The blindness occurring with diabetes, premature birth, age- related macular degeneration, retinal venous occlusion, and sickle cell anemia may be controlled if the growth of new retinal capillaries can be inhibited. This application seeks to study the communication of growth controlling signals generated intracellularly in response to growth-inducing hormones. The study will test the hypothesis that inhibitors of retinal microvascular endothelial cell proliferation in vitro act via inhibition of this intracellular signal transduction. The signals monitored in real time in intact cells will be the free cytoplasmic calcium ion concentration and the cytoplasmic pH. These ions change the activity of many enzymes important for cellular growth, and therefore relate to the readiness of cells to divide. The studies will use cultured retinal microvascular endothelial cells and purified hormones. Two growth-stimulating hormones, acidic and basic fibroblast growth factors are important for these cells in vitro. Two other hormones, transforming growth factor- beta and gamma interferon, inhibit the proliferation of these cells in tissue culture. The hormones will be tested alone and in stimulator-inhibitor combinations. The studies will compare culture growth rates and division of single cells with measurements of intracellular calcium ion pH. The principal method is to monitor the specific changes in fluorescence of intracellularly active dyes that are sensitive to ionic calcium or protons (pH). The same cells will be filmed, after measuring calcium or pH, to document cell division. The objective is to learn not only the interactions of growth controlling hormones at the level of intracellular communication, but also to test the hypothesis that the changes in calcium of pH are important to the control of cell division. This information will potentially help clinicians prevent many forms of blindness, as well as other diseases exacerbated by formation of new capillaries.
