The mechanism of neutrophil (PMN)-mediated endothelial injury involves the generation of oxidants. This proposal is based on the hypothesis that activation of endothelial second messenger pathways by oxidants mediates endothelial injury and thereby increases endothelial permeability. Preliminary data indicate that oxidants, in concentrations which do not lyse endothelial cells, activate the protein kinase C (PKC) pathway, which can increase endothelial permeability. The proposed studies will investigate the role of PKC activation in mediating the increase in endothelial permeability induced by PMN activation products. These studies will also investigate the synergistic action of cytosolic Ca2+ in activating PKC, and thus playing a modulatory role in the increase in permeability, and the potential role of the cyclic nucleotides such as cAMP, in """"""""down-regulating"""""""" PKC activation, and thereby preventing the oxidant-induced increase in permeability. We will also determine whether oxidant- induced increases in endothelial permeability correlate with changes in phosphorylation, content and distribution of endothelial cytoskeletal proteins, and the role of second messengers in mediating these changes. All experiments will be done using pulmonary microvessel endothelial cell monolayers grown on microporous filters, plates or coverslips. The permeability of the monolayers will be measured in a system which was developed in our laboratory, which allows the study of the endothelial barrier in controlled conditions of one variable at a time, in the absence of hemodynamic forces. Other techniques to be used have either been used in our laboratory or are standard techniques. A better understanding of the mechanisms by which oxidants generated following PMN activation induce increases in endothelial permeability will help in preventing permeability increases in inflammatory responses.
| Siflinger-Birnboim, A; Malik, A B (1996) Regulation of endothelial permeability by second messengers. New Horiz 4:87-98 |
| Siflinger-Birnboim, A; Lum, H; Del Vecchio, P J et al. (1996) Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability. Am J Physiol 270:L973-8 |
