Retinal neovascularization is associated with proliferative diabetic retinopathy and retinopathy of prematurity. Clinical studies suggest that insulin like growth factor-1 (IGF-1) is involved in both diseases and has direct effects on retinal vasculature. To understand the mechanisms by which IGF-1 contributes to normal and abnormal vascular physiology, several processes must be understood in greater detail. This includes how IGF-1 receptor (IGF-1R) expression affects both normal and abnormal retinal vascular growth and how expression of IGF binding protein-3 (IGFBP-3) mediates IGF-1 bioavailability and influences endothelial cell behavior. Of the seven known IGFBPs, IGFBP-3 is the primary carrier of IGF-1 in the serum and its direct apoptotic effects have been demonstrated in numerous cells systems including tumor cells. These apoptotic effects of IGFBP-3 are independent of its ability to bind IGF-1. This proposal focuses on manipulating IGF-1R and IGFBP-3 expression to examine their effects on aberrant vascularization of the retina. We will test the following hypothesis: Decreasing the expression of IGF-1R and increasing the secretion of soluble IGFBP-3 will result in the inhibition of retinal neovascularization though the induction of endothelial cell apoptosis.
In Aim 1, we will synthesize IGF-1R mRNA-specific hammerhead ribozymes, infect human retinal endothelial cells with these ribozymes, and characterize ribozyme effects on IGF-1R expression. We will characterize the apoptotic pathways activated by IGFBP-3 and evaluate the action of endogenously produced IGFBP-3 (to induce apoptosis directly) in HREC; the effects of the combined effect of the IGF-1R inhibition with overexpression of IGFBP-3 on HREC apoptosis will also be examined.
In Aim 2, we will package our IGF-1R ribozyme into adeno-associated virus (AAV) for site directed expression. A cell cycle/proliferating endothelial cell-specific promoter will drive expression of the IGF-1R ribozymes. These AAV-packaged constructs will be used to inhibit retinal and pre-retinal neovascularization specifically in two mouse models: the mouse pup model of oxygen-induced retinopathy and an adult mouse model that we have developed.
In Aim 3, we will express IGFBP-3 using a rAAV protein expression vector and test it in the two mouse models described in Aim 2. We will combine the AAV-IGF-1R ribozymes with AAV-IGFBP-3 to determine if additional inhibition of proliferation and apoptosis occurs. Targeting the IGF-IR and IGFBP-3 in this manner will provide a tool for understanding the role of the IGF-1 system in retinal vascular growth and may provide novel ways to inhibit retinal angiogenesis.
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