VCAM-1 (CD106) is a transmembrane glycoprotein and involved in many pathological inflammatory processes. VCAM-1 plays an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (£\4£]1). In our preliminary data, we observed 2-3 fold increase in the expression of VCAM-1 in the side population of ovarian cancer, which exhibits stem cell-like properties in ovarian cancer. In addition, we have also found VCAM-1 is upregulated in many breast cancer epithelial cells and directly correlated with breast tumor progression; however, its mechanism of action in tumor biology remains unknown. Here, we describe the establishment and use of breast cancer cell line model systems to dissect the functional roles of VCAM-1 activation in the manifestation of malignant phenotype of human breast cancer. We show that VCAM-1 overexpression in the NMuMG breast epithelial cells increase cell motility rates and chemoresistance to doxorubicin and cisplatin in vitro, conversely, in an established metastatic breast cancer cell line, MDAMB231, we find that knockdown endogenous VCAM-1 expression by small interfering RNA reduced the migration rate . Furthermore, we also demonstrated that knockdown endogenous VCAM-1 expression in MDAMB231 cells reduced the tumor formation in SCID xenograft mouse model. In conclusion, our findings are consistent with the hypothesis that overexpression of VCAM-1 facilitates breast cancer progression by enhancing the malignant properties of breast cancer cells and suggests that targeting of VCAM-1 induced pathways are attractive strategies for therapeutic intervention.

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Thèse doctorat : Biochimie : INSA LYON : 2001. / Titre provenant de l'écran-titre. Bibliogr. p. 161-198.

Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-activated protein present on endothelial cells (ECs). Our laboratory has provided evidence that DNA methylation, a mark associated with gene silencing, is fundamental for regulating VCAM-1 expression. First, we showed that RNA polymerase II, preferentially associates with VCAM-1 hypomethylated alleles. This finding was confirmed using fluorescence-activated cell sorting (FACS) to sort populations of cytokine-activated ECs with high vs. low cell surface VCAM-1 expression. We found that ECs with high VCAM-1 expression were hypomethylated at the promoter. We then went on to show that populations of cells generated from single ECs exhibit differential VCAM-1 methylation from one another, and from the original founder population. Intriguingly, our data shows that VCAM-1 mRNA levels differ between the clones, and correlate with the observed differences in DNA methylation. Taken together, this data provides exciting evidence that DNA methylation is important in the regulation of VCAM-1 gene expression.

Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-activated protein present on endothelial cells (ECs). Our laboratory has provided evidence that DNA methylation, a mark associated with gene silencing, is fundamental for regulating VCAM-1 expression. First, we showed that RNA polymerase II, preferentially associates with VCAM-1 hypomethylated alleles. This finding was confirmed using fluorescence-activated cell sorting (FACS) to sort populations of cytokine-activated ECs with high vs. low cell surface VCAM-1 expression. We found that ECs with high VCAM-1 expression were hypomethylated at the promoter. We then went on to show that populations of cells generated from single ECs exhibit differential VCAM-1 methylation from one another, and from the original founder population. Intriguingly, our data shows that VCAM-1 mRNA levels differ between the clones, and correlate with the observed differences in DNA methylation. Taken together, this data provides exciting evidence that DNA methylation is important in the regulation of VCAM-1 gene expression.

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No description available.

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.