The main objective of this proposal is to provide Dr. Tolentino a mechanism for rigorous training both technically and conceptually in gene therapy and molecular biology. Dr. Tolentino's long term goal is to become an independent investigator focusing on understanding the molecular mechanisms and developing treatments for pathologic ocular neovascularization seen in blinding conditions such as macular degeneration and diabetic retinopathy. The training plan proposed is essential for Dr. Tolentino to successfully pursue these long-term goals. The broad long-term research objective of this grant proposal is to understand the biochemical mechanisms that lead to choroidal neovascularization (CNV). The development of CNV in age related macular degeneration (AMD) is the leading cause of blindness in the United States. Patients with Sorsby's fundus dystrophy, an autosomal dominant form of macular degeneration, have similar clinical findings to AMD including CNV. These patients have been found to have a mutation in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene.
The specific aims of this study are:
Specific aim 1 : To test the hypothesis that CNV induced by mutant TIMP-3 (mTIMP-3) is caused by loss of functionality or direct stimulation of angiogenesis by mTIMP-3. To accomplish this aim, mTIMP-3 transfected endothelial cell will be assayed for their ability to proliferate, form tubes and migrate to angiogenic stimuli in vitro. Sub retinal injections into normal and transgenic mice of adenovirus expressing mTIMP-3 will be performed to determine mTIMP-3's ability to cause sub retinal neovascularization in vivo.
Specific aim 2 : To test the hypothesis that upregulation of vascular endothelial growth factor (VEGF) and/or down regulation of pigment epithelial derived growth factor (PEDF) is involved in mTlW-3 induced CNV. Both mTIMP-3 transfected retinal pigment epithelial (RPE) cells and a transgenic mouse model with a human TIMP-3 mutation that develops CNV will be assayed for protein and mRNA upregulation using ELISA, northern blot, immunohistochemical and in situ hybridization techniques.
Specific aim 3 : To develop and test gene vectors expressing inhibitors of angiogenesis (angiostatin, endostatin, PEDF) and test their ability to inhibit subretinal neovascularization in the mutant transgenic mouse.

National Institute of Health (NIH)
National Eye Institute (NEI)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (ZEY1-VSN (02))
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Dudley, Peter A
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University of Pennsylvania
Schools of Medicine
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Tolentino, Michael J; Brucker, Alexander J; Fosnot, Joshua et al. (2004) Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina 24:132-8
Ibarra, Michael S; Hsu, Jason; Mirza, Naureen et al. (2004) Retinal temperature increase during transpupillary thermotherapy: effects of pigmentation, subretinal blood, and choroidal blood flow. Invest Ophthalmol Vis Sci 45:3678-82
Prenner, Jonathan L; Rosenblatt, Brett J; Tolentino, Michael J et al. (2003) Risk factors for choroidal neovascularization and vision loss in the fellow eye study of CNVPT. Retina 23:307-14
Auricchio, Alberto; Behling, Kathryn C; Maguire, Albert M et al. (2002) Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents. Mol Ther 6:490-4