The pulmonary endothelium is a critical, semi-selective cellular barrier which prevents the leakage of water, solutes, macromolecules and cells from the vasculature. Disruption of lung endothelial cell integrity results in alveolar flooding, hypoxemia and multi-organ dysfunction due to increases in both endothelial cell permeability and transendothelial leukocyte migration to the lung. Both of these important inflammatory processes can be attributed to increases in paracellular gaps which form between adjacent endothelium. Our previous studies have identified an important role for endothelial cell contractile forces in paracellular gap regulation and we have cloned several novel endothelial cell contractile elements which contribute to this contractile process including a novel endothelial cell myosin light chain kinase (EC MLCK) isoform. In this proposal, we will examine novel biochemical and molecular mechanisms by which endothelial contractile forces are initiated and maintained.
Specific Aim #1 will examine the interaction between p60src kinase, the p85 kD actin-binding protein, cortactin, and EC MLCK in the context of endothelial cell cytoskeletal rearrangement.
Specific Aim #2 will determine the exact contribution of Rho family GTPases to endothelial cell myosin phosphorylation, gap formation and permeability evoked by barrier-disrupting agonists.
Specific Aim #3 will identify the contributing role of the actin- and myosin- binding protein, caldesmon, in sustained endothelial cell contraction and endothelial cell barrier regulation. The very diversity of potential pathogenetic factors involved in vascular barrier dysfunction emphasizes that there is still much to be understood about the pathophysiology of this challenging disease. We speculate that the results obtained in this competitive renewal application will significantly impact our understanding of the initiating phases (SA #1 and SA #2) and the sustained phase (SA #3) of endothelial cell contraction and barrier regulation and allow us to develop new insights into this important aspect of lung inflammation.
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