Ziel der vorliegenden Arbeit war die immunhistologische Charakterisierung der Expression der profibrotischen Wachstumsfaktoren Transforming growth factor-beta-1, -beta2 und -beta3 und des Enzyms Matrixmetalloproteinase-2 (MMP-2) im equinen Endometrium während des Zyklus sowie innerhalb der verschiedenen Erscheinungsformen der equinen Endometrose. Zudem wurde der potentielle Einfluss einer gleichzeitig auftretenden Endometritis auf die glanduläre und stromale Wachstumsfaktor- und Enzym-Expression untersucht. Die Ergebnisse dieser Studie sollten klären, ob und inwieweit den untersuchten Wachstumsfaktoren unter Beteiligung von MMP-2 in der Pathogenese der equinen Endometrose eine mit anderen Organfibrosen vergleichbare Schlüsselrolle zukommt. Zu diesem Zweck standen an definierten Tagen entnommene Endometriumbioptate (n=21) von drei zyklisch aktiven, klinisch und gynäkologisch gesunden Maidenstuten sowie Endometriumbioptate von 60 Stuten mit graduell variabler Endometrose unterschiedlichen Charakters und Endometriumbioptate von 22 Stuten mit mittelgradiger Endometrose und gleichzeitiger mittelgradiger eitriger (n=16) bzw. nichteitriger (n=6) Endometritis aus dem Routineeinsendungsmaterial des Institutes für Veterinär-Pathologie der Universität Leipzig zur Verfügung. Die Wachstumsfaktoren TGF-beta1, -beta2 und -beta3 sowie das Enzym MMP-2 zeigen im Zyklus ein typisches, zellspezifisches Reaktionsmuster, das unterschiedlichen Regulations-mechanismen zu unterliegen scheint. Ein Maximum der TGF-beta1-Expression in den luminalen Epithelzellen, Stroma- und Drüsenzellen kann in der endometrialen Sekretionsphase mit Anstieg bzw. einem Maximum der Serumprogesteron-Konzentration beobachtet werden. Im Gegensatz dazu tritt eine Expression von MMP-2 in den Stromazellen in der Sekretionsphase mit Abfall der Progesteronkonzentration im Serum auf. Das luminale Epithel und die Stromazellen zeigen eine maximale Expression von TGF-beta2 beim Vorliegen hoher Progesteronspiegel im Serum bzw. mit Abfall der Serumprogesteron-Konzentration in der Sekretionsphase. TGF-beta3 weist im luminalen Epithel ein ähnliches Expressionsmuster auf, eine deutliche Abhängigkeit zu den Serumhormon-Konzentrationen lässt sich jedoch nicht feststellen. Die stromale Expression von TGF-alpha unterliegt im equinen Endometrium keinen zyklusabhängigen Variationen. Die Stromazellen innerhalb der verschiedenen Endometroseherde zeigen, im Vergleich zum unveränderten Endometrium, vor allem eine verminderte Expression von TGF-alpha. Das Expressionsmuster der TGF-beta-Wachstumsfaktoren ist grundsätzlich variabel, es fällt jedoch auf, dass die Stromazellen insbesondere in inaktiven Endometrosen eine geringere Expression der TGF-beta-Isoformen aufweisen. Ursache ist möglicherweise eine gestörte hormonelle Stimulation bzw. eine stromale Synthesestörung in Folge veränderter epithelial/stromaler Wechselwirkungen. Das Enzym MMP-2 wird dagegen in den Stromazellen aller Endometroseherde, unabhängig von deren Differenzierung und dem Auftreten glandulärer Alterationen, deutlich vermehrt nachgewiesen. Dies ist sehr wahrscheinlich Folge der Extra-zellularmatrix-Akkumulation innerhalb der Endometroseherde und für die fortschreitende Zerstörung der glandulären Basalmembranen verantwortlich. Die glanduläre Expression innerhalb der Endometroseherde gleicht weitgehend der der unveränderten Drüsenzellen, lediglich in destruierenden Endometrosen werden TGF-alpha, TGF-beta2 und MMP-2 in den involvierten Drüsenzellen vermehrt nachgewiesen. Mögliche Ursachen wären eine Diffusion durch die geschädigte glanduläre Basalmembran bzw. eine Anregung der Synthese im Rahmen der epithelialen Wundheilung. Eine Anregung der glandulären und stromalen Expression der untersuchten Wachstumsfaktoren und des Enzyms MMP-2 im Rahmen der Endometrose durch die Anwesenheit von Entzündungszellen konnte nicht nachgewiesen werden. Eine der Leber- und Lungenfibrose ähnelnde, überschießende Wundheilungsreaktion durch eine primär epithelial bedingte, vermehrte TGF-Wachstumsfaktorproduktion sowie direkte Zusammenhänge zwischen der MMP-2- und TGF-beta-Wachstumsfaktor-Expression waren in der equinen Endometrose nicht festzustellen. Da vor allem die Stromazellen in der Endometrose eine veränderte Expression der Wachstumsfaktoren aufwiesen, ist möglicherweise eine primäre stromale Fehldifferenzierung der Ausgangspunkt für die Entstehung der Endometrose. Eine mit der Leber- und Lungenfibrose vergleichbare Schlüsselrolle der TGF-Wachstumsfaktoren in der Pathogenese der equinen Endometrose konnte nicht eindeutig belegt werden.

During cardiac development and in cardiac disease changes in hemodynamic load initiate events leading to remodeling of the ECM. This study addresses the hypothesis that interactions between Integrins and Metalloprotienases function to modulate cell adhesion in the cultured cardiac fibroblast. The fibroblast is positioned to detect and respond to changes in the mechanical load on the heart. Functionally the cardiac fibroblast is the primary cell type responsible for the production, maintenance, and remodeling of the cardiac interstitium. Matrix Metalloproteinases, specifically the Gelatinases, are expressed in concert during development and in disease with changes in the hemodynamic loading of the heart. Our studies have identified by a complex on the surface of the cardiac fibroblast composed of the a3b1 integrin, MMP-2, and TIMP-2. Putatively, this complex is involved in the maturation of adhesions. Inhibition of MMP-2 was associated with a decrease in the strength of adhesion of cell plated on collagen and fibronectin. Confocal imaging and analysis indicate that a predominate interaction occurs between MMP-2 and the a3 integrin chain. Taken together biochemical, functional, and microscopic data have identified a complex on the surface of the cardiac fibroblast that represents elements of mechanotransduction and matrix metabolism in a single site that functions in the maturation of adhesion

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

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.

Salivary duct carcinoma (SDC) is an uncommon neoplasm showing extensive local invasion and metastasis. Histologically it exhibits ductal differentiation without myoepithelial participation and resembles intraductal and invasive ductal carcinoma of the breast. This study investigates the incidence of intraductal disease within invasive SDC and characterizes the expression of CK19, COX-2, MMP-2, 9 and 11 in intraductal and invasive SDC. 21 conventional SDC, 8 SDC arising as carcinoma ex-pleomorphic adenoma (CaExPA), 2 pure low-grade intraductal SDC, 1 pure high-grade intraductal SDC and 1 intraductal SDC with de-differentiation were studied. The presence of intraductal tumor in SDC is relatively common (52.4%) but is not a prominent component. SDC tumor cells were strongly positive for CK19 and COX-2. MMP-2 and 9 expression showed no significant pattern. MMP-11 was expressed by fibroblasts surrounding invasive tumor and was significantly associated with lymph node positivity (p=0.02).

Salivary duct carcinoma (SDC) is an uncommon neoplasm showing extensive local invasion and metastasis. Histologically it exhibits ductal differentiation without myoepithelial participation and resembles intraductal and invasive ductal carcinoma of the breast. This study investigates the incidence of intraductal disease within invasive SDC and characterizes the expression of CK19, COX-2, MMP-2, 9 and 11 in intraductal and invasive SDC. 21 conventional SDC, 8 SDC arising as carcinoma ex-pleomorphic adenoma (CaExPA), 2 pure low-grade intraductal SDC, 1 pure high-grade intraductal SDC and 1 intraductal SDC with de-differentiation were studied. The presence of intraductal tumor in SDC is relatively common (52.4%) but is not a prominent component. SDC tumor cells were strongly positive for CK19 and COX-2. MMP-2 and 9 expression showed no significant pattern. MMP-11 was expressed by fibroblasts surrounding invasive tumor and was significantly associated with lymph node positivity (p=0.02).

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.