The overall goal of this research project is to characterize changes in cytoskeletal and associated proteins in normoxic and oxygen exposed endothelial cells and relate cytoskeletal alterations with permeability changes across an endothelial monolayer. Our studies will involve all three cytoskeletal filament systems, the microfilaments (actin), intermediate filaments (vimentin, desmin and keratin), and microtubules (tubulin). We will pursue the following specific aims. (1) Quantitate F-actin in normal and oxygen exposed cells and correlate the polymerization state of actin with alteration in monolayer permeability. The ultrastructural state of the interendothelial junction will also be investigated. (2) Determine intracellular calcium concentration in normal and oxygen exposed cells and ask whether the calcium antagonist lanthanum chloride alters actin polymerization in these cells. (3) Determine if actin specific mRNA content, structural alterations of the cytoskeletal proteins, or actin associated proteins are expressed differently in oxygen exposed cells. (4) Characterize expression of the intermediate filaments and quantitate the amount of membrane bound vimentin and actin in normoxic and oxygen exposed cells. (5) Characterize cytoplasmic microtubules, microtubule associated proteins and quantitate cytoskeletal tubulin polymer in normoxic and oxygen exposed cells. Oxygen toxicity is partially mediated through the production of hydroxyl, superoxide and hydrogen peroxide. Parallel experiments will be carried out using dimethylthiourea (hydroxyl radical scavenger) liposome encapsulated superoxide dismutase and catalase as controls. Understanding cytoskeletal alteration in relation vascular permeability may be used to develop specific interventions to interrupt the pathogenesis in many forms of acute lung injury.
