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T. Borras, W. Xue, N. Comes; The Calcification Marker Alkaline Phosphatase (ALP) Is Increased in the Trabecular Meshwork (TM) of Glaucoma Donors and Cells Treated With Glaucomatous Insults. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2055.
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The third most abundantly expressed gene in the humanTM tissue is the inhibitor of calcification Matrix Gla (MGP). The enzyme ALP is a well-established marker of osteogenic differentiation, also activated during pathological vascular calcification and present in atherosclerotic plaques. In this study we sought to determine the presence of calcification markers in the TM tissue from glaucoma donors and in primary TM cells insulted by the glaucomatous associated factors dexamethasone (DEX) and transforming growth factor ß2 (TGFß2). We also investigate the effect of silencing MGP in the TM cells.
Anterior segments from post-mortem human eyes from 8 glaucoma and 9 age/race/gender matched normal individuals were perfused at 3 µl/min constant flow between 1-6 days. Average pressure at 24 h baseline was 18.2 ± 6.2 mmHg. Primary HTM cells were generated from surgical residual corneal rims. HTM cells at passage 4-6 were treated with 0.1 µM DEX for 7 days and with 1-3 ng of TGFß2 for 3 days in serum-free medium. Dissected TM tissues and harvested cells were processed to assay ALP activity, genomic DNA (Hoechst) and RNA extraction. Transfection of MGP siRNA was accomplished by nucleofector electroporation. MGP, γ-carboxylase and 18S cDNAs were quantified by TaqMan real-time PCR.
The normalized ALP levels of TM specimens from normal donors was 7.3 ± 1.6 ALP/µg DNA (n=4) while that of the TMs from glaucoma donors was 37.0 ± 10.7 ALP/µg DNA (n=5) (p≤0.04). DEX and TGFß2 induced significant upregulation of ALP activity in two HTM cell lines. Expression of genes encoding MGP and its activating enzyme γ-carboxylase were reduced in the glaucoma tissue -4.4 ± 1.7 and -20.5 fold respectively. Silencing MGP by siRNA resulted in a 197% increase in ALP activity.
The increased activity the calcification marker in glaucoma TMs might be indicative of an undergoing mineralization process during the development of the disease. The inhibition of calcification mechanism represented by the presence of active MGP appears to be compromised in glaucoma tissues.
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