Biomechanical Rigidity and Proteomics Analyses of Segmental Outflow Regions of the Trabecular Meshwork at Physiologic and Elevated Pressures

Abstract

Purpose : Changes in the extracellular matrix (ECM) of the trabecular meshwork (TM) are associated with glaucoma. The ECM of the TM is involved in regulation of outflow resistance and intraocular pressure homeostasis. Segmental outflow adds complexity to molecular mechanisms regulating outflow facility. It was shown by atomic force microscopy (AFM) that glaucomatous TM is stiffer than age-matched normal TM. We hypothesized that biomechanical and protein expression differences exist between low (LF) and high flow (HF) segmental regions of the TM. Here we investigated stiffness in segmental areas of the TM and identified changes in ECM proteins at physiological (1x) and elevated (2x) pressures.

Methods : Human anterior segments were perfused at 1x or 2x pressure. Cell mask plasma membrane stain was perfused and relative fluorescence was used to distinguish LF and HF areas of the TM. Elastic modulus of TM tissue was measured by AFM. For proteomics analyses TM tissues (n=3) from segmental flow areas were homogenized before trypsin digestion. Peptides were labeled with tandem mass tags (TMT) to quantitate relative protein abundance using an Orbitrap Fusion instrument.

Results : The elastic modulus of LF regions was 14.96 ± 1.56 kPa while that of HF regions was 6.49 ± 0.73 kPa (mean ± SEM) at 1x pressure. At 2x pressure moduli were 2.72 ± 0.08 kPa (HF) and 30.33 ± 2.4 kPa (LF). In glaucomatous TM preliminary results show a similar trend (stiffer LF regions). Proteomics data from HF regions identified multiple ECM proteins enriched under 2x pressure including stromelysin, fibulin-5, galectin-3-binding protein, EGF-containing fibulin-like ECM protein-1, and MMP-2, whereas laminin alpha 3 chain was enriched at 1x pressure. Decorin, collagen alpha-3 (VI) chain, TGFβ-induced protein, lumican, angiopoietin-related protein 7, and thrombospondin 4 were enriched at 2x pressure in HF compared with LF regions.

Conclusions : AFM data demonstrate that LF regions of the TM are more rigid than HF regions in normal and glaucomatous TM. Proteomics data indicate that elevated pressure causes greater changes in protein expression than differences between HF and LF regions. Together this implies that HF regions are more compliant and dynamic in homeostatic response to elevated pressure. These data provide insights into how TM cells may remodel their ECM in response to changes in pressure.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.