We reported that phorbol myristate acetate (PMA) stimulated polymorphonuclear leukocytes (PMN) from healthy humans, but not from persons with chronic granulomatous disease (CGD) increased transfer of albumin across an intact microvasculature (isolated perfused lung) and across monolayers of cultured endothelial cells. Direct observation of endothelial monolayers exposed to PMA-stimulated PMN demonstrated retraction of adjacent endothelial cells from each other. These reports suggested that reactive oxygen molecules, released from inflammatory cells, could alter endothelial cell shape and the transfer of albumin across an endothelium. More recently we observed that a brief exposure of endothelium to oxidants generated by xanthine and xanthine oxidase caused a 100% increase in albumin transfer across endothelium and that transfer returned to normal within 3.5 hours if the endothelium was reincubated in tissue culture medium enriched with 10% fetal bovine serum. The serum was necessary since reincubation in tissue culture medium alone resulted in progressive injury. Serum could not be replaced by a general antioxidant or by antiproteases. The effects of xanthine oxidase on albumin transfer were blocked by lanthanum chloride, and endothelial cell lysis by xanthine oxidase was inhibited by chelation of extracellular calcium. The changes in albumin transfer were associated with reversible retraction of adjacent cells from one another and the retraction was also prevented by LaCl3. This data suggests that oxidants can reversibly alter endothelial cell shape and that Ca++ is important to this process. The proposed studies extend these observations. One group of experiments will determine if the effects of oxidants are reversible in individual endothelial cells. Another group will investigate how oxidants alter endothelial calcium homeostasis. A third group will investigate oxidant stimulation of phospholipase activity and the last group will investigate how serum enables endothelium to reverse the oxidant injury. These investigations should increase our understanding of how oxidants affect endothelium and are relevant to acute inflammation and to atherosclerosis.
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