Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth

Steven S Eamegdool; Ephrem I Sitiwin; Adrian V Cioanca; Michele C Madigan

doi:10.1016/j.freeradbiomed.2019.11.018

Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth

Free Radic Biol Med. 2020 Jan:146:357-371. doi: 10.1016/j.freeradbiomed.2019.11.018. Epub 2019 Nov 18.

Authors

Steven S Eamegdool¹, Ephrem I Sitiwin², Adrian V Cioanca³, Michele C Madigan⁴

Affiliations

¹ Save Sight Institute, University of Sydney, 2000, Australia; Eye Genetics Research Unit, Children's Medical Research Institute, 2145, Australia. Electronic address: seamegdool@cmri.org.au.
² Save Sight Institute, University of Sydney, 2000, Australia; School of Optometry and Vision Science, UNSW, 2052, Australia; Biomedical Imaging Facility, University of New South Wales, Sydney, NSW, 2052, Australia. Electronic address: efroix@gmail.com.
³ Save Sight Institute, University of Sydney, 2000, Australia; The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia. Electronic address: valerin.cioanca@anu.edu.au.
⁴ Save Sight Institute, University of Sydney, 2000, Australia; School of Optometry and Vision Science, UNSW, 2052, Australia. Electronic address: michele.madigan@sydney.edu.au.

PMID: 31751761
DOI: 10.1016/j.freeradbiomed.2019.11.018

Abstract

Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1⁺ macrophages/microglia in human donor eye sections (aged and AMD) using multi-marker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD.

Keywords: Cell biology; Extracellular matrix; Inflammation; Oxidative stress; Pro-inflammatory cytokines; Retinal degeneration; Retinal pigment epithelium.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aged
Bruch Membrane
Extracellular Matrix / metabolism
Humans
Macular Degeneration* / metabolism
Oxidative Stress
Retinal Pigment Epithelium*