Matrix metalloproteinase 2 activation in cultured corneas
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Published on: August 20, 2016
V A Smith1, A El-Rakhawy, D L Easty
1Division of Ophthalmology, Bristol Eye Hospital, University of Bristol, UK.
Purpose:
Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane.
Methods:
Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [(3)H]type I collagen.
Results:
The stromal tissue of fresh, normal corneas produced inactive MMP-2 of M(r) 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of M(r) 62,000 and M(r) 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [(3)H]type I collagen correlated with the appearance of the M(r) 43,000 activity but not the M(r) 62,000 activity.
Conclusion:
Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.
Insights
Activated matrix metalloproteinase-2 (MMP-2) is produced in cultured corneas, potentially damaging the epithelial basement membrane. Corneal grafts stored longer than 4 weeks may have impaired healing due to this MMP-2 activation.
Area of Science:
- Ophthalmology
- Biochemistry
- Tissue Engineering
Background:
- Corneal epithelial cell shedding and poor repopulation after transplantation can result from damage to the epithelial basement membrane.
- Matrix metalloproteinase-2 (MMP-2), produced by corneal keratocytes, is investigated for its role in this damage.
Purpose of the Study:
- To determine if MMP-2 activation occurs during corneal storage in culture medium.
- To investigate if activated MMP-2 cleaves type IV collagen in the epithelial basement membrane.
Main Methods:
- Analysis of fresh and rejected corneas stored for various durations.
- Soluble protein extraction and MMP-2 visualization via zymography.
- Assay of MMP-2 activity against collagen and other substrates.
Main Results:
- Fresh corneas contained inactive MMP-2 (66,000 M(r)).
- Cultured corneas showed additional active MMP-2 forms (62,000 and 43,000 M(r)) that correlated with storage time.
- The 43,000 M(r) MMP-2 form correlated with collagen cleavage activity.
Conclusions:
- Activated MMP-2 is generated in corneas during culture.
- Corneal storage in culture medium should not exceed 4 weeks to preserve graft integrity.
