Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

purposeTo test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents.

methodsCorneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or β-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly.

resultsThree or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and β) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin.

conclusionsThis collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents.