Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a protein with multiple functions that include regulating the turnover of the extracellular matrix (ECM) by inhibiting members of the metzincin family. Extracellular levels of soluble TIMP-3 are low, reflecting its binding to components of the ECM including sulfated glycosaminoglycans (SGAGs) and its endocytosis by low density lipoprotein receptor-related protein 1. Because TIMP-3 inhibits ECM-degrading enzymes, the ability of SGAG mimetics to elevate extracellular concentrations of TIMP3 is of interest for osteoarthritis treatment. However, previous studies of such interactions have utilized immobilized forms of the protein or ligands. Here we have quantified the thermodynamics of the interactions of the inhibitory domain of TIMP-3 with chondroitin sulfate (CS), pentosan polysulfate (PPS) and suramin in solution using isothermal titration calorimetry. All three interactions are driven by a (favorable) negative enthalpy ychange combined with an unfavorable decrease in entropy. The heat capacity change (ΔCp) for the interaction of N-TIMP-3 with CS, PPS, or suramin is essentially zero, indicating an insignificant contribution from the hydrophobic effect. Based on the effects of ionic strength on the interaction of N-TIMP-3 with suramin, their interaction appears to be driven by electrostatic interactions. Modeling supports the view that the negatively charged sulfates of CS, PPS, and suramin interact with a cationic region on N-TIMP-3 that includes Lys -26, -27, -30, and -possibly 76 on the opposite face of TIMP-3 from its reactive site for metalloproteases. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Chronic obstructive pulmonary disease (COPD) is characterized by a non-fully reversible airflow limitation and a chronic inflammatory response accompanied by the development of emphysema. The β2-adrenoceptor agonists and anticholinergic agents are widely used in patients with COPD due to their bronchodilator properties. Today, many studies in vitro and in vivo in experimental animal models have shown that these bronchodilators also exert anti-inflammatory effects, but their protective roles against lung inflammation and the development of emphysema in patients with COPD remain to be determined. The imbalance between the activity of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) is considered to play a key role in the pathogenesis of COPD, especially in the development of pulmonary emphysema. The modulation of inflammatory responses and emphysema via the inhibition of the MMPs activity induced by the use of synthetic inhibitors of MMPs suggests that MMPs may be therapeutic targets for COPD patients. However, very few studies have demonstrated the regulation exerted by β2-adrenoceptor agonists and anticholinergic agents on the activity of MMPs. The combination of β2-adrenoceptor agonists with anticholinergic agents has been found to exert an additive and even synergistic bronchodilator effect, but nothing is known about their combination on the inflammatory pathogenesis and the development of emphysema. Thus, a better knowledge of the activities of β2-adrenoceptor agonists and anticholinergic agents on controlling pulmonary inflammation and emphysema in COPD could provide a new therapeutic approach in this area. The first goal of the present work was to investigate the effects of formoterol, a β2-adrenoceptor agonist and/or ipratropium bromide, an anticholinergic agent, on acute pulmonary inflammation induced by cadmium inhalation in rats. In addition, we examined whether the expected anti-inflammatory effects of formoterol and/or of ipratropium bromide were associated with a modulation of the gelatinase A (MMP-2), gelatinase B (MMP-9) and macrophage metalloelastase (MMP-12) activity. Compared with the data observed in rats exposed to a single dose of cadmium, the pre-administration of formoterol or ipratropium bromide inhibited the cadmium-induced increase in airway resistance. Formoterol significantly reduced the total cell, neutrophil and macrophage counts in bronchoalveolar lavage fluid (BALF), whereas, ipratropium bromide only reduced the neutrophil number. Both bronchodilators administrated alone attenuated significantly the lung lesions associated with parenchyma inflammatory cell influx and congestion observed in cadmium-group. The increased MMP-9 activity was significantly attenuated. A reduction of pulmonary edema was also detected by measuring the lung wet-to-dry weight ratio. However, no additive or synergistic effect was obtained when formoterol was administrated in combination with ipratropium bromide. In conclusion, formoterol and ipratropium bromide partially protect the lungs against inflammation by reducing the neutrophilic infiltration. This protective effect may be related to the reduction of MMP-9 activity which plays an important role in the acute inflammation. Up to now, the impact of a long-term administration of bronchodilators aiming to control the chronic inflammation and the development of emphysema in experimental animal models and in patients with COPD has been poorly investigated. In this context, it was rational to investigate whether the protective role of formoterol and ipratropium bromide identified in acute conditions persists in a rat model of subacute neutrophilic pulmonary inflammation with an enlargement of airspaces. In the second part of this study, we also intended to determine whether these anti-inflammatory effects are related to the modulation of imbalance between MMPs and TIMPs. Though ipratropium induced no effect on the subacute pulmonary inflammation and the airspace enlargement induced by repeated cadmium inhalations during 5 weeks in rats, formoterol elicited marked anti-inflammatory effects on the increase of total cell and neutrophil counts as well as the activity of MMP-9 mainly expressed in alveolar macrophages and epithelial cells. This drug also prevented the inflammatory infiltration in alveoli and in interstitial tissue and significantly inhibited the airspace enlargement as demonstrated by the significant decrease in the mean linear intercept (Lm). The combination of both bronchodilators at inefficient concentrations induced synergistic inhibitory effects on the total cell and neutrophil counts and on the cadmium-induced increased Lm associated with a reduction of MMP-9 activity in BALF. These data suggest that formoterol alone or combined with ipratropium could protect lungs against subacute pulmonary inflammation and the airspace enlargement by inhibiting neutrophilic infiltration via the reduction of MMP-9 activity. To the best of our knowledge, this is the first report which reveals the anti-inflammatory effects of β2-adrenoceptor agonists and anticholinergic agents in an animal model which mimics the main features of COPD. The data obtained in this work contribute to identify new therapeutic targets in COPD for drugs currently used in clinical practice./ La broncho-pneumopathie chronique obstructive (BPCO) est caractérisée essentiellement par une limitation du débit aérien qui n'est pas entièrement réversible et une inflammation chronique pulmonaire accompagnée dun développement demphysème. Les agonistes β2-adrénergiques et les anticholinergiques sont largement utilisés chez les patients atteints de BPCO en raison de leurs propriétés bronchodilatatrices. Aujourdhui, de nombreuses études expérimentales in vitro et in vivo, utilisant des modèles animaux, ont montré que ces bronchodilatateurs exerçaient également des effets anti-inflammatoires. Leur rôle protecteur contre linflammation pulmonaire et le développement d'emphysème chez des patients souffrant de BPCO reste à déterminer. Le déséquilibre entre les métalloprotéinases de la matrice (MMPs) et leurs inhibiteurs tissulaires (TIMPs) est considéré comme un mécanisme clé dans lévolution de la maladie et surtout dans le développement d'emphysème pulmonaire. La modulation des réactions inflammatoires et de lemphysème obtenue grâce à la réduction de lactivité des MMPs induite par des inhibiteurs synthétiques suggère que les MMPs pourraient être des cibles thérapeutiques importantes dans le traitement de la BPCO. Mais jusquà ce jour, très peu détudes ont été consacrées à la régulation de lactivité des MMPs par les agonistes β2-adrénergiques et les anticholinergiques. Lassociation dun agoniste β2-adrénergique avec un anticholinergique donne lieu à une amplification des effets bronchodilatateurs, mais il nest pas certain que leur combinaison débouche sur des effets additifs ou synergiques sur le plan dun meilleur contrôle de la réaction inflammatoire. Ainsi, une meilleure connaissance des activités des agonistes β2-adrénergiques et des anticholinergiques visant au contrôle de l'inflammation pulmonaire et de l'emphysème dans la BPCO pourrait fournir une nouvelle approche thérapeutique dans ce domaine. Lobjectif de la première partie de ce travail était donc d'étudier les effets du formotérol, un agoniste β2-adrénergique et / ou du bromure d'ipratropium, un anticholinergique, sur l'inflammation pulmonaire aiguë provoquée par linhalation de cadmium chez le rat. En outre, nous voulions aussi vérifier si ces effets anti-inflammatoires étaient associés à une modulation de lactivité de la gélatinase A (MMP-2), de la gélatinase B (MMP-9) et de la métallo-élastase du macrophage (MMP-12). Par rapport aux effets observés chez des rats exposés à une dose de cadmium, ladministration préventive de formotérol ou de bromure dipratropium a atténué laugmentation de la résistance des voies aériennes. Le formotérol a induit une diminution significative du nombre de cellules totales, des neutrophiles et des macrophages dans le liquide de lavage broncho-alvéolaire (BALF). Par contre, le bromure dipratropium na entraîné quune diminution du nombre de neutrophiles. Les lésions pulmonaires caractérisées par de la congestion et une réaction inflammatoire du parenchyme ont été significativement inhibées par ces deux bronchodilatateurs administrés séparément. Lélévation remarquable de lactivité de MMP-9 dans le BALF a été significativement atténuée par le prétraitement au formotérol ou au bromure dipratropium. Il en est de même pour ldème pulmonaire évalué par le biais du rapport entre le poids humide et le poids sec du parenchyme. Lorsque les deux principes actifs ont été combinés et administrés préventivement à laction du cadmium, aucun effet synergique ou additif na été constaté. En conclusion, le formotérol et le bromure dipratropium préviennent partiellement linflammation pulmonaire aiguë en réduisant linfiltration neutrophilique du parenchyme pulmonaire faisant suite à une exposition aiguë au cadmium. Cet effet protecteur pourrait être lié à une réduction de lactivité de MMP-9 qui joue un rôle pro-inflammatoire important dans linflammation aiguë. Jusquici, les effets potentiels des bronchodilatateurs contre linflammation chronique et lévolution de lemphysème pulmonaire chez des animaux et chez les patients atteints de BPCO restent mal connus. Il nous restait donc à vérifier si les effets protecteurs du formotérol et du bromure dipratropium révélés par nos premières études au cours desquelles les rats ont été exposés de manière aiguë au cadmium persistent dans un modèle dinflammation pulmonaire subaiguë accompagnée dun élargissement des espaces aériens. Nous voulions également déterminer si ces effets étaient liés à la modulation du déséquilibre entre les MMP-2/9/12 et les TIMP-1/2. Bien que le bromure dipratropium nait aucun effet sur linflammation subaiguë pulmonaire et lélargissement des espaces aériens induits par des inhalations répétées de cadmium chez le rat, le prétraitement par du formotérol a, quant à lui, inhibé significativement laugmentation du nombre de cellules totales et des neutrophiles ainsi que de lactivité de MMP-9 exprimée principalement dans les macrophages et les cellules épithéliales alvéolaires. En outre, une atténuation importante des lésions pulmonaires caractérisées par un élargissement des espaces aériens les plus distaux et une infiltration de cellules inflammatoires dans les alvéoles et le tissu ont été observées. La combinaison des deux bronchodilatateurs, à des concentrations pourtant inefficaces, a provoqué un effet synergique sur la plupart des paramètres étudiés, en particulier sur linfiltration par les neutrophiles et lactivité de MMP-9 dans le BALF. Ce travail suggère que le formotérol, seul ou combiné avec le bromure dipratropium, pourrait protéger partiellement les poumons contre linflammation pulmonaire et lélargissement des espaces aériens en réduisant l'infiltration neutrophilique éventuellement via l'inhibition de lactivité de MMP-9. A notre connaissance, il sagit du premier rapport montrant les effets anti-inflammatoires des agonistes β2-adrénergiques et des anticholinergiques dans un modèle animal mimant les principales caractéristiques physiopathologiques de la BPCO. Les données obtenues dans ce travail pourraient contribuer à identifier de nouvelles cibles thérapeutiques pour cette maladie.

Compelling evidence indicated that major risk factors for atherosclerosis such as oxidatively modified low density lipoprotein (oxLDL), high glucose, and reactive oxygen species promote endothelial cell apoptosis and thereby may contribute to the initiation of atherosclerotic lesion formation. Using fast protein liquid chromatography (FPLC), plasma LDL from familial hypercholesterolemic (FH) patients were separated into five subfractions, L1¡VL5. Among them, L5 subfraction was highly electronegative and suppressed DNA synthesis in cultured bovine aortic endothelial cells (BAEC) and stimulated mononuclear cell adhesion to cultured endothelial cells in vitro. Because impaired angiogenesis plays an important role in the pathogenesis of atherosclerosis, the anti-angiogenic functions of LDL subfractions from FH subjects were examined. Subconfluent BAEC (6 to 10 passages) maintained in DMEM containing 10% serum were treated with LDL subfractions at a dose of 20 £gg/ml, and the effects on anti-angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, and secretion of matrix metalloproteinase (MMP) were determined. Similar to Cu2+ ox-LDL, FH-L4 and FH-L5 inhibited cell proliferation to 80.9¡Ó2.4% (p<0.05) and 58.5¡Ó4.3% of control (p<0.001), respectively, while the other FH (L1-L3) and all subfractions isolated from normocholesterolemic (N) subjects had negligible effects. Similarly, FH-L4 and -L5, but not FH-L1 to -L3, retarded cell migration to 326.9 ¡Ó 19.4 (p<0.05) and 215¡Ó16 cells (p<0.001 with the control values of 402¡Ó34 cells), respectively. FH-L5 induced almost 20% of BAEC to undergo apoptosis; FH-L4 caused very mild effects, and other subfractions did not affect apoptosis In addition, FH-L4 and -L5 perturbed tube formation by BAEC in culture (5.8¡Ó0.2 and 3.4¡Ó0.4, respectively, versus control 8.5¡Ó1.5 tubes). Finally, FH-L4 and -L5 inhibited secretion of MMP-2 by BAEC (72.7¡Ó6.9 and 18.9¡Ó4.8% of control, respectively). The results demonstrate that FH-L5 potently affects multiple processes that are vital to normal angiogenesis, FH-L4 had milder effects, and other FH and N subfractions had negligible effects. In turn, these effects in vitro on processes pivotal to angiogenesis are consistent with potential effects of ox-LDL on endothelial dysfunction during atherogenesis in vivo.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

 Squamous cell carcinoma of the tongue has a low survival rate, with cure rate reduced by half if cervical lymph node metastasis is present. Standard treatment regimen includes surgical resection of the tumor, radiotherapy and chemotherapy. These treatment modalities, however, can result in irreversible side effects including loss of form and function of the tongue. So far, there is no efficient treatment regime targeting migration and invasion of tongue carcinoma. Curcumin is a natural polyphenol extracted from Curcuma longa. Recent studies indicated that curcumin is a potential anti-cancer agent. The anticancer effects have been demonstrated in numerous cancers including lung cancer, liver cancer, breast cancer, prostate cancer and melanoma. In head and neck cancers, the number studies is limited and its inhibitory effects in migration and invasion is rarely explored. To explore the global expression changes in tongue cancer, we used microarray to evaluate the genes responsive to curcumin treatment and focused on genes related to migration and invasion of tongue cancer cell line HN21B. The genes down-regulated by curcumin were validated in HN21B and two other tongue cancer cell line CAL27 and HN96 using qRT-PCR, Western blotting and immunostaining. The identified genes were quantified in tongue carcinoma tissues to examine whether it was up-regulated in human tongue tissues. Scratch wound assay and radial-migration assay were used to assess the degree of inhibition on migration. Adhesion and invasion assays were also performed to assess the adhesion and invasion ability. Transcriptomic analyses showed that MMP-10 was 2.36 fold down-regulated in HN21B in response to curcumin. Curcumin treatment resulted in down-regulation of MMP-10 gene in all the 3 tongue carcinoma cell lines at mRNA and protein levels. Out of 24 tongue carcinoma cases, 55% tumor tissue had obvious up-regulation of MMP-10 expression in comparison with the normal counterpart. Adhesion, migration and invasion ability of tongue carcinoma cell lines was significantly reduced upon IC50 of curcumin treatment in all TSCC cell lines. In conclusion, our results indicated that curcumin could reduce migration, adhesion and invasion in tongue carcinoma cells partly through reducing MMP-10 expression. Further investigations are warranted to explore the potential therapeutic use of curcumin to inhibit migration and invasion of tongue carcinoma cells. / published_or_final_version / Surgery / Master / Master of Philosophy

Η κιρσοκήλη αποτελεί τη συχνότερη αιτία ανδρικής υπογονιμότητας και ορίζεται ως η παθολογική κιρσοειδής διεύρυνση των φλεβών του σπερματικού τόνου (ελικοειδούς πλέγματος). Η επίπτωση της κιρσοκήλης στο γενικό πληθυσμό κυμαίνεται σε ποσοστό 15‐20%, ενώ στους υπογόνιμους άνδρες αγγίζει το 40%. Παρά το γεγονός ότι η κιρσοκήλη είναι σπάνια στα παιδία, πρόσφατες μελέτες έδειξαν πως το ποσοστό της αγγίζει το 6% σε παιδιά ηλικίας 10 ετών, ενώ στους εφήβους το αντίστοιχο ποσοστό ανέρχεται στο 16%. Πιθανότατα, προκαλείται λόγω ανεπάρκειας των φλεβικών βαλβίδων, κάτι που σαν αποτέλεσμα έχει την παλινδρόμηση του αίματος εντός των σπερματικών φλεβών. Η κιρσοκήλη εμφανίζεται κυρίως στην αριστερή πλευρά, εξαιτίας της ανατομίας των φλεβών στην περιοχή αυτή. Παρά την αυξημένη συχνότητα της, η παθοφυσιολογία της παραμένει εν πολλοίς άγνωστη. Πρόσφατες μελέτες σε κιρσοειδείς φλέβες κάτω άκρων έχουν δείξει ότι η μείωση του ρυθμού απόπτωσης σχετίζεται με την εμφάνιση πρωτοπαθούς φλεβίτιδας. Επιπλέον αυξημένη παραγωγή των συστατικών της εξωκυττάριας θεμέλιας ουσίας, οδηγεί στις μορφολογικές αλλοίωσεις και στην απώλεια του φλεβικού τόνου που χαρακτηρίζει τους κιρσούς κάτω άκρων. Σκοπός: Θεωρώντας ότι αντίστοιχοι μηχανισμοί πρέπει να ισχύουν όχι μόνο στους κιρσούς κάτω άκρων αλλά και στην κιρσοκήλη, υποθέσαμε ότι στο τοίχωμα των κιρσοειδών έσω σπερματικών φλεβών θα παρατηρείται κατ’ αναλογία μείωση του ρυθμού απόπτωσης και αύξηση στο ρυθμό σύνθεσης των συστατικών της εξωκυττάριας θεμέλιας ουσίας με μείωση της έκφρασης των MMPs (MMP‐1, MMP‐ 9) και αντίστοιχη αύξηση των TIMPs (TIMP‐1). Μέθοδοι: Το υλικό μελέτης αποτελούσαν 45 δείγματα έσω σπερματικών φλεβών ασθενών με κιρσοκήλη. Σαν μάρτυρες χρησιμοποιήθηκαν κλάδοι των κάτω επιγαστρικών φλεβών που αφαιρέθηκαν από τον κάθε ασθενή κατά τη διάρκεια χειρουργικής επέμβασης, έτσι ώστε ο κάθε ασθενής να αποτελεί και τον δικό του μάρτυρα. Προσδιορίσαμε τις μορφολογικές αλλοιώσεις στο αγγειακό τοίχωμα με ανοσοϊστοχημική χρώση έναντι της ακτίνης. Επιπλέον, μελετήσαμε την έκφραση των MMP‐1 και MMP‐9 καθώς και του TIMP‐1 στο τοίχωμα των κιρσοειδών όσο και των υγιών φλεβών. Τέλος, το σύνολο των δειγμάτων, εξετάσθηκαν ανοσοϊστοχημικά για την ανίχνευση των μεσολαβητών που ρυθμίζουν το ενδογενές (Bcl‐2, Cas‐9) και το εξωγενές (Cas‐8) μονοπάτι της απόπτωσης. Αποτελέσματα: Συγκριτικά με τις φυσιολογικές φλέβες, οι κιρσοειδείς εμφάνισαν πάχυνση του μέσου χιτώνα. Οι MMP‐1 και TIMP‐1 εκφράσθηκαν στο κυτταρόπλασμα των λείων μυϊκών κυττάρων του μέσου χιτώνα των αγγείων, τόσο στις φυσιολογικές όσο και στις κιρσοειδώς διευρυμένες, με την ένταση της έκφρασης όμως, να είναι μεγαλύτερη στους κιρσούς. Η MMP‐9 εντοπίσθηκε στο κυτταρόπλασμα των λείων μυϊκών κυττάρων του μέσου χιτώνα μόνο των κιρσών. Όσον αφορά την έκφραση των αποπτωτικών δεικτών, παρατηρήσαμε έκφραση της Cas‐9 στο πυρήνα των λείων μυϊκών κυττάρων του μέσου χιτώνα τόσο στις κιρσοειδείς φλέβες όσο και στους μάρτυρες, με την ένταση να είναι μεγαλύτερη στους κιρσούς. Σε μικρό αριθμό δειγμάτων παρατηρήσαμε πυρηνική χρώση της Cas‐ 8 στα λεία μυϊκά κύτταρα του μέσου χιτώνα στους κιρσούς. Bcl‐2 δεν ανιχνεύθηκε σε κανένα από τα δείγματα που εξετάσθηκαν.Συμπεράσματα: Τα δεδομένα μας έδειξαν υπερτροφία των λείων μυϊκών κυττάρων του μέσου χιτώνα των αγγείων στις κιρσοειδείς φλέβες. Επιπλέον παρατηρήσαμε αυξημένη έκφραση των δεικτών Cas‐9, MMP‐1, MMP‐9 και TIMP‐1 στις κιρσοκήλες σε σύγκριση με τους μάρτυρες. Η έκφραση των δεικτών αυτών ήταν ανεξάρτητη από παραμέτρους όπως ο βαθμός (Grade) της κιρσοκήλης και η ηλικία των ασθενών. Τα ευρήματα μας αυτά δεν συνάδουν με αντίστοιχα από κιρσούς κάτω άκρων υποδηλώνοντας ότι πιθανώς άλλοι μηχανισμοί εμπλέκονται στην πρόκληση των κιρσοειδώς διευρυμένων έσω σπερματικών φλεβών. / Background: Varicocele is the most common cause of male infertility, defined as the abnormal varicose enlargement of the scrotal portion of the spermatic veins (pampiniform plexus). The incidence of varicocele in the general population ranges from 15% to 20%, while in subfertile men it reaches 40%. In children the frequency of varicocele is considered to be rare but recent studies have shown its presence in 6% of children aged 10 years, while in adolescents this figure rises up to 16%. Probably caused by insufficient venous valves which cause retrograde flow into the spermatic veins. Varicocele occurs predominately on the left side because of the anatomy of the veins in this region. Despite its great frequency, its pathophysiology remains unclear. Recent studies from varicose veins in the legs have shown that the reduction of rate of apoptosis is associated with the occurrence of primary varicose veins. Furthermore an increased production of the components of the extracellular matrix, leads to morphological alterations and loss of venous tone that characterizes varicose veins in the legs. Purpose: Considering that similar mechanisms should apply not only to lower limbs varicose veins but also at vericocele, we hypothesized that the medial wall of internal spermatic veins should show correspondenly a reduction in the rate of apoptosis and an increase in the rate of synthesis of components of the extracellular matrix by reducing expression of MMPs (MMP‐1, MMP‐9) and a corresponding increase in TIMPs (TIMP‐1). Methods: The study group consisted of 45 samples of internal spermatic vein (ISV) from patients with varicocele. Normal subcutaneous veins were harvested from each patient at the time of surgery and used as control specimens, so that each patient would serve as its own control. Both pathological ISVs and normal subcutaneous veins were used to detect the mediators that regulate the instrinsic (Bcl‐2 and Caspase‐9) and extrinsic (Caspase‐8) apoptotic pathways by immunohistochemical staining. We also investigated matrix metalloproteinases (MMPs) and also their inhibitors (TIMPs) expression in the varicose ΙSV, including MMP‐1, MMP‐9 and TIMP‐1. Finally, we examined the morphological alterations of varicose veins by immunohistochemical staining of Anti‐Actin antibody. Results: Compared with normal veins, a thickening of the smooth muscle layer of the ISV, was found in patients with varicocele. MMP‐1 and TIMP‐1 were expressed within cytoplasm of smooth muscle cells in media of both normal and varicose veins, but normal veins expressed less ΜΜP‐1 and TIMP‐1 than varicoceles did. MMP‐9 was localized to cytoplasm of smooth muscle cells only in varicose veins. As for the expression of apoptotic proteins, we showed expression of Cas‐9 within nuclei of smooth muscle cells in media of both normal and varicose veins however the expression was higher in varicoceles. In small number of samples we noticed nuclear staining of Cas‐8 within nuclei of smooth muscle cells in varicoceles. Bcl‐2 was not detected with immunohistochemistry in any specimens examined. Conclusions: Our data showed vascular smooth muscle hypertrophy in the diseased vessels. We also noticed increased expression of Cas‐9, MMP‐1, MMP‐9 and TIMP‐1 in varicoceles compared with normal veins. The expression of these markers was independent on factors such as the degree of varicocele and the age of the patients. Our findings are not consistent with corresponding varicose veins from legs, suggesting that other mechanisms are probably involved in causing vericoceles.

Η έκφραση των MMP και TIMP γονιδίων και η ενεργοποίηση των MMPs έχει συσχετιστεί με την εξέλιξη του καρκίνου του μαστού και η υπερέκφραση αυτών σχετίζεται με φτωχή πρόγνωση για τον ασθενή (Bjorklund et al 2005, Wurtz et al 2005). Σε αυτή την εργασία δείχτηκε με RT-PCR ανάλυση ότι τα MCF-7 κύτταρα εκφράζουν MMP-9, MT1- και MT2-MMP, αποτελέσματα που επιβεβαιώνουν προηγούμενες εργασίες (Kousidou at al 2004, Kousidou et al 2005) και που μπορεί να σχετίζονται με τη χαμηλή δυνατότητα μετάστασης της καρκινικής αυτής σειράς. Εντούτοις MMP2-mRNA δεν ανιχνεύτηκε, παρά το γεγονός ότι η MMP2 συχνά εμφανίζεται σε κακοήθεις ιστούς στο μαστό. Η επώαση των κυττάρων με λεουπρολίδιο για 48h οδήγησε σε χαμηλότερα επίπεδα της ΜΜΡ-9 mRNA. Έχει δεiχτεί ότι, σε αντίθεση με την ΜΜΡ-2, η έκφραση της ΜΜΡ-9 εξαρτάται από την παρουσία αυξητικών παραγόντων, χημοκινών και άλλων μορίων σηματοδότησης (Bjorklund et al, 2005). Σε αυτό το πλαίσιο η υπερέκφραση της ΜΜΡ-9 δεν διατηρείται όταν τα καρκινικά κύτταρα επωάζονται σε περιβάλλον ελεύθερο τέτοιων παραγόντων, υποθέτοντας ότι η παρουσία στον ορό μερικών αυξητικών παραγόντων παρακινούν την έκφραση βασικών MMPs σε φυσιολογικά και καρκινικά κύτταρα. Από τις μεμβρανικές μεταλλοπρωτεϊνάσες οι ΜΤ1- (ΜΜΡ-14) και ΜΤ2 (ΜΜΡ-15) εμφανίζουν παρόμοια συμπεριφορά και αυξάνονται παρουσία του αγωνιστή LHRH, λεουπρολιδίου. Επιπλέον η ΜΤ1-ΜΜΡ και η ΤΙΜΡ-2 συμμετέχουν στην κύρια οδό ενεργοποίησης του ΜΜΡ-2 στην επιφάνεια των κυττάρων με τη δημιουργία ενός τριμοριακού συμπλέγματος ΜΜΡ-2, ΜΤ1-ΜΜΡ και ΤΙΜΡ-2 (Deryugina et al 2001, Lafleur et al 2003). Σε αυτήν την εργασία τα επίπεδα ΤΙΜΡ-2 mRNA επίσης αυξήθηκαν. Έτσι θα μπορούσε να ειπωθεί ότι τα αυξημένα επίπεδα ΜΤ1-ΜΜΡ και ΤΙΜΡ-2 mRNA εκφράζουν την διάθεση των κυττάρων να ενεργοποιήσουν την proMMP-2. Τα αποτελέσματα μας δείχνουν ότι τα γονίδια ΤΙΜΡ-1 και ΤΙΜΡ-3 μειώνονται παρουσία του αγωνιστή LHRH, λεουπρολιδίου. Η αύξηση των ΤΙΜΡs από τα καρκινικά κύτταρα για να εξισοροπήσουν την υψηλή προτεολυτική δράση των MMPs αποτελεί μία πιθανή θεώρηση (Brown et al, 1998). Αυτό θα μπορούσε να θεωρηθεί ως ένας αμυντικός μηχανισμός του ανθρώπινου οργανισμού για να αντιμετωπήσει την ανώμαλη αύξηση των MMPs. Έτσι τα χαμηλότερα επίπεδα του ΤΙΜΡ-1 mRNA μπορεί να σχετίζονται με τα χαμηλότερα επίπεδα ΜΜΡ-9, παρουσία του λεουπρολιδίου, αφού ο ΤΙΜΡ-1 αναστέλλει την ΜΜΡ-9 με μεγάλη εξειδίκευση. Εντούτοις ιστοί με μεγάλη περιεκτικότητα σε ΤΙΜΡ-1 mRNA και πρωτείνες σχετίζονται με κακή πρόγνωση ασθενών με καρκίνο του μαστού, παρά την ανασταλτική δράση των ΜΜΡs. Η πρόσφατη ανακάλυψη της αντι αποπτωτικής και προ αγγειογενετικής δράσης του ΤΙΜΡ-1 μπορεί να είναι μέρος αυτής της υπόθεσης (Wurtz et al, 2005). Έχοντας αυτό υπόψην τα παρατηρούμενα χαμηλά επίπεδα ΤΙΜΡ-1 mRNA μπορεί να είναι αποτέλεσμα και άλλων δράσεων με ωφέλιμο ρόλο στον καρκίνο του μαστού. Γενικά η διαφορετική έκφραση των ΤΙΜΡs στον καρκίνο του μαστού αποτελεί ένα πολύ σημαντικό θέμα, που χρήζει περαιτέρω διερεύνησης. Συμπερασματικά τα αποτελέσματά μας δεικνύουν ότι ο αγωνιστής LHRH, λεουπρολίδιο, μπορεί να τροποποιήσει σημαντικά την έκφραση των γονιδίων ΜΜΡ/ΤΙΜΡ στα κύτταρα MCF-7. Οι MMPs και ΤΙΜΡs είναι πολυδύναμα μόρια με σημαντική επίδραση στην ανάπτυξη του καρκίνου, της μετάστασης και της αγγειογένεσης. Δεδομένου ότι τα ανάλογα LHRH αποτελούν μέρος της θεραπείας ασθενών με προ ή μετά εμμηνοπαυσιακό καρκίνο του μαστού, η επίδραση των παραπάνω αναφερθέντων μεταβολών από τη χρήση του λεουρπολιδίου, χρήζει περαιτέρω διερεύνησης. / Luteinizing hormone-releasing hormone (LHRH) is not only produced by hypothalamus but also by other normal and cancer tissues. LHRH peptide agonists and antagonists inhibit the proliferation of breast cancer cells, but their effect on the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) has not been studied despite the fact that growth and invasiveness of breast cancer cells in adjacent and distant sites is associated with the expression of MMPs. In the present study, the effects of [D-Leu6,Pro9 -NHEt]LHRH, leuprolide, on gene expression of MMPs and TIMPs in the breast cancer cell line MCF-7 were examined with semi-quantitative RT-PCR. Results showed that incubation of MCF-7 with 30μM of the leuprolide for 48h in serum-containing medium resulted in a down-regulation of MMP-9 expression and increase in MT1- and MT-2 MMP mRNA levels. Furthermore, leuprolide induced a significant decrease in TIMP-1 and TIMP-3 mRNA levels and increase in TIMP-2 mRNA levels. The impact of the observed changes on the expression of MMPs and TIMPs warrants further investigation on the effects of LHRH analogues on the invasiveness and metastatic potential of breast cancer cells.

Several diseases, including cancer, have been associated with high membrane type-1 matrix metalloproteinase (MT1-MMP) expression levels. MT1-MMP together with a non-membrane-bound, soluble MMP, MMP-2, also associated with many other biological functions, have been implicated in breast cancer progression, invasion and metastasis, and poor prognosis. Researchers who ran early clinical trials that employed broad-spectrum MMP inhibitors (MMPIs) lacked understanding of the intricate physiological and patho-physiological roles that MMPs play in tissues. In addition, structural similarities between MMPs hamper selective inhibition. Selective inhibition of MT1-MMP is of particular interest as MT1-MMP is overexpressed in target cancer cells relative to normal cells and is key in signalling for invasion. The inhibition profiles of two structurally similar synthetic pyrimidine-class MMPIs, with increased bioavailability and stability compared to hydroxamates tested in earlier clinical trials, TF 17-2 and TF 22d, were assessed. TF 17-2 and TF 22d were applied to a normal MCF-10A breast epithelial cell line and its premalignant H-ras(V12)-transfected MCF-10AneoT derivative to assess their efficacy for inhibiting MT1-MMP-mediated normal and premalignant cell migration, and indirectly, invasion. Both inhibitors form a co-ordination complex with the zinc ion of the catalytic site of MT1-MMP and MMP-2 with greater affinity for MT1-MMP. The computational molecular docking package, AutoDock Vina, was used for in silico predictions of binding affinities that could potentially substitute for in vitro kinetic assays when assessing inhibitor potential for inhibiting target MMPs. The binding of the two MMPIs was assessed using AutoDock Vina and compared to established kinetic data. The AutoDock Vina program was found to be an unreliable predictor for assessing relative efficacy of inhibition. During in vitro applications, analysis of the induction of apoptosis and metabolic effects were assessed using flow cytometry and the MTS assay, respectively. These showed no significant toxicity. Effects of inhibitors on collective and single cell migration in the normal and premalignant cell model, assessed using time lapse live cell imaging, cell morphology and labelling for vinculin and F-actin (for focal adhesions, FAs) showed that the TF 17-2 and TF 22d inhibitors reduced the collective cell migration of MCF-10A cells in scratch assays. Live-cell analysis of single cell migration, however, showed that TF 22d increased cell migration rates, and reduced the size of FAs and actin stability in MCF-10AneoT cells, resulting in a predominently rounded cell morphology in the premalignant cell line. TF 17-2, on the other hand was seen to be a relatively selective inhibitor of premalignant cell migration and resulted in MCF-10AneoT cells re-establishing larger focal - v - adhesions due to more stable F-actin networks resembling those of the non-transfected MCF-10A cell line, but reduced MCF-10AneoT cell migration most markedly. FA size and velocity of movement seemed inversely related in the normal and premalignant cells. The results of the current study suggest that, TF 17-2 seemed to have the greater therapeutic potential than TF 22d for inducing phenotype reversion, inhibition of dissemination, invasion and metastasis. Three promising selective pharmacological actions of TF 17-2 on the premalignant MCF10AneoT cell line include the suppression of proliferation, induction of increased in metabolic activity (possibly indicating cell stress) and a decrease in premalignant cell migration. A lack of cytotoxicity, however, suggests that TF 17-2 would need to be administered with an ancillary chemotherapeutic agent. This study showed that MMPIs directed against MT1-MMP, may still represent an effective strategy for inhibiting the migration of premalignant cells expressing high levels of MT1-MMP, and suggests further studies on this topic may be profitable. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Microorganisms may evade killing by neutrophils (PMNs) by altering signal transduction and hence phagosome maturation. Secreted, active matrix metalloproteinases (MMPs) appear to be required for PMN killing of pseudomonas microorganisms, via an MMP and complement-dependent, but otherwise unknown mechanism. This also depends on the absence of the inhibitor of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1). By altering their particular complement opsonin and hence the PMN complement receptor bound, microorganism may evade killing, as not all PMN complement receptors trigger phagosome maturation and hence killing of microorganisms. C1 inhibitor of the classical complement cascade, required for the exposure of C1q and further assembly of complement factors on the bacterial surface and hence binding to specific PMN receptors, is MMP sensitive. MMP secretion may, therefore, not only facilitate the killing of microorganisms, but inappropriate secretion, induced by pathogens, may prevent complement assembly and killing via complement-mediated pathways. It was, therefore, decided to assess MMP-9 and TIMP-1 secretion in the presence of C1q-opsonized polystyrene beads and subsequently upon stimulation with pseudomonas organisms, and explore the relationship between secretion of PMN MMPs (specifically MMP-9) and TIMP-1 and phagocytic uptake and maturation of the PMN phagosome into a killing body. MMP-9 and TIMP-1 secretion was seen to occur at low levels under most conditions. However, in the presence of serum, and hence complement, MMP-9 secretion was found to be upregulated during uptake of C1q-coated beads. MMP-9 possibly inactivates C1 inhibitor at this stage, causing local tissue swelling (normally associated with the inactivation of C1-inhibitor), entry of various white blood cells and further complement into the area of infection, assisting in the extracellular killing of microorganisms. MMP secretion may simultaneously down-regulate the activation of further PMNs via inactivation of C1q assembly and hence phagocytic uptake and activation of PMNs. Unlike MMP-9, secretion of TIMP-1 was not upregulated by C1q receptor binding, implying that any secreted MMP-9 may, therefore, be in excess and hence uninhibited by TIMP-1. A distinct regulatory mechanism seems to be responsible for the release of TIMP-1, though TIMP-1 secretion was upregulated by extracellular calcium levels, partially contradicting previous findings which suggested that TIMP-1 was not calcium regulated. It seems unlikely that extracellular calcium levels would be the only mechanism by which TIMP-1 is regulated, however, and further surface receptor mediated agonists should be explored. Levels of MMP-9 and TIMP-1 secretion in the presence of pseudomonas microorganisms now need to be assessed to see whether these secretion patterns are altered to favour the evasion of opsonization by C1q. Uptake of C1q-opsonized beads was also increased by the presence of serum, possibly due to presence of complement. MMP-9 and TIMP-1 secretion patterns still need to be correlated with phagosomal uptake and killing of microorganisms, before their role in killing of microorganisms becomes fully evident. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.