While formation of new blood vessels is a necessary aspect of such normal processes as wound healing and embryological development, their formation following corneal trauma or inflammation and during proliferative diabetic retinopathy can have devastating consequences. Building upon our earlier studies of actively growing corneal vessels, we propose to examine further the processes occurring in this region, as the potential for local control of this process is suggested by some of our recent findings. Because both the cell attachment factor laminin and the protease urokinase-like plasminogen activator (u-PA) are associated with the new nessel tips, the effect in vivo of inactivating these two molecules will be examined using the corneal pocket system. An analog of laminin will be used to examine the role of laminin in new vessel growth, and both antibodies against u-PA and drugs which effect its activity will be tested in this system to examine the role of this enzyme in stromal invasion by new nessel tips. Because preliminary evidence suggests that endothelial cells in new corneal vessels can bind fibroblast growth factor in situ, these new vessels will be probed for the presence of important mitogenic factor using the techniques of both light and electron microscopic immunohistochemistry. In situ hybridization techniques will be used to more carefully examine the corneal new vessels, using DNA probes for plasminogen actiators as well as an inhibitor of this enzyme known to be released by endothelial cells PAI- 1). In a third group of experiments, we will take advantage of f recently developed in vitro model to examine the effect on endothelial cell behavior of culturing on sections of avascular corneal stroma. Because this stroma supports the growth of apparently normal vessels following prostaglandin E1 implantations, we will examine the effect of this three- dimensional growth substrate on endothelial cell attachment, migration, extracellular matrix production, and the secretion of both plasminogen activators and their inhibitor. To do this, we will use the combined complementary techniques of scanning electron microscopy, immunohistochemistry and transmission electron microscopy to examine these processes in the carefully controlled experimental conditions this model will allow. Examination of endothelial cell behavior in this new model system will hopefully shed light on the activities occurring at the newly forming tips, a region not easily accessible to examination.
