No description available.

This study investigated the expression and activity of tissue transglutaminase (tTG) in human umbilical vein endothelial cells (HUVEC) and vessel wall. tTG was located in the SMC and sub-endothelium of normal vessels. Cross-linking activity was also in this area. Vessels with atherosclerotic plaque showed increased staining for tTG and cross-links. Positive staining for tTG was located in the SMC, neointima, macrophages and the fibrous cap. Most cross-linking activity was observed in the fibrous cap, and cross-linking was observed around macrophages and smooth muscle cells. Cross-linking activity was also observed with incorporation of a labelled cross-linking substrate into vessel sections. Free tTG could be extracted from the vessel wall. HUVEC expressed 10 g tTG/mg total protein. tTG was detected in cell lysate and extracellular matrix, but not in the culture supernatant. Thrombin up-regulated tTG expression at both the mRNA and protein level. Optimal up-regulation was at a thrombin concentration of 1 U/ml The up-regulation by thrombin was dependent on thrombin activity, and was mediated through the thrombin receptor, protease-activated receptor 1 (PAR-1). Cross-linking activity was also increased after thrombin treatment, measured with a microtitre plate assay and an in situ assay. The specific activity of tTG increased after thrombin treatment. Thrombin treatment increased the level of tTG in the HUVEC ECM. Treatment of HUVEC with PMA reduced the expression of tTG mRNA, reduced the level of tTG protein, but increased the tTG cross-linking activity compared to untreated cells.

No description available.

L’athérosclérose est un processus d’évolution lente, dont les premières étapes sont marquées par le dépôt de lipoprotéines de basse densité (LDL) dans l’intima des artères. Les études histologiques montrent que les cellules musculaires lisses (CML) sont prépondérantes dans l’intima des lésions précoces et qu’elles ont un rôle critique dans le développement des lésions. A l’inverse des CML saines de la média qui présentent un phénotype contractile, certaines CML de l'intima sont caractérisées par la synthèse de matrice extracellulaire, associée à une migration et une prolifération augmentées. Comme les macrophages, une fraction de ces CML peut internaliser les LDL et participe à la formation de cellules spumeuses.Dans ce travail, nous avons cherché à déterminer le rôle du syndécan-1 dans la modulation du phénotype de la CML. Nous avons mis en évidence une surexpression du syndécan-1 par les CML intimales lors des stades précoces de l'athérosclérose. Dans le but de déterminer l'effet de cette surexpression, nous avons élaboré un vecteur lentiviral codant le syndécan-1 afin de le surexprimer dans les CML en culture. Nos résultats montrent qu'en réponse au transforming growth factor beta (TGF-β), les CML surexprimant le syndécan-1 présentent une augmentation amplifiée des gènes du collagène de type I et de certains marqueurs de fonction contractile incluant la SM α-actine, la calponine et la smootheline.Un autre volet de l'étude s'est intéressé à l'origine des cristaux de cholestérol (CCs) dans les lésions. Fréquemment retrouvés dans les lésions avancées, les études montrent que ces CCs pourraient induire mécaniquement la rupture de la plaque. Par l'analyse de coupes d'aortes fraîches, nous avons observé que les CCs apparaissent au stade de la lésion fibrolipidique et qu'ils sont associés à la présence de CML mortes. In vitro, les CML chargées en cholestérol sont capables de produire des cristaux, un processus qui est accéléré par la présence de collagène de type I et par l'inhibition de l'autophagie et de l'estérification du cholestérol. Nous avons montré que le collagène de type I entraine une diminution de p62 et une augmentation de Niemann-Pick C1, suggérant qu'il module l'expression de gènes associés à l'autophagie et au trafic du cholestérol.L’ensemble de ce travail ouvre ainsi de nouvelles perspectives quant à l’identification des mécanismes initiant la modulation du phénotype de CML, et aux conséquences de cette modulation au niveau de la lésion. / Atherosclerosis is a chronic pathological process that is characterized, in the earliest stage, by low density lipoproteins (LDL) accumulation within the arterial intima. Evidences show that smooth muscle cells (SMCs) are preponderant in the intima of early lesions and play a major role in lesions development. Whereas medial SMCs are involved in the contractile function, intimal SMCs are characterized by increased proliferation and migration, loss of contractile markers, and extracellular matrix production. Like macrophages, these SMCs have the capacity to internalize LDL and become foam cells. In this work, we explored syndecan-1 role in SMCs phenotypic modulation. We show that syndecan-1 expression is increased in intimal SMCs of early lesions. To determine the effect of this overexpression in SMCs in vitro, we elaborated the construction of a lentiviral vector encoding syndecan-1 cDNA. Our results demonstrate that syndecan-1 amplifies the up-regulation of type I collagen, SM α-actin, calponin, and smoothelin mRNA expression, induced by TGF-β.In the second part of our work, we investigated cholesterol crystals (CCs) formation in atherosclerotic lesions. Besides to being a major component of the atheromatous core, studies have shown that CCs could promote plaque rupture by causing mechanical damage. Our observations of fresh aortas revealed that CCs first appeared at the fibroatheroma transition and are associated with the death of SMC. Cholesterol-loaded human SMCs were capable of producing CCs in vitro, a process that was enhanced by type I collagen and by inhibition of autophagy and cholesterol esterification. We found that type I collagen leads to a decrease in the expression of p62, and an increase in Niemann-Pick C1, suggesting that collagen induces changes in genes relating to regulation of intracellular cholesterol levels and localization.In conclusion, this work described a novel candidate potentially implicated in SMCs phenotypic modulation, and the important consequences of this modulation in lesions development.

Les maladies cardiovasculaires (MCV), résultant de complications de l'athérosclérose, restent la principale cause de morbidité et de mortalité dans le monde. L'athérosclérose, considérée comme une maladie inflammatoire chronique, implique à la fois les systèmes immunitaires inné et adaptatif. Les macrophages jouent un rôle majeur dans l'initiation de la lésion, sa progression et les complications thrombotiques potentiellement dévastatrices. Un grand nombre de données rapporte l'implication du stress oxydatif, conséquence d'un déséquilibre entre antioxydants et espèces réactives de l'oxygène, dans les maladies cardiovasculaires. En outre, ces pathologies sont fréquemment associées à des changements dynamiques de l'activation des macrophages, soit vers le phénotype pro-inflammatoire M1 (activation classique), soit vers le phénotype anti-inflammatoire M2 (activation alternative). Parmi les antioxydants endogènes, la thiorédoxine-1 (Trx1) est une protéine ubiquitaire qui exerce différents effets physiologiques. Outre son rôle dans l'homéostasie redox cellulaire et comme puissant antioxydant, cette protéine est également impliquée dans le métabolisme énergétique, les réponses inflammatoires, la croissance cellulaire et la survie. En revanche, sa forme tronquée (Trx80) exerce des effets opposés. Il est à noter que plusieurs études ont rapporté le rôle bénéfique du système de la Trx1 dans les MCV mais les mécanismes moléculaires n'ont toujours pas été décrits. Par conséquent, notre étude porte sur le rôle des Trx1 et Trx80 dans le développement et/ou la régression de l'athérosclérose, en particulier dans la modulation de la polarisation des macrophages et dans les différentes voies de signalisation impliquées dans ces processus. Nos principaux résultats obtenus in vitro sur des cultures primaires de macrophages humains ou de macrophages péritonéaux murins, ont révélé d'une part que la Trx1 induit la polarisation des macrophages vers le phénotype anti-inflammatoire M2 suite à la régulation négative de p16INK4a et l’inhibition de la translocation nucléaire de la protéine activatrice-1(AP-1) et de Ref-1; d'autre part, que la Trx1 inhibe la polarisation des macrophages vers le phénotype pro-inflammatoire M1 induit par le lipopolysaccharide (LPS). Par contre, la Trx80 inhibe la polarisation anti-inflammatoire M2 induite par IL-4 ou IL-4/IL-13 mais potentialise le phénotype M1 induit par le LPS. Pour valider in vivo les résultats obtenus in vitro, nous avons utilisé comme modèles expérimentaux, des souris C57Bl/6.ApoE2.ki hyperlipoprotéinémiques et des vaisseaux athérosclérotiques de patients ayant subi une chirurgie vasculaire. Injectées en intraveineuse, la Trx1 et la Trx80 affectent le phénotype des macrophages dans le thymus, le foie et les lésions athérosclérotiques. Chez la souris, la Trx1 réduit la surface des lésions aortiques alors que la Trx80 l’augmente. Enfin le traitement aussi bien par la Trx1 que par la Trx80 n’affecte pas les niveaux de cholestérol et de triglycérides plasmatiques. Pour explorer davantage nos résultats, nous avons étudié les voies de signalisation impliquées dans ces processus. Nos résultats montrent que la Trx1 et la Trx80 activent toutes les deux Akt. La Trx80 utilise la voie de signalisation mTOR pour exercer ses effets dans la polarisation M1 des macrophages puisqu'elle active mTOR de manière dose-dépendante comme l’indique l'augmentation de la phosphorylation de p70S6K. De plus, la Trx1 antagonise l'athérosclérose alors que la Trx80 la potentialise par suite des changements des phénotypes M1/M2, ce qui fait de la Trx1 une cible thérapeutique prometteuse. / The cardiovascular diseases (CVDs), resulting from complications of atherosclerosis, remain the leading cause of morbidity and death worldwide. Atherosclerosis as a chronic inflammatory disease, involves both innate and adaptive arms of immunity in which macrophages play the orchestral role in modulating lesion initiation, progression, and potentially devastating thrombotic complications. Available evidences support the notion of a central role of oxidative stress, due mainly to the imbalance between antioxidants and reactive oxygen species (ROS) in CVDs. Furthermore, the pathology is frequently associated with dynamic changes in macrophage activation, with classically activated M1 cells implicated in initiating and sustaining inflammation and M2 or M2-like cells associated with resolution or smoldering chronic inflammation. Among endogenous antioxidants, the thiordoxine-1 (Trx1) plays a central role in several diseases including CVD. Thus, the ubiquitous Trx1 has been reported to exert a myriad of beneficial roles. Indeed, it regulates not only cellular redox homeostasis and acts as a principal antioxidant defense system, but it also affects energy metabolism, modulates the immunological and inflammatory responses, and controls cell growth and survival. In contrast, its truncated form (Trx-80), exerts an opposite effects. However, several studies reported the beneficial role of Trx system in CVDs but the detailed molecular mechanism is not addressed yet. Therefore, the present study aims to investigate the role of both Trx1 and Trx80 in the biology of atherosclerosis through the modulation of macrophage polarization and the implicated signaling pathways as well. Our in vitro major findings, using human macrophages and murine peritoneal macrophages, revealed that Trx1 on one hand promoted the polarization of anti-inflammatory M2 macrophages through downregulation of p16INK4a and suppressing nuclear translocation of activator protein-1 (AP-1) and Ref-1 as evidenced by the expression of the CD206 and IL-10 markers. On the other hand Trx1 also reduced the lipopolysaccharide (LPS)-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α (TNF-) and monocyte chemoattractant protein-1 (MCP¨-1). By contrast, Trx80 treatment attenuated the polarization of anti-inflammatory M2 macrophages induced by IL-4 or IL-4/IL-13 even it potentiated LPS-induced M1 activation. To validate our obtained in vitro results, hyperlipoproteinemic C57Bl/6.ApoE2.ki mice and human atherosclerotic vessel specimens from patients undergoing vascular surgery were used. Consistently, Trx1 and Trx80 affected macrophage phenotype in thymus, liver and atherosclerotic lesions. As a consequence, Trx1 reduced whereas Trx80 increased the aortic lesion area in mice. Plasma levels of cholesterol and triglycerides did not changed by the treatment. To further explore our results, the implicated signaling pathways has been studied and it was found that both Trx1 and Trx80 activated Akt. Furthermore, Trx80 uses mTOR signaling pathway to exert its effect in polarizing macrophages toward M1 phenotype since it activated mTOR in a dose-dependent manner as demonstrated by the increased phosphorylation of P70S6K. Based on our results, Trx1 antagonizes whereas Trx80 potentiates atherosclerosis through changing M1/M2 phenotypes. Therefore, Trx1 represents a promising target for therapeutic interventions.

Atherosclerosis is a term coined to describe a state in which arterial wall thickens due to the accumulation of fatty materials like cholesterol. Though not completely understood, it is believed to occur due to the accumulation of macrophage white blood cells and promoted by low density lipoprotein. Increase in accumulation of plaque leads to enlargement of arteries as arterial wall tries to remodel itself. But eventually the plaque ruptures, letting out its inner content to blood stream. The ruptured plaque clots and heals and shrinks down as well but leaves behind stenosis - narrowing of cross section. Depending on the degree of stenosis blood supply from the artery to its respective organ could decrease and even get blocked completely. Frequently, as the vulnerable plaques rupture, thrombus formed as such could flow through bloodstream towards smaller vessels and block them, leading to a sudden death of tissues fed by that vessel. If the plaques do not rupture and artery gets enlarged to a great extent then it results in an aneurysm. Such blockage of coronary arteries in heart can lead to myocardial infarction - heart attack, in carotid arteries in brain can lead to what is called a stroke, in peripheral arteries in legs can lead to ulcers, gangrene (death of tissue) and hence loss of leg, in renal arteries can lead to kidney malfunction. The most disturbing fact about atherosclerosis is the inability to detect the disease in preliminary stages. As stated by Miller (2001), most of the times coronary artery disease (CAD) gets diagnosed only after 50-75 percent occlusion of arteries.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

The cardiovascular diseases (CVDs), resulting from complications of atherosclerosis, remain the leading cause of morbidity and death worldwide. Atherosclerosis as a chronic inflammatory disease, involves both innate and adaptive arms of immunity in which macrophages play the orchestral role in modulating lesion initiation, progression, and potentially devastating thrombotic complications. Available evidences support the notion of a central role of oxidative stress, due mainly to the imbalance between antioxidants and reactive oxygen species (ROS) in CVDs. Furthermore, the pathology is frequently associated with dynamic changes in macrophage activation, with classically activated M1 cells implicated in initiating and sustaining inflammation and M2 or M2-like cells associated with resolution or smoldering chronic inflammation. Among endogenous antioxidants, the thiordoxine-1 (Trx1) plays a central role in several diseases including CVD. Thus, the ubiquitous Trx1 has been reported to exert a myriad of beneficial roles. Indeed, it regulates not only cellular redox homeostasis and acts as a principal antioxidant defense system, but it also affects energy metabolism, modulates the immunological and inflammatory responses, and controls cell growth and survival. In contrast, its truncated form (Trx-80), exerts an opposite effects. However, several studies reported the beneficial role of Trx system in CVDs but the detailed molecular mechanism is not addressed yet. Therefore, the present study aims to investigate the role of both Trx1 and Trx80 in the biology of atherosclerosis through the modulation of macrophage polarization and the implicated signaling pathways as well. Our in vitro major findings, using human macrophages and murine peritoneal macrophages, revealed that Trx1 on one hand promoted the polarization of anti-inflammatory M2 macrophages through downregulation of p16INK4a and suppressing nuclear translocation of activator protein-1 (AP-1) and Ref-1 as evidenced by the expression of the CD206 and IL-10 markers. On the other hand Trx1 also reduced the lipopolysaccharide (LPS)-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α (TNF-) and monocyte chemoattractant protein-1 (MCP¨-1). By contrast, Trx80 treatment attenuated the polarization of anti-inflammatory M2 macrophages induced by IL-4 or IL-4/IL-13 even it potentiated LPS-induced M1 activation. To validate our obtained in vitro results, hyperlipoproteinemic C57Bl/6.ApoE2.ki mice and human atherosclerotic vessel specimens from patients undergoing vascular surgery were used. Consistently, Trx1 and Trx80 affected macrophage phenotype in thymus, liver and atherosclerotic lesions. As a consequence, Trx1 reduced whereas Trx80 increased the aortic lesion area in mice. Plasma levels of cholesterol and triglycerides did not changed by the treatment. To further explore our results, the implicated signaling pathways has been studied and it was found that both Trx1 and Trx80 activated Akt. Furthermore, Trx80 uses mTOR signaling pathway to exert its effect in polarizing macrophages toward M1 phenotype since it activated mTOR in a dose-dependent manner as demonstrated by the increased phosphorylation of P70S6K. Based on our results, Trx1 antagonizes whereas Trx80 potentiates atherosclerosis through changing M1/M2 phenotypes. Therefore, Trx1 represents a promising target for therapeutic interventions.

Oxidative stress (OS) plays a critical role in the pathogenesis of atherosclerosis potentially through interaction with nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) pathway. Although considerable literature supports a pro-atherogenic role for iNOS-induced NO, recent evidence suggest an anti-atherogenic property for this enzyme where iNOS-induced NO attenuates atherosclerotic lesions after immune injury, enhancing endothelial integrity, survival, protecting against OS-induced apoptosis and necrosis. We therefore hypothesize that iNOS may have a cardio-protective role in the atherosclerotic vessel and that under conditions of OS, expression and function of this enzyme may be impaired, thus contributing to the deleterious consequences of OS. Experiments have therefore been conducted to establish whether pro-oxidants regulate iNOS expression/function in rat cultured aortic smooth muscle cells (RASMCs). These cells were induced for 24 hours with LPS and IFN-γ to mimic inflammatory conditions. Oxidative stress inducers may modulate iNOS-induced NO production through alteration of the expression and/or function of the inducible L-arginine-NO pathway. We examined the effects of hydrogen peroxide (H2O2), antimycin A and diethyl maleate (DEM) on this pathway in vascular smooth muscle cells. H2O2 had little effect on NO production or L-arginine transport while antimycin A and DEM independently caused a concentration dependent inhibition of both processes. Only DEM induced hemeoxygenase-1 (HO-1) expression, monitored by western blotting as a marker of OS. The effects of statins on NO synthesis and L-arginine transport in the presence and absence of OS were also investigated. The benefits of statins therapy in cardiovascular medicine are ascribed in part to their lipid-lowering effect by inhibiting 3-hydroxy-3-methoxyglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme for cholesterol synthesis. However, statins may possess anti-inflammatory properties and are able to improve endothelial function, stabilize atherosclerotic plaque, and inhibit platelet aggregation, vascular smooth muscle cells proliferation and vessel wall inflammation. These effects may be exerted through novel actions of statins that include interaction with specific signalling pathways in cells which may be associated with the induction of iNOS and/or cationic amino acid transporters (CATs). Thus, we have extended our investigations to include an examination of the effects of statins on both iNOS and CAT function and expression under control conditions and following exposure of cells to OS. Atorvastatin caused a bell shaped response on NO production and iNOS expression and also enhanced L-arginine transport but in a non-concentration dependent manner. Simvastatin only affected NO synthesis without altering transporter activity. Pravastatin was without effect on either system. Further studies demonstrated that that atorvastatin was able to reverse the effects of antimycin A and DEM but only on NO production. These findings confirm that the inducible L-arginine-NO pathway can be downregulated by pro-oxidants. This mechanism may therefore contribute to the deleterious effects observed in disease states associated with OS. Moreover, statins (in particular atorvastatin) appear to be effective in reversing the inhibition of NO production caused by inducers of OS. This, together with the fact that atorvastatin and simvastatin can potentiate iNOS-induced NO production and indeed L-arginine transport (with atorvastatin), highlights a potential novel mechanism through which the cardio-protective actions of these compounds could be mediated.