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Determining MMP‐2 and MMP‐9 reductive activities of bovine and equine amniotic membranes homogenates using fluorescence resonance energy transfer
Author(s) -
Capistrano da Silva Erotides,
Gibson Daniel J.,
Jeong Sunyoung,
Zimmerman Kelli L.,
SmithFleming Kathryn M.,
Martins Bianca da C.
Publication year - 2021
Publication title -
veterinary ophthalmology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.594
H-Index - 50
eISSN - 1463-5224
pISSN - 1463-5216
DOI - 10.1111/vop.12888
Subject(s) - matrix metalloproteinase , in vitro , chemistry , förster resonance energy transfer , membrane , serial dilution , matrix (chemical analysis) , microbiology and biotechnology , biochemistry , fluorescence , chromatography , biology , pathology , medicine , physics , alternative medicine , quantum mechanics
Matrix metalloproteinases (MMPs)‐2 and ‐9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP‐2 and MMP‐9 activity. Objective To determine if bovine and equine AMH reduce in vitro MMP‐2 and MMP‐9 activities associated with the presence of TIMPs. Procedures Undiluted and diluted twofold series (0‐fold to 16‐fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme‐linked immunosorbent assays for TIMPs 1–4 were applied to quantify TIMPs in the samples. Results AMH from both species were able to inhibit MMP‐2 and MMP‐9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP‐2 and MMP‐9 in vitro. TIMPs −2 and −3 were present in EAMH and BAMH. TIMP‐1 was detected only in BAMH, and TIMP‐4 was not detected in any samples. Conclusion Both EAMH and BAMH directly inhibited MMP‐2 and MMP‐9 in vitro without dilution, and BAMH showed better inhibition of MMP‐2 and MMP‐9 before and after dilution compared to EAMH.