The central hypothesis of this proposal (based on our preliminary data) is that TNF primes the lung for the development of edema, at least in part, by activating endothelial PKC. TNF, upon binding to its endothelial surface receptor, stimulates the translocation of specific PKC isotypes from the cytosol to the membrane. The """"""""TNF-activated PKC"""""""" (i.e. the isotypes of PKC activated by TNF in the lung endothelium) phosphorylates specific cytoskeletal proteins, and also decreases the generation of endothelial-derived nitrovasodilators. These actions could occur as a direct effect of TNF, or indirectly, via the induction of reactive oxygen species (ROS) and/or the depletion of glutathione. The net effect of the TNF-induced activation of PKC is both endothelial barrier dysfunction and altered vasoreactivity, predisposing to the development of pulmonary edema. In this proposal, the pathogenesis of the pulmonary edema induced by TNF will be studied using both in situ (the isolated lung) and in vitro (pulmonary endothelial monolayers) models. Initially, the mechanisms by which TNF induces pulmonary endothelial PKC translocation in vivo will be investigated using immunoblotting in vitro and immunofluorescence in situ, focusing on the roles of isotype-specificity, and the site of endothelial origin (i.e. arterial vs. microvascular). The effects of TNF-activated PKC on nitrovasodilators will be evaluated using pharmacologic intervention and direct mediator assay, emphasizing mechanisms related to changes in ROS and glutathione. The mechanisms of the vascular effects of TNF will be probed using immunoblotting and immunofluorescence, focusing on the endothelial cytoskeletal changes induced by, and the phosphorylation substrate(s) of, TNF-activated PKC. Finally, the edemagenic effects of TNF-activated PKC on vasoreactivity (isolated lung) and permeability (capillary filtration coefficient, pulmonary critical capillary pressure in the lung, 125I-albumin clearance in vitro) will be studied, emphasizing mechanisms related to changes in nitrovasodilators, ROS, glutathione and the endothelial cytoskeleton. Successful completion of the proposed studies may eventually result in progress in the prevention and treatment of the Adult Respiratory Distress Syndrome, which is believed to be partly mediated by TNF.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL048406-04
Application #
2224470
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1991-09-06
Project End
1998-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Albany Medical College
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Albany
State
NY
Country
United States
Zip Code
12208
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Gertzberg, N; Clements, R; Jaspers, I et al. (2000) Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation. Am J Respir Cell Mol Biol 22:105-15
Ferro, T; Neumann, P; Gertzberg, N et al. (2000) Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha. Am J Physiol Lung Cell Mol Physiol 278:L1107-17
Ferro, T J; Gertzberg, N; Selden, L et al. (1997) Endothelial barrier dysfunction and p42 oxidation induced by TNF-alpha are mediated by nitric oxide. Am J Physiol 272:L979-88
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Phelps, D T; Ferro, T J; Higgins, P J et al. (1995) TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C. Am J Physiol 269:L551-9
Johnson, A; Phelps, D T; Ferro, T J (1994) Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C. Am J Physiol 267:L318-25
Serfilippi, G; Ferro, T J; Johnson, A (1994) Activation of protein kinase C mediates altered pulmonary vasoreactivity induced by tumor necrosis factor-alpha. Am J Physiol 267:L282-90
Ferro, T J; Parker, D M; Commins, L M et al. (1993) Tumor necrosis factor-alpha activates pulmonary artery endothelial protein kinase C. Am J Physiol 264:L7-14
Ferro, T J; Hocking, D C; Johnson, A (1993) Tumor necrosis factor-alpha alters pulmonary vasoreactivity via neutrophil-derived oxidants. Am J Physiol 265:L462-71

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