The pathogenesis of pulmonary edema resulting from ischemia/reperfusion injury or bacterial infection involves increased microvascular permeability. The goals of this project are to investigate increased pulmonary microvascular endothelial monolayer permeability caused by reoxygenation after hypoxia (hypoxia/reoxygenation). the hypothesized mechanism involves increased degradation of the extracellular matrix by proteases released from endothelial cells, as well as changes in cytoskeletal conformation. Changes in matrix composition then effect changes in endothelial cytoskeletal structure and alter production of matrix components. This proposed research will explore the role of endothelial cells in remodelling the extracellular matrix in hypoxia/reoxygenation, and the involvement of the matrix changes in increased endothelial monolayer permeability. In particular, this project will characterize a gelatinolytic protease from pulmonary microvascular endothelial cells that is activated upon hypoxia/reoxygenation, and determine its role in matrix degradation. The model system to be used consists of bovine pulmonary microvascular endothelial (BPMVE) cells grown on gelatinized filters glued to plastic cylinders to form wells. The BPMVE cells are exposed to hypoxia/reoxygenation in a gas-tight chamber, and monolayer permeabilities are determined by measuring transfer of 125I- albumin from the luminal to abluminal sides of the filters. Pulse-chase techniques will be used to determine alterations in production of matrix components, and release of radioactivity from metabolically radiolabelled matrices will be measured to assess degradation. The permeabilities and cytoskeletal structures of monolayers seeded on matrices from BPMVE exposed to hypoxia/reoxygenation will be assessed to determine influence of matrix alterations. Other experiments will utilize immunofluorescent techniques to determine changes in relative quantities and configuration of matrix components. These components will be further examined by extraction followed by SDS-PAGE, densitometry, and western blotting with specific antibodies. It is expected that these studies will provide many useful insights into the mechanisms behind increased endothelial permeability in pulmonary edema, and will provide a basis for formulating more effective treatment strategies.
| Partridge, C A; Phillips, P G; Niedbala, M J et al. (1997) Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts. Am J Physiol 272:L813-22 |
| He, N G; Singhal, S S; Chaubey, M et al. (1996) Purification and characterization of a 4-hydroxynonenal metabolizing glutathione S-transferase isozyme from bovine pulmonary microvessel endothelial cells. Biochim Biophys Acta 1291:182-8 |
| Partridge, C A (1995) Hypoxia and reoxygenation stimulate biphasic changes in endothelial monolayer permeability. Am J Physiol 269:L52-8 |
