ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure

Abstract

PurposeSulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial–stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial–stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing.MethodsRabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3–100 nmol in 20 μL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays.ResultsNitrogen mustard–induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial–stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial–stromal attachment.ConclusionsMustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial–stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial–stromal separation.

The molecules in the corneal basement membrane zone affected by mustard exposure suggest potential therapies

“…Therefore, inhibitors of ADAM17 should also reduce mustard injury of the cornea by preserving the epithelial–stromal junction. This was also found to be true in NM‐exposed corneal organ cultures treated with ADAM17‐inhibiting hydroxamates …”

“…With the evidence that epithelial–stromal separation was very likely a consequence of mustard activating MMP‐9 and ADAM17, we tested for activity of these enzymes in corneal organ cultures exposed to NM . Corneas were dissected from the eyes retaining ∼2 mm of scleral rim, then laid epithelial‐side down in the curved wells of spot plates.…”

“…We next addressed whether preventing cleavage of the other known hemidesmosomal component, collagen XVII, would help retain the integrity of epithelial–stromal attachment. One of the enzymes known to cleave collagen XVII, ADAM17 (also known as tumor necrosis factor α–converting enzyme), was specifically assessed . As shown in Figure A, NM exposure produced an ADAM17 immunofluorescent signal that was not seen in the unexposed control.…”

“…Hydroxamates are known to inhibit ADAM17; therefore, four hydroxamates were synthesized for evaluation as potential therapies for ocular mustard exposure . Retro olvanil‐8 (i.e., NDH4417) was found to reduce the ADAM17 levels to more than half that in exposed corneas receiving no therapy (Fig.…”

“…This was also found to be true in NM-exposed corneal organ cultures treated with ADAM17-inhibiting hydroxamates. 28 The corneal organ culture system Mice, rabbits, and pigs are among the animals that have been used for mustard studies. Rabbits were our choice of ocular models.…”