DEVELOPING VISION--THE MECHANISM OF TISSUE REMODELING

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It is our long-term goal to understand the mechanism of developmentally programmed capillary regression. As a model system we use the pupillary membrane (PM), a transient capillary network situated in the anterior chamber of the eye. It has the advantage that it is accessible to manipulation through trans- corneal injection, is easily examined in whole-mount when dissected from the eye and regresses postnatally. We have previously shown, (1) that macrophages are required for capillary regression, (2) that macrophages induce programmed cell death in endothelial cells and pericytes to drive capillary regression, and (3) that endothelial cells and pericytes die as a consequence of a macrophage signal received in G1-phase of cell cycle. In the current application, we will pursue the broad question of how macrophages mediate capillary regression with the following aims. Aim 8: To determine whether pericytes have a role in regulating PM regression. Pericytes are thought to have a role in maintaining capillaries, perhaps through trophic support. Thus, we will test whether macrophage-induced pericyte death is a distinct and essential step in PM regression. Aim 9: To determine whether macrophages influence cell-cycling independent of the induction of apoptosis. Macrophages are a source of endothelial cell mitogens and suppressors of cell-cycle. This raises the possibility that they may regulate endothelial cell- cycle independent of their role in inducing cell death. Aim 10: To determine whether TGFbeta signaling is required for macrophage-induced cell-cycle dependent programmed cell death. TGFbeta can have a pro-apoptotic activity on endothelial cells and is a recognized regulator of cell-cycle. We have shown that there is active TGFbeta in the aqueous that bathes the PM and will determine, using a multiple strategies, whether this is required for PCD or regulation of cell-cycle. Aim 11: To determine whether macrophage-induced PCD is dependent upon the action of matrix proteases. A cell-cycle state dependent cell death suggests that inhibition of essential signaling at the restriction point of cell-cycle may be the cause. We will investigate the possibility that matrix proteases produced by macrophages may cause PCD by degrading essential matrix ligands. There are few systems where the mechanism of capillary regression can be studied in detail. With the methods we have developed and the strategies outlined in this proposal, we have an excellent opportunity to understand this clinically important process. There is every possibility that what we learn about developmentally programmed capillary regression will be applicable to the capillaries that support the growth of tumors or those that are the central feature of some vascular diseases.