Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors.
OBJECTIVE
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/microg DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 +/- 10.7 ng ALP/microg genomic DNA (n = 5; P </= 0.04). DEX and TGFbeta2 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.
Xue
Wei
W
Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7041, USA.
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Núria
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Borrás
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D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000469:Alkaline Phosphatase / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism*; D002114:Calcinosis / Q000201:enzymology*; D002135:Calcium-Binding Proteins / Q000235:genetics; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D018274:Electroporation / Q000379:methods; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005260:Female; D020868:Gene Silencing; D005901:Glaucoma / Q000201:enzymology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000494:pharmacology; D014019:Tissue Donors; D014129:Trabecular Meshwork / Q000187:drug effects / Q000201:enzymology*; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology; D015854:Up-Regulation; D000097568:Matrix Gla Protein
D000469:Alkaline Phosphatase; D015415:Biomarkers; D002135:Calcium-Binding Proteins; D003907:Dexamethasone; D016326:Extracellular Matrix Proteins; D034741:RNA, Small Interfering; D053781:Transforming Growth Factor beta2; D000097568:Matrix Gla Protein