Corneal ulceration occurs when the balance between proteinase and inhibitors in the cornea is disrupted. Understanding the means by which the cornea controls proteinase inhibitor levels is critical for the development of ways to prevent corneal ulceration, promote corneal healing and to maintain and/or restore transparency. We have shown that the cornea contain and synthesizes alpha1-proteinase inhibitor (alpha1- antitrypsin), alpha1-antichymotrypsin and alpha2-macroglobulin. Further, alpha1-proteinase synthesis is stimulated by the inflammatory cytokine interleukin-1Beta (IL-1Beta), basic fibroblast growth factor and retinol. The long term goal is to characterize, at the biochemical level, the process of corneal ulceration by identifying not only the proteinase involved but other factors influencing the degradation process. The general and specific hypotheses are the following: The cornea has the ability to respond to an injury and/or inflammation by the synthesis of molecules which protect the cornea thereby minimizing damage and promoting healing. Given that the cytokine IL-1Beta regulates the response of cells to injury and inflammation, we hypothesize that proteinase inhibitor synthesis in the cornea is induced by IL-1Beta as part of a coordinated early response to injury/and or inflammation. This hypothesis will be tested using both a human corneal organ culture system and a human stromal cell culture system.
The specific aims of this proposal are the following: 1. To determine if IL-1Beta selectively induces the synthesis of alpha1-proteinase inhibitor by the cornea or whether it coordinates an increase in other proteinase inhibitors, other protective proteins or target proteinase in the same time frame. Synthesis of the proteins will be assays will be used to assess the relative levels of proteinase synthesized by the cornea. 2. To determine the effects of IL-1Beta function modulators on the synthesis of alpha1-proteinase inhibitor and other corneal proteinase retinoid, IL- 6, TGF-Beta, INFgamma, IL-2 and bFGF on the effects of IL-1Beta on proteinase inhibitor synthesis. 3. To determine the mechanism by which IL-1Beta and its modulators regulate the levels of alpha1-proteinase inhibitor and other proteinase inhibitors in the cornea. Inhibitors and specific agonists will be used to elucidate which signal transduction pathways are important. The effect of IL-1Beta and its modulators on the steady state mRNA levels of the inhibitors will be determined by Northern analysis. The rates of mRNA synthesis will be determined using a generated will be determined using PCR methodology. Finally the synthesis and stability of the protein product will be assayed in pulse and pulse chase experiments.
| Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin). Invest Ophthalmol Vis Sci 35:458-62 |
| Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Alpha 2-macroglobulin is present in and synthesized by the cornea. Invest Ophthalmol Vis Sci 35:3226-33 |
| Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Alpha 1-antichymotrypsin is present in and synthesized by the cornea. Curr Eye Res 13:433-9 |
| Twining, S S; Kirschner, S E; Mahnke, L A et al. (1993) Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34:2699-712 |
