The long term objective of this proposal is to determine the influence of the mechanical and chemical environment of the endothelium on the transport properties of this barrier that controls the flux of materials between blood and tissue.
The specific aims of the proposed research are: 1. To develop methods for reducing endothelial permeability to LDL under physiological (pressurized) conditions. We will test the hypothesis that apoptosis and mitosis rates dictate endothelial permeability to LDL under physiological (pressurized) conditions using in vitro models of the endothelial tranpsort barrier. We will reduce rates of endothelial cell apoptosis and mitosis pharmacologically and determine the effect of these manipulations on endothelial monolayer permeability to LDL under pressure. 2. To determine the influence of smooth muscle cells on the transport properties of the endothelium. We will test the hypothesis that paracrine interactions between smooth muscle cells (SMC) and endothelial cells (EC) affect the transport characteristics of the endothelium. We will co-culture bovine aortic endothelial cells on the surface of a collagen gel laden with smooth muscle cells and measure transport properties under static and shear stress conditions. We will explore mechanisms mediating the EC/SMC interactions by quantifying intercellular junction proteins in ECs and Ang-1, VEGF and TGF-b1 gene expression in SMCs. 3. To determine the role of the glycocalyx in transducing fluid shear stress stimuli into responses of the EC transport barrier. Using in vitro models, we will investigate the hypothesis that the EC glycocalyx is the mechanosensor/transducer that initiates alterations in EC transport properties. This will be accomplished by digesting specific components of the glycosaminoglycan (GAG) surface layer with enzymes and determining the influence on shear-induced EC transport properties, and by using a chimeric syndecan-1 protein that has its cytoplasmic tail deleted to study mechanotransduction by the dominant proteoglycan on the EC surface. .
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