**3. Results**

#### *3.1. Confirmation of VITD Receptor Expression in ARPE-19 and HREC Cell Lines*

In this study, we demonstrated that ARPE-19 and HREC cell lines expressed the machinery for vitamin D3 and could produce 1,25(OH)2D3. This is the first time that the expression of Vit D3-synthesizing components has been reported in HREC cells.

We performed conventional polymerase chain reaction (PCR) experiments in order to determine the expression of the following genes involved in VITD synthesis. ARPE-19 cell line highly expressed the genes cytochrome P450 *(CYP)2R1*, *CYP27B*, and *CYP24A* and showed a low expression of vitamin D receptor (*VDR*), *CYP27A*, and *cubilin* genes; however, they did not express the *megalin* gene. HREC cell line highly expressed the genes *CYP2R1* and *CYP27B*. HREC showed a lower expression of *VDR*, *CYP27A*, *cubilin*, and *megalin* genes and did not express the *CYP24A* gene (Table 1). Ribosomal 18S was used as an internal PCR control (Figure 1).

**Table 1.** Summary of the main vitamin D (VITD) synthesizing genes in human retinal pigment epithelial cells (ARPE-19) and human retinal endothelial cells (HREC).


+++: high expression; ++: medium expression; −: no expression.

**Figure 1.** Conventional PCR was carried out using total RNA extracted from ARPE-19 (lines 1 and 2) and HREC cells (line 3). We analyzed the gene expression of *VDR*, *CYP27B1*, *CYP24A1*, *CYP27A1*, *CYP2R1*, *cubilin*, and *megalin*. Ribosomal 18S was used as an internal PCR control. Results are representative of at least three independent experiments. Molecular sizes (base pairs [bp]): *18S* (400), *VDR* (421), *CYP27B1* (302), *CYP24A1* (485), *CYP27A1* (292), *CYP2R1* (259), *cubilin* (518), and *megalin* (290).

#### *3.2. Validation of the Cell Lines: Stable Phenotypic Characterization*

We performed immunofluorescence for specific ARPE-19 and HREC cell lines' markers. We used RPE65 protein (Abcam 78036) for ARPE-19 cells and caveolin protein (Cell Signalling 3238S) for HREC cells. No changes in the phenotypic characteristics were found, and the cells expressed all the selected markers (Figure 2).

**Figure 2.** ARPE-19 and HREC cells' phenotyping. Immunofluorescence of RPE65 (green) and caveolin (green) for ARPE-19 and HREC cell lines' labelling, respectively. Upper panel shows cells at bright-field microscopy and down panel shows cells under fluorescence microscopy. Nuclei were labeled with 4-,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 20 μm.

#### *3.3. E*ff*ect of VITD Addition on Cytoxicity and Proliferation*

VITD at 1 nM and for 1 h did not show cytotoxic effects in ARPE-19 cells and HREC cells (Figure 3A,B). Moreover, proliferation was not affected by VITD addition at 1 nM and 1 h of exposure time (Figure 3C,D).

**Figure 3.** Graphs showing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (**A**,**B**) and Bromodeoxyuridine (BrdU) (**C**,**D**) results for ARPE-19 (**A**,**C**) and HREC (**B**,**D**) cells treated with different VITD concentrations (1, 5, 10, and 50 nM) for 1 h. Any VITD dose showed cytotoxic effects on ARPE-19 cells compared to saline group. HREC showed significant alterations in MTT D50 compared to the rest of the groups (\* *p* < 0.05). ARPE-19 proliferation was statistically reduced (\*\*\* *p* < 0.001) in the highest dose analyzed (50 nM) compared to saline and to all the remaining doses. HREC cells' proliferation was not affected by any VITD dose.

VITD doses ranged from 1 to 50 nM for both cell types tested. All doses were safe for ARPE-19 cells at any time measured, and the highest dose used significantly reduced proliferation (*p* < 0.001). HREC cells showed an increase in viability, with the highest vitamin D dose (*p* < 0.05) response being in the highest dose at 50 nM. In addition, proliferation was reduced in ARPE-19 cells subjected to a 50 nM VITD dose. To be sure that the effects observed in the subsequent analysis were caused by vitamin D itself and that they were not masked by deleterious effects on cell viability and proliferation, we decided to use 1 nM of VITD, which did not affect proliferation and viability.

#### *3.4. E*ff*ect of VITD Addition on Integrity and Apoptosis of Cells*

ARPE-19 cells' integrity was conserved after adding VITD at 1 nM. Hydrogen peroxide and LPS induced an increase in tortuosity of the junction contacts in ARPE-19 cells that were stabilized in oxidative and inflammatory conditions by the addition of VITD to the media (Figure 4).

**Figure 4.** Integrity of ARPE-19 cells evaluated by zonula occludens-1 (ZO-1) (red) and caspase-3 (green) immunofluorescence. VITD (1 nM, 1 h; (**D**)) did not affect ZO-1 structure compared to saline (**A**,**G**). Lipopolysaccharide (LPS) (**B**,**H**) and H2O2 (**E**,**K**) addition damaged tight junctions and concomitant incubation with VITD (1 nM, 1 h; (**C**,**I**) and (**F**,**L**)) restored the altered structure. (**G–L**) show the apical junction in higher magnification. Caspase-3 was highly observed in the H2O2 group (**E**) compared to saline (**A**) and VITD (**D**). VITD addition showed restoration, and caspase-3 activation was absent (**F**). Nuclei were labeled with DAPI (blue). Scale bar: 20 μm. Densitometry of ZO-1 expression in ARPE-19 cells under oxidative stress (**M**) and inflammatory (**N**) conditions. Although a tendency to reduce the ZO-1 expression was observed, no statistical differences were found. VITD restored values similar to saline group. *n* = 3.

LPS and H2O2 addition showed a tendency to reduce ZO-1 expression, although that difference was not significant. Moreover, the supplementation with VITD partly increased the ZO-1 expression, but this result did not reach statistical significance.

Early (caspase-3, Figure 4) and late (TDT- mediated dUTP-biotin nick end-labeling, TUNEL, Figure 5) apoptosis markers revealed that VITD (1 nM) addition did not affect cell death processes. After inflammatory and oxidative induction, ARPE-19 cells and HREC cells showed alterations and an increase labelling for both markers. VITD (1 nM) addition was able to restore those alterations (Figure 5).

**Figure 5.** Late apoptosis measured in ARPE-19 (**A**–**D**) and HREC (**E**–**H**) cells by TDT- mediated dUTP-biotin nick end-labeling (TUNEL) and analyzed by fluorescence. TUNEL-positive ARPE-19 and HREC cells were observed after H2O2 stimulation (2 h; (**B**,**F**)) compared to saline (**A**,**E**). VITD (1 nM, 1 h; (**C**,**G**)) showed similar results to saline groups for both cell types. VITD, in concomitance with H2O2 (1 h; (**D**,**H**)), showed a reduction in altered nuclei, especially in ARPE-19 cells (**D**), and absence of TUNEL labeling. Nuclei were labeled with DAPI (blue). Scale bar: 20 μm.

#### *3.5. Antioxidative and Anti-Inflammatory Properties of VITD Addition*

Figure 6 shows that oxidative stress induction by H2O2 significantly (*p* < 0.001) increased 8-OHdG in supernatants from ARPE-19 cells. VITD alone did not modify oxidative damage compared to saline. VITD was able to significantly (*p* < 0.001) reduce 8-OHdG production under oxidative-induced conditions.

Figure 7 shows that LPS induction increased IL-8, IFN-γ, IL-1β, MCP-1, TNF-<sup>α</sup>, IL-10, IL-18, IL-6, and IL-12p70 in ARPE-19 cells. Under inflammatory conditions, VITD was able to significantly (*p* < 0.05) reduce IL-8, IFN-γ, MCP-1, TNF-<sup>α</sup>, and IL-6.

**Figure 6.** Graph showing 8-OHdG results for ARPE-19 cells measured by ELISA. VITD was able to significantly reduce the 8-OHdG levels elevated by H2O2 (\*\*\* *p* < 0.001). *n* = 3.

**Figure 7.** Multiplex inflammatory cytokine array in ARPE-19 cells. All inflammatory cytokine levels were increased by adding LPS (\* *p* < 0.05), and Interleukin (IL)-8, Interferon (IFN)-γ, Monocyte chemoattractant protein (MCP)-1, Tumor necrosis factor (TNF)-<sup>α</sup>, and IL-6 levels were reduced (\* *p* < 0.05) with the addition of VITD in concomitance. *n* = 3.

HREC cells subjected to LPS induction showed an increase in IL-8, IFN-γ, IL-1β, MCP-1, IL-10, IL-6, and IL-12p70 cytokines. VITD significantly (*p* < 0.05) decreased IL-8, IFN-γ, IL-1β, MCP-1, TNF-<sup>α</sup>, IL-6, and IL-12p70 under inflammatory conditions in HREC cells (Figure 8).

**Figure 8.** Multiplex inflammatory cytokine array in HREC cells. IL-8, IFN-γ, IL-1β, MCP-1, IL-10, IL-6, and IL-12p70 inflammatory cytokine levels were increased by adding LPS (\* *p* < 0.05), and IL-8, IFN-γ, IL-1β, MCP-1, TNF-<sup>α</sup>, IL-6, and IL-12p70 levels were reduced (\* *p* < 0.05) with the addition of VITD in concomitance. *n* = 3.
