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Updated: Aug 13, 2026
Ex Vivo Corneal Organ Culture Model for Wound Healing Studies
Published on: February 15, 2019
Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant
1Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Florida, USA.
Purpose:
To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens.
Methods:
Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system.
Results:
MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants.
Conclusions:
MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.
Insights
Matrix metalloproteinase (MMP)-9 and TIMP-1 are elevated in ocular rosacea tear fluid. MMP-3 activates MMP-9 on the ocular surface, and doxycycline reduces MMP-9 activity in corneal epithelial cultures.
Area of Science:
- Ophthalmology
- Biochemistry
- Cell Biology
Background:
- Matrix metalloproteinases (MMPs) play crucial roles in tissue remodeling and inflammation.
- MMP-9 and its regulators are implicated in various ocular surface diseases.
Purpose of the Study:
- To investigate activators and inhibitors of MMP-9, MMP-3, and TIMP-1 in human corneal epithelial culture supernatant and tear fluid.
- To determine the effect of MMP-3 on MMP-9 activation in these specimens.
Main Methods:
- Collected tear fluid from ocular rosacea patients and controls.
- Quantified MMP-9, MMP-3, and TIMP-1 using ELISA and immunoblotting.
- Assessed MMP-9 activation by MMP-3 in tear fluid and epithelial cell supernatants, with and without doxycycline, using gelatin zymography.
Main Results:
- Significantly higher concentrations of MMP-9 and TIMP-1 were found in rosacea tear fluid compared to controls.
- MMP-3 was exclusively detected in tear fluid of ocular rosacea patients with corneal disease.
- MMP-3 induced MMP-9 activation, while doxycycline reduced MMP-9 concentration and activity in corneal epithelial cultures.
Conclusions:
- Human corneal epithelium produces MMP-9 and TIMP-1, present in tear fluid.
- MMP-3 is sufficient to activate MMP-9 on the ocular surface.
- Doxycycline does not directly inhibit MMP-3-mediated MMP-9 activation but reduces MMP-9 activity in corneal epithelial cultures.
