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Lucia Sobrin, Zuguo Liu, Dagoberto C. Monroy, Abraham Solomon, Marie G. Selzer, Balakrishna L. Lokeshwar, Stephen C. Pflugfelder; Regulation of MMP-9 Activity in Human Tear Fluid and Corneal Epithelial Culture Supernatant. Invest. Ophthalmol. Vis. Sci. 2000;41(7):1703-1709.
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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.
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