Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

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

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.

Zinc Nutrition and Inflammation in the Aging Retina.

Zinc is an essential nutrient for human health. It plays key roles in maintaining protein structure and stability, serves as catalytic factor for many enzymes, and regulates diverse fundamental cellular processes. Zinc is important in affecting signal transduction and, in particular, in the development and integrity of the immune system, where it affects both innate and adaptive immune responses. The eye, especially the retina-choroid complex, has an unusually high concentration of zinc compared to other tissues. The highest amount of zinc is concentrated in the retinal pigment epithelium (RPE) (RPE-choroid, 292 ± 98.5 µg g-1 dry tissue), followed by the retina (123 ± 62.2 µg g-1 dry tissue). The interplay between zinc and inflammation has been explored in other parts of the body but, so far, has not been extensively researched in the eye. Several lines of evidence suggest that ocular zinc concentration decreases with age, especially in the context of age-related disease. Thus, a hypothesis that retinal function could be modulated by zinc nutrition is proposed, and subsequently trialled clinically. In this review, the distribution and the potential role of zinc in the retina-choroid complex is outlined, especially in relation to inflammation and immunity, and the clinical studies to date are summarized.

On the origin of proteins in human drusen: The meet, greet and stick hypothesis.

Retinal drusen formation is not only a clinical hallmark for the development of age-related macular degeneration (AMD) but also for other disorders, such as Alzheimer's disease and renal diseases. The initiation and growth of drusen is poorly understood. Attention has focused on lipids and minerals, but relatively little is known about the origin of drusen-associated proteins and how they are retained in the space between the basal lamina of the retinal pigment epithelium and the inner collagenous layer space (sub-RPE-BL space). While some authors suggested that drusen proteins are mainly derived from cellular debris from processed photoreceptor outer segments and the RPE, others suggest a choroidal cell or blood origin. Here, we reviewed and supplemented the existing literature on the molecular composition of the retina/choroid complex, to gain a more complete understanding of the sources of proteins in drusen. These "drusenomics" studies showed that a considerable proportion of currently identified drusen proteins is uniquely originating from the blood. A smaller, but still large fraction of drusen proteins comes from both blood and/or RPE. Only a small proportion of drusen proteins is uniquely derived from the photoreceptors or choroid. We next evaluated how drusen components may "meet, greet and stick" to each other and/or to structures like hydroxyapatite spherules to form macroscopic deposits in the sub-RPE-BL space. Finally, we discuss implications of our findings with respect to the previously proposed homology between drusenogenesis in AMD and plaque formation in atherosclerosis.

A new perspective on lipid research in age-related macular degeneration.

There is an urgency to find new treatment strategies that could prevent or delay the onset or progression of AMD. Different classes of lipids and lipoproteins metabolism genes have been associated with AMD in a multiple ways, but despite the ever-increasing knowledge base, we still do not understand fully how circulating lipids or local lipid metabolism contribute to AMD. It is essential to clarify whether dietary lipids, systemic or local lipoprotein metabolismtrafficking of lipids in the retina should be targeted in the disease. In this article, we critically evaluate what has been reported in the literature and identify new directions needed to bring about a significant advance in our understanding of the role for lipids in AMD. This may help to develop potential new treatment strategies through targeting the lipid homeostasis.

The effects of zinc supplementation on primary human retinal pigment epithelium.

Population-based and interventional studies have shown that elevated zinc levels can reduce the progression to advanced age-related macular degeneration. The objective of this study was to assess whether elevated extracellular zinc has a direct effect on retinal pigment epithelial cells (RPE), by examining the phenotype and molecular characteristics of increased extracellular zinc on human primary RPE cells. Monolayers of human foetal primary RPE cells were grown on culture inserts and maintained in medium supplemented with increasing total concentrations of zinc (0, 75, 100, 125 and 150 µM) for up to 4 weeks. Changes in cell viability and differentiation as well as expression and secretion of proteins were investigated. RPE cells developed a confluent monolayer with cobblestone morphology and transepithelial resistance (TER) >200 Ω*cm2 within 4 weeks. There was a zinc concentration-dependent increase in TER and pigmentation, with the largest effects being achieved by the addition of 125 µM zinc to the culture medium, corresponding to 3.4 nM available (free) zinc levels. The cells responded to addition of zinc by significantly increasing the expression of Retinoid Isomerohydrolase (RPE65) gene; cell pigmentation; Premelanosome Protein (PMEL17) immunoreactivity; and secretion of proteins including Apolipoprotein E (APOE), Complement Factor H (CFH), and High-Temperature Requirement A Serine Peptidase 1 (HTRA1) without an effect on cell viability. This study shows that elevated extracellular zinc levels have a significant and direct effect on differentiation and function of the RPE cells in culture, which may explain, at least in part, the positive effects seen in clinical settings. The results also highlight that determining and controlling of available, as opposed to total added, zinc will be essential to be able to compare results obtained in different laboratories.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

Purpose: Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods: Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results: Apparently functional primary RPE cells, when cultured on 10-µm-thick inserts with 0.4-µm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions: The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Effects of non-toxic zinc exposure on human epidermal keratinocytes.

Zinc is an essential microelement; its importance to the skin is highlighted by the severe skin symptoms in hereditary or acquired zinc deficiency, by the improvement of several skin conditions using systemic or topical zinc preparations and by the induced intracellular zinc release upon UVB exposure, which is the main harmful environmental factor to the skin. Understanding the molecular background of the role of zinc in skin may help gain insight into the pathology of skin disorders and provide evidence for the therapeutic usefulness of zinc supplementation. Herein, we studied the effects of zinc chloride (ZnCl2) exposure on the function of HaCaT keratinocytes, and the results showed that a non-toxic elevation in the concentration of extracellular zinc (100 µM) facilitated cell proliferation and induced significant alterations in the mRNA expression of NOTCH1, IL8, and cyclooxygenase-2. In addition, increased heme oxygenase-1 (HMOX1) expression and non-toxic generation of superoxide were detected in the first 4 h. Regarding the effects on the UVB-induced toxicity, although the level of cyclobutane pyrimidine dimers in the keratinocytes pre-treated with zinc for 24 h was reduced 3 h after UVB irradiation, significantly enhanced superoxide generation was observed 10 h after UVB exposure in the zinc pre-exposed cells. The overall survival was unaffected; however, there was a decrease in the percentage of early apoptotic cells and an increase in the percentage of late apoptotic plus necrotic cells. These results suggest that the exposure of human keratinocytes to non-toxic concentrations of ZnCl2 impacts gene expression, cell proliferation and the responses to environmental stress in the skin. It would be important to further examine the role of zinc in skin and further clarify whether this issue can affect our thinking regarding the pathogenesis of skin diseases.