The limited capacity that human corneal endothelium has for in vivo proliferation could be a factor in the development of many corneal diseases, including aphakic bullous keratopathy, pseudophakic bullous keratopathy, and Fuchs' dystrophy. Long term growth of human corneal endothelial cells in culture has been difficult, with high rates of success being reported primarily for normal endothelium from younger donors. Given the limited availability of these cells, it is not surprising that little is known regarding the regulation of corneal endothelial cell growth. Wider availability of growth factors and the development of sensitive techniques, such as the polymerase chain reaction (PCR), have provided the opportunity for approaching these problems. The long term objective of this study is to examine the levels of messenger RNAs coding for growth factors, growth factor receptors, and hormone receptors in cultured human corneal endothelial cells during proliferation, confluence, senescence, and serum starvation in order to appreciate and manipulate the factors controlling the proliferation of these cells. In preliminary studies, PCR was used to demonstrate that messenger RNAs coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha are produced by human corneal endothelial cells in primary culture. In subsequent experiments, PCR will be used to determine whether messenger RNAs coding for other growth factors, growth factor receptors, and hormone receptors are produced by human corneal endothelial cells. Quantitative PCR assays will be applied to examine variations that occur in the levels of these endogenously-produced modulators of cell growth in proliferating, confluent, senescent, and serum-starved human corneal endothelial cells. Immunoblotting techniques will be used to determine whether variations in the levels of messenger RNAs coding for these modulators are paralleled by variations in the levels of the specific proteins. The effects of exogenous growth factors and interleukin 1-alpha antisense oligonucleotide on cell morphology, cell proliferation, and the levels of messenger RNAs coding for endogenous growth factors, growth factor receptors, and hormone receptors will also be examined. Finally, methods will be developed for stimulating the proliferation of human corneal endothelial cells in vitro by transfection with plasmids containing constitutive and inducible Simian virus 40 (SV40) large T antigen coding oncogene sequences. Physiologic characteristics related to collagen type, growth factor production, and responses to exogenous growth factors will be examined in immortalized endothelial cells to determine the applicability of this in vitro model to the study of normal human corneal endothelial cells.
Showing the most recent 10 out of 16 publications
