Disease and damage at the level of Descemet's membrane/corneal endothelium may result in production of retrocorneal fibrous membrane (RCFM). Although it is known that the corneal endothelium responds to various insults by conversion to a fibroblast-like cell, definitive experimental support for this phenomenon is still lacking. However, our data suggest that polymorphonuclear leukocytes (PMN) may play an important role in endothelial modulation. The modulation process induced by PMN-conditioned medium appears to be composed of several stages: selection of sensitive cells, induction to type-I synthesizing cells and enhancing their growth until they reach irreversible metaplasia. One objective is to purify and characterize factors specific for endothelial modulation, which is an essential step to study how the modulation is initiated, progresses and is regulated. Furthermore, PMN-conditioned medium is effective on only a minor fraction of cells, suggestive of heterogeneity in endothelial cells. This raises a question whether the responding cells are genetically different from the nonresponder. In order to answer the question, a second goal is to synthesize and clone complementary DNA (cDNA) for the proAlpha1(I) and Alpha1(IV) mRNAs. The apparent difference of collagen types in RCFM (type I) and in normal endothelium (type IV) will be the basis for analyzing endothelial modulation. We will use a combination of protein and recombinant DNA technology. The factors specific for endothelial modulation will be purified by chromatography (ion-exchange, molecular sieve, and affinity) and characterized by their biological activities; the cDNAs will be cloned from the libraries from corneal endothelial mRNAs and modulated endothelial cell mRNAs. The DNA probes will be used as hybridization probe to detect and quantitate the mRNAs for proAlpha1(I) and Alpha1(IV) during modulation induced by the purified factors in vivo and in vitro. In addition, in situ hybridization, which can detect the location and abundance of RNA transcripts in single cells, will be used to distinguish the responding cells from those that do not respond to PMN-factors. The long-term objective to understand how the expression of collagen genes is modulated during wound-healing. Understanding the mechanisms of the change in endothelial cells that inevitably leads to RCFM formation, provide a basis not only for a rational therapy for the prevention of RCFM, but also for understanding mechanisms of atypical wound-healing in cornea.
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