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W. Xue, E. Davis, R. Wallin, T. Borrás; Matrix GLA (MGP) Decreased Bone Morphogenetic Protein 2 (BMP2)–Induced Calcification in the Human Trabecular Meshwork (HTM) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1158.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous data from our laboratory indicated the presence of a calcification mechanism in aged HTM cells. Here we investigated the involvement of MGP in HTM calcification. We tested whether overexpression of BMP2 in HTM cells induced calcification and whether this process could be reduced by overexpression and binding of MGP to the BMP2 protein. Methods: MGP mRNA levels were evaluated by RQ–RT–PCR normalized to 18S RNA. Adenoviral vectors carrying full coding human BMP2 or MGP cDNAs driven by CMV promoters were constructed by standard methods (Ad5BMP2 and AdhMGP). HTM primary cells at 70% confluency were infected at passage 3 to 5 with either Ad5BMP2 alone or co–infected with Ad5BMP2 plus AdhMGP at a viral particle ratio of 1:8. Calcification was evaluated on cell extracts, at several post–infection times, by the fluorofotometric assay of a well–established marker of bone calcification (endogenous alkaline phosphatase, ALP), using the AttoPhos AP Fluorescent Substrate System (Promega). ALP values were normalized to total cellular genomic DNA, measured by real time TaqMan PCR with a telomerase reverse transcriptase 5’fluorescence–3’quencher labeled specific probe (Applied Biosystems). Immunocytochemistry was performed on single and co–infected cells using goat anti–rat BMP2 (1:200) and rabbit anti–bovine MGP (1:200) followed by Cy2 conjugated donkey anti–goat (1:200) and Cy3–conjugated donkey anti–rabbit (1:200) antibodies. Results: Normalized MGP mRNA levels were reduced 1.5X ± 0.3 in aged (pasage5, incubated for 2 months) vs. young (fresh passage 4) HTM cells. Infection with 2X108 Ad5BMP2 viral particles increased cell’s normalized ALP levels 2.8X ± 0.02 (day 1), 8.7X ± 0.03 (day 3) and 14.0X ± 0.02 (day 5) over uninfected control cells. Co–infection experiments showed that, at conditions of 3–fold ALP induction by Ad5BMP2, addition of AdhMGP at 1:4 and 1:8 ratios, reduced the normalized ALP values by 51 % ± 5 and 45% ± 1 respectively. Confocal immunocytochemistry showed colocalization in some of the co–infected cells. Conclusions: Overexpression of BMP2 induces calcification in HTM cells. MGP overexpression counteracts the calcification process induced by BMP2. Expression of MGP in the TM could be representative of a mechanism which would protect the TM tissue from hardening as a result of old age and calcification. In turn, preventing TM calcification could contribute to prevent increased resistance and elevated IOP.
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