Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors

Authors:
Xue W, Comes N, Borras T
In:
Source: Invest Ophthalmol Vis Sci
Publication Date: (2007)
Issue: 48(7): 3184-94
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Trabecular meshwork, human
Species: human
Tissue Origin: eye
Platform:
Nucleofectorâ„¢ I/II/2b
Abstract
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 beta2 (TGFbeta2), 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/mug DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 +/- 10.7 ng ALP/mug genomic DNA (n = 5; P