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W Goossens, R Wertz, AM Miller, J Choi, RR Allingham, PA Knepper; MMP-9 in Aqueous of Normal and Primary Open-Angle Glaucoma . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1056.
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Purpose: The extracellular matrix of the trabecular meshwork is stabilized by multiple interactions and by remodeling. Matrix turnover is crucial for repair of the trabecular meshwork; matrix metalloproteinases (MMP's) are key enzymes involved in matrix degradation. Methods: Aqueous humor of normal and primary open-angle glaucoma (POAG) patients was assayed for latent and active MMP-9 by gelatin B zymography and western analysis. Latent MMP-9 was identified by p-aminophenylmercuric acetate (APMA) activation. Aqueous humor CD44 was co-immunoprecipitated by incubation with anti-CD44 antibody for 15 hours at 4ºC, followed by incubation with a secondary antibody conjugated to agarose for 90 minutes at 23ºC. After centrifugation and elution of the CD44-bound material from the agarose using glycine-HCl pH 2.5, western analysis was performed using anti-CD44 antibody (BU52 [Binding Site]), and anti-MMP-9 antibody (MAB 936 [R&D]). The immunoprecipitate was then subjected to gelatin zymography to detect MMP activity. Results: Western analysis of the precipitate indicated several distinct CD44 isoforms coprecipitate with MMP-9. Zymography studies revealed that normal and POAG express active MMP-9. Some CD44 isoforms are closely associated with the active form of MMP-9 whereas other CD44 are associated with inactive forms of MMP-9. A latent MMP-9 was activated by APMA in POAG. Conclusion: The results indicated that certain CD44 isoforms serve to anchor active MMP-9 and other CD44 isoforms may associate with inactive/latent MMP-9. MMP-9 is regulated in part, by their activation from a latent state and through the influence of endogenous tissue inhibitors of metalloproteinases.
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