published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Periodontitis is a chronic inflammatory disease that affects over 116 million adults in the United States. A shift in the normal microflora occurs as periodontal disease develops resulting in a larger number of Gram-negative anaerobes and spirochetes. An increase in the oral spirochete, Treponema denticola, is highly correlated with periodontal disease progression and severity. The ability of this periopathogen to thrive in the subgingival crevice is dependent on complement evasion mechanisms. Earlier analyses demonstrated that the primary mechanism of T. denticola serum resistance is binding of the human complement regulatory protein, Factor H (FH), to the factor H-binding protein (FhbB). FH serves as cofactor in the Factor-I mediated cleavage of C3b and accelerates the decay of the C3 convertase complex, leading to downregulation of C3b production. Several pathogens bind FH, and a number of these bacterial binding proteins have been shown to bind plasminogen. Plasminogen is a plasma glycoprotein that circulates as a zymogen. Its active form, plasmin, degrades components of the extracellular matrix and cleaves complement proteins C3b and C5 inhibiting complement pathway progression. Through molecular and biochemical analyses, this study demonstrates that FhbB simultaneously binds plasminogen and FH to residues located on its positively and negatively charged surfaces, respectively, and that the two ligands do not compete for binding. This study also shows that the surface-bound plasminogen is available for proteolytic cleavage into the active serine protease plasmin. The activated plasmin could break down components of the periodontal tissue leading to increased nutrient availability and creation of a larger anaerobic environment where the bacteria can flourish, thereby promoting periodontal disease.

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<p>Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function.</p><p>A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression.</p><p>Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice.</p><p>Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice.</p><p>In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.</p>

The plasminogen activator (PA) system has been shown to be intimately involved in wound healing. However, the role of this system in the initiation and resolution of inflammation during healing process remained to be determined. The aims of this thesis were to investigate the molecular mechanism underlying the interaction between the PA system and the inflammatory system during wound healing and to explore the therapeutic potential of plasminogen in various wound-healing models. The role of plasminogen in the inflammatory phase of the healing process of acute and diabetic wounds was studied first. Our data showed that administration of additional plasminogen to wild-type mice accelerates the healing of acute wounds. After injury, both endogenous and exogenous plasminogen are bound to inflammatory cells and are transported to the wound site, which leads to activation of inflammatory cells. In diabetic db/db mice, wound-specific accumulation of plasminogen does not take place and the inflammatory response is impaired. However, when additional plasminogen is injected, plasminogen accumulates in the wound, the inflammatory response is enhanced, the signal transduction cascade is activated and the healing rate is significantly increased. These results indicate that administration of plasminogen may be a novel therapeutic strategy to treat different types of wounds, especially chronic wounds in diabetes. The role of plasminogen at the later stage of wound healing was also studied in plasminogen-deficient mice. Our data showed that even if re-epithelialization is achieved in these mice, a prolonged inflammatory phase with abundant neutrophil accumulation and persistent fibrin deposition is observed at the wound site. These results indicate that plasminogen is also essential for the later phases of wound healing by clearing fibrin and resolving inflammation. The functional role of two physiological PAs during wound healing was further studied in a tympanic membrane (TM) wound-healing model. Our data showed that the healing process was clearly delayed in urokinase-type PA (uPA)-deficient mice but not in tissue-type PA (tPA)-deficient mice. Less pronounced keratinocyte migration, abundant neutrophil accumulation and persistent fibrin deposition were observed in uPA-deficient mice. These results indicate that uPA plays a central role in the generation of plasmin during the healing of TM perforations. Finally the therapeutic potential of plasminogen in the TM wound-healing model was studied. Our data showed that local injection of plasminogen restores the ability to heal TM perforations in plasminogen-deficient mice in a dose-dependent manner. Plasminogen supplementation also potentiates healing of acute TM perforations in wild-type mice, independent of the administration method used. A single local injection of plasminogen in plasminogen-deficient mice can initiate healing of chronic TM perforations resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. Three plasminogen injections lead to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer that can start to reorganize and further mature to its normal appearance. In conclusion, our results suggest that plasminogen is a promising drug candidate for the treatment of chronic TM perforations in humans.  Taken together, our data indicate that plasminogen is a novel inflammatory regulator that promotes wound healing.

El sistema d'activació del plasminogen (PA) és un grup de serina-proteases que participen en nombrosos processos en els quals té lloc de degradació de la matriu extracel·lular (ECM). El plasminogen, mitjançant l'acció dels seus activadors específics, dóna lloc a la plasmina activa. La plasmina és una potent proteasa d'ampli espectre de substrat. El seu paper clàssic ha estat la degradació dels coàguls de fibrina, tot i que també actua sobre altres components de l'ECM directament, i de manera indirecta mitjançant l'activació de diverses metal·loproteases. Tanmateix, la plasmina pot activar les formes latents de diversos factors de creixement. Aquest potent sistema de proteases està regulat a diferents nivells: a nivell d'inhibidors i a nivell de receptors que concentren l'activitat proteolítica a l'espai pericel·lular. Existeixen diverses proteïnes situades a la membrana cel·lular que posseeixen l'habilitat d'unir plasminogen, entre elles cal destacar l'alfa-enolasa. L'alfa-enolasa és un enzim glucolític que es transloca a la membrana cel·lular mitjançant un mecanisme desconegut, on actua com a receptor del plasminogen. La unió del plasminogen als seus receptors facilita la seva activació a plasmina i la protegeix de l'acció dels seus inhibidors.El sistema PA participa en un elevat nombre de processos fisiopatològics que requereixen un elevat grau de proteòlisi. Entre aquests processos cal destacar la remodelació tissular i, en particular, la remodelació del múscul esquelètic. El teixit muscular és un bon exemple de proteòlisi extracel·lular massiva després d'un miotraumatisme. Resultats previs demostraren que l'activador del plasminogen de tipus urokinasa (uPA) i el plasminogen desenvolupen un paper fonamental durant el procés de regeneració muscular. El principal objectiu d'aquesta tesi fou l'anàlisi del paper de la unió plasminogen/alfa-enolasa durant la miogènesi in vitro i la regeneració muscular in vivo. Aquest treball es va dur a terme mitjançant l'ús d'inhibidors de la unió plasminogen/alfa-enolasa (MAb 11G1, contra l'alfa-enolasa; i EACA, contra el plasminogen) i l'ús d'un inhibidor de l'activitat proteolítica de la plasmina lliure (aprotinina) en models de miogènesi in vitro i regeneració muscular in vivo. El cultiu de mioblasts murins primaris en presència dels tres inhibidors va demostrar una inhibició sobre la diferenciació de l'ordre d'un 50% després del tractament amb MAb 11G1 i EACA, mentre que l'aprotinina no tingué cap efecte. En canvi, quan s'analitzava el procés de fusió, la inhibició fou del 90%. Aquest resultat indica que, després del bloqueig de la unió del plasminogen al seu receptor, els mioblasts inicien el procés de diferenciació, però no arriben a fusionar-se en cap moment, demostrant que la unió del plasminogen a l'alfa-enolasa és un mecanisme clau durant la miogènesi. A continuació es va analitzar el paper de la unió plasminogen/alfa-enolasa durant la regeneració del múscul esquelètic in vivo, mitjançant l'ús de dos models animals: un model de regeneració muscular mitjançant una injecció amb cardiotoxina (CTX) i el ratolí distròfic mdx, el model animals per a la Distròfia Muscular de Duchenne (DMD). Els resultats obtinguts mostraren que els inhibidors de la unió plasminogen/alfa-enolasa bloquegen el procés de regeneració muscular després d'una lesió amb CTX, mentre que l'aprotinina no té cap efecte, mostrant que l'activitat proteolítica de la plasmina unida a la membrana cel·lular és necessària durant la regeneració muscular. Quan s'analitzà l'efecte dels diferents inhibidors en els ratolins distròfics mdx la patologia esdevingué més severa, indicant que el bloqueig de la unió del plasminogen al seu receptor provoca un important agreujament en la distròfia dels ratolins mdx. Tots aquests resultats demostren que l'activitat proteolítica de la plasmina associada a l'alfa-enolasa desenvolupa un paper fonamental en el procés de regeneració muscular in vivo, mentre que l'activitat proteolítica de la plasmina lliure no participa en aquest procés. / Plasminogen Activation (PA) System is a group of serin-proteases that participates in biological processes in which extracellular matrix (ECM) remodeling takes place. Plasmin, generated by plasminogen activation, is an extracellular protease specialized in the degradation of the ECM components. Several proteins able to bind plasmin(ogen) to the cell surface have been described. In particular, alpha-enolase acts as a plasmin(ogen) receptor in several cell types concentrating proteolytic activity on the cell surface. PA system participates in a high number of biological processes, including skeletal muscle remodelling. Previous results showed an overexpression of alpha-enolase during myoblasts differentiation in vitro, and in skeletal muscle regeneration in vivo, suggesting an important role of this protein during these processes. So, the main goal of this work was the analysis of the role of plasmin(ogen)/alpha-enolase binding during myogenesis and skeletal muscle regeneration. For this goal, we used inhibitors of plasminogen/alpha-enolase binding: MAb 11G1 and EACA. Using primary cultures or Muscle Precursor Cells (MPCs), our results showed that myogenic differentiation, fusion and cell migration were abrograted in the presence of inhibitors of plasmin(ogen)/alpha-enolase binding. The effect of plasmin(ogen)/alpha-enolase binding inhibitors were evaluated in a regeneration model in mice after an injury. Muscle regeneration was blocked by plasmin(ogen)/alpha-enolase binding inhibitors, as assessed by histological and morphological parameters. When the mdx mice (the animal model for Duchenne Muscular Dystrophy, DMD) were treated with plasminogen/alpha-enolase binding inhibitors the myopathology became more severe. Our results demonstrate that plasmin(ogen)/alpha-enolase association is necessary for myogenesis and muscle regeneration to take place correctly.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 110-133). Also issued on the Internet.

Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function. A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression. Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice. Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice. In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.

Hematopoietic cells are recruited to and co-opted by the growing tumor making expansive tumor growth possible. Although several cell types become associated with the growing tumor, macrophages play a fundamental role. The movement of macrophages across the basement membrane and through the extracellular matrix to the tumor site requires the activation of proteases, such as plasmin, at their cell surface. The proteolytic aspect of macrophage recruitment may represent an exploitable aspect of tumor growth in terms of therapeutic strategies. Here I show that the S100A10 protein facilitates the infiltration of macrophages into the site of tumor growth by stimulating the generation of the protease plasmin at their surface. Using a mouse model in which wild-type (WT) and S100A10-null mice are inoculated with tumor cells, a decrease in tumor-associated macrophages (TAMs) and greatly diminished tumor growth in tumors grown in S100A10-null mice was observed. Although tumor growth in S100A10-null mice could be restored by intraperitoneal injection of WT macrophages, S100A10-null macrophages only restored tumor growth when directly injected into the tumor. Lastly, selective depletion of macrophages from a WT mouse by liposome encapsulated clodronate treatment resulted in similar tumor growth deficits as in the S100A10-null mouse. These results highlight a new role for the S100A10 protein in the recruitment of TAMs to the tumor site and demonstrate a potential therapeutic strategy in which the tumor associated cells may be targeted.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.