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

Glaucoma is a complex ocular disease that results in irreversible blindness. It is the second leading cause of blindness worldwide and, in the United States, exhibits a higher prevalence among African Americans and probably also among Hispanic Americans.1,2 It is widely accepted that failure to regulate elevated intraocular pressure (IOP) is the major risk factor for the development of glaucoma.3 Similarly, it is well established that disruption of trabecular meshwork function leads to increased resistance to the aqueous humor outflow and to the generation of elevated IOP. Aqueous humor flows out of the eye through the trabecular meshwork, a soft spongiform tissue located in the anterior segment at the corner of the iris and cornea. To maintain physiological aqueous humor resistance, the trabecular meshwork uses a variety of cellular functions and mechanisms. Functions such as secretion, phagocytosis, cytoskeletal reorganization, cell-matrix adhesion, ion channel transport, signal transduction, and proteosome involvement have all been shown to have a role in outflow facility. Among them, extra-cellular matrix (ECM) deposition as well as its quality4 has been shown to have a strong correlation with increased resistance and glaucoma.5 Identification of genes expressed in the human trabecular meshwork under different conditions has revealed the gene encoding the inhibitor of calcification Matrix Gla (MGP) as the third most abundant expressed gene in adult trabecular meshwork tissue6,7 and among the 20 highest genes in infant-cultured cells.8 In addition, we have shown that MGP expression is regulated by elevated IOP in a model of perfused human anterior segments from postmortem donors using contralateral eyes as controls for identical genetic backgrounds.9 In the same study, MGP was downregulated in primary human trabecular meshwork (HTM) cultures by transforming growth factor-β1 (TGFβ1), a growth factor found elevated in patients with glaucoma.9 MGP is an 84-amino acid protein that contains five γ-carboxyglutamic acid (Gla) residues. The Gla residues are produced by γ-carboxylation of glutamic acid residues by γ-carboxylase, a vitamin K–dependent enzyme. The only known function of Gla residues is to bind calcium ions.10 Matrix Gla was originally isolated from demineralized bovine bone matrix, but it is also known to be expressed in cartilage, vascular smooth muscle cells (VSMCs), and kidney.10 MGP plays a key role in the formation of atherosclerotic plaques and vascular calcification.11 Protection of soft tissue calcification is thought to be partly attributed to the complexes of calcium to inhibitors of calcification such as MGP.10 A number of studies have drawn parallels between VSMCs and trabecular meshwork cells and between their corresponding pathologic conditions of atherosclerosis and glaucoma. For example, one of the VSMC markers is smooth muscle actin, whose promoter directs specific expression to these cells in the arterial wall.12 Smooth muscle actin is also a well-characterized protein in the trabecular meshwork,13 where it was shown to be responsible for the contractile properties of tissue.14 Similarly, the first reported glaucoma marker, endothelial leukocyte adhesion molecule-1 (ELAM-1),15 happens to be the earliest marker for the atherosclerotic plaque. Other genes that are abundant in the trabecular meshwork, such as cartilage Gp-39,6 are also known to be strongly induced during atherosclerosis.6 Previous studies from our laboratory have shown that, in addition to very high MGP expression, cells of the trabecular meshwork exert very high γ-carboxylase activity, and their MGP protein is indeed γ-carboxylated.16 We also showed that overexpression of MGP reverses bone morphogenetic protein 2 (BMP2)–induced calcification in HTM cells.16 These findings indicate that MGP has the ability to function in the trabecular meshwork as a calcification inhibitor. In this study, we continued our investigation on the potential calcification of the trabecular meshwork tissue under conditions associated with glaucoma. As a marker of calcification, we used the presence and upregulation of the activity of the enzyme alkaline phosphatase (ALP), a well-established marker of osteogenic differentiation17-23 and vascular calcification.21,23,24 ALP is an enzyme that hydrolyzes a great variety of substrates that lead to increases of Pi and is activated when a pluripotent, undifferentiated mesenchymal cell becomes committed to differentiate into bone-forming cells (osteoblasts).18 ALP further allows inactivation of mineralization inhibitors18 and has been detected in atherosclerotic plaques.23 For the source of tissue, we obtained intact trabecular meshworks from matched donors with and without glaucoma. To revive and stabilize postmortem eyes, we perfused them on arrival by an organ culture system that mimics physiological aqueous humor flow through an intact trabecular meshwork. For the source of conditioned cells, we treated primary HTM cells with two common glaucoma-associated molecules, dexamethasone (DEX) and TGFβ2. Elevated IOP develops in 40% of patients treated with DEX,25 and increased concentrations of TGFβ2 in the aqueous humor have undoubtedly been associated with the pathogenesis of glaucoma.26,27 We observed an increase in ALP activity on all glaucomatous conditions that was accompanied by a decrease in the expression of MGP. Silencing the MGP encoding gene by siRNA in normal HTM cells also led to an increase in ALP activity. We believe these results support the hypothesis that protection against mineralization of the trabecular meshwork by MGP may be a relevant factor in the maintenance of the soft nature of the tissue and thereby may affect resistance to aqueous humor outflow. Most important, our observations revealed that increased mineralization of the trabecular meshwork is concomitant with the presence of glaucomatous conditions and may be a contributing factor to the disease.