L’activateur tissulaire du plasminogène (tPA) est une protéase initialement découverte dans le sang pour son rôle fibrinolytique. C’est pour cette fonction que le tPA recombinant est utilisé pour traiter la phase aigüe de l’accident vasculaire cérébral (AVC) ischémique, même s’il présente quelques limites. Le tPA exogène peut passer du compartiment vasculaire au parenchyme cérébral où il peut influencer des processus physiologiques, et participer au devenir neuronal, notamment aggraver la mort neuronale lors d’un AVC ischémique. Le laboratoire a montré que le tPA peut traverser la barrière-hémato-encéphalique (BHE), par transcytose au travers des cellules endothéliales de la BHE et cela sous le contrôle des récepteurs LRP1 (Low density lipoprotein receptor-related protein 1). D’autres barrières existent au sein du système nerveux central notamment la barrière sang-liquide cérébrospinal (BSLCS), formée par les plexus choroïdes (PCs). Les PCs sont une route de migration pour les cellules inflammatoires et le LCS peut véhiculer des solutés, via les espaces péri-artériels, vers le parenchyme cérébral. Ainsi, dans notre première étude, nous avons testé l’hypothèse d’un passage du tPA vasculaire par les PCs. Pour cela, nous avons produit un tPA traçable in vivo et in vitro. Nous avons commencé par étudier la distribution du tPA suite à une injection intraveineuse (IV) avec comme focus les PCs et le LCS. Nos résultats montrent que le tPA exogène, suite à une injection IV, est retrouvé de manière séquentielle dans les PCs puis dans le LCS. Le tPA est donc capable de traverser les PCs. Nous avons alors développé un modèle de culture primaire de cellules épithéliales de PCs (CPECs) de souris pour disséquer le(s) mécanisme(s) sous-jacents à l’internalisation du tPA par les CPECs. Ce modèle nous a permis de montrer que l’internalisation du tPA par les CPECs est un phénomène actif, médié par un membre de la famille des récepteurs LRP, mais qui n’est ni LRP1, ni LRP2. Nous avons également mis en évidence la nécessité du domaine Finger du tPA pour son internalisation par les CPECs. Une étude préliminaire dans un modèle d’AVC suggère que l’ischémie modifie la cinétique de passage du tPA, puisqu’il y a plus de tPA dans les PCs des souris ischémiées que les souris non ischémiées.Dans une deuxième étude nous nous sommes intéressés à l’effet du tPA endogène sur les PCs. Nous montrons que l’absence de tPA endogène n’influence ni la morphologie des PCs, ni la diffusion du LCS. De plus, nous montrons que cette absence de tPA n’influence pas le nombre de macrophages et de lymphocytes T dans les PCs en conditions basales. / Tissue-type plasminogen activator (tPA) is a protease initially discovered in the blood for its fibrinolytic role. Accordingly, recombinant tPA has become the gold standard to treat the acute phase of ischemic stroke, despite some limitations. Exogenous tPA can switch from the vascular compartment to the brain parenchyma, where it can influence physiological processes, and participate in neuronal fate, including a worsening of neuronal death during ischemic stroke. In the team, it has been shown that tPA can cross the blood-brain barrier (BBB), by a transcytosis through BBB endothelial cells, under the control of LRP1 receptors (Low density lipoprotein receptor-related protein 1). The central nervous system has other barriers, including the blood-cerebrospinal fluid barrier (BCSFB), that relies on choroid plexuses (CPs). CPs are a migration route for inflammatory cells and a major source of CSF, which carries solutes to the cerebral parenchyma, via peri-arterial spaces. Thus, in a first study, we tested the hypothesis of a passage of vascular tPA through CPs. We thus produced a fluorescent tPA that can be tracked in vivo and in vitro. We first studied the distribution of tPA following intravenous (IV) injection, focusing on CPs and CSF. We show that after an IV injection, exogenous tPA is sequentially found in the CPs and then in the CSF. tPA is therefore able to cross the CPs. We then developed a model of primary culture of mouse choroid plexus epithelial cells (CPECs) to dissect the mechanism (s) underlying the internalization of tPA. This model allowed us to demonstrate that the internalization of tPA by CPECs is an active phenomenon, mediated by a member of the family of LRP receptors, but which is neither LRP1 nor LRP2. We also highlight the requirement for the Finger domain of tPA for its internalization by CPECs. A preliminary study in a murine stroke model suggests that ischemia alters the tPA passage kinetics, since there is more tPA in ischemic CPs than non-ischemic CPs.In a second study, we investigated the effect of endogenous tPA on CPs. We show that the absence of endogenous tPA influences neither CPs morphology nor CSF diffusion . Moreover we show that the absence of tPA does not influence the number of macrophages and T cells in the stroma of PCs under basal conditions.

L'anevrysme intracrânien (AIC) est une anomalie morphologique spécifique des artères cérébrales exposant au risque, particulièrement grave, de saignement intracrânien. Malgré les progrès établis dans le traitement curatif , aucun traitement préventif de la formation des AIC n’a été prouvé chez l’Homme. L’absence de traitement non invasif et l’absence de consensus sur le traitement des AIC non rompus sont le corolaire direct d’un manque de connaissance des mécanismes physiopathologiques de la maladie anévrysmale.L’ensemble du travail exposé ici a eu pour but de mieux comprendre les mécanismes de la pathologie anévrysmale intracrânienne pouvant être la base de nouvelles stratégies préventive améliorant le traitement des AIC.Nous avons tout d'abord étudié l'influence des anomalies du tissus de soutien des artères cervicales sur la formation des AIC. Au cours de cette étude, nous avons réalisé une étude cas-témoins dont l’objectif principal était d’étudier l’association entre les anomalies morphologiques des artères cervicales et la présence d’un AIC. Les objectifs secondaires étaient premièrement d’examiner si cette association variait selon le caractère rompu ou non de l’anévrisme et deuxièmement d’examiner si la sévérité des anomalies artérielles cervicales était liée à la sévérité de la pathologie anévrismale. Nous rapportons que la prévalence des anomalies angiographiques chez les patients porteurs d’AIC est élevée. De plus l’incidence d’une pathologie rare, la dysplasie fibro-musculaire est très élevée chez les patients porteurs d’AIC comparativement à la population générale. La présence des anomalies angiographiques n’a aucun impact sur l’évolution naturelle des AIC vers la rupture, ni sur d’autres aspect comme leur taille, leurs nombres ou leur forme. Dans le cadre de notre étude, nous pensons que l’association entre les anomalies angiographiques et les AIC semble être expliquée par l’hypothèse d'une vulnérabilité du tissu de soutien pariétal (condition pathologique particulière de la paroi artérielle à l’origine de la formation des AIC). Cette condition pathologique, affecterait de manière diffuse la vascularisation cérébrale et les artères cervicales. Les AIC seraient ainsi des manifestations cliniquement « bruyantes » de pathologies vasculaires plus silencieuses affectant la paroi de l’ensemble des vaisseaux. Les anomalies morphologiques des artères cervicales témoignent de façon sensible mais très peu spécifique de l’association des pathologies du tissu de soutien avec la présence d’AIC.Dans une deuxième partie nous essayons de décrire et de caractériser une voie inédite de la formation et de la croissance des AIC, la voie de la fibrinolyse via l'activateur de plasminogène de type tissulaire (tPA). Nos données suggèrent que le tPA présent dans la circulation sanguine est suffisant pour favoriser la formation et la rupture des anévrismes. Nous avons donc proposé que le tPA vasculaire était un des responsables de la formation des AIC. Nous avons également constaté une certaine continuité dans le temps de l'influence du tPA sur le remodelage matriciel. Nous avons donc proposé le tPA vasculaire comme une nouvelle cible possible pour prévenir la progression et la rupture des AIC. Différentes expériences ont été entreprise pour inhiber sélectivement le tPA et les résultats préliminaires sont encourageants et ouvrent la voie à une stratégie thérapeutique non invasive inédite. On peut aussi imaginer que ces différentes approches puissent être combinées entre elles et avec des agents matriciels ciblant directement l’activité du tPA dans la paroi des AIC. L’amélioration des stratégies thérapeutiques dans la pathologie anévrysmale intracrânienne est définitivement un axe de recherche dont les possibilités sont immenses et les résultats nécessaires et attendus. / Intracranial aneurysm (IA) is a specific morphological abnormality of the cerebral arteries that exposes to devastating intracranial bleeding. Despite the progress made in the curative treatment, no preventive treatment of IA formation has been proven in humans. The lack of non-invasive treatment and consensus on the treatment of unruptured IA are the consequences of the lack of knowledge of the physiopathological mechanisms of aneurysmal disease. All of the work presented here aims to better understand the mechanisms of intracranial aneurysmal pathology, which may be the basis of new preventive strategies improving the treatment of IA.We first studied the influence of cervical artery abnormalities on IA formation. In this study, we performed a case-control study whose main objective was to study the association between morphologic abnormalities of cervical arteries and the presence of IA. The secondary objectives were first to examine whether this association varied according to whether or not the aneurysm was broken, and secondly to examine whether the severity of the cervical arterial abnormalities was related to the severity of the aneurysmal pathology. We report that the prevalence of angiographic abnormalities in patients harboring IA is high. In addition, the incidence of a rare pathology, fibro-muscular dysplasia is very high in patients with IA compared to the general population. The presence of angiographic abnormalities has no impact on the rupture of the IA, nor on other aspects such as their size, numbers or shape. In our study, we believe that the association between angiographic abnormalities and IA seems to be explained by the hypothesis of a vulnerability of the arterial wall (a particular pathological condition of the arterial wall at the origin of IA formation). This pathological condition would affect the whole cerebral vasculature and cervical arteries. IA would thus be the clinical manifestations of more silent vascular pathologies affecting the wall of all vessels. The morphological abnormalities of the cervical arteries testify sensitively but not very specifically of the association of the arterial wall diseases with the presence of IA.In a second study, we try to describe and characterize an unprecedented pathway of formation and growth of IA : the pathway of fibrinolysis via tissue-type plasminogen activator (tPA). Our data suggest that tPA present in the bloodstream is sufficient to promote formation and rupture of aneurysms. We therefore proposed that vascular tPA was one of those responsible for training IA. We also noted a certain continuity in the time of the influence of the tPA on the matrix remodeling. We therefore proposed vascular tPA as a possible new target to prevent progression and rupture of IA. Various experiments have been undertaken to selectively inhibit tPA and the preliminary results are encouraging and open the way to an unprecedented non-invasive therapeutic strategy. It is also conceivable that these different approaches could be combined with each other and with matrix agents directly targeting tPA activity in the AIC wall. The improvement of therapeutic strategies in intracranial aneurysmal pathology is definitely an topic of ​​research whose possibilities are huge and the results necessary and expected.

L’activateur tissulaire du plasminogène est une sérine protéase initialement découverte dans le compartiment vasculaire et qui joue un rôle prépondérant dans le processus de fibrinolyse. De manière intéressante, le tPA est également présent dans le parenchyme cérébral, où il est notamment exprimé par les neurones. Le tPA est impliqué dans de nombreuses fonctions cérébrales dont la plasticité synaptique, les processus de mémoire et d’apprentissage ainsi que dans la survie et la mort neuronales. Le tPA est capable d’augmenter la signalisation calcique induite par une activation des récepteurs N-Méthyl-D-Aspartate (NMDAR) : un mécanisme à la base de la plasticité synaptique mais également de la mort neuronale excitotoxique. Cependant, il peut également activer les récepteurs du facteur de croissance épidermique (EGFR) pour induire un effet anti-apoptotique sur les neurones. Afin de mieux comprendre les différentes fonctions du tPA sur les neurones, nous nous sommes intéressés à la distribution et au trafic intracellulaire du tPA. Pour cela, nous avons créé un nouvel outil afin d’imager le tPA dans les neurones en temps réel: un plasmide codant pour une protéine fusion, le tPA-HaloTag®.Premièrement, nos résultats montrent que le tPA est présent dans les axones et les dendrites des neurones corticaux matures en culture et qu’il est majoritairement présent dans le compartiment post-synaptique. Cette étude a également permis de voir que le tPA est stocké et libéré par des vésicules d’exocytose VAMP2, qu’il peut être endocyté par des vésicules Rab5, recyclé par des vésicules Rab11 et dégradé par des vésicules Rab7. Deuxièmement, nous avons montré que le tPA est présent dans les mêmes vésicules synaptiques que le facteur neurotrophique issu du cerveau (BDNF) : une neurotrophine importante pour le bon fonctionnement cérébral et dont la maturation dépend de l’activité protéolytique du tPA. Ce travail fournit une meilleure compréhension du rôle et de la distribution du tPA dans les neurones et ouvre de nouvelles voies de recherche dans l’implication de du tPA et du BDNF dans la survie neuronale. / Tissue-type Plasminogen Activator (tPA) is a serine protease, firstly discovered for its fibrinolytic role in the vascular compartment. Interestingly, tPA is also present in the brain parenchyma, being notably expressed by neurons. tPA displays important roles in synaptic plasticity(Danny Baranes et al., 1998; Melchor and Strickland, 2006), learning, memory processes(R Madani et al., 1999; R Pawlak et al., 2002), neuronal survival and death. tPA is able to promote N-Methyl-D-Aspartate Receptors (NMDAR)-induced calcium influx, promoting synaptic plasticity or excitotoxic neuronal death. tPA is also able to activate Epidermal Growth Factor Receptors (EGFR), a mechanism mediating its anti-apoptotic effect. To better understand the different functions of tPA on neurons, we studied the pattern of distribution and trafficking of neuronal tPA. For that, we designed a new tool to image tPA in living neurons: a plasmid encoding for a tPA-HaloTag® fusion protein. We first found that tPA is present in both axons and dendrites of mature cultured cortical neurons and preferentially at the post-synaptic part. Our results also showed that tPA is stored and released by VAMP2 exocytotic vesicles, and can be endocytosed by Rab5 vesicles, recycled by Rab11 vesicles and degraded by Rab7 vesicles. Furthermore, tPA is localized and sorted in the same vesicles than Brain-Derived Neurotrophic Factor (BDNF), one of the most important neurotrophins, Interestingly, BDNF maturation is dependent of tPA proteolytic activity. This work provides a better understanding of the role and distribution of tPA in living neurons and opens new avenues into the involvement of tPA and BDNF in neuronal survival.

Background: Clinical factors associated with exclusion from recombinant tissue plasminogen activator in both men and women are not completely understood. The aim of this study is to determine whether there is a gender difference in clinical risk factors that excluded ischemic stroke patients with a history of smoking from recombinant tissue plasminogen activator. Methods: Retrospective data from a stroke registry were analyzed, and multivariable linear regression models were used to determine gender differences. Logistic regression models determined exclusion clinical risk factors for thrombolysis in male and female acute ischemic stroke patients with a history of smoking, while sequentially adjusting for sociodemographic, clinical, and stroke-related variables. The Kaplan–Meier survival analysis was used to determine the exclusion probabilities of men and women with a history of smoking within the stroke population. Results: Of the 1,446 acute ischemic stroke patients eligible for recombinant tissue plasminogen activator, 379 patients with a history of smoking were examined, of which 181 received recombinant tissue plasminogen activator while 198 were excluded from receiving recombinant tissue plasminogen activator. Of the 198 patients, 75 females and 123 males were excluded from receiving recombinant tissue plasminogen activator. After multivariable adjustment for age, National Institutes of Health scores, and stroke-related factors, females who present with weakness/paresis on initial examination (OR = 0.117, 95% CI, 0.025–0.548) and men who present with a history of previous transient ischemic attack (OR = 0.169, 95% CI, 0.044–0.655), antiplatelet medication use (OR = 0.456, 95% CI, 0.230–0.906), and weakness/paresis on initial examination (OR = 0.171, 95% CI, 0.056–0.521) were less likely to be excluded from recombinant tissue plasminogen activator (thrombolysis therapy). Conclusions: In an ischemic stroke population with a history of smoking, female smokers are more likely to be excluded from thrombolysis therapy in comparison to men, even after adjustment for confounding variables.

No description available.

No description available.

Motivation: Cardiovascular heart disease (CVD) is the leading cause of death worldwide. Risk markers for CVD include, amongst others, the haemostatic factors tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), factor VII (FVII) and fibrinogen and more recently, C-reactive protein (CRP), a sensitive marker of inflammation. Epidemiological studies have demonstrated an inverse association between dietary fibre (DF) consumption and risk factors for CVD and CVD prevalence. Some research indicates that this protection may be related to favourable changes in the haemostatic profile and inflammatory markers. This is applicable for the consumption of total DF, as well as soluble and insoluble fibre. However, clinical intervention trials report conflicting data on the effects of DF on t-PA, PAI-1, FVII, fibrinogen and CRP. In addition, available literature is not clear on the mechanisms through which DF may have favourable effects. Objective: The main objective of this study was to review the results of randomised controlled trials systematically on the effects of DF on the above-mentioned selected haemostatic variables and CRP in healthy adults and subjects with hypertriglyceridaemia and the metabolic syndrome. Methods: Human adult intervention trials, at least two weeks in duration, with an increased and measurable consumption of DF were included. Electronic databases were searched from the earliest record to May/July 2006 and supplemented by crosschecking reference lists of relevant publications. From the literature search, two reviewers identified studies that were rated for quality based on the published methodology. No formal statistical analysis was performed due to the large differences in the study designs of the dietary intervention trials. The primary outcome measures were percentage changes between intervention and control groups, or baseline to end comparisons for t-PA, PAI-1, FVII, fibrinogen and CRP. Results t-PA activity increased significantly (14-167%) over the short and long-term following increased fibre intakes. PAI-1 activity decreased significantly between 15-57% over periods ranging from two to six weeks. These favourable changes in t-PA and PAI-1 occurred in healthy, hypertriglyceridaemic and metabolic syndrome subjects following consumption of diets containing ≥3.3 g/MJ DF and ≥4.5 g/MJ DF respectively. Mechanisms through which DF may affect t-PA and PAI-1 include its lowering effect on insulinaemic and glycaemic responses, decreasing triglycerides which are a precursor of very-low-density lipoproteins, fermentation of DF to short-chain fatty acids, which may reduce free fatty acid concentrations, as well as the role of DF in promoting weight loss. High DF intakes did not have a significant effect on fibrinogen concentrations possibly because of relatively little weight loss, too low DF dosages and maintaining a good nutritional status. Inadequate study designs deterred from meaningful conclusions. Significant decreases in FVll coagulant activity (6-16%) were observed with DF intakes of ≥3.3 g/MJ and concomitant decreased saturated fat intakes and weight loss in healthy and hypertriglyceridaemic subjects. Confounding factors include weight loss and a simultaneous decreased intake of saturated fats. The type of fibre seems to play a role as well. Mechanisms through which DF may reduce FVll concentrations include its effects on triglyceride-rich lipoproteins, insulin and weight loss. Increased DF consumption with dosages ranging between 3.3-7.8 g/MJ were followed by significantly lower CRP concentrations (25-54%), however, simultaneous weight loss and altered fatty acid intakes were also present in all the studies. Mechanisms are inconclusive but may involve the effect of DF on weight loss, insulin, glucose, adiponectin, interleukin-6, free fatty acids and triglycerides. Conclusions: Epidemiological evidence indicates an association between DF and the CVD risk factors t-PA, PAI-1, FVII, fibrinogen and CRP. In general, the risk of CVD may improve with high-fibre intakes as indicated by the favourable changes in some of the parameters. However, simultaneous reduced fat intakes and weight loss presented difficulties in separating out the effects of specific components. Furthermore, DF is consumed in a variety of different forms and different dosages that may have different effects. Overall, the study designs used in the intervention trials prevented significant conclusions. DF did, however, play a role in modifying t-PA, PAI-1, FVII and CRP. Potential effects on fibrinogen were not quantifiable. Recommendations: The results from this investigation provide the motivation for additional controlled clinical research to establish the effect and mechanisms of DF on haemostatic variables and CRP. A critical aspect of future studies would be to set up suitable protocols. The amount of subjects, duration of the trials, confounding factors such as weight loss and altered fat intakes and differentiation between types and dosage of DF are important. DF supplemental studies are recommended as they may be the most suitable method to reach meaningful conclusions. / Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007

Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

Motivation: Cardiovascular heart disease (CVD) is the leading cause of death worldwide. Risk markers for CVD include, amongst others, the haemostatic factors tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), factor VII (FVII) and fibrinogen and more recently, C-reactive protein (CRP), a sensitive marker of inflammation. Epidemiological studies have demonstrated an inverse association between dietary fibre (DF) consumption and risk factors for CVD and CVD prevalence. Some research indicates that this protection may be related to favourable changes in the haemostatic profile and inflammatory markers. This is applicable for the consumption of total DF, as well as soluble and insoluble fibre. However, clinical intervention trials report conflicting data on the effects of DF on t-PA, PAI-1, FVII, fibrinogen and CRP. In addition, available literature is not clear on the mechanisms through which DF may have favourable effects. Objective: The main objective of this study was to review the results of randomised controlled trials systematically on the effects of DF on the above-mentioned selected haemostatic variables and CRP in healthy adults and subjects with hypertriglyceridaemia and the metabolic syndrome. Methods: Human adult intervention trials, at least two weeks in duration, with an increased and measurable consumption of DF were included. Electronic databases were searched from the earliest record to May/July 2006 and supplemented by crosschecking reference lists of relevant publications. From the literature search, two reviewers identified studies that were rated for quality based on the published methodology. No formal statistical analysis was performed due to the large differences in the study designs of the dietary intervention trials. The primary outcome measures were percentage changes between intervention and control groups, or baseline to end comparisons for t-PA, PAI-1, FVII, fibrinogen and CRP. Results t-PA activity increased significantly (14-167%) over the short and long-term following increased fibre intakes. PAI-1 activity decreased significantly between 15-57% over periods ranging from two to six weeks. These favourable changes in t-PA and PAI-1 occurred in healthy, hypertriglyceridaemic and metabolic syndrome subjects following consumption of diets containing ≥3.3 g/MJ DF and ≥4.5 g/MJ DF respectively. Mechanisms through which DF may affect t-PA and PAI-1 include its lowering effect on insulinaemic and glycaemic responses, decreasing triglycerides which are a precursor of very-low-density lipoproteins, fermentation of DF to short-chain fatty acids, which may reduce free fatty acid concentrations, as well as the role of DF in promoting weight loss. High DF intakes did not have a significant effect on fibrinogen concentrations possibly because of relatively little weight loss, too low DF dosages and maintaining a good nutritional status. Inadequate study designs deterred from meaningful conclusions. Significant decreases in FVll coagulant activity (6-16%) were observed with DF intakes of ≥3.3 g/MJ and concomitant decreased saturated fat intakes and weight loss in healthy and hypertriglyceridaemic subjects. Confounding factors include weight loss and a simultaneous decreased intake of saturated fats. The type of fibre seems to play a role as well. Mechanisms through which DF may reduce FVll concentrations include its effects on triglyceride-rich lipoproteins, insulin and weight loss. Increased DF consumption with dosages ranging between 3.3-7.8 g/MJ were followed by significantly lower CRP concentrations (25-54%), however, simultaneous weight loss and altered fatty acid intakes were also present in all the studies. Mechanisms are inconclusive but may involve the effect of DF on weight loss, insulin, glucose, adiponectin, interleukin-6, free fatty acids and triglycerides. Conclusions: Epidemiological evidence indicates an association between DF and the CVD risk factors t-PA, PAI-1, FVII, fibrinogen and CRP. In general, the risk of CVD may improve with high-fibre intakes as indicated by the favourable changes in some of the parameters. However, simultaneous reduced fat intakes and weight loss presented difficulties in separating out the effects of specific components. Furthermore, DF is consumed in a variety of different forms and different dosages that may have different effects. Overall, the study designs used in the intervention trials prevented significant conclusions. DF did, however, play a role in modifying t-PA, PAI-1, FVII and CRP. Potential effects on fibrinogen were not quantifiable. Recommendations: The results from this investigation provide the motivation for additional controlled clinical research to establish the effect and mechanisms of DF on haemostatic variables and CRP. A critical aspect of future studies would be to set up suitable protocols. The amount of subjects, duration of the trials, confounding factors such as weight loss and altered fat intakes and differentiation between types and dosage of DF are important. DF supplemental studies are recommended as they may be the most suitable method to reach meaningful conclusions. / Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.