The incidence rate of chronic hepatopathy in Taiwan is high, which afflicts the patients by progressively developing irreversible cirrhosis. Hepatic fibrosis is the intermediate and crucial stage of this process, characterized by reversibility. If treated properly in this stage, cirrhosis can be successfully prevented. In the liver, activated stellate cells are the key mediators of fibrosis. Transforming growth factor-

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

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.

This study examined the efficacy of continuous nicotinamide (NAM) administration on recovery of function in rats following traumatic brain injury (TBI). TBI was induced via controlled cortical impact (CCI) bilaterally in the prefrontal cortex (+1.5, 0.0 relative to bregma) or sham surgeries were performed. Rats were then treated with either NAM (150 mg/kg/day) or vehicle (saline). Rats were tested behaviorally on the bilateral tactile adhesive removal task, locomotor placing task, novel exploratory behavior and the Morris water maze (MWM). Rats were also assessed histologically by looking at lesion size, GFAP expression (as a measure of active astroctyes) and MMP-9 expression (as a measure of inflammatory response) at time points of 24 and 48 hours and 30 days. The behavioral assessments showed significant improvements in the NAM-treated animals on the bilateral tactile adhesive removal, locomotor placing and MWM. The histological assessments showed significant lesion reduction at 30 days in the NAM-treated group. There were no differences between NAM-treated and vehicle groups on either GFAP or MMP-9 expression. These results indicate that NAM treatment after TBI can significantly improve recovery of function in rats.

Abstract Breast carcinoma is the most common malignancy in females in western countries. Classical prognostic factors such as the size of a primary tumor and the presence or absence of axillary lymph node metastases, malignancy grade and hormone receptor status reflect the subsequent risk of disease recurrence after primary therapy and the need for adjuvant therapies. However, most breast carcinomas are detected in the early stage of the disease and the value of these classical prognostic factors is limited. There is also a great need to find new factors predicting the clinical efficacy of the anticancer drugs available. In this thesis tumor suppressor gene p53, oncogene c-erbB-2 and matrix metalloproteinase-9 were evaluated for their prognostic relevance in breast carcinoma patients treated in Oulu University Hospital, and matrix metalloproteinase-9 was also analyzed in women with premalignant lesions in the breast tissue in order to examine its role in breast carcinogenesis. Histological analyses were carried out from formalin-fixed, paraffin-embedded primary tumor specimens and p53, c-erbB-2 and matrix metalloproteinase-9 (MMP-9) statuses were systematically analyzed by immunohistochemistry. P53 expression correlated with disease-free survival and overall survival in patients with early-stage breast carcinoma, regardless of adjuvant antiestrogen therapy. The co-expression of p53 and c-erbB-2 characterizes a tumor type with a clinically aggressive course of breast carcinoma. The clinical efficacy of anthracyline-based chemotherapy in metastatic carcinoma might be limited in patients with p53 expression in a primary tumor. When postmenopausal patients with lymph node metastases and receiving adjuvant antiestrogen therapy were examined, MMP-9 expression indicated a slightly greater risk of breast carcinoma recurrence in patients with estrogen receptor negative tumors. Hyperplastic breast tissue and invasive breast carcinoma lesions expressed some MMP-9 immunopositivity. However, the strongest positivity was seen in ductal carcinoma in situ samples, suggesting that MMP-9 participates in breast carcinogenesis in the preinvasive phase.