Choroidal neovascularization (CNV) is the primary cause of severe vision loss in age-related macular degeneration (AMD), but little is known about the complex mechanisms leading to CNV growth into the neurosensory retina. Human histopathology of occult CNV in AMD has shown that contact between the endothelial cells (EC) of the choriocapillaris and the retinal pigment epithelium (RPE) leads to compromise of the RPE barrier, which precedes the growth of CNV into the neurosensory retina. We will test the hypothesis that RPE-EC contact causes a functional change in the RPE monolayer that permits the transmigration of EC across the RPE. Specifically, we will determine whether 1) EC contact with RPE causes compromised RPE barrier function and structure; 2) EC contact with RPE causes a major change in the ratio of angiogenic stimulators to inhibitors (i.e., VEGF: PEDF) in the RPE; and 3) RPE-EC contact specifically activates cell-associated RPE VEGF isoforms that enable transmigration of EC across the RPE monolayer. Methods will include measurement of transepithelial electrical resistance (TER), cell counts, viability tests, and staining for ZO-1, n-cadherin, and actin in human cell co-cultures of RPE and EC to evaluate RPE barrier structure and function; ELISA, Western blot, and Northern blot to quantitate VEGF:PEDF ratios under various culture conditions; staining for actin and barrier proteins, TER, permeability, and RT-PCR to quantitate soluble and cell-associated VEGF isoforms; and fluorescent labeling of RPE and EC to follow transmigration of EC. Approximately 90% of the legal blindness that occurs in patients with AMD is the result of CNV that originates from the choriocapillaris and grows beyond the natural boundaries of Bruch's membrane and the outer blood-retinal barrier of the RPE into the neurosensory retina. Increased knowledge of the mechanisms that cause this destructive phenomenon may lead to future means to prevent CNV in the neurosensory retina, and thus, reduce vision loss in AMD.
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