The proposed investigations will examine the effects of inflammation on endothelial P-selectin, PECAM-1, and integrin-mediated adhesion. Adhesion molecules will be studied in cultured human endothelial cells that are exposed to recombinant human cytokines or culture medium conditioned by human bronchial epithelial cells infected by viruses. These in vitro models of inflammation will allow the study of adhesion molecule responses in isolation from the variations in growth factors, cytokine levels, and substrate delivery that complicate whole organ and whole animal studies. The transcription, post-translational processing, cytoskeletal interactions, and signal transduction pathways associated with these major endothelial cell adhesion molecules will be studied. Preliminary data indicate that TNFalpha induces P-selectin transcription in human endothelial cells.
Specific aim 1 is to characterize the kinetics and mechanisms of cytokine induction of P-selectin in human vascular endothelium. This will be accomplished by stimulating human umbilical vein and pulmonary artery endothelial cells with inflammatory mediators and determining the kinetics of upregulation of P-selectin transcription using northern blotting, in situ hybridization and protein assays. Stimulus-specific promoter regions and transcription factors for P-selectin induction will then be identifies using truncated promoter/reporter constructs, gel shift mobility assays, and nuclear run-on studies. Our previous work and preliminary data demonstrated that inflammatory cytokines alter the intercellular localization and cytoskeletal association of PECAM-1 in human endothelial cells.
Specific aim 2 is to characterize the mechanisms of the redistribution. PECAM-1 redistribution will be spatially defined using immunofluorescence and immuno-electron microscopy. The cytoskeletal association of PECAM-1 from endothelial cells exposed to inflammatory mediators will be quantified, and interactions with specific cytoskeletal ligands will be determined. Cytokine-induced changes in PECAM-1 phosphorylation and associated changes in cytoskeletal ligand-binding affinities will be measured.
Specific aim 3 is to examine the effects of inflammatory mediators on FAK signalling and endothelial cell adhesion to extracellular matrix proteins. The activation state of FAK during inflammatory cytokine exposure will be compared with tyrosine phosphorylation of FAK and other cytoskeletal proteins. Focal adhesion dynamics will be measured by confocal microscopy in endothelial cells with normal and altered FAK activity during cytokine treatment. Direct interactions between cytokine-responsive transcription factors and FAK expression will be measured by gel shift mobility analysis. The long term goal of the research program is to define the mechanisms underlying changes in vascular endothelial cell adhesion that occur during viral respiratory infections. These investigations may lead to the therapeutic modulation of inflammatory lung injury due to respiratory viral infection in childhood
|Mellow, Thomas E; Murphy, Paula C; Carson, Johnny L et al. (2004) The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium. Exp Lung Res 30:43-57|
|Thai, C H; Gambling, T M; Carson, J L (2002) Freeze fracture study of airway epithelium from patients with primary ciliary dyskinesia. Thorax 57:363-5|
|Veness-Meehan, Kathleen A; Pierce, Richard A; Moats-Staats, Billie M et al. (2002) Retinoic acid attenuates O2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol 283:L971-80|
|Carson, Johnny L; Reed, William; Lucier, Thomas et al. (2002) Axonemal dynein expression in human fetal tracheal epithelium. Am J Physiol Lung Cell Mol Physiol 282:L421-30|
|Kazachkov, Mikhail Y; Hu, P C; Carson, Johnny L et al. (2002) Release of cytokines by human nasal epithelial cells and peripheral blood mononuclear cells infected with Mycoplasma pneumoniae. Exp Biol Med (Maywood) 227:330-5|
|Noah, Terry L; Ivins, Sally S; Murphy, Paula et al. (2002) Chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis. Clin Immunol 104:86-95|
|Price, W A; Lee, E; Maynor, A et al. (2001) Relation between serum insulinlike growth factor-1, insulinlike growth factor binding protein-2, and insulinlike growth factor binding protein-3 and nutritional intake in premature infants with bronchopulmonary dysplasia. J Pediatr Gastroenterol Nutr 32:542-9|
|Volberg, T; Romer, L; Zamir, E et al. (2001) pp60(c-src) and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions. J Cell Sci 114:2279-89|
|Noah, T L; Becker, S (2000) Chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus. Clin Immunol 97:43-9|
|Veness-Meehan, K A; Bottone Jr, F G; Stiles, A D (2000) Effects of retinoic acid on airspace development and lung collagen in hyperoxia-exposed newborn rats. Pediatr Res 48:434-44|
Showing the most recent 10 out of 15 publications