PURPOSE: Matrix metalloproteinases (MMPs) play a central role in the wound-healing process. The objective of this study was to identify and characterize the levels and molecular forms of human tear fluid collagenase-2 (MMP-8) and membrane type 1-MMP (MT1-MMP or MMP-14) in patients who had undergone excimer laser photorefractive keratectomy (PRK) and in healthy subjects. Whether MT1-MMP activates pro-MMP-8 was also determined. METHODS: Tear fluid samples were collected with scaled and blunted microcapillaries from healthy control subjects and, on the second postoperative day, from patients who had undergone PRK. Time and the volume collected were registered. Molecular forms and levels of pro and active MMP-8 and MT1-MMP in these samples were determined by Western immunoblot analysis, quantitated by computer scanning. The concentration of MMP-8 was also determined by immunofluorescence assay. The conversion of pure human polymorphonuclear neutrophil (PMN) pro-MMP-8 to the active form by the catalytic domain of MT1-MMP was studied by Western immunoblot analysis. RESULTS: The tear fluid flow was increased after PRK. Tear fluid flow-corrected excretion of MMP-8 was significantly higher in PRK-treated patients, as assessed by immunofluorescence assay and quantitative Western immunoblot analysis. The major MMP-8 species detected in tears of both PRK-treated patients and healthy control subjects represented latent and active 75- and 65-kDa highly glycosylated MMP-8 isoforms. The less-glycosylated 45- to 55-kDa MMP-8 isoform was not detectable. Tear fluid flow-corrected secretion of MT1-MMP was significantly higher in PRK-treated patients. Soluble 80-kDa MT1-MMP immunoreactivities were detected in tears of both healthy control subjects and PRK-treated patients, and may represent a complex captured by tissue inhibitor of metalloproteinase (TIMP)-2. Human PMN pro-MMP-8 was converted to the active form by MT1-MMP, and TIMP-2 prevented this activation. CONCLUSIONS: Corneal renewal eventually occurs at a high rate and is affected by the rate of corneal collagen and other matrix protein breakdown. Accordingly, tear fluid MMP-8 and MT1-MMP levels were shown to be constantly high in normal subjects. With PRK, a fast wound-healing process was associated with even higher MMP-8 and MT1-MMP levels and their activation. The results suggest a role for a MMP-8 and MT1-MMP network in the corneal wound-healing cascade. Furthermore, MT1-MMP (MMP-14) seems to activate pro-MMP-8.
PURPOSE: Matrix metalloproteinases (MMPs) play a central role in the wound-healing process. The objective of this study was to identify and characterize the levels and molecular forms of human tear fluid collagenase-2 (MMP-8) and membrane type 1-MMP (MT1-MMP or MMP-14) in patients who had undergone excimer laser photorefractive keratectomy (PRK) and in healthy subjects. Whether MT1-MMP activates pro-MMP-8 was also determined. METHODS: Tear fluid samples were collected with scaled and blunted microcapillaries from healthy control subjects and, on the second postoperative day, from patients who had undergone PRK. Time and the volume collected were registered. Molecular forms and levels of pro and active MMP-8 and MT1-MMP in these samples were determined by Western immunoblot analysis, quantitated by computer scanning. The concentration of MMP-8 was also determined by immunofluorescence assay. The conversion of pure human polymorphonuclear neutrophil (PMN) pro-MMP-8 to the active form by the catalytic domain of MT1-MMP was studied by Western immunoblot analysis. RESULTS: The tear fluid flow was increased after PRK. Tear fluid flow-corrected excretion of MMP-8 was significantly higher in PRK-treated patients, as assessed by immunofluorescence assay and quantitative Western immunoblot analysis. The major MMP-8 species detected in tears of both PRK-treated patients and healthy control subjects represented latent and active 75- and 65-kDa highly glycosylated MMP-8 isoforms. The less-glycosylated 45- to 55-kDa MMP-8 isoform was not detectable. Tear fluid flow-corrected secretion of MT1-MMP was significantly higher in PRK-treated patients. Soluble 80-kDa MT1-MMP immunoreactivities were detected in tears of both healthy control subjects and PRK-treated patients, and may represent a complex captured by tissue inhibitor of metalloproteinase (TIMP)-2. Human PMN pro-MMP-8 was converted to the active form by MT1-MMP, and TIMP-2 prevented this activation. CONCLUSIONS: Corneal renewal eventually occurs at a high rate and is affected by the rate of corneal collagen and other matrix protein breakdown. Accordingly, tear fluid MMP-8 and MT1-MMP levels were shown to be constantly high in normal subjects. With PRK, a fast wound-healing process was associated with even higher MMP-8 and MT1-MMP levels and their activation. The results suggest a role for a MMP-8 and MT1-MMP network in the corneal wound-healing cascade. Furthermore, MT1-MMP (MMP-14) seems to activate pro-MMP-8.
Authors: Gordon W Laurie; Leslie A Olsakovsky; Brian P Conway; Robert L McKown; Kazuko Kitagawa; Jason J Nichols Journal: Optom Vis Sci Date: 2008-08 Impact factor: 1.973
Authors: Carolin Steinl; Mike Essl; Thomas D Schreiber; Konstanze Geiger; Lea Prokop; Stefan Stevanović; Oliver Pötz; Harald Abele; Johannes T Wessels; Wilhelm K Aicher; Gerd Klein Journal: Stem Cells Dev Date: 2013-02-12 Impact factor: 3.272