Multidrug-Resistant Pseudomonas aeruginosa Keratitis Associated With Artificial Tear Use.

PURPOSE: Keratinization of the mucosal epithelia develops in severe ocular surface disorders, causing severe visual loss. This study elucidates the molecular mechanisms of keratinization in conjunctival epithelial cells (CjECs) and investigates the involvement of the vitamin A pathway.

METHODS

Keratinized conjunctival epithelial sheets were generated by a closed system culture of human CjECs and confirmed by immunostaining. Comprehensive gene expression analysis and quantitative real-time PCR (qRT-PCR) were used to examine whether the cells could be used as an in vitro keratinization model. Moreover, immunostaining and qRT-PCR were used to examine alterations of vitamin A pathway-related genes in the cells and also the effect of adding all-trans retinoic acid (ATRA) and retinoic acid receptor alpha/beta (RARA/RARB) agonist Am80. Knockdown of RARA or RARB was also performed using transfection of small interfering RNA to identify receptors for retinoic acid.

RESULTS

Immunostaining revealed that CjECs cultured in a closed system had increased expression of keratinization markers. Comprehensive gene expression analysis and qRT-PCR revealed expression changes in vitamin A pathway genes, in addition to keratinization. In the closed system culture, immunostaining revealed that conjunctival epithelial keratinization was suppressed or partially ameliorated by ATRA or Am80, and qRT-PCR revealed that vitamin A pathway-related genes were significantly altered. Moreover, knockdown of RARA or RARB induced an increase in keratinization marker involucrin.

CONCLUSIONS

Keratinization of CjECs involves RARA or RARB-mediated pathways, and ATRA and Am80 alter the expression of vitamin A pathway gene and suppress keratinization.