ENDOTHELIAL CELL DERIVED NEUTROPHIL CHEMOATTRACTANTS

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The overall objective of the proposed research is to investigate the potential of the endothelial cell to regulate its own environment in conditions associated with lung vascular injury, an important cause of mobidity and mortality. Endothelial cell injury and neutrophil accumulation may be important events in the pathogenesis of this disorder. We have developed preliminary evidence that exposure to histamine, angiotensin II, talc, or a hypoxic environment stimulates endothelial cells in tissue culture to release substances that stimulate neutrophil migration in a modified Boyden chamber assay. Further studies with cyclo-oxygenase and lipoxygenase inhibitors and initial assays with reverse phase high performance liquid chromatography (HPLC) suggest that these substances are lipoxygenase metabolites of arachidonic acid. To characterize, purify and identify the endothelial cell derived neutrophil chemoattractant activity, we will examine: 1) heat stability, 2) solubility in aqueous or non-aqueous solvents, 3) stability to proteases, 4) ability to incorporate 3H-arachidonic acid metabolites, 6) further purification of reverse phase HPLC of peaks of chemoattractant activity resolved on the initial HPLC analysis, and 7) scanning ultraviolet spectrum analysis of the purified fractions as compared to the spectrum of known standards. To define the conditions associated with generation of neutrophil chemoattractant, we will determine: 1) the effect of endothelial cell type on production by using bovine aortic, bovine pulmonary arterial, and human umbilical vein endothelial cells; 2) the specificity for endothelial cells by testing vascular smooth muscle cells and IMR-90 fibroblasts, and 3) the effect of the degree of confluence of the endothelial cell monolayer. In addition, we will compare the nature of endothelial cell derived neutrophil chemoattractant induced by the four agents - histamine, angiotensin II, talc, and hypoxia - which likely influence endothelial cells by different means in order to provide insights into the events which might initiate the metabolic process associated with release of neutrophil chemoattractants. Better understanding of the ability of the endothelial cell to regulate its own environment obtained by these studies might lead to more effective methods of prevention and treatment of various forms of vascular injury.