Non-cardiac pulmonary edema is a frequent affliction in pulmonary medicine and is caused by endothelial barrier dysfunction. It is now recognized that this dysfunction may be initiated by programmed cell signaling as opposed to non-specific disruption of the vascular barrier. Since the endothelial barrier is known to be altered by exposure to hypoxia leading to pulmonary edema, we have utilized a rat pulmonary artery microvascular endothelial cell (EC) monolayer culture model to study the effects of hypoxia on EC barrier function. Preliminary studies have led to the hypothesis that exposure to hypoxia initiates cell signaling through the p38 MAP kinase pathway involving MK2 and HSP27 activation that leads to actin cytoskeleton and hic-5 redistribution. These signaling events are associated with an alteration in biomechanical """"""""tethering"""""""" and """"""""stiffness"""""""" and enhanced permeability of the EC monolayer. """"""""Tethering"""""""" may be regulated by the p38 MAP kinase pathway while """"""""stiffness"""""""" is regulated by Rho/Rho kinase and myosin light chain phosphorylation. We plan in the present proposal to study these responses more thoroughly in the rat pulmonary artery microvascular EC monolayer and to perform confirmatory studies in similar cells from human pulmonary microvasculature. In addition, we will investigate these signaling pathways in mice in vivo using pharmacological inhibitors, as well as mice in which the expression of MK2, a key kinase in the p38 pathway, has been """"""""knocked-out"""""""". Specifically, we plan to:
Aim 1 : Define signaling pathways important for mediating cytoskeletal changes in endothelial cell monolayers exposed to hypoxia;
Aim 2 : Determine the relevance of the biomechanical endpoints """"""""stiffness"""""""" and """"""""tethering"""""""", which increase with exposure to hypoxia, to physiological determinants of endothelial barrier function;
and Aim 3 : Evaluate the signaling events that lead to increased pulmonary endothelial permeability in hypoxia in vivo. The findings of this study are expected to further our understanding of mechanisms involved in the development of endothelial barrier dysfunction, in general, and to suggest targets for treatment of pulmonary edema.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Respiratory Integrative Biology and Translational Research Study Section (RIBT)
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Moore, Timothy M
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Tufts University
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