Endothelial barrier dysfunction is a hallmark of lung inflammation and the proximate cause of pulmonary edema. Although, myosin light chain kinase (MLCK)-dependent contractile mechanisms result in edema formation (Project 1), additional MLCK-independent mechanisms may be equally important in producing barrier dysfunction. For example, phorbol esters and pertussis toxin induce MLCK-independent endothelial cell monolayer permeability, which is dramatically attenuated by CAMP elevation. Based on our preliminary data, we will test the HYPOTHESIS that MLCK-independent endothelial cell barrier dysfunction may result from loss of barrier protective cyclic nucleotides, activation of the contractile apparatus, and/or disruption of cell/cell and cell/matrix adhesions. SA#1 will characterize the contribution of reduced cAMP/PKA activity to MLCK- independent barrier dysfunction. We will determine if MLCK-independent agonists (PMA, pertussis toxin, and ionomycin) decrease [cAMP via reduction on adenylate cyclase activity (particularly type III and type VI isotypes), or PKA activity, activation of phosphodiesterase activity. We will link decreased cAMP/PKA activity with agonist-induced alterations in endothelial gap formation, electrical resistance, force development, and albumin permeability. SA#2 will explore potential mechanisms of MLCK- independent activation of the EC contractile apparatus by examining agonist effects on phosphorylation of the actin-, myosin-, and calmodulin- binding protein, caldesmon. We will identify the specific kinases involved in situ and in vitro and link caldesmon phosphorylation to altered cytoskeletal protein interactions and to agonist-induced barrier dysfunction. SA#3 will determine the contribution of altered tethering forces (focal adhesions and adherens junctions) to MLCK-independent barrier dysfunction. We will examine agonist effects on the cellular organization and phosphorylation status of target focal adhesion and adherens junction proteins. We will examine the link between altered tethering force function and endothelial barrier regulation. This in depth analysis of novel MLCK-independent mechanisms that likely contribute to endothelial barrier dysfunction will improve understanding of edema formation relevant to diverse inflammatory lung syndromes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Johns Hopkins University
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