Abstract
Purpose: :
Tissue engineering provides a means to rebuild damaged or diseased corneal tissues. The gap junction protein Connexin31.1 (Cx31.1) appears to form non-functional channels which are expressed in the outer layers of the corneal epithelium prior to epithelial cell apoptosis and shedding. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to knockdown Cx31.1 protein levels to further investigate the impact of regulation of this protein on epithelial cell survival.
Methods: :
Deoxyribozymes (Dzs) were designed and tested for their ability to cleave Cx31.1 mRNA in vitro. Based upon the in vitro cleavage results, antisense ODNs were produced and applied to ex vivo rat and human corneal organotypic culture models to assess the effect of Cx31.1 knockdown. Cx31.1 protein levels, epithelial cell proliferation, and apoptosis were analysed immunohistochemically after three antisense treatments over 24 hours, and corneal structure examined histologically.
Results: :
Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are routinely shed into the tear film. Dzs cleavage efficacy results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 protein knockdown in both rat and human corneal organotypic culture models, leading to a 72% reduction in apoptosis and up to 50% increase in epithelial cell layers (p = 0.0045) without any change in the rate of cell proliferation.
Conclusions: :
Cx31.1 appears to play a role in triggering cell death and shedding in the outer epithelial layers. Down-regulation of Cx31.1 using antisense treatment results in reduced apoptosis and a thickened corneal epithelium. Knocking down Cx31.1 may provide a novel approach for tissue repair and corneal engineering.
Keywords: gap junctions/coupling • apoptosis/cell death • cornea: epithelium