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(Investigative Ophthalmology and Visual Science. 2007;48:3184-3194.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1403
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Presence of an Established Calcification Marker in Trabecular Meshwork Tissue of Glaucoma Donors

Wei Xue, Núria Comes, and Teresa Borrás

From the Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, North Carolina.

PURPOSE. To determine the presence of calcification markers in the trabecular meshwork tissue from glaucoma donors and in trabecular meshwork cells insulted by dexamethasone (DEX) and transforming growth factor ß2 (TGFß2), factors associated with glaucoma. To investigate as well the effect of silencing the inhibitor of calcification matrix Gla (MGP) in the trabecular meshwork cells.

METHODS. Trabecular meshwork tissue was obtained from perfused postmortem anterior segments of glaucomatous and normal eyes. Primary trabecular meshwork cells were obtained from residual corneal rims after surgical corneal transplantation. Calcification marker alkaline phosphatase (ALP) enzyme activity was assayed by fluorescence produced after substrate cleavage. DNA quantification was evaluated by fluorescence produced after binding to the Hoechst dye. Transfection of siRNA to primary cells was accomplished by nucleofector electroporation with trabecular meshwork–optimized conditions. cDNA quantification was performed with the use of TaqMan real-time PCR.

RESULTS. Human trabecular meshworks from glaucoma donors exhibited significantly higher levels of ALP activity than their matched counterparts with normal eyes. The normalized ALP of the control specimens was 7.3 ± 1.6 ng ALP/µg DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 ± 10.7 ng ALP/µg genomic DNA (n = 5; P ≤ 0.04). DEX and TGFß2 significantly induced the upregulation of ALP activity in two trabecular meshwork primary cell lines. Expression of the gene encoding MGP was reduced in the glaucomatous tissue by –4.4 ± 1.7-fold (n = 9; P ≤ 0.006). Silencing MGP by siRNA resulted in ALP activity that was increased by 197% ± 8.4% (P ≤ 0.0003).

CONCLUSIONS. The increased activity of the calcification marker, ALP, in glaucomatous trabecular meshworks might be indicative of an undergoing mineralization process during development of the disease. Inhibition of the calcification mechanism represented by the presence of active MGP appears to be compromised in glaucomatous tissue.






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Copyright © 2007 by the Association for Research in Vision and Ophthalmology