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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY009379-03
Application #
2162993
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1992-01-01
Project End
1994-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Wilson, S E; Weng, J; Blair, S et al. (1995) Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated high-proliferative capacity. Invest Ophthalmol Vis Sci 36:32-40
Wilson, S E; Klyce, S D (1994) Screening for corneal topographic abnormalities before refractive surgery. Ophthalmology 101:147-52
Wilson, S E; Schultz, G S; Chegini, N et al. (1994) Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res 59:63-71
Wilson, S E; Lloyd, S A; He, Y G (1994) Glucocorticoid receptor and interleukin-1 receptor messenger RNA expression in corneal cells. Cornea 13:4-8
Wilson, S E; He, Y G; Weng, J et al. (1994) Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells. Exp Eye Res 59:665-78
Wilson, S E; Lee, W M; Murakami, C et al. (1994) Mooren-type hepatitis C virus-associated corneal ulceration. Ophthalmology 101:736-45
Wilson, S E (1994) Growth factor and receptor messenger RNA production in human lacrimal gland tissue. Adv Exp Med Biol 350:197-204
Ruiz-Montenegro, J; Mafra, C H; Wilson, S E et al. (1993) Corneal topographic alterations in normal contact lens wearers. Ophthalmology 100:128-34
Wilson, S E; Lloyd, S A; He, Y G (1993) Fibroblast growth factor-1 receptor messenger RNA expression in corneal cells. Cornea 12:249-54

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