Increased endothelial monolayer permeability is mediated by actomyosin filament formation and contraction resulting in disruption of cell-cell junctions and formation of intercellular gaps. Additional changes include increases in adhesive forces at cell-cell and cell-extracellular matrix contacts. While much progress has been made in understanding molecular mechanisms regulating barrier dysfunction, little is known about the intracellular signaling necessary for maintenance of endothelial barrier integrity. We have shown that PKC( overexpression enhanced microvascular endothelial basal barrier function by augmenting focal adhesions. In addition, inhibitor studies support a role for PKC( in enhancing barrier function in unstimulated endothelial monolayers by stabilizing focal adhesions and actomyosin filaments. Our data suggests that PKC( modulates endothelial barrier function through modulation of stress fiber and focal adhesion formation through a RhoA pathway. Also, our data shows that PKC( inhibition diminishes cytoskeletal and focal adhesion dynamics;effects which were reversed upon exposure to thrombin. Additionally, thrombin-induced barrier dysfunction was exacerbated by pretreatment with rottlerin. Results also suggest that PKC( regulates RhoA activity through p190RhoGAP. Finally, we demonstrate an induction of lung edema in vivo upon inhibition of PKC(. Thus, our work has demonstrated a crucial role for the PKC( isoform in regulating endothelial barrier function integrity in in vitro and in vivo. The overall goal of this proposal is to elucidate the molecular mechanisms by which PKC( regulates endothelial basal monolayer permeability. The emphasis on cell-ECM complexes and the investigation of mechanisms of maintenance of basal permeability are unique and novel aspects of the proposed research.
Aim I : To determine the role of PKC( in focal adhesion assembly/disassembly in endothelial basal barrier function and agonist-induced barrier dysfunction;
Aim II : To determine if PKC( maintains endothelial barrier integrity by preserving a basal level of active RhoA through modulation of p190RhoGAP activity;
and Aim III : To determine the role of PKC( in maintaining lung vascular barrier function in vivo. Understanding mechanisms of maintenance of pulmonary endothelial barrier integrity may be useful in developing therapies for acute lung injury to limit the extent of lung injury and speed recovery to normal lung function. Lay Description: Blood vessels in the lung become leaky upon injury or trauma, resulting in fluid transported by the blood vessels to leak into the lung causing difficulty breathing. Currently, no effective treatments for preventing or resolving this condition are available. We hope that identification of proteins important in maintaining a non-leaky state will assist in discovering a treatment for patients suffering from lung edema.
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