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Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

ABSTRACT: Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell--likely accounting for increased outflow resistance--that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness--and its biomechanical effects on pore formation--as a therapeutic target in glaucoma.

SUBMITTER: Overby DR

PROVIDER: S-EPMC4183270 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

Overby Darryl R DR Zhou Enhua H EH Vargas-Pinto Rocio R Pedrigi Ryan M RM Fuchshofer Rudolf R Braakman Sietse T ST Gupta Ritika R Perkumas Kristin M KM Sherwood Joseph M JM Vahabikashi Amir A Dang Quynh Q Kim Jae Hun JH Ethier C Ross CR Stamer W Daniel WD Fredberg Jeffrey J JJ Johnson Mark M

Proceedings of the National Academy of Sciences of the United States of America 20140908 38

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous huma ...[more]

PMID: 25201985

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Project description:PurposeSchlemm's canal (SC) endothelial cells derived from donors with or without glaucoma showed different mechanical properties and gene expression. As an important contributor to the regulation of intraocular pressure (IOP) and pathogenesis of primary open-angle glaucoma (POAG), the heritable key epigenetic changes, methylation may play an important role in the physiologic function of SC cells. This study aims to identify differentially methylated CpG sites (DMSs) in primary cultures of human SC cells with or without glaucoma.MethodsWe examined the methylation pattern of seven strains of primary human cells (two glaucoma and five normal SC cell samples), which were isolated and characterized using established protocols. DNA methylation was profiled using Illumina Human Methylation 450 BeadChip. Raw data were extracted and exported using Illumina GenomeStudio software. After quantile normalization, DNA methylation data were analyzed using R package RnBeads in Bioconductor. DMSs were filtered with p ≤ 1E-5, methylation change ≥ 0.1, and false discovery rate ≤ 0.05. The closest genes and the location of each CpG site were annotated using R package FDb.InfiniumMethylation.hg19. Gene Ontology and pathway analysis was performed using WebGestalt. Selected DMSs were validated using the Zymo qMethyl kit.ResultsWe used five non-glaucoma and two glaucomatous SC cell samples to profile genome-wide DNA methylation using Illumina Infinium Methylation BeadChips. Principle component analysis showed the separation between the glaucoma and control samples. After quality control and differential analysis, we identified 298 highly significant DMSs (p ≤ 1E-5). Among them, 221 DMSs were within 1 kb of a nearby gene. Gene Ontology analysis demonstrated significant enrichment in positive regulation of cell migration, negative regulation of endothelial cell proliferation, and stress fiber and actin filament bundles. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in cell adhesion and gap junctions. Several glaucoma-related genes were identified, including TGFBR3, THBS1, PITX2, DAXX, TBX3, TNXB, ANGPT1, and PLEKHA7. We also examined differentially methylated regions (DMRs) near these CpG sites and identified significant DMRs in TBX3, TNXB1, DAXX, and PITX2.ConclusionsThis study represents the first genome-wide DNA methylation profiling in cultured human primary SC cells. The DMSs were enriched in the pathways related to outflow resistance. Several DMRs were validated in glaucoma-associated genes, further suggesting the role of DNA methylation in glaucoma development. This study could provide comprehensive understanding of DNA methylation in glaucoma and its effect on aqueous humor outflow.

Project description:Aqueous humour transport across the inner wall endothelium of Schlemm's canal likely involves flow through giant vacuoles and pores, but the mechanics of how these structures form and how they influence the regulation of intraocular pressure (IOP) are not well understood. In this study, we developed an in vitro model of giant vacuole formation in human Schlemm's canal endothelial cells (HSCECs) perfused in the basal-to-apical direction (i.e., the direction that flow crosses the inner wall in vivo) under controlled pressure drops (2 or 6 mmHg). The system was mounted on a confocal microscope for time-lapse en face imaging, and cells were stained with calcein, a fluorescent vital dye. At the onset of perfusion, elliptical void regions appeared within an otherwise uniformly stained cytoplasm, and 3-dimensional reconstructions revealed that these voids were dome-like outpouchings of the cell to form giant vacuole-like structures or GVLs that reproduced the classic "signet ring" appearance of true giant vacuoles. Increasing pressure drop from 2 to 6 mmHg increased GVL height (14 ± 4 vs. 21 ± 7 ?m, p < 0.0001) and endothelial hydraulic conductivity (1.15 ± 0.04 vs. 2.11 ± 0.49 ?l min?¹ mmHg?¹ cm?²; p < 0.001), but there was significant variability in the GVL response to pressure between cell lines isolated from different donors. During perfusion, GVLs were observed "migrating" and agglomerating about the cell layer and often collapsed despite maintaining the same pressure drop. GVL formation was also observed in human umbilical vein and porcine aortic endothelial cells, suggesting that giant vacuole formation is not a unique property of Schlemm's canal cells. However, in these other cell types, GVLs were rarely observed "migrating" or contracting during perfusion, suggesting that Schlemm's canal endothelial cells may be better adapted to withstand basal-to-apical directed pressure gradients. In conclusion, we have established an in vitro model system to study giant vacuole dynamics, and we have demonstrated that this system reproduces key aspects of giant vacuole morphology and behaviour. This model offers promising opportunities to investigate the role of endothelial cell biomechanics in the regulation of intraocular pressure in normal and glaucomatous eyes.

Project description:The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (?65,000 ?m(2) per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm(2) at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular "I-pores" and paracellular "B-pores" both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 ?m for I-pores (N = 79 pores) and 0.67 ?m for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

Dataset Information

Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

Publications

Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

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