Research article
Pressure-induced expression changes in segmental flow regions of the human trabecular meshwork

https://doi.org/10.1016/j.exer.2016.06.009Get rights and content

Highlights

  • ECM genes are differentially expressed in high flow versus low flow regions.

  • A set of ECM genes is differentially expressed in response to elevated pressure.

  • ECM genes in segmental flow regions have differential responses to pressure.

  • ECM turnover in response to elevated pressure is critical to IOP homeostasis.

  • ECM remodeling in response to elevated pressure is part of IOP homeostasis.

Abstract

Elevated intraocular pressure (IOP) is thought to create distortion or stretching of the juxtacanalicular and Schlemm’s canal cells and their extracellular matrix (ECM) leading to a cascade of events that restore IOP to normal levels, a process termed IOP homeostasis. The ECM of the trabecular meshwork (TM) is intricately involved in the regulation of outflow resistance and IOP homeostasis, as matrix metalloproteinase (MMP)-initiated ECM turnover in the TM is necessary to maintain outflow facility. Previous studies have shown ECM gene expression and mRNA splice form differences in TM cells in response to sustained stretch, implicating their involvement in the dynamic process of IOP homeostasis. The observation that outflow is segmental around the circumference of the eye adds another layer of complexity to understanding the molecular events necessary to maintaining proper outflow facility. The aim of this work was to identify molecular expression differences between segmental flow regions of the TM from anterior segments perfused at either physiological or elevated pressure. Human anterior segments were perfused in an ex vivo model system, TM tissues were extracted and quantitative PCR arrays were performed. Comparisons were made between high flow and low flow regions of the TM from anterior segments perfused either at normal (8.8 mmHg) or at elevated (17.6 mmHg) perfusion pressure for 48 h. The results are presented here as independent sets: 1) fold change gene expression between segmental flow regions at a single perfusion pressure, and 2) fold change gene expression in response to elevated perfusion pressure in a single flow region. Multiple genes from the following functional families were found to be differentially expressed in segmental regions and in response to elevated pressure: collagens, ECM glycoproteins including matricellular proteins, ECM receptors such as integrins and adhesion molecules and ECM regulators, such as matrix metalloproteinases. In general, under normal perfusion pressure, more ECM genes were enriched in the high flow regions than in the low flow regions of the TM, whereas more ECM genes were found to be enriched in low flow regions of the TM in response to elevated perfusion pressure. Thus it appears that a limited subset of ECM genes is differentially regulated in both high and low flow regions and in response to elevated pressure. Some of these same ECM genes have previously been shown to be involved in the pressure response of stretched TM cells supporting their central role in IOP homeostasis. In general, different ECM gene family members are called upon to produce the response to elevated pressure in different segmental regions of the TM.

Introduction

Glaucoma is one of the leading causes of blindness affecting over 67 million people worldwide (Quigley, 1996, Quigley, 2011). Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and is targeted for all current glaucoma therapies. Aqueous humor flows out of the anterior chamber primarily via the conventional outflow pathway through the trabecular meshwork (TM) tissue to Schlemm’s canal (SC) and then into the episcleral venous system (Acott and Kelley, 2008, Acott et al., 2014, Brubaker, 1991). IOP is generated primarily by creating resistance to aqueous humor outflow in the TM (Johnson, 2006, Tamm, 2009). This resistance is believed to reside predominantly within the juxtacanalicular (JCT) region of the TM and the inner wall of Schlemm’s canal (Acott and Kelley, 2008, Inomata et al., 1972, Johnson et al., 1990, Overby et al., 2009).

Section snippets

Segmental outflow

Aqueous humor outflow has been shown to be segmental in nature around the circumference of the eye. Regions of relatively high, intermediate or mixed, and low flow have been demonstrated in many studies using tracers of different composition and size to visualize the outflow patterns (Buller and Johnson, 1994, Chang et al., 2014, de Kater et al., 1989, Ethier and Chan, 2001, Hann et al., 2005, Keller et al., 2011, Vranka et al., 2015). In addition, non-uniform patterns of aqueous outflow have

Extracellular matrix of the TM and IOP homeostasis

The probable primary site of outflow resistance is located within the deepest portion of the JCT and Schlemm’s canal inner wall basement membrane (Acott and Kelley, 2008, Ethier, 2002, Johnson, 2006, Stamer and Acott, 2012). The extracellular matrix (ECM) of the TM is thought to play a significant role in modulating aqueous humor outflow resistance, since modulating or disrupting it has been shown to have a direct effect on outflow resistance (Acott and Kelley, 2008, Bradley et al., 1998,

Molecular components of segmental flow

One of the goals of a newly published study was to correlate patterns of ECM gene expression with high and low flow regions of the TM in human anterior segments perfused at physiological pressure in organ culture (Vranka et al., 2015). Standard physiological flow rates were in the range of 1–9 μl/min when perfused at physiologic pressure of 8.8 mmHg, which is similar to normal physiological rate and pressure (minus episcleral venous pressure) in vivo. A number of ECM and adhesion genes were

Elevated pressure-induced effects in segmental regions of the TM

In the normal physiologic response to elevated pressure the TM undergoes ECM turnover and remodeling to correct the outflow resistance and reduce IOP (Acott et al., 2014). TM cells under mechanical stretch increased MMP14 and MMP2, while TIMP2 was decreased (Bradley et al., 2001). We previously conducted microarray gene expression studies after mechanical stretching of TM cells and identified many ECM genes that exhibited increased or decreased mRNA levels in response to stretch at varied times

Discussion

The observation of aqueous humor outflow segmentation has dramatic implications on the resistance adjustments that occur during IOP homeostasis under normal conditions, as well as during sustained pressure increases in the eye. The ECM of the JCT and SC is intricately involved in this complex process presumably through a series of steps including sensing of distortion or stretch relayed to cells triggering signaling pathways and resulting in ECM degradation and remodeling (Acott and Kelley, 2008

Disclosure

J. Vranka, none; T. Acott, none.

Acknowledgments

The authors would like to thank their funding sources: the BrightFocus Foundation (Vranka, G2014058), NIH/National Eye Institute grants EYOO3279, EY008247, EY010572 (TSA), and an unrestricted grant to the Casey Eye Institute from Research to Prevent Blindness. We would also like to thank Lions VisionGift for procuring human donor eyes.

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      Although experimental [38,68,119–123] and numerical [40,124,125] studies, as well as review articles [10,16,31,126–132], have significantly contributed to our understanding of the mechanism of outflow resistance in the conventional outflow pathway, gaps in our knowledge of outflow tissue biomechanics remain. Conventional outflow tissues are continuously subjected to a variety of static and dynamic mechanical stresses and strains that may influence their function, morphology [38,133], and outflow resistance [134]; so there is likely a coupling (fluid-structure interaction) between outflow hydrodynamics and the mechanical behavior of the TM, JCT and SC inner wall [18]. Experimental studies also showed a correlation between aqueous outflow facility and the biomechanics of the outflow connective tissues [38,39], implying that the conventional outflow resistance is actively regulated.

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