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Rachel Genova, Andrew Pieper, Matthew Harper; The role of neutral endopeptidase in ocular surface homeostasis and corneal wound healing. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4376.
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© ARVO (1962-2015); The Authors (2016-present)
Neutral endopeptidase (NEP) modulates inflammation by metabolizing signaling peptides. Recently NEP was identified in the human cornea, a tissue that expresses many of its neuropeptide substrates. However, the role of NEP in homeostatic maintenance and wound healing of the cornea is unknown. Here we investigate the effect of genetic disruption of NEP activity in the murine cornea under naive and injured conditions with the goal of identifying novel management strategies for ocular surface injury.
The corneas of injury-naive WT and NEP-/- mice were imaged with slit-lamp biomicroscopy (n = 5/group) and optical coherence tomography (n = 7-9/group) to evaluate in vivo morphology. Corneas were collected for NEP, βIII-tubulin, and CD31 histology (n = 3-6/group); NEP Western blots (n = 3-4/group), and NEP activity assays (n = 5/group). Another cohort receiving topical 0.5 M NaOH or saline (sham) to induce corneal injury were followed for 7 d with slit-lamp biomicroscopy. Rose bengal instillation aided in visualization and quantification of corneal reepithelialization (n = 12-18/group).
NEP ran at 100 kDa on Western blot and immunolocalized to the superficial epithelium in WT corneas. In NEP-/- corneas, the 100 kDa band was absent, but diffuse cytoplasmic immunostaining was present. However, NEP enzyme activity was greater in WT corneas (4152.43±142.04 RFU) compared to NEP-/- corneas (260.71±46.89 RFU, p<0.0001), which did not differ from background activity. No differences in total corneal thickness, or in the thickness of epithelial or stromal layers individually, were seen with OCT between naive groups. Furthermore, NEP-/- mice showed no evidence of spontaneous corneal neovascularization on clinical exam or with CD31 immunostaining, but had increased corrected total βIII-tubulin immunofluorescence compared to WT mice (3.10±0.13 vs 2.19±0.12 AU, p=0.0006). In our injury model, NEP-/- mice had increased corneal reepithelialization at 3 and 7 d post-injury, compared to WT mice (47.52±5.01 vs 88.23±3.67% rose bengal staining at 3 d, p<0.0001; 27.79±3.16 vs 65.77±5.64% staining at 7 d, p<0.0001).
NEP is functionally expressed in the mouse cornea where its constitutive genetic disruption leads to accelerated reepithelialization following chemical corneal injury. By contrast, loss of NEP activity appears to have minimal effect on homeostatic maintenance of the adult mouse cornea.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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