Abstract
Purpose :
Altered tissue biomechanics have long been known to be associated with glaucoma, though the underlying molecular mechanisms are poorly understood. Recent studies have shown that glaucomatous trabecular meshwork (TM) tissue is markedly stiffer than age-matched control, and this stiffness difference appears to be localized in the low flow regions of the TM. The goal of this study was to determine the underlying mechanism of the stiffness differences by measuring crosslinking in segmental flow cells and tissues from the TM.
Methods :
Primary cells were cultured from high (HF) and low (LF) flow regions of human TM tissues from multiple donor eyes (n = 4 non-glaucoma, n= 3 glaucoma donors). Cells were grown to confluence for up to 2 weeks and serum-free conditioned medium was collected. Total protein concentration was determined by BCA assay and samples were normalized to total protein levels. Samples were subsequently subjected to tissue transglutaminase (TGM2) and lysyl oxidase (LOX) enzyme activity assays to compare crosslinking activities. Immunohistochemistry and western blots were also performed to assess crosslinking protein levels. Quantitative RT-PCR was performed to measure TGM2 and LOX gene levels in HF and LF cells.
Results :
Conditioned medium from HF and LF cell strains (n = 4 biological replicates) showed differences in TGM2 and LOX enzyme activities, as well as some differences in overall crosslinking gene and protein expression levels. Differences in measured enzyme activities between cells from HF and LF cells were also evaluated from glaucomatous eyes. Differences observed in protein expression in tissues ex vivo was modest in comparison with those observed between cells in vitro.
Conclusions :
Our data demonstrate that differences in crosslinking enzyme activities observed between segmental cells exist and correlate with tissue stiffness reported previously. HF and LF TM cell strains are a new tool used here to dissect the molecular mechanisms of segmental flow and its relationship to extracellular matrix crosslinking and biomechanics. Further studies are required to dissect their roles in homeostasis and outflow regulation in pathology.
This is a 2020 ARVO Annual Meeting abstract.