1. From: Development of a Gene Therapy Virus with a Glucocorticoid-Inducible MMP1 for the Treatment of Steroid Glaucoma
Invest. Ophthalmol. Vis. Sci ;51(6): doi: /iovs
Figure Legend:
Enzymatic activity of secreted recombinant MMP1 in HTM cells. Subconfluent primary HTM cells were infected with wild-type or mutant adenovirus vectors (AdhGRE.MMP1 and AdhGRE.mutMMP1) and were treated with 0.1 μM DEX at t = 0. Media were concentrated 40×. (A) Ten microliters of media, collected 5 days after wild-type infection, were incubated with 1 mM APMA for 3 hours to cleave pro-MMP1 enzyme-inhibitor complex (51 kDa) and release the active MMP1 (41 kDa). Commercial purified pro-MMP1 protein was used as positive control. Western blot analysis was probed with an anti-MMP1 antibody that detected both the latent and the active form. (B) Five microliters of serum-free media, collected 7 days after infection, untreated and treated with DEX, were activated and incubated with rat tail native collagen type I (10 μg) for 2 hours. Samples were run on a 4% to 15% PAGE gel and were stained with Coomassie blue. (C) Schematic representation of the FRET assay: a MMP substrate peptide labeled with a 5-FAM (fluorophore) and QXL52 (quencher) releases the fluorophore after cleavage of the peptide by MMP1. Fluorescence is read using 490/520 nm EX/EM filters. (D) Ten microliters of serum-containing media, collected 5 days after infection, untreated or treated with DEX, were activated and incubated with the FRET peptide for 40 minutes at 37°C (n = 3 independent experiments). MMP1 activity was expressed in relative fluorescence units of the sample with higher activity. Although the MMP1 produced by the mutant was inactive, the MMP1 produced by the wild-type adenovirus was activated by APMA, degraded native collagen type I, and cleaved the FRET peptide with high efficiency. *P = 1 × 10−9.
Date of download: 12/25/2017
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