This application describes a comprehensive vascular research program designed to develop Dr. Childs into an independent investigator. Through course work in molecular biology, research design, and statistics, he will acquire the necessary analytical skills to achieve this goal. He will also be required to attend several annual conferences and a monthly journal club. An Advisory Board of experienced investigators in vascular and cellular biology has been established for Dr. Childs to obtain hands on experience in laboratory techniques. Development of grantsmanship is emphasized throughout the duration of the K01 award, beginning with applications for local funds, graduating to an American Heart Association grant, and culminating in a R01 submission to the NIH in the third year of this award. The research plan focuses on the regulation of endothelial cell-cell adherens junctions to prevent microvascular hyperpermeability following hemorrhagic shock. Fluid loss from the circulation is a major factor in compromising cardiovascular homeostasis following trauma, burns, sepsis and hemorrhagic shock. Preventing microvascular barrier opening or reestablishing barrier function is crucial in limiting the deleterious effects on the cardiovascular, pulmonary and nervous systems. Hemorrhagic shock-induced hyperpermeability results in alteration of the endothelial cell-cell adherens junction complex. Site-specific alterations in the structure of (3-catenin have been implicated as an important modulator of these junctions. The applicant hypothesizes that hemorrhagic shock-induced activation of caspases results in a (3- catenin-dependent modification of endothelial adherens junction. To address this hypothesis, the applicant proposes two interlocking specific aims: (1) define the relationship between the apoptotic cascade and hemorrhagic shock-induced hyperpermeability and (2) identify the interaction(s) of caspase-3 with the adherens junction complex. Intravital microscopy with a novel in vivo protein transfection technique in parallel with cell monolayers will be utilized to determine changes in microvascular hyperpermeability. Atomic force microscopy will be used to assess endothelial cell adhesive forces.
