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Royce Mohan, John Wizeman; Expression and Inhibition of Peptidyl arginine Deiminase (PAD) Enzymes in Eye Injury. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1208.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the expression of peptidyl arginine deiminase (PAD) enzymes in the injured retina after alkali injury to the cornea.
The corneal alkali injury model was employed using equal numbers of 129SvEV male and female mice as described (Mol Vis. 2016 22:1137). Injured mice recovered for different periods of time and RNA and protein extracted from retina of noninjured and injured cohorts. Quantitative PCR was performed for GAPDH, PAD2, PAD3 and PAD4 transcripts. Tissue sections were subjected to immunohistochemistry (IHC) and protein analyzed by western blot (WB) using antibodies against GFAP, PAD2, PAD4, β-III Tubulin, GAPDH and F95 antibody for citrullinated proteins. In other experiments, mice were injured in vivo and the enucleated eye was subjected to intravitreal injection with PAD4 inhibitor streptonigrin. Mouse eyes were maintained in buffered saline for 5 h at 37 oC in 5% CO2 and retinal extracts subjected to WB. Statistical differences were identified by two-tailed t-test.
PAD2 transcripts detected in the uninjured retina decreased by 7 days post-injury. PAD3 transcript was absent in the retina at all stages. PAD4 transcript was detected in both the injured and uninjured retina, showing a 4-fold increase 1 h post-injury and greater than 24-fold by 7 days. By WBs, PAD4 protein showed increased expression (Unj =1.91 ± 0.38; 7d injury =3.59 ± 0.42; P<0.05). PAD4 immunoreactivity by IHC was not detected in the uninjured retina, but at 7 days post-injury, PAD4 expression was observed throughout the layers of the inner retina. In injured retina, PAD4 expression aligned with GFAP expression in Müller glial cells and exhibited a filament like expression pattern, with PAD4-positive processes beginning at the vitreous. GFAP expression was restricted to the ganglion cell layer astrocytes in the uninjured eye, but extended to the outer nuclear layer in the injured retina. In the injured enucleated eye model, 25 nM streptonigrin inhibited citrullination as detected by the F95 antibody, as well as, reduced GFAP expression by WB (Veh =2.78 ± 0.57; streptonigrin =1.56 ± 0.12; P<0.05).
PAD4 expression increases in a timely manner after retinal injury and its expression localizes to Müller glial cells where GFAP is a major target of citrullination. PAD4 may be a likely target for attenuation of glial reactivity, especially where chronic gliosis can lead to scar formation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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