No description available.

<p>Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). </p><p>In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes.</p><p>Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.</p>

Small diameter synthetic vascular grafts have yet to be clinically successful due to luminal narrowing from thrombosis and intimal hyperplasia. Current attempts to address this issue include the development of materials that support endothelialisation and protein modification to the material surfaces that reduce thrombosis. The extracellular matrix protein elastin has been found to be one of the least thrombogenic components of blood vessels, and its purified and recombinant forms have shown reduced thrombogenicity in both in vitro and in vivo models. Biomaterial coatings of elastin-like polypeptides (ELPs) recombinantly produced in the Woodhouse laboratory showed reduced fibrinogen adsorption, platelet adhesion, and platelet activity. However, the reason for their relative non-thrombogenicity is still not fully understood. In this work, the leukocyte response to ELP-coated materials was investigated. In particular, ELP1 and ELP4, which differ in molecular weight and sequence length, were physically adsorbed to a polyethylene terephthalate surface (MylarTM), yielding 0.22 ± 0.13 μg/cm2 and 0.37 ± 0.19 μg/cm2 surface coverage, respectively, as determined by the colorimetric assay, FastinTM Elastin. These surfaces were exposed to flowing citrated whole blood for surface and bulk evaluation of leukocyte activity using scanning electron microscopy and flow cytometry, respectively. Little leukocyte activation was observed on the surface of the controls, low-density polyethylene and uncoated MylarTM. In the bulk, tissue factor (TF) expression (monocytes: ELP1 = 38.6 ± 16.3 %, ELP4 = 33.9 ± 18.1 %) and platelet-leukocyte aggregates determined by CD61 (monocytes: ELP1 = 63.1 ± 17.1 %, ELP4 = 61.8 ± 16.8 %; granulocytes: ELP1 = 62.7 ± 17.0 %, ELP4 = 60.5 ± 20.1 %) were both decreased compared to uncoated MylarTM, while CD11b upregulation (monocytes: ELP1 = 18.7 ± 2.2 %, ELP4 = 19.7 ± 2.7 %; granulocytes: ELP = 21.4 ± 3.7 %, ELP4 = 22.0 ± 3.2 %) was increased. The statistical dependence of TF expression and platelet-monocyte aggregates was tested; however, no correlation was found. Overall, platelet-leukocyte aggregate formation was reduced and there were conflicting results with regards to the reduction of leukocyte activation for the ELP coatings on MylarTM. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-10-10 15:34:51.802

Rationale: Tissue Factor (TF) is a transmembrane glycoprotein that canonically functions as the initiator of the coagulation cascade. Increased levels of TF have been associated with inflammatory airway diseases. Since lipopolysaccharide (LPS) is known to elicit and inflammatory response in airway epithelium, we hypothesized that airway epithelial cells release TF when exposed to LPS. Since TF aids in local wound healing, we also hypothesized that inhibition of TF would decrease NHBE growth. The specific aim of this work was to evaluate the effects of LPS exposure on TF production and release from airway epithelia and determine the signaling pathways involved. A secondary aim was to evaluate the effects of TF inhibition on NHBE growth. Methods: Normal human bronchial epithelial cells were grown in submerged cell culture and exposed to LPS as well as several intracellular signaling pathway agonist and inhibitors. Measurements: Tissue Factor mRNA and protein were measured in culture media and cell lysate by reverse-transcriptase polymerize chain reaction and enzyme-linked immunosorbent assay, respectively. Signaling pathways were evaluated using selective agonists and inhibitors. Main results: TF protein levels increased nearly two-fold in cell media after exposure to LPS (p < 0.01). This did not occur in the presence of an MEK/ERK inhibitor (PD98059) or a SMAD inhibitor (SB431542). TF protein levels also increased nearly ten-fold in the presence of TGF-beta (p < 0.05). mRNA of TF and TGF-beta was not altered by LPS or TGF-beta exposure. NHBE grown in the presence of Tissue Factor Pathway Inhibitor grew significantly slower than those grown in standard media (P < 0.05). Conclusions: NHBE release TF when exposed to LPS. This phenomenon is post-translational and may be mediated by an autocrine mechanism involving MEK/ERK signaling that increases TGF-beta which then leads to the release of TF. Our data suggest that this airway epithelium release of TF serves as a local repair function.

L’hémostase est un processus physiologique permettant de préserver l’intégrité du système vasculaire et de prévenir une perte de sang en réponse à une blessure. En situation pathologique, comme dans le cas de cancers, d’infections ou de maladies cardiovasculaires, il peut y avoir activation de la cascade de coagulation entraînant la formation d’un thrombus.Dans cette étude, nous avons utilisé la microscopie intravitale dans un modèle de blessure au rayon laser pour comprendre les mécanismes cellulaires et moléculaires mis en jeu dans la formation d’un thrombus plaquettaire en conditions physiologiques et pathologiques lors du développement d’un cancer. La première partie de ce travail a consisté à décrire l’implication des polynucléaires neutrophiles dans la formation d’un thrombus. Nous montrons que les neutrophiles sont les premières cellules à s’accumuler au niveau du site de blessure et représentent la principale source de facteur tissulaire menant à la génération de fibrine et à la formation du thrombus. Ces neutrophiles sont nécessaires au recrutement de cellules endothéliales progénitrices (Endothelial colony- forming cells, ECFCs), qui sont des cellules capables de jouer un rôle important dans la réparation vasculaire. Dans une seconde partie, nous avons déterminé, dans des modèles murins adaptés, l’implication de l’activation du FT et des plaquettes dans la thrombose associée au cancer. En conclusion, notre travail donne de nouvelles perspectives dans la compréhension du rôle pathophysiologique des neutrophiles, des cellules progénitrices endothéliales et des plaquettes. / Hemostasis is a physiological process to preserve the integrity of the vascular system and to prevent blood loss in response to injury. In pathological conditions, such as cancers, infections or cardiovascular diseases, the blood coagulation cascade can be activated, leading to the formation of a platelet thrombus.Using a laser-injury model coupled with a high-definition, high-speed camera, we explored the cellular and molecular mechanisms involved in thrombus formation in physiological and in pathological conditions associated with the development of a cancer. The first part of this work describes the role of polymorphonuclear neutrophils (PMNs) in thrombus formation. We show that PMNs are the first cells to accumulate at the site of injury and represent the main source of blood-borne tissue factor (TF), leading to the generation of fibrin and thrombus formation. We also show that once present at the site of injury, PMNs recruit Endothelial Progenitor Cells (endothelial colony-forming cells, ECFCs), which play a key role in vascular repair. The second part of this work we determined, in dedicated mouse models, the involvement of TF and platelet activation in thrombosis associated with cancer. Together, our findings provide new perspectives in the understanding of the pathophysiological role of polymorphonuclear neutrophils, Endothelial Progenitor Cells and platelets.

L'objectif de ce travail est d'évaluer le rôle du facteur tissulaire (FT) dans l'initiation de la coagulation d'un circuit d'hémodialyse. A partir de l'analyse des membranes d'hémodialyse, nous avons observé que les leucocytes et, majoritairement les polynucléaires neutrophiles (PNN) adhéraient aux membranes hémocompatibles (de type AN69ST). Ces cellules expriment un FT fonctionnel. Nous avons développé différents modèles d'étude de l'expression du FT par les PNN. A partir de PNN humains obtenus chez des sujets sains, nous montrons que les PNN expriment le FT après stimulation par le TNF. L'IL-8, chemokine chimioattractante des PNN augmentent, par un effet de priming, l'expression de FT en réponse au TNF. L'inhibition de l'adhésion par un anticorps dirigé contre les β2-intégrines, induit une diminution de l'expression de FT en réponse au TNF. L'inhibition de la voie de signalisation MEK1/2, la p38 MAPK, et des radicaux libres oxygénés, inhibe également cette expression. A partir de PNN provenant de péritonites secondaires à une dialyse péritonéale, nous avons mis en évidence une forte expression de FT par ces PNN (ARNm et protéine). Le FT possède un fort potentiel pro-coagulant. Ce modèle physiopathologique est la conséquence d'une migration et d'une activation inflammatoire comparable au modèle que nous avons développé in-vitro. Dans l'objectif de faire la preuve de ce nouveau concept, nous avons évalué un facteur VII humain recombinant inactivé (FVIIai) dans un modèle d'hémodialyse chez le mouton. Nos résultats sont en faveur d'un effet anticoagulant du circuit d'hémodialyse, sans effet anticoagulant mesurable chez l'animal. / The objective of this study is to analyse the role of Tissue Factor, the unique physiological trigger on thrombin generation. Analysing haemodialysis membranes, we found that leukocytes, mainly polymorphonuclear neutrophils (PMN) adhere to hemocompatible (AN69ST) membranes. These cells express a functional TF. We next showed that human PMN obtained from healthy subjects expressed TF in response to TNF. IL-8, a major chemokine involved in PMN chemoattraction primed TNF-induced TF by PMN. TF expression was down regulated when 2 integrins were blocked by a potent antibody. The inhibition of MEK1/2, p38 MAPK and free radicals reduced TF expression. We observed, that PMN obtained from patients experiencing peritonitis, expressed high levels of TF (mRNA and protein). Functional assays measuring Xa generation and kinetics of thrombinn generation (thrombinography) indicate the stong procoagulant potential of these cells. This physiopathological model is close to our in vitro model as it results from PMN migration and inflammatoty activation. For proof of concept, we evaluated the effect of an inactivated human recombinant factor VIIa ( FVIIai) in a sheep model of hemodialysis. Our results show that FVIIai limits haemodialysis circuit coagulation without any measurable systemic anticoagulant effect

Acidentes pela serpente Bothrops jararaca (Bj) causam distúrbios hemostáticos em pacientes. Sabe-se que a fisiopatologia desses distúrbios é complexa, porém não se conhece a relevância das duas principais famílias de enzimas presentes no veneno de Bj com atividade anti-hemostática, as metaloproteinases e serinaproteases, para promover esses distúrbios. Além disso, a injúria local induzida no local da inoculação do veneno poderia estimular a liberação de fator tissular (TF) na circulação sanguínea, favorecendo a coagulopatia. Assim, o objetivo deste projeto foi investigar a contribuição das metaloproteinases e serinaproteases do veneno de Bj e a expressão de TF para a gênese dos distúrbios hemostáticos, utilizando um modelo experimental em ratos. O veneno de Bj foi previamente incubado com Na2-EDTA 13 mM ou AEBSF 4 mM para inibir as metaloproteinases e serinaproteases, respectivamente, e administrado pelas vias s.c. ou i.v. Após 3 e 6 h, os parâmetros hemostáticos e de expressão proteica de TF e isomerase de dissulfeto proteico (PDI) foram avaliados. Os níveis de veneno circulante se elevaram mais rapidamente no grupo injetado pela via i.v., e o tratamento do veneno com Na2-EDTA ou AEBSF não reduziu os níveis circulantes de veneno em comparação ao grupo controle com veneno. Em comparação com animais tratados com salina, houve uma queda abrupta na contagem plaquetária em todos os grupos e tempos administrados com veneno de Bj, sendo o grupo administrado pela via i.v. o que apresentou uma queda de maior intensidade. O pré- tratamento do veneno com o AEBSF não impediu o consumo plaquetário e somente o Na2-EDTA parcialmente reverteu a plaquetopenia. Por outro lado, o veneno não tratado causou consumo de fibrinogênio plasmático, geração de produtos de degradação de fibrinogênio e fibrina, prolongamento do tempo de protrombina (TP) e hemorragia no local de inoculação do veneno. No entanto, o Na2-EDTA, e não o AEBSF, bloqueou completamente esses parâmetros. Não houve redução dos níveis de fator VII ao longo do envenenamento, e seus níveis apresentou um notável aumento nos animais envenenados no grupo 6 h s.c. Ratos envenenados apresentaram notável aumento dos níveis do TF plasmáticos, que foi inibido pelo pré-tratamento com o Na2-EDTA. Houve também aumento da expressão de TF no pulmão e pele. Nos grupos injetados com veneno de Bj em 6 h ocorreu uma redução da expressão de PDI. Os resultados mostram que as metaloproteinases são componentes essenciais para o desencadeamento da coagulopatia do envenenamento. No entanto, as metaloproteinases e serinaproteases não estão diretamente envolvidas na gênese da plaquetopenia induzida pelo veneno de Bj e outros mecanismos/toxinas parecem estar envolvidos. Os resultados também demonstraram o aumento dos níveis de TF plasmático durante o envenenamento, similar àquele observado na coagulação intravascular disseminada, o que sugere a importância da geração de TF como um mecanismo para promover distúrbios sistêmicos da coagulação / Bites by Bothrops jararaca (Bj) snakes evoke hemostatic disturbances in patients. The pathophysiology of such disturbances is complex, but the importance of the major enzyme families with anti-hemostatic activity, found in Bj venom. i.e., metalloproteinases and serine proteinases, to promote them is not known. Moreover, the local injury induced at the site of venom inoculation might also stimulate tissue factor (TF) release into bloodstream, favoring the coagulopathy. The aim of this study was to investigate the contribution of metalloproteinases and serine proteinases of Bj venom, as well the TF expression to the genesis of hemostatic disturbances, using an experimental model in rats. Crude Bj venom was previously incubated with 13 mM Na2-EDTA or 4 mM AEBSF to inhibit metalloproteinases and serine proteinases, respectively, and administered s.c. or i.v. into rats. After 3 and 6 h, hemostatic parameters and TF and protein disulfide isomerase (PDI) expression were evaluated in plasma and samples of skin and lungs. Circulating venom levels increased more rapidly in i.v. group, and neither Na2-EDTA nor AEBSF treatment reduced the circulating venom levels in comparison with the control group. Platelet counts showed a marked decrease in all groups administered with Bj venom in comparison with saline-treated rats; the fall in platelet counts was more intense in animals administered i.v. with Bj venom. The pre-treatment of venom with AEBSF failed to block the fall in platelets count, and only Na2-EDTA minimally reversed thrombocytopenia. Nonetheless, non-treated venom provoked plasma fibrinogen consumption, generation of fibrin(ogen) degration products, prolongation of prothrombin time (TP), and hemorrhage at the site of venom inoculation. However, Na2-EDTA, but not AEBSF, completely blocked these parameters. Factor VII levels were not reduced during envenomation, and they showed a marked increase in envenomed rats at 6 h in the s.c. group. Envenomed rats showed a marked increase in plasma TF levels, which was also blocked by Na2-EDTA. In addition, TF expression was increased in the lung and skin samples. PDI expression in skin was reduced at 6 h in all groups treated with venom. These findings demonstrate that metalloproteinases are essential venom components involved in the Bj-induced coagulopathy. Nonetheless, metalloproteinases and serine proteases had no direct involvement in the genesis of Bj-induced thrombocytopenia and other venom mechanisms/toxins seem to be associated therein. High levels of TF in plasma may occur during snake envenomation, so that the etiopathogenesis of coagulopathy in snake envenomation resembled that of true disseminated intravascular coagulation syndrome

Acidentes pela serpente Bothrops jararaca (Bj) causam distúrbios hemostáticos em pacientes. Sabe-se que a fisiopatologia desses distúrbios é complexa, porém não se conhece a relevância das duas principais famílias de enzimas presentes no veneno de Bj com atividade anti-hemostática, as metaloproteinases e serinaproteases, para promover esses distúrbios. Além disso, a injúria local induzida no local da inoculação do veneno poderia estimular a liberação de fator tissular (TF) na circulação sanguínea, favorecendo a coagulopatia. Assim, o objetivo deste projeto foi investigar a contribuição das metaloproteinases e serinaproteases do veneno de Bj e a expressão de TF para a gênese dos distúrbios hemostáticos, utilizando um modelo experimental em ratos. O veneno de Bj foi previamente incubado com Na2-EDTA 13 mM ou AEBSF 4 mM para inibir as metaloproteinases e serinaproteases, respectivamente, e administrado pelas vias s.c. ou i.v. Após 3 e 6 h, os parâmetros hemostáticos e de expressão proteica de TF e isomerase de dissulfeto proteico (PDI) foram avaliados. Os níveis de veneno circulante se elevaram mais rapidamente no grupo injetado pela via i.v., e o tratamento do veneno com Na2-EDTA ou AEBSF não reduziu os níveis circulantes de veneno em comparação ao grupo controle com veneno. Em comparação com animais tratados com salina, houve uma queda abrupta na contagem plaquetária em todos os grupos e tempos administrados com veneno de Bj, sendo o grupo administrado pela via i.v. o que apresentou uma queda de maior intensidade. O pré- tratamento do veneno com o AEBSF não impediu o consumo plaquetário e somente o Na2-EDTA parcialmente reverteu a plaquetopenia. Por outro lado, o veneno não tratado causou consumo de fibrinogênio plasmático, geração de produtos de degradação de fibrinogênio e fibrina, prolongamento do tempo de protrombina (TP) e hemorragia no local de inoculação do veneno. No entanto, o Na2-EDTA, e não o AEBSF, bloqueou completamente esses parâmetros. Não houve redução dos níveis de fator VII ao longo do envenenamento, e seus níveis apresentou um notável aumento nos animais envenenados no grupo 6 h s.c. Ratos envenenados apresentaram notável aumento dos níveis do TF plasmáticos, que foi inibido pelo pré-tratamento com o Na2-EDTA. Houve também aumento da expressão de TF no pulmão e pele. Nos grupos injetados com veneno de Bj em 6 h ocorreu uma redução da expressão de PDI. Os resultados mostram que as metaloproteinases são componentes essenciais para o desencadeamento da coagulopatia do envenenamento. No entanto, as metaloproteinases e serinaproteases não estão diretamente envolvidas na gênese da plaquetopenia induzida pelo veneno de Bj e outros mecanismos/toxinas parecem estar envolvidos. Os resultados também demonstraram o aumento dos níveis de TF plasmático durante o envenenamento, similar àquele observado na coagulação intravascular disseminada, o que sugere a importância da geração de TF como um mecanismo para promover distúrbios sistêmicos da coagulação / Bites by Bothrops jararaca (Bj) snakes evoke hemostatic disturbances in patients. The pathophysiology of such disturbances is complex, but the importance of the major enzyme families with anti-hemostatic activity, found in Bj venom. i.e., metalloproteinases and serine proteinases, to promote them is not known. Moreover, the local injury induced at the site of venom inoculation might also stimulate tissue factor (TF) release into bloodstream, favoring the coagulopathy. The aim of this study was to investigate the contribution of metalloproteinases and serine proteinases of Bj venom, as well the TF expression to the genesis of hemostatic disturbances, using an experimental model in rats. Crude Bj venom was previously incubated with 13 mM Na2-EDTA or 4 mM AEBSF to inhibit metalloproteinases and serine proteinases, respectively, and administered s.c. or i.v. into rats. After 3 and 6 h, hemostatic parameters and TF and protein disulfide isomerase (PDI) expression were evaluated in plasma and samples of skin and lungs. Circulating venom levels increased more rapidly in i.v. group, and neither Na2-EDTA nor AEBSF treatment reduced the circulating venom levels in comparison with the control group. Platelet counts showed a marked decrease in all groups administered with Bj venom in comparison with saline-treated rats; the fall in platelet counts was more intense in animals administered i.v. with Bj venom. The pre-treatment of venom with AEBSF failed to block the fall in platelets count, and only Na2-EDTA minimally reversed thrombocytopenia. Nonetheless, non-treated venom provoked plasma fibrinogen consumption, generation of fibrin(ogen) degration products, prolongation of prothrombin time (TP), and hemorrhage at the site of venom inoculation. However, Na2-EDTA, but not AEBSF, completely blocked these parameters. Factor VII levels were not reduced during envenomation, and they showed a marked increase in envenomed rats at 6 h in the s.c. group. Envenomed rats showed a marked increase in plasma TF levels, which was also blocked by Na2-EDTA. In addition, TF expression was increased in the lung and skin samples. PDI expression in skin was reduced at 6 h in all groups treated with venom. These findings demonstrate that metalloproteinases are essential venom components involved in the Bj-induced coagulopathy. Nonetheless, metalloproteinases and serine proteases had no direct involvement in the genesis of Bj-induced thrombocytopenia and other venom mechanisms/toxins seem to be associated therein. High levels of TF in plasma may occur during snake envenomation, so that the etiopathogenesis of coagulopathy in snake envenomation resembled that of true disseminated intravascular coagulation syndrome

La thèse décrit, dans le premier chapitre, les effets opposés sur la régulation de l'expression du gène du facteur tissulaire (TF) par la voie PI3K/Akt et la voie Erk1/2 in vitro. TF est une molécule clé pour initier la coagulation du sang. Son rôle est maintenant connu au développement embryonnaire, le maintien de l'intégrité vasculaire et la réparation tissulaire. De fait que divers cancers expriment des niveaux aberrantes du TF qui sont corrélés avec le pronostic, TF pourrait réellement favorise la croissance tumorale, l'angiogenèse et la métastase. En utilisant une lignée cellulaire épithéliale de cancer du sein MDA-MB-231, nous avons quantifié l'expression du gène du TF par le test luminescent, qPCR, western blot, et d'activité TF associée aux cellules in vitro. Nous avons constaté que 1) PI3K/Akt est la principale voie qui active l'expression des gènes TF. 2) L'activité Erk1 / 2 inhibe l'expression du gène TF; 3) le blocage de la voie Erk1/2par PD98059 induit une expression aberrante du gène du TF via sur-activation du récepteur du EGF; 4) cette sur-expression du TF peut être neutralisé par le blocage de l'EGFR et de la voie PI3K/Akt; 5) cette sur-expression induite par l'inhibition de Erk1/2est une caractéristique commune pour les lignées de cellules épithéliales cancéreuses testés, comme SKOV-3-3 et OVCAR ; 6) la forme soluble du TF suite à l'épissage alternatif représente une faible proportion de l'ARNm de TF totale et 7) Le niveau d’expression duTF des cellules MDA-MB-231 est corrélée à une activité procoagulante cellulaire et à l'invasivité des cellules in vitro. Cette étude a révélé une boucle de régulation négative de l’Erk1/2 vis-à-vis du l’activité du EGFR, ce qui suggère un effet indésirable des agents thérapeutiques ciblant l’Erk dans la clinique.La thèse décrit, dans le deuxième chapitre, les éléments de preuve de la fonction angiogénique de Hypb, une H3K36 méthyltransférase avec Hypb-/ - knockout souris.Ces souris ont montré une létalité embryonnaire à E10.5-E11.5 et de graves anomalies vasculaires dans le sac vitellin d'embryons et le placenta. Les expériences avec des cellules endothéliales HMEC-1 in vitro utilisant l’anti-Hypb siARN ont démontré défaut de migration et d’invasion cellulaire. En outre, les cellules traitées ont perdu la capacité de former des vaisseaux. Ces données sont bien cohérente avec l'analyse histologique de embryons Hypb-/- de souris dont le réseau complexe de ramification vaisseaux embryonnaires et la circulation sanguine étaient absentes. L'analyse génétique sur sac vitellin avec microarray ont suggéré une association entre le défaut de l'angiogenèse dans Hypb-/ - souris et la déréglementation de la sécrétion des protéines Angptl3 etCyr61, qui pourraient se lier à avß3. Il a également souligné le rôle de l'angiogénine,Angptl3 et Gja4 dans ce défaut, parce que lien de ces gènes à l'angiogenèse étaient démontrés. Mécanismes plausibles sont explorés. La régulation épigénétique de l'angiogenèse est une question importante parce qu'elle contrôle la régulation spatiale et temporelle de l'expression de milliers de gènes. Notre étude suggère clairement un rôle clé de Hypb et H3K36 méthylation et Hypb mécanismes liés aux processus de vascularisation chez les mammifères (vasculogenèse et l'angiogenèse). L'angiogenèse est importante pour la recherche fondamentale et application médicale dans les maladies cardio-vasculaires et de la thérapie anti-cancereuse. Nous espérons que de nouvelles stratégies thérapeutiques ciblant les voies épigénétiques permettra d'offrir de nouvelles voies thérapeutiques. / The thesis described, in the first chapter, an opposite regulatory effects of PI3K/Akt pathway and Erk1/2 pathway on tissue factor(TF) gene expression in vitro. TF is a keymolecule required to initiate blood coagulation, and is now accepted to be essential forembryo development, maintenance of vascular integrity and tissue repair. Since a variety of cancers show aberrant prognostics-correlated high levels of TF expression, it is believed that TF promotes tumor growth, angiogenesis and metastasis. Using an epithelial breast cancer cell line MDA-MB-231, we quantified TF gene expression by luminescent test, qPCR, western blot, and cell-associated TF activity in vitro. We found that 1) PI3K/Akt is the major pathway that activates TF gene expression. 2) Erk1/2activity inhibits TF gene expression; 3) blocking Erk1/2 by PD98059 aberrant lyupregulates TF gene expression via enhancing EGFR activity; 4) this enhanced TF expression can be neutralized by blocking EGFR and PI3K/Akt pathway activation; 5) TF upregulation induced by Erk inhibition is a common feature in the tested epithelial cancer cell lines SKOV-3 and OVCAR-3; 6) Soluble form of TF due to alternative splicing represents a small proportion of total TF mRNA and 7) The level of TF gene expression in MDA-MB-231 cells is correlated to cell procoagulant activity and cell invasiveness in vitro. This study revealed a negative feedback loop of Erk-mediated EGFR inhibitions,suggesting an undesirable effect of the agents targeting Erk in clinic.The thesis described, in the second chapter, the evidence of angiogenic function of Hypb,a H3K36 methyltransferase with Hypb-/- knockout mice. These mice demonstrated embryonic lethality at E10.5-E11.5 and severe vascular defects in the Hypb−/− embryo,yolk sac, and placenta. The experiments with endothelial cells HMEC-1 in vitro using anti-Hypb siRNA demonstrated defective cell migration and invasion. Furthermore, the treated cells lost the capacity of vessel formation. These data were well coherent with histological analysis of Hypb-/- mice embryos that lacked the intricate network of branching embryonic vessels and showed disrupted blood flow. The genetic microarray analysis on yolk sac suggested an association between the defect of angiogenesis inHypb-/- mice and deregulated Angptl3 and Cyr61 protein release, since both of which could bind to αvβ3. It also suggested the roles of angiogenin, Angptl3 and Gja4 in this defect because these angiogenesis-related genes were downregulated. Plausible mechanisms are discussed. The epigenetic regulation of angiogenesis is an important issue because it controls the spatial and temporal regulation of expression of thousands of genes. Our study clearly suggests a key role of Hypb and H3K36 methylation and Hypb-related mechanisms in the processes of mammalian vascularization (vasculogenesis and angiogenesis). Angiogenesis is important for both basic researchand medical application in cardiovascular diseases and cancer therapeutics. We believe that novel therapeutic strategies targeting epigenetic pathways will achieve real benefit in medical practices.

<p>The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term.</p><p>Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue.</p><p>The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta.</p><p>No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.</p>