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Marion Gordon, Andrea Rodrigues, Rita Hahn, Donald Gerecke, Kathy Svoboda, Ned Heindel; Corneal Wound Healing is Facilitated by Hydroxamates that Reduce EMMPRIN Expression. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3910.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate mechanisms of corneal wound healing, rabbit corneas in air lifted organ cultures were exposed to nitrogen mustard (NM) to determine whether hydroxamate compounds facilitate recovery. NM is a vesicating agent that causes separation at the epithelial-stromal junction, and can lead to sloughing of the epithelium 1-2 days after exposure. Separation of the cell layers involves, in part, cleavage of hemidesmosomal collagen XVII by ADAM17. Hydroxamates inhibit ADAM17 via binding to the enzyme’s catalytic zinc. Our goal was to evaluate two hydroxamates for their ability to inhibit ADAM17 and to explore other mechanisms for how they favor epithelial-stromal integrity after injury.
Rabbit corneas in organ culture were exposed to 100 nmol NM (in 10 ul) for 2 hr. Corneas were then washed, medium was changed, and 3 nmol of each hydroxamate (in 20 ul) was applied to NM-exposed corneas 4 times over the course of 22 hrs. The hydroxamate compounds used as therapies were olvanil OH (NDH4409) and retro olvanil 8 (NDH4417).
Without hydroxamate treatment, ADAM17 was activated in the cornea immediately upon exposure to nitrogen mustard (NM). Activation persisted for a minimum of 24 hr post exposure, correlating with epithelial-stromal separation. With the application of either hydroxamate, epithelial sloughing was reduced at 24 hr post exposure, and the overall histology of the basement membrane zone was greatly improved. Surprisingly, the improvement of histology did not correlate with the ability of the hydroxamates to inhibit ADAM17. NDH4417 was a much better inhibitor than NDH 4409 in activity assays. Since matrix metalloproteinases (MMPs) also contain catalytic Zn ions, both NDH4417 and NDH4409 were tested for their ability to reduce MMP9 levels, and both were found to be very effective. Next, to investigate an upstream step in MMP activation, the expression of the MMP inducer, EMMPRIN, was evaluated. EMMPRIN was found to be upregulated by mustard exposure. Both NDH4409 and NDH4417 treatment after NM exposure reduced EMMPRIN expression levels to values lower than unexposed controls, despite that EMMPRIN is not known to contain a zinc moiety.
The hydroxamates employed not only reduce the activity of ADAM17 and MMPs, but they also lower MMP levels by attenuating expression of the MMP inducer, EMMPRIN. The mechanism behind this latter activity is as yet unknown.
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