Evidence for a calcification process in the trabecular meshwork

Abstract

The human trabecular meshwork (TM) expresses many genes that have been associated with physiological (bone, cartilage, teeth) and pathological (vascular systems, kidney) calcification. In particular, the TM highly expresses the inhibitor of calcification Matrix Gla (MGP) gene, which encodes a vitamin K-dependent protein that requires post-translational activation to inhibit the formation of calcium precipitates. TM cells have high activity of the activating gamma-carboxylase enzyme and produce active MGP. Silencing MGP increases the activity of alkaline phosphatase (ALP), an enzyme of the matrix vesicles and marker of calcification. Overexpressing MGP reduces the ALP activity induced by bone morphogenetic 2 (BMP2), a potent inducer of calcification. In this review we gathered evidence for the existence of a mineralization process in the TM. We selected twenty regulatory calcification genes, reviewed their functions in their original tissues and looked at their relative abundance in the TM by heat maps derived from existing microarrays. Although results are not yet fully conclusive and more experiments are needed, examining TM expression in the light of the calcification literature brings up many similarities. One such parallel is the role of mechanical forces in bone induction and the high levels of mineralization inhibitors found in the constantly mechanically stressed TM. During the next few years, examination of other calcification-related regulatory genes and pathways, as well as morphological examination of knockout animals, would help to elucidate the relevance of a calcification process to TM's overall function.

Figure 1. Heat map of twenty calcification regulatory genes in the human TM

Human U133Plus2.0 Affymetrix Genechips analyzed with the GeneSpring software package. Expression signals in each chip are compared to a median value. Change from the median is visually represented by a color assignment (scale at right). The custom calcification gene list was generated from the Gene Ontology lists on Gene Spring. A: human TMs from perfused post-mortem eyes used as control in several experiments. Ages of donors were between 62–92 years old (all Caucasians and all but one males). Perfusion was conducted at constant flow for 3 to 6 days under standard, serum free conditions. Chips 112 – 113 were untreated; chips 116–129 were treated with a control recombinant adenovirus carrying no gene (AdNull). B: primary HTM cells from control cultures. Primary cells were all from the same line (donor 43 year old Caucasian male), at passage 4 and grown on 10% FBS. Each chip’s RNA originated from an independent experiment. Chips 13–15 were untreated; chips 17–23 were treated with a control recombinant adenovirus carrying no gene (AdNull).

Figure 2. Evidence of calcification in aged HTM cells in cultures

Measurements of normalized calcium content (top) and alkaline phosphatase activity (middle) in young (1 week in culture) and old (4 weeks in culture) primary HTM cells at passage 6. Bottom panel shows alizarin red staining of calcification nodules on cells aged in culture for 12 weeks (Xue et al., 2006).

Figure 3. Evidence of calcification in HTM cells treated with Dexamethasone (DEX)

Measurements of normalized calcium content (top, 10 days post-treatment) and alkaline phosphatase activity (middle) in untreated and DEX-treated (0.1µM) primary HTM cells. Bottom panel shows alizarin red staining of calcification nodules on DEX-treated cells for 4 days. (Xue et al., 2007 and unpublished).