Effect of excess synthesis of extracellular matrix components by trabecular meshwork cells: Possible consequence on aqueous outflow

Abstract

The extracellular matrix (ECM) of the trabecular meshwork (TM) is an important determinant of its functional properties. This study was performed to investigate whether overexpression of ECM components, laminin (LM) and collagen type IV (Col) by TM cells may play a role in the development of outflow resistance. To determine the effect of excess LM and Col expression on cell monolayer permeability, an in vitro cell culture model was used in which overexpression of the two ECM components, LM and Col, was induced by high glucose (HG) (30 mM) or 0.1 μM dexamethasone (D) in bovine and human trabecular meshwork (BTM and HTM) cells. Western blot analysis and immunofluorescence staining confirmed increased LM and Col synthesis in cells exposed to HG or D. Increased level of LM and Col protein resulted in reduced cell monolayer permeability. Transfection with antisense oligos (AS-oligos) targeted against LM or Col inhibited HG- or D-induced LM and Col gene overexpression in TM cells with concomitant increase in permeability. The AS-oligo strategy was effective in reducing LM or Col level in the TM cells in all conditions tested in this study. These findings suggest that increased LM and Col deposition in the outflow pathway may cause resistance to aqueous outflow and contribute to the development of primary open angle glaucoma (POAG).

Introduction

Elevated intraocular pressure (IOP), which is closely associated with the development of POAG, is maintained through a delicate balance between aqueous humor production and its drainage (Rauz et al., 2003, Marquis and Whitson, 2005). Aqueous humor is continuously produced in the ciliary body and circulated through the anterior chamber. After percolating through the TM, the aqueous humor enters the Schlemm's canal in the chamber angle and drains out through intrascleral and episcleral venous plexuses. Studies suggest that increased production of ECM components in the chamber angle by TM cells may be partly responsible for the development of increased IOP (Lutjen-Drecoll et al., 1989, Tripathi et al., 1994, Lutjen-Drecoll, 1999). Recent study indicates that increased fibronectin (FN) synthesis could result in concomitant increase in IOP (Fleenor et al., 2006). Qualitative analysis of a series of TM samples of trabeculectomy specimens from glaucomatous eyes showed abundance of collagen compared to those in the control samples (Rohen et al., 1993). In steroid-induced glaucoma, ECM component synthesis is also increased and is associated with elevated IOP. Overall, the build up of excess ECM in the TM is believed to impart resistance to aqueous outflow and result in elevated IOP.

The TM is a specialized tissue composed of various ECM components including LM and Col (Murphy et al., 1987, Dickerson et al., 1998, Sato and Roy, 2002, Ueda and Yue, 2003, Filla et al., 2004). These proteins assemble together in a highly organized manner to provide a cross-linked, integrated, scaffold-like structure (Paulsson, 1992, Abrams et al., 2000, Furuyama and Mochitate, 2000, Antikainen et al., 2002). LM and Col are prominent ECM components in the TM that interacts to form a network and contribute to the meshwork formation. These findings suggest that LM and Col are important components of the TM and that their composition can influence the meshwork ultrastructure as well as influence the meshwork's function including outflow facility.

Our previous study investigating the role of another ECM component, FN, suggested that FN upregulation in the TM cells may potentially contribute to reduced aqueous outflow (Li et al., 2004). Also, since FN interacts with LM and Col, and because FN synthesis is increased in glaucomatous eyes, it is important to determine whether overexpression of LM and Col may affect aqueous outflow. Other studies have suggested that glucocorticoids, such as dexamethasone can induce excess accumulation of ECM proteins in TM tissues and result in resistance to aqueous outflow and contribute to increased IOP (Rohen, 1983, Babizhayev and Brodskaya, 1989, Furuyoshi et al., 1997). The excess ECM accumulation in dexamethasone-treated TM cells mirrors the increased ECM accumulation in the aqueous outflow pathway that has been shown to occur in elderly and glaucomatous eyes (Steely et al., 1992, Rohen et al., 1993, Dickerson et al., 1998, Zhou et al., 1998, Zhou et al., 1998). However, there is no direct evidence showing excess ECM accumulation can contribute to outflow resistance.

The increased association of diabetes with glaucoma is controversial. However, individuals with diabetes are reported to experience higher frequencies of glaucoma and elevated IOP compared to the normal population (Klein et al., 1984, Katz and Sommer, 1988). Because glucose content is significantly higher in the aqueous humor of diabetic individuals (Davies et al., 1984), and because aqueous humor is in constant contact with the TM cells, it is conceivable that these cells can be influenced by the high glucose content in the aqueous humor. High glucose conditions have been reported to increase ECM synthesis in different cell types including endothelial cells and TM cells (Sato and Roy, 2002, Roy et al., 2003, Li et al., 2004, Oshitari et al., 2005, Oshitari et al., 2006). Studies on the effects of dexamethasone, a glucocorticoid, have shown increased ECM gene expression and ECM deposition (Hernandez et al., 1985, Johnson et al., 1990, Steely et al., 1992) as well as increased ocular tension in a third of the dexamethasone-perfused human eye organ cultures (Clark et al., 1995). Similarly an increase in Col expression has been documented in TM cell cultures and bovine eye organ cultures treated with dexamethasone suggesting a role for increased ECM synthesis in the development of outflow resistance (Zhou et al., 1998, Zhou et al., 1998). Increased LM and Col synthesis by TM cells exposed to ascorbic acid, a prominent component in the aqueous humor, was also demonstrated (Zhou et al., 1998, Zhou et al., 1998). However, there are no reports that show reduced aqueous outflow facility may be caused by excess synthesis and accumulation of LM and Col components either in glaucomatous individuals or in diabetic individuals with POAG. In this study, we have examined whether HG- or D-induced increased synthesis of LM and Col plays a role in TM cell monolayer permeability. In particular, we have determined whether the antisense oligo strategy can prevent HG- or D-induced LM and Col overexpression and its possible contribution towards excess permeability in TM cells.