Identification of Molecules That Are Potentially Involved in the Angiogenic Pathway in the Cornea of Corn1 Mice

Abstract

Abstract: : Purpose: Corneal disease–1 (corn1) mice, with a deletion of the destrin (actin depolymerizing factor) gene, develop corneal epithelial hyperproliferation and subsequent corneal stromal neovascularization. Because these two events occur without external manipulation, corn1 mice offer a unique model to evaluate the regulatory factors responsible for these processes. The purpose of this study was to identify molecules that are potentially involved in the angiogenic pathway in corn1 mice. Methods: Subtractive hybridization analysis was performed using double stranded cDNA created from the cornea of 4 weeks old corn1 and wild–type control mice. Subtracted clones were amplified by PCR, subcloned and sequenced. Genes identified by subtractive hybridization were subsequently tested by real time PCR analysis. Immunohistochemistry was performed using leukocyte markers to test whether infiltration of inflammatory cells is observed in the corn1 cornea. Results: Through subtractive hybridization and real time PCR analyses, 10 clones were identified as being upregulated in the corn1 cornea. They include molecules with angiogenic properties and/or chemotactic activity for inflammatory cells. Only 1 clone was downregulated in corn1 mice. This clone was destrin, which is not expressed in corn1 mice, providing a validation for the analysis. The marker analysis showed that infiltration of leukocytes is observed in the corn1 cornea as early as 3 weeks of age (earliest time point tested). Conclusions: We identified a set of genes differentially expressed in the corn1 cornea that can be evaluated as candidate molecules that may participate in the induction of neovascularization. Infiltration of leukocytes suggests a possible involvement of these cells in the angiogenic mechanism.

Keywords: cornea: basic science • neovascularization • gene/expression