Abstract
Purpose: :
Mustard vesicants are blister-causing agents that induce epithelial-stromal separation. We have found that vesicant injury increases ADAM17 activity. ADAM17 (aka TNFα converting enzyme, or TACE) is induced upon injury, and clips collagen XVII, a transmembranous component of the anchoring complex that rivets the epithelium to the stroma. The cleavage of collagen XVII allows epithelial migration for wound closure. Our goal was to determine whether rabbit corneal organ cultures treated with vesicants would show faster epithelial-stromal junction recovery if an ADAM-17/TACE inhibitor were applied 2 hr after exposure.
Methods: :
Corneas were dissected from rabbit eyes (PelFreez) and placed in air lifted organ cultures. Medium was added up to the corneal-scleral junction. 200 nmoles of half mustard (aka CEES) or 100 nmoles of nitrogen mustard were dripped onto central corneas. After 2 hr, contaminated medium was replaced with either fresh medium or with medium plus an ADAM-17/TACE inhibitor, applied dropwise to the central cornea. Medium plus or minus inhibitor was applied a total of 4 times over the course of 22 hours. Corneas were then analyzed by light and immunofluorescence microscopy and by western blotting.
Results: :
Sections of unexposed corneas showed a continuous line of immunofluorescent signal at the basement membrane zone with a collagen XVII antibody. 24 hr after vesicant exposures, the antibody showed few reactive areas, indicating cleavage of collagen XVII. H&E staining of CEES- and nitrogen mustard-exposed corneas confirmed epithelial-stromal separations. In contrast, corneas treated for 22 hr with medium plus an ADAM17/TACE inhibitor, delivered starting at 2 hr post exposure, showed few separations. In these sections the collagen XVII antibody pattern was similar to that of controls, being a mostly continuous line of fluorescence at the basement membrane zone.
Conclusions: :
Vesicant exposure leads to activation of ADAM17/TACE, which cleaves collagen XVII at the epithelial-stromal junction, and causes formation of microbullae. The application of ADAM17/TACE inhibitors 2 hr after exposure attenuates detachment of the epithelium from the stroma.
Keywords: wound healing • cornea: epithelium • ocular irritants