Endothelial Cell VCAM-1 Signal Transduction

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DESCRIPTION (provided by the applicant): The long term objective of this proposal is to understand molecular mechanisms for vascular cell adhesion molecule- 1 (VCAM- 1) signal transduction in endothelial cells. Disruption of the VCAM- 1 gene results in embryonic death. VCAM- 1 is expressed on endothelial cells in sites predisposed to atherosclerotic lesion formation and by endothelial cells during atherosclerosis, infection, and allograft rejection. VCAM- 1 binds to leukocytes to promote leukocyte extravasation into tissues. However, little is known regarding mechanisms for signal transduction in endothelial cells via VCAM- 1. Study of VCAM- 1 signaling has been impeded by complications from signals for induction of VCAM- 1 expression and by simultaneous interactions of other endothelial cell adhesion molecules with leukocytes. To circumvent these problems in studying VCAM- 1 signaling, we developed and characterized endothelial cell lines that constitutively express VCAM-1 and not other known adhesion molecules. Using these endothelial cells lines and primary endothelial cell cultures, we demonstrated that VCAM- 1 stimulates endothelial cell NADPH oxidase-catalyzed production of reactive oxygen species (ROS). This production of ROS is required for endothelial cell shape changes and for endothelial cell promotion of leukocyte migration. Another report indicates that antibody cross linking of VCAM- 1 activates a calcium flux and phospholipase release of inositol triphosphate. However, it is not known what effects these signals have on endothelial cell function. It has been established that phospholipase metabolites stimulate NADPH oxidase in neutrophils. This information led us to hypothesize that VCAM- 1 activation of a phospholipase/calcium/protein kinase C cascade activates endothelial cell NADPH oxidase production of ROS for the modulation of endothelial cell actin restructuring and leukocyte migration. Based on the reports that low levels of ROS activate matrix metalloproteinases (MMPs) and inhibit pbosphatases, VCAM- 1-stimulated ROS production may signal endothelial cell shape changes via these enzymes. To address our hypothesis, we will use biochemical, genetic, and pharmacological approaches to determine whether a signaling cascade involving a calcium flux (Aim 1), phospholipase activity (Aim 2), and protein kinase C activity (Aim 3) is required for VCAM-l stimulation of NADPH oxidase activity in endothelial cells. Then, we will determine whether VCAM-1 -stimulated NADPH oxidase production of ROS activates MMPs (Aim 4) and modulates phosphatase activity (Aim 5) during localized endothelial cell shape changes. It is anticipated that the proposed experiments will provide new insight into the mechanisms by which VCAM- I regulates endothelial cell signal transduction. This information may provide novel targets for therapies designed to modulate vascular function during atherosclerosis, infection, or transplantation.