A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

Abstract

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

A Potential New Role for Zinc in Age-Related Macular Degeneration through Regulation of Endothelial Fenestration

“…Zinc supplementation was in the form of zinc sulfate (ZnSO 4 ). As reported earlier, bioavailable zinc was below 100 nM even as high as 175 μM added ZnSO 4 in bEND.5 cell culture media [ 7 , 13 ]. We assessed the toxicity of zinc using propidium iodide and calcein and did not find signs of cell damage in the concentration of added zinc we used (data not shown).…”

“…All in all, maintaining normal zinc levels in the peripheral circulation and local tissues is becoming essential for maintaining the outer blood-retinal barrier. This study on endothelial cells and previous works on epithelial cells show that maintaining sufficient zinc concentrations at the RPE/choroid interface is likely to be critical to maintaining healthy cellular functions and cell-to-cell communication and transport [ 7 , 13 ]. Both forms of end-stage AMD (neovascular AMD and geographic atrophy) are preceded by common pathological changes, namely sub-RPE deposit formation.…”

“…In our experiments, we supplemented zinc in the form of ZnSO 4 , a water-soluble salt that did not precipitate in the culture medium. While we added up to 200 μM of zinc to the culture medium, it is important to emphasize that the bioavailable zinc concentration remained below 100 nM due to the buffering capacity of the culture medium [ 7 , 13 , 16 ]. However, it was not established in this study if the zinc buffering capacity of the media could be saturated at higher concentrations >200 μM.…”

“…Zinc is a ubiquitous trace element present throughout the human body [ 6 ]. Many of the functions of zinc are enabled by its ability to interact with amino acids and binding to proteins [ 7 ]. One of the highest tissue concentrations of zinc in the human body is found in the retina/choroid complex in the eye [ 6 ].…”

Dietary Supplements and Natural Products: An Update on Their Clinical Effectiveness and Molecular Mechanisms of Action During Accelerated Biological Aging

“…Zinc has many biological effects and functions, including antioxidant, anti-inflammatory, immune modulation, DNA damage response, protein synthesis, apoptosis, etc., Nonetheless, zinc insufficiency, which presents as various physiological alterations in organs and cellular function, is often observed throughout accelerated aging, and contributes to human age-related disorders. This close association between zinc and age has made dietary zinc supplementation a therapeutic option for controlling age-related health conditions, including neurological disorders, infectious diseases, age-related macular degeneration, etc., ( de Almeida Brasiel, 2020 ; Emri et al, 2020 ). In summary, a lot of animal literature proposes that zinc and calcium intake positively control accelerated cellular aging.…”