Switch to Autophagy the Key Mechanism for Trabecular Meshwork Death in Severe Glaucoma

Abstract

Purpose

The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma.

Design

In-vitro laboratory study on surgical specimens and primary cell lines.

Methods

Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H₂O₂ (400–1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma.

Results

Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were up-regulated at 48–72 hours of exposure in contrast to apoptosis-related genes, showing down-regulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG.

Conclusion

A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.

Keywords:

• trabecular meshwork
• cell death
• autophagy
• apoptosis
• glaucoma

Abbreviations

POAG, primary open angle glaucoma; PACG, primary angle closure glaucoma; TM, trabecular meshwork; HTM, human trabecular meshwork, refers to primary cell lines, refer to text for details; IOP, intraocular pressure; PBMC, peripheral blood mononuclear cells. Gene abbreviations listed separately in Supplemental Methods and Appendix of abbreviations.

Data Sharing Statement

All data generated or analysed during this study are included in this published article (and its supplementary information files). Protein database is uploaded at proteome exchange with the dataset identifier PXD010752. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010752-Submission Reference: 1-20180810-113504.

Ethics Approval and Consent to Participate

This study was approved by the institutional review board of LV Prasad Eye Institute, MTC campus, Bhubaneswar, India.

Acknowledgments

We would like to thank all patients who participated in the study, Dr Ravindran and Birender Prusty for helping with cytokine analysis at ILS, Bhubaneswar. We would also like to thank Mr Bikash Sahoo for helping us with the FACS analysis on PBMC and HTM cells in KSBT, KIIT.

Author Contributions

APR – conception, design of the work; the acquisition, analysis, interpretation of data; funding, supervision, administrative, the creation of new software used in the work; drafted the work and substantively revised it.

PS – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

MC – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

BP – design of the work; the acquisition, analysis, the creation of new software used in the work, interpretation of data; substantively revised it.

SS – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

GDJ – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

PM – the acquisition, interpretation of data; substantively revised it.

RM – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

MS – design of the work; the acquisition, analysis, interpretation of data; substantively revised it.

All authors have read and approved the manuscript.

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests in this work.

Additional information

Funding

This study was partly funded by the DBT Wellcome Trust grant: ref no IA/CPHI/15/1/502031, Role of funder - for HTM in-vitro based experiments.