Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Janice A Vranka,Kate E Keller,Ted S Acott,John M Bradley,Yong-Feng Yang,Sanjoy Bhattacharya

doi:10.1371/journal.pone.0122483

Abstract

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm’s canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

Highlights

In the human eye, the majority of aqueous humor fluid exits the anterior chamber via the conventional outflow pathway, which drains aqueous fluid through the filterlike trabecular meshwork (TM) tissue to Schlemm’s canal.[1]
The TM can be divided into 3 separate regions based on location and structure: 1) the outer uveal meshwork, and 2) the deeper corneoscleral meshwork, both regions containing fenestrated beams of lamellae and large, open intertrabecular spaces, and 3) the juxtacanalicular tissue (JCT) that is directly adjacent to the inner wall endothelium of Schlemm’s canal. [2,3,4] It is composed of JCT cells embedded in a loosely arranged extracellular matrix
Frontal sections of tissues were imaged using confocal microscopy to compare their distributions across the various layers of the TM within high and low flow regions (S1 Fig)

Summary

Introduction

The majority of aqueous humor fluid (approximately 90%) exits the anterior chamber via the conventional outflow pathway, which drains aqueous fluid through the filterlike trabecular meshwork (TM) tissue to Schlemm’s canal.[1]. [2,3,4] It is composed of JCT cells embedded in a loosely arranged extracellular matrix. Intraocular pressure (IOP) is generated by building resistance to aqueous humor outflow in the TM.[2,5] Aqueous humor outflow resistance is believed to reside within the 7–14 μm of the inner wall of Schlemm’s canal, which is the approximate thickness of the JCT.[2,5,6,7,8] The extracellular matrix (ECM) of the JCT is thought to be integrally and extensively involved in generating the outflow resistance, since disrupting it by several methods has been shown to affect outflow resistance.[2,9,10,11] For example, perfusion of MMPs or their inhibitors resulted in increased or decreased outflow, respectively[9], whereas over-expression of MMP in a steroid-inducible adenovirus increased outflow in perfused anterior segments.[12] In anterior segment organ culture, outflow facility (C) is defined as the flow rate divided by the perfusion pressure, and is inversely proportional to outflow resistance. Glycosaminoglycans (GAGs) and proteoglycans were implicated as integral molecular components of the resistance by perfusion of GAG-degrading enzymes, which increased outflow facility in animals [13,14,15] Studies using RNAi gene silencing and chemical inhibitors to modify the GAG biosynthesis and structure elicited similar responses on outflow in humans.[3,11,16]

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