The effects of zinc supplementation on primary human retinal pigment epithelium

Abstract

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*cm 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.

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

“…The majority of complement genes are expressed in the liver, resulting in an abundance of complement proteins in the blood. However, the RPE expresses several key complement genes which may modulate the complement attack on RPE and drusen (Chen et al, 2007;Kim et al, 2009;Pao et al, 2018). Interestingly, locally produced CFH is, at least in cultured RPE cells, secreted apically, and not basally (Kim et al, 2009;Pao et al, 2018).…”

“…However, the RPE expresses several key complement genes which may modulate the complement attack on RPE and drusen (Chen et al, 2007;Kim et al, 2009;Pao et al, 2018). Interestingly, locally produced CFH is, at least in cultured RPE cells, secreted apically, and not basally (Kim et al, 2009;Pao et al, 2018). Consequently, the potential regulating role of locally produced CFH in vivo, and other complement factors, potentially involved in the complement attack on drusen, needs to be further investigated.…”

“…This network contains annexin A2 (ANXA2), a relatively small calcium and phospholipid-binding protein involved in multiple intra-cellular transport functions. The RPE secretion data set, called RPE-IVS (Table 3; STable 1) reveals that this protein is indeed secreted basally by the RPE (Pao et al, 2018). The protein was initially assigned to drusen (Crabb et al, 2002).…”

“…Interestingly, in an RPE cell culture model that mimics drusen formation, Pao (2018) and coworkers found that APOE is secreted basally by these cells. Subsequent exposure of these cultures to human serum led to heterogeneous sub-RPE-BL space deposits, some of which were rich in serum-derived proteins such as vimentin, clusterin and amyloid P (Pao et al, 2018). In addition to ANXA1 and APOE, the serum amyloid proteins S100A7, S100A8 and S100A9 are part of this functional cluster and the drusen proteome.…”

“…COL6A1 is secreted basally by the RPE in vitro (Pao et al, 2018) (Table 3), and is part of the BrM (Booij et al, 2010a). Consequently, the protein may end up in the drusen dataset either as a contamination, or as a remnant of the turnover of BrM components.…”