May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Lectin- and Cytokine-induced in vitro Activation of Gelatinase A by Trabecular Cells is Associated with Increased Expression of Membrane Type-1 MMP
Author Affiliations & Notes
  • B.A. Pfeffer
    Bausch & Lomb, Rochester, NY, United States
  • S.P. Bartels
    Bausch & Lomb, Rochester, NY, United States
  • Footnotes
    Commercial Relationships  B.A. Pfeffer, Bausch and Lomb E, P; S.P. Bartels, Bausch and Lomb E, P.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4234. doi:
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      B.A. Pfeffer, S.P. Bartels; Lectin- and Cytokine-induced in vitro Activation of Gelatinase A by Trabecular Cells is Associated with Increased Expression of Membrane Type-1 MMP . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4234.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: To demonstrate activation of latent Gelatinase A ("GelA"; MMP-2) and to assess quantitatively the expression of MT1-MMP by human and monkey trabecular meshwork (TM) in cell and organ culture, following incubation with Concanavalin A or interleukin-1α. Resistance to aqueous outflow has been attributed to extracellular matrix (ECM) components in the TM, and an abnormal accumulation of ECM may underlie the pathology of glaucoma. Since MMPs play a role in the degradation and remodeling of ECM, a sensitive, specific, and reproducible assay for TM cell-associated MMP activity was developed. Methods: Cell cultures of macaque TM, enriched in the juxtacanalicular meshwork component, as well as anterior segment organ culture-derived human TM explants, were utilized. Routine culture medium contained 1% calf serum, and 48 hr incubations with Con A (10 µg/ml), or IL-1α (0.1 or 1 ng/ml), were performed in defined serum-free medium. Substrate-gel zymography was used to detect expression of both active and latent GelA. After exposure to lectin or cytokine, TM monolayers were incubated with a thiopeptolide substrate preferentially cleaved by gelatinases. The resulting proteolytic product of membrane-associated MMP activity reacted with a thiol reagent to yield increased absorbance at 410 nm. The colorimetric assay also was performed using TM strips, dissected from organ cultures exposed to the active agents. Results: In the thiopeptolide assay, 10 µg/ml Con A elicited a significant increase in MT-MMP activity for cultured TM of 402% (n=5) over constitutive (control) levels; for IL-1α the increase was 395% and 510% for 0.1 ng/ml and 1 ng/ml, respectively (n=6). Exogenous proGelA post-incubated with stimulated TM also evinced activation by means of this assay. Zymograms of conditioned medium from the treated cultures demonstrated the appearance of endogenously activated GelA, in contrast to control cultures, which produced only the proenzyme. A specific increase in MT1-MMP was documented by Western blot. Results obtained with inhibitors of transcription, translation, and proteases, as well as with cells permeabilized by digitonin treatment, further confirmed the specificity of the assay. MT-MMP activity detected in the human TM strips showed a dose-response to Con A, with a maximum at 100 µg/ml (199% over control). Conclusions: TM can upregulate its expression of MT1-MMP, the physiological activator of GelA, in response to stimulatory agents. Our assay could be used to screen antiglaucoma compounds, whose potential mode of action would be to increase breakdown of ECM in the TM.

Keywords: outflow: trabecular meshwork • enzymes/enzyme inhibitors • extracellular matrix 

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