The proposed research is designed to test the hypothesis that increased expression of gelatinase B (MMP-9) during monocyte transmigration of vascular endothelium results in an increase in endothelial permeability to macromolecules. Such an effect may contribute to diminished endothelial barrier function and initiation of atherosclerotic lesion through facilitating the non-specific entry and accumulation of plasma molecules, including circulating lipoproteins.
Specific aims i nclude measuring permeability of endothelial monolayers to macromolecular tracers during transmigration of both wild type and MMP-9 knockout monocytes, and investigating the mechanism of MMP-9 induction during monocyte/endothelial cell interactions. We propose to investigate permeability changes during monocyte transmigration using an in vitro coculture system consisting of mouse bone marrow monocytes and mouse aortic endothelium grown on matrix-covered cell culture inserts. To investigate the specific role of MMP-9 in transmigration, we will use monocytes and endothelial cells derived both from wild type and from MMP-9 knockout mice. Endothelial permeability will be measured using flourescently labeled dextrans. Tracer concentrations will be measured fluorimetrically. Expression of MMP-9 protein and enzymatic activity will be determined by means of Western blotting and SDS-PAGE zymography, respectively. To explore the mechanism of MMP-9 induction during monocyte transmigration of endothelium, we will examine the role of specific interactions between monocyte integrins and endothelial cell adhesion molecules. Specifically, we propose to assess the role of monocyte binding to endothelial VCAM-1 using VCAM-1 knockout mouse endothelial cells in transmigration assays. These studies should further our understanding of the early events that contribute to the development of athersclerotic lesions and could provide insight into the use of MMP-9 inhibitors to treat endothelial barrier dysfunction.
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