιασμό καρκινικών κυττάρων του μαστού, εφόσον ο καρκίνος του μαστού αποτελεί κύρια αιτία θανάτου σε γυναίκες, χρησιμοποιήσαμε τις κυτταρικές σειρές MCF-7 και MDA-MB-231 ενώ μετρήθηκε και η επίδραση στην διηθητικότητα της κυτταρικής σειράς MDA-MB-231. Μετά από επώαση 24 ωρών, η κροκετίνη ανέστειλε σημαντικά όχι μόνο τον πολλαπλασιασμό αλλά επίσης την διήθηση, στις συγκεντρώσεις 1 και 10 μM. Μελέτες όσον αφορά στις μοριακές αλλαγές στην έκφραση των MMPs και των TIMPs, έδειξαν σημαντική μείωση στη γονιδιακή και πρωτεϊνική έκφραση των MT1-MMP και MT2-MMP και των ζελατινασών. / Among the different species of Crocus, only C. sativus has been extensively studied for the composition and the biological properties of its styles, since these constitute the well-known spice saffron, which is widely used in the Mediterranean, Indian and Chinese diet. Throughout centuries, saffron is attributed with many therapeutic uses. Crocetin is a carotenoid dicarboxylic acid, which in nature is esterified with glucose or gentiobiose units forming the crocins, abundant components of saffron (a spice with many reputed medicinal uses). Although studies have demonstrated that crocetin and crocins from saffron have various biological functions, issues concerning the route and way of saffron administration, the absorption and metabolism of saffron carotenoids in humans have not been answered yet. In the present study, an isocratic reversed-phase liquid chromatographic method was developed and validated for the determination of crocetin in plasma. Samples were pre-treated by solid phase extraction (recoveries >72%) and were chromatographed on a Luna C-18 column (4.6 mm x 250 mm, 5 μm) with a mobile phase consisting of methanol/water/trifluoroacetic acid (75.0/24.5/0.5, % v/v/v) at a flow rate of 1.0 mL min-1. The HPLC method developed resulted in sharp peaks at 10.7 (trans-crocetin) and 18.6 min (cis-crocetin), whereas the calibration curve of total crocetin in plasma displayed a good linearity for concentrations of 0.020 to 20 μΜ (R2=0.999). Specificity, precision, accuracy and stability were also studied with spiked plasma samples and were acceptable. The developed method was applied to the determination of crocetin levels in plasma of four healthy human volunteers before and after consumption of one cup of saffron infusion (200 mg of saffron in water 80°C for 5 min). Results showed that the concentration of crocetin was high after 2h (1.24 -3.67 μΜ) and still determined after 24 h (0.10-0.24). Interestingly, the percentage of the cis-isomer ranges from 25 to 50%, suggesting in vivo isomerization. With high performance liquid chromatography (HPLC) and UV/vis spectroscopy we show for the first time, the presence of hydrophilic carotenoids in the styles of three other Crocus taxa, endemic in Greece: C. boryi ssp. tournefortii, C. boryi ssp. boryi and C. niveus. In order to investigate the effect of the methanolic extracts and the constituents of saffron on breast cancer cell proliferation, since breast cancer is a major cause of death among women, we used the cell lines MCF-7 and MDA-MB-231 cells. Incubation for 24 and 48 h showed an inhibitory effect on cell proliferation. Studies on the effect of constituents of C. sativus styles showed that the antiproliferative effect is mainly attributed to crocetin. In order to further study the effect of crocetin, the active metabolite of crocins, on breast cancer cells, we used the highly invasive MDA-MB-231 cells and measured the viability with the WST-1 assay and the invasiveness through a reconstituted basement membrane. After 24 h incubation, crocetin inhibited not only proliferation but also invasion significantly at the physiological relevant concentrations of 1 and 10 μM. Studies in the molecular changes of expression of MMPs and their endogenous inhibitors (TIMPs), were performed following reversed transcription and polymerase chain reaction, whereas protein expression and gelatinolytic activity were determined with Western blotting and zymography. The gene and protein expression of MT1-MMP and MT2-MMP were greatly attenuated by both crocetin and all-trans-retinoic acid (ATRA, used as control). Incubation with 10 μM crocetin for 24 h in serum free conditions reduced pro-MMP-9 levels, whereas when cultured in media with sera 2 and 5%, it also reduced gelatinase protein expression. Crocetin and ATRA also upregulate TIMP-1 and TIMP-3 mRNA expression and downregulate TIMP-2 expression.

Osteopontin (OPN) plays an important role in left ventricular (LV) remodeling after myocardial infarction (MI) by promoting collagen synthesis and accumulation. This study tested the hypothesis that MMP inhibition modulates post-MI LV remodeling in mice lacking OPN. Wild-type (WT) and OPN knockout (KO) mice were treated daily with MMP inhibitor (PD166793, 30 mg/kg/day) starting 3 days post-MI. LV functional and structural remodeling was measured 14 days post-MI. Infarct size was similar in WT and KO groups with or without MMP inhibition. M-mode echocardiography showed greater increase in LV end-diastolic (LVEDD) and end-systolic diameters (LVESD) and decrease in percent fractional shortening (%FS) and ejection fraction in KO-MI versus WT-MI. MMP inhibition decreased LVEDD and LVESD, and increased %FS in both groups. Interestingly, the effect was more pronounced in KO-MI group versus WT-MI (P < 0.01). MMP inhibition significantly decreased post-MI LV dilation in KO-MI group as measured by Langendorff-perfusion analysis. MMP inhibition improved LV developed pressures in both MI groups. However, the improvement was significantly higher in KO-MI group versus WT-MI (P < 0.05). MMP inhibition increased heart weight-to-body weight ratio, myocyte cross-sectional area, fibrosis and septal wall thickness only in KO-MI. Percent apoptotic myocytes in the non-infarct area was not different between the treatment groups. Expression and activity of MMP-2 and MMP-9 in the non-infarct area was higher in KO-MI group 3 days post-MI. MMP inhibition reduced MMP-2 activity in KO-MI with no effect on the expression of TIMP-2 and TIMP-4 14 days post-MI. Thus, activation of MMPs contributes to reduced fibrosis and LV dysfunction in mice lacking OPN.

As metaloproteinases da matriz (MMPs) são enzimas proteolíticas dependentes de zinco que degradam componentes da matriz extracelular, facilitando a remodelação tecidual e a migração celular. MMPs secretadas por osteoclastos exercem papel central na absorção óssea fisiológica e estão também associadas a processos de degradação patológica do osso. No entanto, o papel essencialmente degradador de osso das MMPs, particularmente da MMP-2, vem sendo questionado em anos recentes por estudos que evidenciam sua importância na diferenciação de células da linhagem osteoblástica e na formação de tecido ósseo em cultura. Neste sentido, é possível que a MMP-2 exerça um papel importante na formação de tecido ósseo em processo de reparação. O objetivo do presente trabalho foi investigar a pretensa ação osteo-estimulatória da rhMMP-2 ligada à monoleína (usada como carreador) implantada em defeito confeccionado na calvária de ratos. Foram confeccionados defeitos ósseos unilaterais de 4 mm de diâmetro na calvária de ratos adultos; nos animais controles o defeito ósseo foi mantido com o preenchimento natural de coágulo sanguíneo e nos animais implantados o defeito foi preenchido com monoleína ou com rhMMP-2 ligada à monoleína. Os animais foram eutanasiados após 2 e 4 semanas e a taxa de neoformação óssea foi estimada em cortes histológicos por um método histométrico de contagem diferencial de pontos. A taxa de neoformação óssea foi semelhante nos animais dos grupos controle e monoleína e significativamente maior nos animais do grupo MMP-2, em ambos os períodos analisados. Os resultados permitem concluir que a monoleína não interferiu com o processo reparacional e pareceu eficaz como carreador da rhMMP-2, e adicionam evidências á hipótese da importância da atividade da MMP-2 para a formação óssea, em um modelo experimental in vivo de reparo ósseo. / Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade extracellular matrix components, facilitating cell migration and tissue remodeling. MMPs secreted by osteclasts are important in the physiological bone resorption as in pathological bone degradation. However, the essentially bone absorbing hole of MMPs, particularly of the MMP-2, has been questioned in recent years by studies that show its importance in osteoblastic cells differentiation and in vitro bone formation. Therefore, the MMP-2 may have also an important hole in reparational bone formation. The purpose of the present study was to investigate the pretense osteostimulatory effect of the rhMMP-2 linked to monoolein (used as a carrier) implanted into rat calvarial defects. Bone defects of 4mm in diameter were created unilaterally in rats calvaria and filled with natural blood clot (control), monoolein or rhMMP-2 linked to monoolein. The animals were killed 2 and 4 weeks postoperatively and the rate of new bone formation was estimated in histological sections by a histometric differential point-counting method. The rate of reparational bone formation was similar in the animals from control and monoolein groups and was significantly greater in the MMP-2 group, in both periods. From the results it may be concluded that monoolein did not interfere with the reparacional process and seemed effective as a rhMMP-2 carrier. In addition, the results add evidence to the importance of MMP-2 activity for bone formation, in an in vivo bone healing experimental model.

As metaloproteinases da matriz (MMPs) são enzimas proteolíticas dependentes de zinco que degradam componentes da matriz extracelular, facilitando a remodelação tecidual e a migração celular. MMPs secretadas por osteoclastos exercem papel central na absorção óssea fisiológica e estão também associadas a processos de degradação patológica do osso. No entanto, o papel essencialmente degradador de osso das MMPs, particularmente da MMP-2, vem sendo questionado em anos recentes por estudos que evidenciam sua importância na diferenciação de células da linhagem osteoblástica e na formação de tecido ósseo em cultura. Neste sentido, é possível que a MMP-2 exerça um papel importante na formação de tecido ósseo em processo de reparação. O objetivo do presente trabalho foi investigar a pretensa ação osteo-estimulatória da rhMMP-2 ligada à monoleína (usada como carreador) implantada em defeito confeccionado na calvária de ratos. Foram confeccionados defeitos ósseos unilaterais de 4 mm de diâmetro na calvária de ratos adultos; nos animais controles o defeito ósseo foi mantido com o preenchimento natural de coágulo sanguíneo e nos animais implantados o defeito foi preenchido com monoleína ou com rhMMP-2 ligada à monoleína. Os animais foram eutanasiados após 2 e 4 semanas e a taxa de neoformação óssea foi estimada em cortes histológicos por um método histométrico de contagem diferencial de pontos. A taxa de neoformação óssea foi semelhante nos animais dos grupos controle e monoleína e significativamente maior nos animais do grupo MMP-2, em ambos os períodos analisados. Os resultados permitem concluir que a monoleína não interferiu com o processo reparacional e pareceu eficaz como carreador da rhMMP-2, e adicionam evidências á hipótese da importância da atividade da MMP-2 para a formação óssea, em um modelo experimental in vivo de reparo ósseo. / Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade extracellular matrix components, facilitating cell migration and tissue remodeling. MMPs secreted by osteclasts are important in the physiological bone resorption as in pathological bone degradation. However, the essentially bone absorbing hole of MMPs, particularly of the MMP-2, has been questioned in recent years by studies that show its importance in osteoblastic cells differentiation and in vitro bone formation. Therefore, the MMP-2 may have also an important hole in reparational bone formation. The purpose of the present study was to investigate the pretense osteostimulatory effect of the rhMMP-2 linked to monoolein (used as a carrier) implanted into rat calvarial defects. Bone defects of 4mm in diameter were created unilaterally in rats calvaria and filled with natural blood clot (control), monoolein or rhMMP-2 linked to monoolein. The animals were killed 2 and 4 weeks postoperatively and the rate of new bone formation was estimated in histological sections by a histometric differential point-counting method. The rate of reparational bone formation was similar in the animals from control and monoolein groups and was significantly greater in the MMP-2 group, in both periods. From the results it may be concluded that monoolein did not interfere with the reparacional process and seemed effective as a rhMMP-2 carrier. In addition, the results add evidence to the importance of MMP-2 activity for bone formation, in an in vivo bone healing experimental model.

Le développement dentaire est orchestré par une série de signalisations inductives réciproques entre l'épithélium dentaire et le mésenchyme, qui conduit à la formation de la dentine et de l'émail. EMMPRIN/CD147 est un INducteur des MetalloPRoteinases de la Matrice Extracellulaire (MMPs) qui régule les interactions épithélio-mésenchymateuses dans le cancer et d'autres processus pathologiques et est exprimé lors du développement dentaire. Ainsi, nous avons utilisé des souris KO pour EMMPRIN pour déterminer le rôle d'EMMPRIN dans la formation des tissus dentaires. Nous avons démontré que l’absence d’EMMPRIN conduisait dans le germe dentaire à une diminution de l’expression de MMP-3 et de MMP-20, à un retard de la dégradation de la membrane basale, à un retard de la formation de l’émail bien visible dans l'incisive à croissance continue, à une diminution du volume et de l'épaisseur d'émail, mais à une maturation amélaire normale. Ces résultats indiquent qu'EMMPRIN est impliqué dans le dialogue épithélio-mésenchymateuse pendant le développement dentaire, principalement par la régulation de l'expression de certaines MMPS. Nous avons ensuite essayé d'évaluer le rôle potentiel d'EMMPRIN dans le processus de réparation dentaire en comparant la cicatrisation de blessures pulpaires des souris KO pour EMMPRIN à des souris WT. Enfin, dans un souci de transfert vers la clinique, nous avons évalué la capacité d’extraits de pépin de raisin (connu pour être des inhibiteurs naturels de MMPs) à empêcher la dégradation de la matrice dentinaire humaine déminéralisée et traitée par MMP-3. / Tooth development is regulated by a series of reciprocal inductive signalings between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissues formation. We demonstrated that EMMPRIN deficiency results in decreased in MMP-3 and MMP-20 expressions, delayed in basement membrane degradation in tooth germ, delayed in enamel formation well distinguishable in incisor, and in decreased enamel volume and thickness but normal maturation. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. Then we tried to investigate the potential role of EMMPRIN in the pulp dentin repair process by comparing the healing of injured pulps of EMMPRIN KO and WT mice. Finally, we evaluated the capacity of grape-seed extracts (known to be natural inhibitors of MMPs and used in new daily mouthrinse) to prevent the degradation of human demineralized dentin matrix by MMP-3.

La maladie d’Alzheimer (MA) est la maladie neurodégénérative la plus commune et reste à ce jour incurable. Elle se caractérise par l’accumulation dans le cerveau du peptide neurotoxique bêta-amyloïde (Aβ), par une neuroinflammation et des atteintes neurovasculaires, qui ensemble induisent la mort des neurones et des déficits cognitifs. En raison de leurs activités multiples, les métalloprotéases matricielles (MMPs) émergent comme des acteurs importants dans la MA. Mes travaux ont permis de mieux comprendre l’implication de 3 de ces MMPs dans la MA et soulignent le caractère spécifique et complémentaire de MT1- et MT5-MMP, directement impliquées dans la production d’Aβ, et le rôle de MMP-12 dans la neuroinflammation et dans la perte d’intégrité de la barrière hémato-encéphalique, un système vasculaire particulier qui protège efficacement le cerveau. Mes travaux ouvrent des perspectives dans le développement de nouvelles stratégies thérapeutiques basées sur la modulation de ces MMPs. / Alzheimer's disease (AD) is the most common neurodegenerative disease and remains to this day incurable. It is characterized by the accumulation in the brain of the beta-amyloid (Aß) neurotoxic peptide, by neuroinflammation and neurovascular damage, which together induce neuronal death and cognitive deficits. Because of their multiple activities, matrix metalloproteinases (MMPs) are emerging as important players in AD. My work has provided insight into the involvement of 3 of these MMPs in AD and highlight the specific and complementary nature of MT1- and MT5-MMP, directly involved in the production of Aß, and the role of MMP-12 in neuroinflammation and in the loss of integrity of the blood-brain barrier, a particular vascular system, which effectively protects the brain. My work opens perspectives in the development of new therapeutic strategies based on the modulation of these MMPs.

Metastasis is one of the main causes of death in cancer patients. Metastatic melanoma is a death sentence, as chemotherapeutic agents have a 5% success rate or do not extend survival beyond 10 months. The lack of effective chemotherapeutic agents for treating metastatic melanoma indicates a dire need to identify new drug targets and develop new therapies. Our lab has previously shown that the kinase activity of Abelson family of non-receptor tyrosine kinases (c-Abl and Arg) is elevated in invasive breast cancer cell lines as compared to non-invasive cell lines. Previous studies from our lab have shown that Abl kinases are convergent point of ErbB2 and Src Kinases in melanoma cells and Abl kinases promote invasion by an undefined mechanism. Although Abl kinases promote invasion, it is not known whether they are important for metastastic potential. For the first time, we report that Abl kinases promote melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. To investigate the mechanism by which Abl kinases promote invasion, we found out that active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3àMMP-1 pathway. In contrast, active Arg drives invasion in a STAT3-independent manner, and upregulates the expression of MMP-3 and MT1-MMP, in addition to MMP-1. We also found that Abl kinases promote invasion via lysosomal degradation of a metastasis suppressor, NM23-H1 by activating lysosomal cathepsins B and L, which directly cleave and degrade NM23-H1. Furthermore, c-Abl and Arg are activated in primary melanomas and cAbl/Arg activity is inversely correlated with NM23-H1 expression both in primary melanoma and human melanoma cells. We also demonstrate, for the first time that active Abl kinases promote metastasis in vivo, as inhibition of c-Abl/Arg with nilotinib, dramatically inhibits lung colonization/metastasis in a mouse model using two different melanoma cell lines. In summary, we identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and our data indicate that nilotinib may be useful in preventing metastasis in a select group of patients, harboring active Abl kinases.