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Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model.

ABSTRACT: Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of glaucomatous HTM. This disease model exhibits HTM marker expression, ECM overproduction, impaired HTM cell phagocytic activity and outflow resistance, which represent characteristics found in steroid-induced glaucoma. In particular, steroid-induced ECM alterations in the glaucomatous model can be modified by a ROCK inhibitor. Altogether, this work presents a novel in vitro disease model that allows for physiological and pathological studies pertaining to regulating outflow, leading to improved understanding of steroid-induced glaucoma and accelerated discovery of new therapeutic targets. Biotechnol. Bioeng. 2016;113: 1357-1368. © 2015 Wiley Periodicals, Inc.

SUBMITTER: Torrejon KY

PROVIDER: S-EPMC4854669 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model.

Torrejon Karen Y KY Papke Ellen L EL Halman Justin R JR Stolwijk Judith J Dautriche Cula N CN Bergkvist Magnus M Danias John J Sharfstein Susan T ST Xie Yubing Y

Biotechnology and bioengineering 20151230 6

Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of g ...[more]

PMID: 26615056

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Project description:Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment.Statement of significanceExtracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.

Project description:The goal of this study was to contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells, to that of control and primary open angle glaucoma (POAG) HTM tissues. Total RNA from cultured HTM cells and HTM tissue dissected from control and POAG anterior segments fixed in RNA laterâ¢ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using the GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by Quantitative real-time PCR in nonamplified RNA. Cultured HTM cells retained the expression of some genes characteristic of the HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants, paraoxonase 3 and ceruloplasmin. Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma. Experiment Overall Design: Total RNA from three cultured HTM cells and HTM tissue dissected from three pairs of control and two pairs of POAG anterior segments fixed in RNA laterâ¢ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays.

Dataset Information

Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model.

Publications

Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model.

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