The long range goal of the proposal is to understand the mechanism that regulates the transformation of RPE cells from the stationary epithelial cell to the migratory fibroblastic state in order to design rational interventions in PVR. The applicant hypothesizes that modulations of cell-cell adhesion are the critical early step in the vitreous-induced, phenotypic transformation of RPE cells that allows the stimulated RPE to escape the constraints of the epithelium. The applicant further hypothesizes that the molecular triggers that are responsible for initiating the loss of the epithelial cell-cell adhesion are to be found in the extracellular matrix and growth factor milieu of the vitreous. First, the applicant will address the question of the role of cadherins in regulating the RPE phenotypic transformation. Function-disrupting N-terminal cadherin antibodies will be used to block cadherin function experimentally in RPE monolayers to demonstrate that a specific loss of cadherin adhesion can lead directly to RPE transformation. Alterations in cell-cell adhesion, and in the epithelial cytoskeleton will be evaluated by immunofluorescent and ultrastructural methods. Metabolic labeling coupled with lectin precipitation methods will be utilized to measure changes in secretory and cell surface glycoprotein expression following the loss of cadherin expression. A comparison between vitreous-induced changes and antibody induced changes in RPE cellular phenotype will allow the applicant to determine which of these parameters are initiated by the breakdown of the RPE adherens junction. Determining how cadherin function is regulated during vitreous-induced RPE transformation is the focus of the second section of the proposal. Tyrosine phosphorylation levels of the cadherin and catenin protein components of the adherens junction complex, immunoprecipitated from control and vitreous-exposed RPE cultures, will be quantitated from blots. These samples will be probed with antiphosphotyrosine antibodies. Next, the applicant will prevent the loss of cadherin function in vitreous exposed RPE cultures by blocking changes in tyrosine phosphorylation using specific tyrosine kinase inhibitors. The applicant will examine cadherin expression down- regulation following vitreous exposure by quantitating protein and mRNA levels using immunoblot and Northern blot analysis. Finally, the applicant will identify fractions of vitreous that are responsible for initiating RPE transformation. Platelet-derived growth factor and fibronectin will be specifically removed from vitreous by affinity chromatography utilizing antibodies to these two components in order to determine their respective contributions to the loss of cell-cell adhesion accompanying vitreous-induced RPE transformation. Vitreous fractionation will be accomplished with a combination of ultrafiltration molecular size selection, and affinity and ion exchange chromatography. These fractions will be tested for their RPE transformation-inducing capacity. With a thorough understanding of the regulation of RPE cell adhesion resulting from these investigations, the applicant will be able to design clinical strategies to pharmacologically prevent RPE adhesion loss, thereby preventing RPE involvement in traction membrane formation.
| Feng, Wei; Zheng, Jing J; Lutz, Douglas A et al. (2003) Loss of RPE phenotype affects phagocytic function. Graefes Arch Clin Exp Ophthalmol 241:232-40 |
| Wilt, S D; Greaton, C J; Lutz, D A et al. (1999) Mannose receptor is expressed in normal and dystrophic retinal pigment epithelium. Exp Eye Res 69:405-11 |
