The long-term objective of this research is to understand how RPE growth control is maintained. Such control is vital to normal epithelial function in supporting photoreceptor cells. Furthermore, dysregulation of RPE cell number, such as occurs in age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), constitutes a significant threat to retinal health. The extreme longevity of these cells, and their limited regenerative capacity under normal conditions, highlight the fact that epithelial cell survival is a paramount strategy. Consequently, these studies focus on survival mechanisms, using an in vitro system whereby conditions may be precisely controlled. Single RPE cells, deprived of all exogenous survival factors, undergo a form of programmed cell death in culture. Furthermore, the same cells can be rescued from cell death by attachment to, and spreading on, substrates coated with extracellular matrix (ECM) proteins. Using attachment to fibronectin as a model of the cell's interaction with its natural substratum, the proposed studies aim to identify and control the individual cell biological components contributing to attachment-mediated RPE cell survival. Experiments will define the roles of cell-surface integrin (fibronectin receptor) activation and cell shape changes as triggering stimuli for cell survival. One potential signal accompanying cell-ECM interaction, protein tyrosine phosphorylation, will be examined in detail in combined biochemical and cell viability studies. In addition, the role of the small GTP-binding protein RhoA in mediating the survival effects of cell spreading will be examined by microinjection into cells of a specific inactivator of this protein. By defining specific molecules as targets for promoting or inhibiting cell survival or death, information gained from these studies may form the basis for designing therapeutic strategies to intervene in RPE proliferation and atrophy.
