Dataset Information

Autophagy Mediates MMP-2 Expression in Glaucomatous Trabecular Meshwork Cells.

ABSTRACT:

Purpose

To investigate the effect of 3-methyladenine (3-MA) and starvation on the expression of matrix metalloproteinase (MMP-2) in patients with primary open-angle glaucoma.

Methods

Primary TM cells were cultured and divided into three groups. The control group was treated with a normal medium, the 3-MA group was stimulated with 3-MA, and the starvation group received nutrient depletion by replacing the normal media with Earle's balanced salt solution. Cellular mRNA and protein were measured at different 3-MA concentrations and starvation time periods. The level of autophagy was accessed by monodansylcadaverine fluorescent staining and expression of specific autophagy-related genes, light chain 3 (LC3), and Beclin1. The effects of 3-MA and starvation on cell proliferation were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay kit. The mRNA and protein expression of LC3-II, Beclin1, and MMP-2 were measured by reverse transcription-polymerase chain reaction and western blot, respectively.

Results

Compared to the control group, starvation significantly upregulated LC3-II and Beclin1 in TM cells after 3 h of stimulation, which peaked at 6 h and 9 h, respectively. Increased MDC-labeled cells were also observed. Starvation downregulated the expression of MMP-2. On the contrary, 3-MA suppressed the activation of autophagy, as shown by the marked downregulation of LC3-II and Beclin1. The expressions of MMP-2 were higher in the 3-MA group compared to the control group, reaching a peak at a concentration of 5 mM.

Conclusion

Autophagy may be involved in the pathogenesis of POAG via regulating the expression of MMP-2 and, subsequently, the deposition of the extracellular matrix.

SUBMITTER: Xiao YL

PROVIDER: S-EPMC9536984 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Autophagy Mediates MMP-2 Expression in Glaucomatous Trabecular Meshwork Cells.

Xiao Yan-Ling YL Wang Xiao-Rui XR Ye Kejing K Chen Wan-Zhu WZ Zheng Bing-Ru BR Huang Yi-Hong YH Wu Yu-Yu YY

Journal of ophthalmology 20220910

<h4>Purpose</h4>To investigate the effect of 3-methyladenine (3-MA) and starvation on the expression of matrix metalloproteinase (MMP-2) in patients with primary open-angle glaucoma.<h4>Methods</h4>Primary TM cells were cultured and divided into three groups. The control group was treated with a normal medium, the 3-MA group was stimulated with 3-MA, and the starvation group received nutrient depletion by replacing the normal media with Earle's balanced salt solution. Cellular mRNA and protein w ...[more]

PMID: 36211598

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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.

Dataset Information

Autophagy Mediates MMP-2 Expression in Glaucomatous Trabecular Meshwork Cells.

Purpose

Methods

Results

Conclusion

Publications

Autophagy Mediates MMP-2 Expression in Glaucomatous Trabecular Meshwork Cells.

Similar Datasets

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