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The overall goal of this research is to elucidate the effects of stiffness on the activation of pulmonary endothelial cells by inflammatory cytokines. The hypothesis tested is that increasing matrix stiffness in the (patho) physiological range will exacerbate the response of cultured endothelial cells to inflammatory stimuli. To test this hypothesis, we are culturing control and TNF-a stimulated rat lung microvascular endothelial cells (RLMVECs) on hydrogels with tunable stiffnesses of 5, 20, and 45 kPa (measured using compression testing), modeling the stiffness of healthy, intermediate and fibrotic lung tissue respectively. The cellular readout was assessed through RT-qPCR, microscopy, and monocyte adhesion for basal expression and upregulation of vascular cell adhesion molecule-1 (VCAM-1) in quiescent and TNF-a stimulated cultured endothelial cell. This model of microvascular pulmonary inflammation, mimicking a normal, intermediate, and fibrotic lung, is aimed at establishing a correlation between substrate stiffness and inflammation. This research demonstrates the significant increase of basal VCAM-1 gene expression as well as monocyte adhesion as substrate stiffness increases. When using inhibition, it was also found that VCAM-1 is partially activated through the Rho/ROCK, YAP/TAZ, and NF-kB pathway. Our results contribute to a mechanistic understanding of disease pathologies such as idiopathic pulmonary fibrosis, in which treatment is just about limited to a full lung transplant and facilitate testing of new drug therapies. / Bioengineering

Alors que les phases de résorption et de couplage du cycle de remodelage de l’os sont de plus en plus connues et ont permis le développement d’agents thérapeutiques, la phase d’activation reste peu étudiée. L’objectif global de ce travail est d’analyser les évènements cellulaires mis en jeu au cours de la phase d’activation du remodelage de l’os. Les objectifs spécifiques ont été 1- de caractériser le trafic cellulaire dans le périoste au cours de la phase d’activation du remodelage et 2- d’étudier le rôle d’une enzyme, la nicotinamide phosphorybosyl transférase (Nampt) dans ces évènements. Dans notre premier travail, nous montrons dans un modèle de remodelage synchronisé de l’os alvéolaire, une expression précoce de ICAM-1 par les vaisseaux qui serait impliquée dans la diapédèse observée de monocyte-macrophages CD68+. Ces cellules migreraient à travers le compartiment non ostéogénique puis ostéogénique, guidées par des cellules de type fibroblastes puis des OB exprimant VCAM-1. Le nombre des cellules RANKL+ dans le compartiment ostéogénique augmente graduellement lors de la phase d’activation. En parallèle, l’expression de la sémaphorine 3a, qui inhibe l’ostéoclastogénèse, diminue chez les OB et les ostéocytes superficiels. Dans notre second travail, nous trouvons que l’expression basale de la Nampt est accrue dans les cellules de la couche ostéogénique au cours de la phase d’activation du remodelage. Inhiber son activité via le FK866 permet de diminuer l’ostéoclastogenèse indiquant que la Nampt serait impliquée dans le recrutement et l’activité des OC. En culture primaire d’ostéoblastes murins, nous montrons que son expression augmente au cours de la différentiation et qu’elle régule l’expression de marqueurs tardifs de différentiation. L’ensemble de ces données montre une série d’évènements coordonnés qui servent au recrutement des précurseurs ostéoclastiques et à leur migration vers la surface osseuse à résorber. La Nampt semble jouer un rôle dans l’acquisition des ostéoblastes d’un phénotype favorable à ces évènements. / Resorption and inversion phases of bone remodeling are well understood, which have permitted the development of therapeutic agents. At the opposite, activation phase remains poorly characterized. This work aims to analyze cellular events involved in the activation phase of bone remodeling. Specific goals were: 1- To characterize cellular traffic in the periosteum during the activation phase of bone remodeling. 2- To study the role of NicotinAMide Phosphorybosyl Transférase (NAMPT) enzyme during activation. In the first study, we show an early expression of ICAM-1 by vessels in a synchronized alveolar-bone-remodeling model. The ICAM-1 expression may be involved in the observed diapedesis of monocytes – macrophages CD68+. These cells migrate through non osteogenic and osteogenic layers, steered by fibroblast-like cells and then by VCAM+ osteoblasts (OB). The number of RANKL+ cells in osteogenic layer gradually increases during the activation phase. Simultaneously, the expression of semaphorine 3a inhibiting osteoclastogenesis, decreases in osteoblasts and superficial osteocytes. In the second study, we show that basal expression of NAMPT increases in osteogenic-layer cells during the activation phase of bone remodeling. Inhibiting its activity with FK866 enhables to decrease osteoclastogenesis, suggesting an involvement of NAMPT in osteoclast recruitment and activity. In primary culture of murine OB, we show that NAMPT expression increases during differentiation. It also regulates OB late-differentiation markers expression. All these data show a series of coordinated events which serve in osteoclasts precursors’ recruitment and migration towards bone surface. NAMPT seems to contribute to acquiring an OB phenotype more favorable to OC recruitment.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The chemokine, Monocyte Chemoattractant Protein (MCP-1), enhances integrin mediated monocyte adhesion to the vascular endothelium during inflammation. In this study, we demonstrate that MCP-1 promotes rapid sub-second adhesion of THP-1 cells to Vascular Cell Adhesion Molecule-1 (VCAM-1), but not to Intercellular Cell Adhesion Molecule-1 (ICAM-1). MCP-1 activates membrane tethered Very Late Antigen 4 (VLA-4, α4β1), but not necessarily cytoskeleton anchored VLA-4. Activated tethered VLA-4 bonds tremendously increased the period of time monocytes remain bound from hundreds of milliseconds to several seconds and also increased the distance over which immunologic surveillance occurs from several microns up to 20 microns along the endothelium. Lastly at the single molecule level, MCP-1 stimulated tethered VLA-4 bonds exhibit increased resistance to pulling force. In conclusion MCP-1 increased tethered VLA-4 bond resistance to force providing a mechanism for monocyte recruitment to the endothelium.

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Made available in DSpace on 2014-06-12T15:51:29Z (GMT). No. of bitstreams: 2 arquivo2776_1.pdf: 6833514 bytes, checksum: de6f82bea549e57112eae403a110a117 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / O estado nutricional tem influências importantes no desenvolvimento fetal e a alteração na qualidade e/ou quantidade da alimentação materna durante a gravidez tem consequências sobre a saúde posterior da prole, mudando suas respostas aos desafios ambientais e, portanto, sua predisposição a doenças. Ácidos graxos ômega-6 e ômega-9 são importantes nutrientes para o crescimento e desenvolvimento e parecem desempenhar um importante papel na modulação da doença inflamatória cardiovascular e hepática. O objetivo deste estudo foi investigar o efeito de dietas hiperlipidêmicas sobre o endotélio e o fígado de matrizes, bem como, avaliar em matrizes hipercolesterolêmicas o efeito do consumo de ômega-6 e ômega-9 sobre o fígado e o endotélio aórtico da prole. Na primeira etapa, avaliamos, em camundongas Swiss, o efeito de uma dieta hiperlipidêmica, rica em ômega-6 e colesterol, sobre o endotélio e o fígado. Análises bioquímicas mostraram que as dietas experimentais causaram dislipidemia, inflamação e peroxidação lipídica; a análise ultraestrutural, por sua vez, evidenciou que as dietas hiperlipidêmicas promoveram patogênese endotelial e acúmulo de lipídeo hepático. Entretanto, a dieta se mostrou mais hepatotóxica quando suplementada com colesterol. Em uma segunda etapa deste trabalho, com o intuito de se caracterizar a influência da dieta de matrizes sobre o endotélio e o fígado da prole, camundongas da espécie C57BL/6J, deficientes de receptor LDL, foram submetidas a uma dieta rica em ômega-6 ou ômega-9 durante 45 dias antes do acasalamento até o nascimento da prole. Matrizes alimentadas com dieta rica em ômega-6 apresentaram aumento sérico de colesterol total (CT) e seus filhotes, por sua vez, apresentaram elevação sérica de CT, triglicérides (TG) e proteína quimiotática de monócito-1 (MCP-1) circulante e diminuição de lipoproteína de alta densidade (HDL). Por outro lado, filhotes de matrizes alimentadas com dieta rica em ômega-9 apresentaram diminuição de TG e aumento de lipoproteína de baixa densidade (LDL). A análise morfológica do fígado de filhotes de matrizes alimentadas com dieta rica em ômega-6 mostrou esteatose, infiltrado leucocitário e aumento da expressão de MCP-1, enquanto que na análise ultraestrutural foram observadas gotículas de lipídeos e miofibroblastos. Tais alterações não foram observadas nos filhotes de matrizes alimentadas com ômega-9. Em análises morfológicas da aorta ascendente, filhotes de matrizes alimentadas com dieta rica em ômega-6 apresentaram um aumento da expressão de VCAM-1 e MCP-1 e alterações ultraestruturais severas como lâmina elástica descontínua, desprendimento de células endoteliais e presença de mitocôndrias degeneradas. De forma semelhante, estas alterações não foram observadas nos filhotes de matrizes alimentadas com dieta rica em ômega-9. Nosso estudo sugere que dietas hiperlipidêmicas promovem dano endotelial e hepático em matrizes. E que matrizes hipercolesterolêmicas alimentadas com dieta rica em ômega-6 predispõem seus filhotes à disfunção endotelial e hepática. Por outro lado, uma dieta rica em ômega-9 tem um efeito não lesivo

Systemisk lupus erythematosus (SLE) är en kronisk autoimmun sjukdom där immunförsvaret angriper kroppens egen vävnad och förorsakar inflammation. Den drabbar främst kvinnor i fertil ålder och antalet nya fall av SLE är 2–8 per 100 000 invånare årligen i Sverige. Sjukdomens orsak är okänd men tros bero på ett samspel mellan genetiska faktorer, miljöfaktorer och hormonpåverkan. Ökad risk för hjärtinfarkt och stroke syns hos SLE patienter. Markörer som är förknippade med SLE och kardiovaskulär sjukdom och presenterades i detta arbete är IFN-α2a, vaskulär celladhesionsmolekylen (VCAM-1) och S100A8/A9. Sjukdomen kännetecknas av bildandet av stora mängder autoantikroppar mot proteiner med nukleärt ursprung och dubbelsträngat DNA. Anti-fosfolipidantikroppar (aPL) är autoantikroppar som binder till strukturer på fosfolipider eller till komplex av proteiner och fosfolipider. Antikroppar mot kardiolipin (aCL) och mot β2-glykoprotein (aβ2GP1) är exempel på aPL och förekommer hos 20–30 % av SLE patienterna. En ytterligare aPL är anti-fosfatidylserin/protrombin (aPS/PT). aPL förknippas med högre risk för kardiovaskulär sjukdom. Syftet med arbetet var att mäta koncentrationen av IFN-α2a, VCAM-1, S100A8/A9 och aPL, och därefter analysera hur de förhåller sig till varandra samt förekomst av kardiovaskulär sjukdom. Koncentrationer av nämnda markörer och aPL mättes med olika immunoassays i 199 prover som tagits vid olika tidpunkter från 66 patienter, och korrelation analyserades med icke-parametriska metoder. Resultatet visar förväntade signifikanta korrelationer mellan sjukdomsaktivitet och IFN-α2a, VCAM-1 samt S100A8/A9. Alla undersökta aPL korrelerade med varandra. IgG antikroppar korrelerade bättre än IgM med IFN-α2a, VCAM-1, S100A8/A9 och sjukdomsaktiviteten. IFN-α2a hade en signifikant korrelation med VCAM-1, aCL-IgG och aPS/PT-IgG. VCAM-1 korrelerade däremot med IFN-α2a, aCL-IgG, aβ2GP1-IgG och aPS/PT-IgG. Ingen association mellan kardiovaskulär sjukdom och de undersökta markörerna samt aPL i patienternas första prov kunde påvisas. / Systemic lupus erythematosus (SLE) is a chronical autoimmune disease in which the body’s immune system attacks healthy tissue and causes inflammation. The disease affects mainly women of childbearing age with 2 to 8 new cases per 100 000 inhabitants yearly in Sweden. One main feature of SLE is the expression of autoantibodies specific to autoantigens with nuclear origin. The cause of SLE is unknown but it is thought to involve a combination of genetic factors, environmental factors, and hormonal influence. Risk of myocardial infarction and stroke is increased in SLE. Markers that are associated with SLE and cardiovascular disease and got presented in this paper are IFN-α2a, Vascular cell adhesion molecule-1 (VCAM-1) and the complex S100A8/A9. Antiphospholipid antibodies (aPLs) are a type of antibodies which binds to structures on phospholipids or to complex of proteins and phospholipids. Antibodies against cardiolipin (aCL) and β2-glycoprotein (aβ2GP1) are two aPLs which can be found in 20-30 % of SLE patients. Another example of aPLs is antiphosphatidylserine/prothrombin (aPS/PT). aPLs are associated with higher risk for CVD. The aim of this study was to study mentioned markers and aPLs to acquire better understanding of how they relate to each other and to CVD. The concentrations of these markers and aPLs were measured in 199 different samples which were taken from 66 patients and correlations were analyzed with non-parametric statistical methods. Results have shown as expected significant correlations for the biomarkers IFN-α2a, VCAM-1 and S100A8/A9 with disease activity. All aPLs have shown strong correlation to each other. IgG correlated better than IgM with the different biomarkers and disease activity. IFN-α2a had strong correlation to VCAM-1, aCL-IgG, and aPS/PT-IgG. VCAM-1 on the other hand had significant correlation to IFN-α2a, aCL-IgG, aβ2GP1-IgG and aPS/PT-IgG. No association could be found in this study between CVD and the studied markers, and aPLs in the first sample of each patient.