Motivation: The detrimental effects of obesity and insulin resistance in Caucasians and African-Americans have been the focus of many recent publications, and the association between PAI-1act and markers of the metabolic syndrome is well established but data on African subjects are still lacking. Objectives: To investigate possible differences between the association of PAI-1act with markers of the metabolic syndrome in Caucasian and African women. Methods We used cross-sectional data from the POWIRS I and II studies, involving 95 African and 114 Caucasian women respectively in the Potchefstroom district of the North West Province, South Africa. Results: Mean plasma PAI-1act was significantly higher in the Caucasian than in the African subjects (p < 0.001). Markers for the metabolic syndrome explained 60% of the variance of PAI-1act in the Caucasian group, but only 2.8% of the variance of PAI-1act in the African group. Waist circumference emerged as the strongest independent predictor of PAI-1act in the Caucasian (34%) as well as the African subjects (11%). Conclusion: This study showed clear differences in PAI-1act between African and Caucasian subjects, along with differences in the association of PAI-1act with markers of the metabolic syndrome. Apparent genetic differences between the two groups (especially the role of the 4G/5G genotype) may have an important influence on PAI-1act The role of PAI-1act in the metabolic syndrome may differ between Caucasians and Africans. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2005.

Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few datas are available concerning their role in lymphangiogenesis. We successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiments. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

This study identified striking increase in plasma of plasminogen activator inhibitor 1(PAI-I), a major inhibitor of fibrinolysis levels in septic patients who are non-neutropenic. Neutropenic patients show less striking changes. Where shock occurs both groups of patients show very high levels of PAI-1. These observations suggest a role for leucocytes in PAI production. In the second section neutrophils are identified as containing PAI-1 in normal subjects, the levels rising significantly in sepsis. Monocytes contain no PAI-1 but do contain Plasminogen activator inhibitor 2(PAI-2) levels of which inhibitor also rise in sepsis. Normal neutrophils contained no PAI-2 but neutrophils from septic patients contained significant quantities of this inhibitor. In the third section mononuclear cells from septic patients are identified as enhancing PAI-1 production in cultured endothelial cell (EC). Septic neutrophils have a more complex effect on EC. Mononuclear cells and neutrophils therefore, both contribute to the fibrinolytic inhibition of septic disorders but by different mechanisms. Each cell type contains one of the major inhibitor of plasminogen activator and levels of these rise in sepsis. Both cell types from septic patients promote greater release of PAI-1 from endothelial cells than do cells from normal individuals. Inhibition of fibrinolysis by leucocytes may contribute to fibrin persistence in sepsis. This may be useful in localizing infection. If generalized, it may contribute to vascular occlusive complications of sepsis such as shock lung, acute renal failure or digital gangrene. Absence of leucocytes may account for the apparent reduction of vascular occlusive complications in leucopenic septic patients.

Motivation: The detrimental effects of obesity and insulin resistance in Caucasians and African-Americans have been the focus of many recent publications, and the association between PAI-1act and markers of the metabolic syndrome is well established but data on African subjects are still lacking. Objectives: To investigate possible differences between the association of PAI-1act with markers of the metabolic syndrome in Caucasian and African women. Methods We used cross-sectional data from the POWIRS I and II studies, involving 95 African and 114 Caucasian women respectively in the Potchefstroom district of the North West Province, South Africa. Results: Mean plasma PAI-1act was significantly higher in the Caucasian than in the African subjects (p < 0.001). Markers for the metabolic syndrome explained 60% of the variance of PAI-1act in the Caucasian group, but only 2.8% of the variance of PAI-1act in the African group. Waist circumference emerged as the strongest independent predictor of PAI-1act in the Caucasian (34%) as well as the African subjects (11%). Conclusion: This study showed clear differences in PAI-1act between African and Caucasian subjects, along with differences in the association of PAI-1act with markers of the metabolic syndrome. Apparent genetic differences between the two groups (especially the role of the 4G/5G genotype) may have an important influence on PAI-1act The role of PAI-1act in the metabolic syndrome may differ between Caucasians and Africans. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2005.

Kyoto University (京都大学) / 0048 / 新制・論文博士 / 博士(工学) / 乙第12494号 / 論工博第4048号 / 新制||工||1503(附属図書館) / 28244 / (主査)教授 村上 正浩, 教授 杉野目 道紀, 教授 濵地 格 / 学位規則第4条第2項該当

L’invasion de la matrice extracellulaire est le premier obstacle que les tumeurs solides rencontrent lors de leur dissémination métastatique. Il existe deux mécanismes principaux: 1) la transition épithélio-mésenchymateuse au cours de laquelle les cellules épithéliales, à l’échelle individuelle, perdent leur cohésion intercellulaire et acquièrent des capacités de motilité pour quitter leur site d’origine ; 2) l’invasion collective dans laquelle des cellules en groupe coopèrent pour migrer collectivement en se frayant un passage à travers le microenvironnement. Dans les modèles murins, on sait maintenant que des groupes ou agrégats de cellules tumorales circulantes issus du détachement de tumeurs primaires présentent une capacité accrue à coloniser des organes à distance. Cependant, les facteurs et mécanismes sous-jacents conduisant au détachement des cellules tumorales en groupe sont peu connus. Pour étudier ces mécanismes, nous avons utilisé des sphéroïdes tumoraux, modèle expérimental tri-dimensionnel (3D), qui mime le microenvironnement ainsi que les caractéristiques morphologiques et fonctionnelles de la tumeur primaire. Nos résultats indiquent que l'inhibiteur de l’activateur du plasminogène (PAI-1), une protéine matricellulaire trouvée en forte concentration dans les cancers invasifs, favorise le détachement en groupe et l’invasion collective de cellules du cancer du sein (lignée cellulaire MCF7) et du colon (lignée cellulaire HCT116) organisées en sphéroïdes tumoraux. PAI-1 a des propriétés dé-adhésives sur des cellules étalées en monocouche sur des matrices pro-adhésives et génère l’agrégation des cellules entre-elles. Ces cellules conservent leurs caractéristiques épithéliales malgré une modification importante de la dynamique du réseau d'actine.Nos résultats suggèrent que les récepteurs cellulaires de la famille des lipoprotéines de basse densité (LDL-R) sont impliqués dans les propriétés dé-adhésives de PAI-1. Dans la cellule, la transduction du signal passe par les kinases ROCK (kinase associée à la GTPase RhoA) et Janus (JAK), ce qui conduit à une phosphorylation de la chaîne légère de la myosine II (MLC2), nécessaire pour l’activité contractile de la myosine et des facteurs de transcription STAT3. En outre, ROCK, un régulateur connu de la contraction du cytoskelette d’actomyosine, est également impliqué dans l'activation de STAT3. L'inhibition de ROCK ou JAK rétablit l’adhésivité des cellules en 2D et réduit leur migration en 3D. Nos données suggèrent que PAI-1 génère des zones de forte activité contractile membranaire dans les cellules en périphérie de la tumeur via ROCK-MLC2 et JAK-STAT, ce qui promeut le détachement de groupe de cellules hautement invasives. Ce nouvel axe de signalisation fonctionnelle de PAI-1 représente une cible anti-métastatique potentielle. / Invasion of the extracellular matrix is the first obstacle that solid tumours encounter during their metastatic dissemination. There are two main mechanisms: 1) epithelial to mesenchymal transition wherein epithelial cells lose their intercellular cohesion and convert to individual migratory behaviours to escape their point of origin; 2) invasion in a collective manner, in which a group or cluster of cohesive cancer cells detaches from the tumour mass and progressively, pushes its way through the microenvironment. It is now known that circulating tumour cell clusters may result from the evasion of cohesive small groups of cells from tumours and such tumour cell clusters display an increased propensity to colonize distant organs in mouse models. However, the extracellular factor/s and the underlying mechanism that enable cell detachment, as clusters are largely unknown. To study the process of tumour cell detachment and invasion, we used a three-dimensional (3D) multicellular tumour spheroid (MCTS) model, which mimics the microenvironment as well as morphological, functional and symmetric geometry features of the primary tumour. Our results strongly indicate that the plasminogen activator inhibitor-1 (PAI-1), a matricellular protein found in high concentration at the invasive front of most cancers, promotes cancer cell cluster detachment and a collective invasion phenotype within MCTS of breast cancer MCF7 and colon cancer HCT116 cell lines. We found that PAI-1 has a de-adhesive effect which induced a multilayered cell clustering of cells spread out on a pro-adhesive matrices in 2D monolayer cultures. Cells retained their epithelial characteristics and membrane-localized E-cadherin despite significant modification of actin dynamics. PAI-1 functions as a de-adhesive molecule most likely involved low-density lipoprotein receptors at the cell surface. We report that the downstream intracellular events may be mediated through the Rho-associated kinase (ROCK) and Janus kinases (JAK) signalling pathway, resulting in phosphorylation of either myosin light chain 2 (MLC2), which is required for myosin II-mediated contractility or STAT3 transcription factors. In addition, ROCK, a known contributor to actomyosin contractility is involved in STAT3 phosphorylation and activation. Inhibition of ROCK and JAK restored adhesion of cells on 2D substratum and reduced their migration/invasion within 3D MCTS model. Our data support a model in which PAI-1 generates actomyosin contractility and high membrane activity at the tumour periphery in a JAK-STAT/ROCK-MLC2 dependent manner promoting the detachment of highly invasive cell clusters. This novel axis of functional signalling of PAI-1 is a potential anti-metastasis target.

No description available.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in blood in a complex with vitronectin (VN). These two proteins are also found localized together in the extracellular matrix in many different pathophysiological conditions. Both of these proteins are involved with a number of physiologically important processes. Though PAI-1 is a well-known inhibitor of serine proteases, more emphasis is now geared towards its protease independent functions. VN, on the other hand, is a binding protein that exists in the circulation in a preferred monomeric conformation. However, in the extracellular matrix, VN exists as multimer with altered conformation. Though the exact reason for such conformational alterations and compartmentalization is unknown, there are a number of biomolecules, including PAI-1 that are proposed to cause such alterations. In last few years, sufficient experimental evidence has been gathered to confirm this protease- independent effect of PAI-1 by which it induces multimerization of VN in a concentration-dependent fashion. It has been observed also that PAI-1 remains associated with this multimeric complex for several hours. A major focus of this dissertation work was to extend our understanding of the mechanism of the interaction between these proteins and to explore the physiological relevance of the multimeric complexes formed by their interaction on cellular adhesion and migration. In our study, emphasis has been given to the presence of an appropriate microenvironment so that the role of the multimeric complexes could be investigated in a relevant biological setting. Our findings indicate the importance of the surrounding microenvironment in establishing the specific role of the VN/PAI-1 complex in cell-matrix interactions. In a previous study from our lab, it was found that vitronectin knock-out mice were more resistant to Candida infection compared to wild type C57Bl/6 mice. One of the goals of this dissertation work was to provide a mechanistic explanation for their increased survival of the vitronectin knock-out mice upon Candida infection. Another important aspect of this work was to establish biophysical methods for understanding the structural changes that happen in PAI-1 naturally or due to ligand binding.

Major Depressive Disorder (MDD) is one of the most common psychiatric conditions affecting adults and afflicts approximately 5% of the world's population. MDD is highly co-morbid with cardiovascular disease, a major source of morbidity and mortality. Although a number of pathological changes observed in MDD may impact cardiovascular health, no single mechanism has been identified that can explain this association. Both MDD and CVD are more common in women, and pregnancy may represent a period of elevated risk for both depression and changes in cardiovascular health that may never fully resolve in the years following pregnancy. This study of 61 pregnant women during their third trimester of pregnancy investigated whether depressive symptoms (as measured by the Edinburgh Postnatal Depression Scale, or EPDS) are associated with decreased heart rate variability, a well-known marker of cardiovascular risk. Additionally, this study will investigate whether Plasminogen Activator Inhibitor-1 and 2 (PAI-1 and PAI-2) mediate this association. These pro-thrombotic proteins have long been linked to the presence and severity of cardiovascular disease, and an emerging body of evidence suggests that plasma concentrations of these proteins may also be elevated in Major Depressive Disorder. Heart rate variability was significantly reduced among participants who had clinically significant depressive symptoms during the third trimester (EPDS >14), although adjustment for age, body mass index, smoking, education level and use of psychiatric medication fully attenuated this relationship. PAI-1 and PAI-2 measured via ELISA assay in a subset of the study population (n=23) was found to not mediate this association. This study is the first of its kind to evaluate the role of PAI-1 in psychiatric illness during pregnancy, and may serve as the impetus for further research aimed at elucidating the relationship between mental health and cardiovascular risk during gestation. / Thesis / Master of Science (MSc)

Une coagulopathie aigue endogène (EAC) est présente chez 25% des patients de traumatologie dès leur arrivée. Des résultats d’études récentes montrent que cette EAC est liée à l’activation de la voie de la protéine C (aPC). Quelques heures après, se développe un état pro-coagulant associant un niveau abaissé d’aPC et un taux plasmatique élevé de l’inhibiteur de l’activateur du plasminogene (PAI-1). Nous trouvons que l’incidence des pneumopathies associées à la ventilation est significativement augmentée chez ces patients sans toutefois connaître le rôle exact de ces anomalies de coagulation. Basé sur cette hypothèse central de susceptibilité augmentée a l’infection et plus particulièrement aux pneumopathie a P.aeruginosa (PA) le but de ce travail est d’identifier les mécanismes par lesquels PAI-1 et aPC peuvent moduler la perméabilité de la barrière alveolo capillaire et ceci a travers 3 objectifs spécifiques1 – Objectif 1 : déterminer les mécanismes par lequel PA augmente la perméabilité endothéliale. 2 – Objectif 2 : déterminer le rôle d’aPC dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.3 – Objectif 3 : déterminer le rôle de PAI-1 dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.En utilisant un inhibiteur spécifique des petites GTPases nous démontrons le rôle centrale joué par RhoA dans le développement de l’œdème pulmonaire induit par PA. PAI-1 et aPC sont impliquées dans le mécanisme lésionnel pulmonaire. aPC et l’inhibition de la voie du RhoA attenue le développement de l’œdème pulmonaire et diminue la dissémination systémique bactérienne. Cependant le blocage invivo de la voie de PAI-1 est associé à une surmortalité et à une augmentation de la charge bactérienne suggérant un rôle de PAI-1 dans l’activation de la réponse inflammatoire nécessaire a l’éradication de PA / A clinically significant acute endogenous coagulopathy (EAC) is present in 25% of major trauma patients upon arrival in the emergency department, before any fluid resuscitation. Results from recent clinical studies indicate that EAC is primarily caused by the activation of the anticoagulant protein C pathway. Several hours later, there is the development of a systemic procoagulant activity associated with low plasma levels of activated protein C (aPC) and an inhibition of the fibrinolysis caused by elevated plasma levels of plasminogen activator inhibitor 1 (PAI-1). We have found that the incidence of ventilator-associated pneumonia (VAP) is significantly increased in trauma patients with these coagulation abnormalities [6, 9]. However, whether these coagulation abnormalities play a mechanistic role in the increased susceptibility to nosocomial lung infection observed after severe posttraumatic hemorrhage is unknown. Thus, the central hypothesis is that the increased susceptibility to P. aeruginosa (PA) pneumonia following severe trauma with tissue hypoperfusion is mediated in part by these posttraumatic coagulation abnormalities within the airspaces of the lung. Specifically, in this work, we will identify through 3 specific aims the mechanisms by which PAI-1 and aPC modulate PA–mediated increase in alveolar-capillary barrier permeability.1 - Specific Aim 1: To determine the mechanisms by which PA increases lung endothelial permeability.2 - Specific Aim 2 : To determine the Role of aPC in modulating the effect of PA on the lung endothelial barrier function3 - Specific Aim 3 : To determine the Role of PAI-1 in modulating the effect of PA on the lung endothelial barrier functionIn the present work, we demonstrated the central role small GTPases RhoA plays in the increase of permeability induced by pseudomonas infection. PAI-1 and aPC are deeply involved in the control of early lung inflammation. aPC and inhibition of the RhoA pathway attenuates the development of pulmonary edema and decrease in the systemic dissemination of P. aeruginosa. However, in vivo disruption of PAI-1 signalling is associated with higher mortality at 24 h and significant increase in the bacterial burden suggesting that PAI-1 is required for the activation of the innate immune response necessary for the eradication of PA from the distal airspaces of the lung