Keratocyte-Extracellular Matrix Adhesion Regulates Structural Integrity of Cornea

Abstract

Purpose: : The function of keratocytes in adult cornea remains unknown. Their location in a structure that is under constant stress due to intraocular pressure indicates a role for them in sustaining corneal structural integrity, but is unproven. We hypothesized that keratocyte-extracellular matrix (ECM) interaction has a role in maintaining corneal structural integrity, deficiency of which could cause changes in keratocyte activity and point to their actual functional role in a normal cornea. Since cells interact with their ECM mainly through integrin receptors we conditionally deleted integrin beta 1 (Itgb1) gene in keratocytes to test our hypothesis.

Methods: : Mice with exon 3 of Itgb1 gene flanked by loxP sites (Raghavan et al., 2000, J Cell Biol., 150:1149-60) were mated with mice expressing tamoxifen-dependent cre recombinase under the control of fibroblast-specific regulatory sequence from the pro alpha 2 (I) collagen gene (Zheng et al., 2002, Am J Pathol., 160:1609-17). Mice homozygous for loxP Itgb1 and hemizygous for cre were administerd tamoxifen (1mg/mouse for 5 days) at 21 days of age to delete Itgb1 gene specifically in fibroblasts. Control mice administered with corn oil / tamoxifen were maintained.

Results: : Using fibroblast-specific expression of cre we were able to delete Itgb1 gene in the keratocytes of the cornea. Protein expression of Itgb1 was almost completely absent 10 days after gene deletion. By 40 days there is decrease in corneal thickness: the stroma is thinner with loss of epithelial cell layers. At later stages the thinning of cornea resulted in erosions and scarring.

Conclusions: : This study confirms our hypothesis and shows for the first-time that keratocyte-ECM interaction is necessary for maintenance of corneal structural integrity. This study could have implications in understanding many corneal diseases that results in loss of corneal structural integrity and thus impairs vision.

Keywords: cornea: stroma and keratocytes • cornea: basic science • cornea: clinical science