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

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/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.

Figure 1

Comparison of alkaline phosphatase activity in the trabecular meshwork of anterior segments of normal and glaucomatous eyes. Top: information on the age, sex, race, and disease state of the nine donors used in this study (normal, n = 4; glaucoma, n = 5). Glaucomatous eyes used in matches 4 and 5 were matched against the normal donor. Bottom: anterior segments were perfused at constant flow between 1 to 6 days. The averaged perfused pressure of the eyes at 24 hours baseline was 19.6 ± 7.1 mm Hg. Extracts of dissected trabecular meshwork tissues were divided into aliquots and evaluated for endogenous ALP and DNA content. ALP values were normalized to total genomic DNA. Normalized ALP of normal and glaucomatous samples were averaged (mean ± SEM) and analyzed by the Student's t-test. *P ≤ 0.04. ALP activity was significantly elevated in glaucomatous trabecular meshworks.

Figure 2

Effect of DEX treatment on calcification of primary HTM cells. Top, middle: HTM-63 cells in passage 4 were grown to confluence, treated with 0.1 μM DEX every other day for 7 days, and stained with 2% alizarin red. DEX-treated cells showed show positive red/orange staining for calcification. Top: general view with standard digital camera. Middle: Brightfield images obtained with a microscope (CK40; Olympus) equipped with a CCD DP70 digital camera (original magnification, ×200). Left: stained-untreated cells. Middle: unstained-treated cells. Right: stained-treated cells showing positive nodules. Bottom: parallel untreated (n = 3) and DEX-treated HTM-63 cells were harvested at 3, 5, and 7 days after treatment (n = 2 each). Cell extracts were divided into aliquots and evaluated for endogenous ALP and DNA content. ALP values were normalized to cell number, represented by total genomic DNA (mean ± SEM). *P ≤ 0.0004 (day 5). *P ≤ 0.001 (day 7). Experiments were repeated in HTM-70 and gave similar results. HTM cells treated with DEX form calcification nodules and show time-dependent increases of ALP activity in the primary HTM cells.

Figure 3

TGFβ2 effect on the alkaline phosphatase activity of primary HTM cells. Confluent HTM-63 cells in passage 5 were treated with 1 ng/mL and 3 ng/mL TGFβ2 for 3 days in serum-free medium. Extracts from harvested cells were divided into aliquots and evaluated for endogenous ALP and genomic DNA content. ALP levels were normalized to cell number, represented by total genomic DNA (mean ± SEM). *P ≤ 0.0002 (1 ng/mL, n = 3). *P ≤ 0.001 (3 ng/mL, n = 3). TGFβ2 significantly induces ALP in a dose-dependent manner in the primary HTM cells.

Figure 4

Expression of MGP and γ-carboxylase enzyme (γ-GGCX) in the trabecular meshwork tissue from normal and glaucoma donors. Top: information on the age, sex, race, and disease state of the eight donors used in this study (normal, n = 4; glaucoma, n = 4). Glaucomatous eye used in match 9 was from the same person used in match 5 in Figure 1. Anterior segments were perfused at constant flow between 1 to 5 days. Average perfused pressure of the eyes at 24 hours baseline was 15.9 ± 3.8 mm Hg. Trabecular meshwork tissues were dissected from frozen or RNA-later perfused anterior segments, or both, and were processed for RNA, RT, and exponential amplification with MGP, γ-GGCX, or 18S TaqMan PCR probes, as described in Methods. Normalization was achieved with the 18S TaqMan probe hybridized to 10⁻⁴ diluted RTs. Left middle, left lower: individual and average (mean ± SEM) fold changes of MGP and γ-GGCX in POAG versus normal specimens, normalized to 18S cDNA. After normalization, normal samples are given a value of 1.0. Right: representative C_T logarithmic curves of the real-time TaqMan hybridizations of MGP, γ-GGCX, and 18S to cDNAs from POAG and normal. *P ≤ 0.006 (n = 9). MGP and its activating enzyme γ-GGCX are downregulated in intact trabecular meshworks obtained after death from donors with glaucoma.

Figure 5

Expression of MGP and γ-carboxylase enzyme (γ-GGCX) in primary trabecular meshwork cells treated with TGFβ2. Primary HTM cells, passage 6, were treated with 1 ng or 3 ng/mL TGFβ2 for 3 days in serum-free medium. Controls cells were left untreated. Wells were processed for RNA, RT, and exponential amplification with MGP, γ-GGCX, or 18S TaqMan PCR probes, as described in Methods. Normalization was achieved with the 18S TaqMan probe hybridized to 10⁻⁴ diluted RTs. Untreated samples were normalized to a value of 1.0, and fold change values are expressed as the mean ± SEM. Left: fold changes of MGP (top) and γ-GGCX (bottom) cDNAs in TGFβ2-treated versus untreated samples were normalized to 18S cDNA. Right: representative C_T logarithmic curves of the real-time TaqMan hybridizations of MGP, γ-GGCX, and 18S to cDNAs from TGFβ2-treated and untreated cells. *P ≤ 0.006 (MGP; 1 ng/mL; n = 3). *P ≤ 0.0001 (MGP; 3 ng/mL; n = 3). *P ≤ 0.004 (γ-GGCX; 1 ng/mL; n = 3). *P ≤ 0.003 (γ-GGCX; 3 ng/mL; n= 3). TGFβ2 downregulates MGP and its activating enzyme γ-carboxylase in human trabecular meshwork cells.

Figure 6

Effect of silencing the MGP gene in human trabecular meshwork cells. Primary human trabecular meshwork cells were transfected by nucleofector electroporation with either MGP siRNA (40 nM and 80 nM final concentrations) or scrambled negative control siRNA (40 nM final). Forty-eight hours after transfection, cells were harvested and homogenized, and aliquots were taken for total RNA, endogenous ALP, and genomic DNA extraction and determination. RNA was reverse transcribed, analyzed, and normalized for MGP expression by real-time TaqMan PCR using probes, as described in Methods. Scrambled negative control siRNA samples are given a value of 100. Top: percentage of remaining transcripts in cells treated with MGP siRNA compared with those treated with the scrambled negative control siRNA. Bottom: normalized endogenous ALP values in MGP and scrambled negative control siRNA-treated samples (n = 3). *P ≤ 0.0003. MGP siRNA silences the MGP gene efficiently in the human trabecular cells. Silencing MGP results in increased endogenous ALP activity.

Figure 7

Schematic illustration depicting the transition of vascular precursor cells to osteoblasts, together with the hypothesis presented here of trabecular meshwork cells undergoing similar osteogenic differentiation.