The goal of this proposal is an understanding of the role of Wnt signaling in regression of the temporary vascular networks of the eye. This work has important implications for therapies aimed at ocular vascular disease, including retinopathy of prematurity and diabetic retinopathy, but also has broader implications for the treatment of many diseases that might be alleviated through pro- or anti-vascular therapy. The eye is ideally suited as a focus for this analysis: it is one of the few structures where vascular networks can readily be examined in their entirety unobstructed by surrounding cells, and where (in rodents) a programmed vascular regression occurs postnatally. Indeed, the eye is one of the few organs in which this analysis could reasonably be executed. We will execute three specific aims:
Aim 1 : Is cell-cycle progression required for endothelial cell (EC) death? Preliminary Results and prior work suggest that EC apoptosis may depend upon Wnt-stimulated entry to the cell cycle and transit through the so-called Restriction Point in mid-G1 phase. We will test this hypothesis by first assessing the cell cycle characteristics of Wnt responsive cells (1.1) and then determining whether imposition of a cell cycle arrest also prevents cell death (1.2).
Aim 2 : How are Angiopoietin and Wnt signaling integrated in programmed vascular regression? The strong genetic interaction between Ang2 and Lrp5 mutations in vascular regression suggests that Tie2 and Wnt signaling are closely integrated. We will test this hypothesis by determining whether Ang2 is a Transcriptional target of the Wnt pathway (2.1), by determining whether Wnt signaling is modified in the absence of Lrp5, or Ang2 (2.2) and by injecting recombinant Wnt agonists and antagonists (2.3).
Aim 3 : Do macrophages kill ECs by producing Wnt ligands? Preliminary evidence suggest that macrophages drive programmed regression by producing Wnt ligands. We will test this possibility directly by asking whether macrophage absence down-modulates EC Wnt responses (3.1), by determining which Wnt ligands are expressed by macrophages (3.2), and by obtaining existing, or generating new Wnt ligand nullmice for phenotypic analysis (3.3). Completion of this work will provide important information on how the Wnt and Angiopoietin signaling pathways interact and furthermore, will indicate how we might modulate these signaling pathways in an effort to treat vascular diseases of the eye and other organs.
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