: In this renewal application we propose a new hypothesis to explain the long- standing controversy concerning the -contribution of contractile mechanisms to the regulation of venular micro-vascular permeability. We suggest that prior exposure to injury or inflammation induces a more contractile phenotype in endothelial cells. Thus we will test the overall hypothesis that actin-myosin contraction is the dominant mechanism to increase permeability in endothelial barriers that have had prior exposure to injury or inflammatory conditions. Much of the conflicting data in the literature may be explained by the presence of micro vessels having endothelium with a more contractile phenotype in the same micro-vascular bed where 80 percent of micro vessels have a non-contractile phenotype. The proposed experiments enable a new understanding of the link between experiments in cultured endothelial cells and acute and sustained increases in permeability in intact micro-vessels. In particular, many endothelial cells in culture express a contractile phenotype, possibly the result of harvesting and culturing. Thus the mechanisms, which regulate permeability in cultured endothelial cells, may be more characteristic of those that regulate permeability in micro-vessels with prior exposure to inflammatory conditions than those that regulate acute increases in permeability. The hypothesis in Specific Aim 1 is that actin-myosin contraction similar to that described in cultured cells is the dominant mechanism to increase permeability in micro-vessels with a sustained high permeability or localized sites of macromolecular leakage.
In Aim 2 we will directly test the hypothesis that one mechanism leading to the development of a contractile endothelial phenotype in a micro-vessel, which initially has a normal permeability is exposure to inflammatory conditions.
In Aim 3 we will test the hypothesis that a common mechanism to modify the balance of contraction and adhesion in intact micro-vessels and cultured endothelium is modification of the signaling pathways that regulate myosin light chain kinase. To test these hypotheses we will use our methods to cannulate and perfuse individual venular micro-vessels in the mesentery of rats and mice and in a new mouse muscle preparation, and measure the micro-vessel permeability after the signaling pathways modulating contraction have been modified by pharmacological inhibitors, specific peptides introduced into endothelial cells, or targeted mutations in mice. These methods are the most direct experimental approach to evaluate the hypotheses. The new information may lead to strategies to selectively attenuate high permeability states, without significant effects on normal endothelial barriers
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