**1. Introduction**

Diabetic retinopathy (DR) is the main cause of blindness among adults of working age globally [1]. An e ffective managemen<sup>t</sup> of diabetes reduces the risk of complications, however, poor control of the condition can result in microvascular complications [2]. Nevertheless, even patients with intensive glycemic control have rate of progression over 7% [3]. The Diabetes Control and Complications Trial (DCCT) found that intensive glycemic control can e ffectively reduce or slow down the development or progression of DR by 76% in patients with type 1 diabetes, while the U.K. Prospective Diabetes Study (UKPDS) came to a similar conclusion in patients with type 2 diabetes [2,4].

The investigation of the underlying mechanisms of DR is of grea<sup>t</sup> importance and may provide potential new alternative treatments. Several studies have shown that diabetes can lead to DR by several mechanisms including the polyol pathway [5], non-enzymatic glycation [6], activation of protein kinase C [7], oxidative stress [8–12], and inflammation [13]. Oxidative stress is considered to be one of the crucial causative factors in the development of DR, in combination with other biochemical imbalances, leading to both structural and functional changes and also promoting an increased loss of capillary cells in the microvasculature of the retina [11,12]. In addition, a significant body of evidence supports the role of proinflammatory cytokines, chemokines, and other inflammatory mediators in the pathogenesis of DR, leading to chronic low-grade inflammation of the retina and eventually to neovascularization [14].

Vitamin D (VITD) is a fat-soluble molecule that is found in two forms: vitamin D2 and vitamin D3 [15]. Vitamin D3 is well recognized as a secosteroid hormone that regulates many cellular signaling activities through its nuclear VITD receptor (VDR) in target cells [15]. Previous studies reported that VITD treatment can protect cells and tissues from oxidative damage [16] and it has also been found to prevent oxidative stress damage in DR in a high-glucose environment [17,18] and in diabetic rats [19]. In addition, some polymorphisms in the VITD receptor gene are associated with increased risk of DR [20–23] and VITD-deficient patients have increased risk of DR [24,25]. VITD also appears to be beneficial in other retinopathies such as age-related macular degeneration (AMD) [26–28]. Therefore, VITD may be suggested as a useful candidate for diabetic patients to reduce the pathological complications of diabetes. However, further research needs to be made to clarify the possible therapeutic potential of VITD in the DR.

In the current study, we investigated the safety of VITD in adult retinal pigment epithelium (ARPE-19) and human retinal endothelial (HREC) cell lines, focusing on its antioxidant and anti-inflammatory e ffect on cell integrity, oxidative stress, and cytokines release. This study constitutes an in vitro evaluation of the molecular pathways by which VITD might tackle the oxidative stress and inflammation observed in patients su ffering from retinal pathologies such as DR.

#### **2. Materials and Methods**

#### *2.1. Expression of Genes Related to VITD Metabolism*

Total RNA was isolated from cell lines using an ABI PRISM 6100 Nucleic Acid PrepStation (Life Technologies, Carlsbad, CA, USA). Subsequently, the quantity and quality of purified messenger RNA (mRNA) was checked using a NanoDrop spectrophotometer (Nanodrop Technologies, Montchanin, DE, USA) at 260/280. Using the qScript cDNA Supermix Kit (Quanta Biosciences, Inc., Gaithersburg, MD, USA), we reverse-transcribed 1000 ng of each mRNA under the manufactured conditions. The primers of the relevant genes are as follows: cytochrome P450 *(CYP)27A1* (Unigene ID-Hs.516700, 5--GGCAAGTACCCAGTACGG-3- and 5--AGCAAATAGCTTCCAAGG-3-), *CYP27B1* (Unigene ID-Hs.524528, 5--CACCTGACCC ACTTCCTGTT-3- and 5--TCTGGGACACGAGAATTTCC-3-), *CYP2R1* (Unigene ID-Hs.371427, 5--AGAGACCCAGAAGTGTTCCAT-3- and 5--GTCTTTCAGCACAGATGAGGTA-3-), *CYP24A1* (Unigene ID-Hs.89663, 5--CCCACTAGCCACCTCGTACCAAC-3- and 5--CGTAGCCCTTCTTT GCGGTAGTC-3-), *VDR* (Unigene ID-Hs.524368, 5--CGCTCCAATGAGT CCTTCACC-3and 5--GCTTCATGCTGCACTCAGGC-3-), *Cubilin* (Unigene ID-Hs.166206, 5--GCGGCTTCACTGC TTCCTA-3- and 5--GAGTGATGGTGTGCCCTTGT-3-), *Megalin* (Unigene ID-Hs.657729, 5--TAAGT CAGTGCCCAACCTTT-3and 5--GCGGTTGTTCCTGGAG-3-). A 2720 Thermal Cycler (Life Technologies, Gaithersburg, MD, USA) was used for amplification with the following protocol: 10 min at 95 ◦C, 40 cycles of 30 s at 95 ◦C, 1 min 58 ◦C, and extension of 45 s at 72 ◦C. Two housekeeping genes, *18S* (Unigene ID-Hs.99999901\_s1, and glyceraldehyde 3-phosphate dehydrogenase, *GAPDH* (Unigene ID-Hs.99999905\_m1), Life Technologies, Gaithersburg, MD, USA) were used as internal controls, and 18S (5--GTTGGTGGAGCGATTTGTCT-3- and 5--GGCCTCACTAAACCATCCAA-3-) was selected as the best control.

## *2.2. Cell Culture*

Human retinal pigment epithelial cells, ARPE-19 (CRL-2302, ATCC, Manassas, VA, USA), and human retinal endothelial cells, HREC (p10880, Innoprot, Vizcaya, Spain), were used. ARPE-19 cells (three passages) were grown to confluence (37 ◦C, 5% CO2) in Dulbecco's modified Eagle's medium (DMEM; D6429, Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; 10270106 Gibco ThermoFisher, Paisley, UK), 1% fungizone (Gibco, Carlsbad, CA, USA), and penicillin–streptomycin (Gibco, Carlsbad, CA, USA). HREC cells were seeded in T75 flasks (353136, Falcon, Corning Life Science, Tewksbury, MA, USA) covered with 1 mg/mL of fibronectin (Innoprot, p8248, Vizcaya, Spain) and grown to confluence in a standard incubator at 37 ◦C under humidified 5% CO2 conditions in Endothelial Cell Medium (Innoprot, p60104, Vizcaya, Spain) containing 5% FBS (Innoprot, Vizcaya, Spain), 1% Endothelial Cell Grow Supplement (ECGS, Innoprot, Vizcaya, Spain), and penicillin–streptomycin solution (Innoprot, Vizcaya, Spain).

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

To verify that ARPE-19 and HREC cells preserved their phenotype, we performed retinoid isomerohydrolase *RPE65* (RPE65) (1:100, 78036, Abcam, Cambridge, MA, USA) and caveolin (1:250, 3238S, Cell Signaling, Danvers, MA, USA) staining by immunofluorescence. Briefly, 100,000 ARPE-19 and 50,000 HREC cells were seeded on a 10 mm dish (Menzel-Glaser, Waltham, MA, USA). Cold methanol was used for cellular fixing. Afterward, cells were washed with 1% phosphate bu ffer saline (PBS) and then incubated with blocking bu ffer containing 1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.2% sodium azide, and 1% fetal bovine serum (FBS) for 1 h at 4 ◦C. Cells were incubated with the primary antibodies, diluted in blocking bu ffer at 4 ◦C for 24 h, and washed once more with PBS and then incubated with the secondary fluorescent antibodies goa<sup>t</sup> anti-mouse 488 (1:250, A11029, Life technologies, Gaithersburg, MD, USA) and donkey anti-rabbit 488 (1:250, A21206, Invitrogen, Carlsbad, CA, USA) for RPE65 marker diluted in blocking bu ffer during 1 h in the dark. Nuclei were labelled with 4-,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA). The morphology of cells was observed under an inverted phase-contrast microscope (Olympus CKX41, Tokyo, Japan) and photographed by a digital camera, and fluorescent images were obtained using a confocal microscope (LSM800, Zeiss, Oberkochen, Germany).

#### *2.4. Treatments and Experimental Design: Oxidative Stress and Inflammation-Like Conditions*

ARPE-19 and HREC cell lines were treated with VITD (1 nM; C9756-1G, Sigma-Aldrich, St. Louis, MO, USA) for 1 h to test its e ffect on cells. To induce in vitro oxidative stress, we subjected cells to H2O2 (1000 μM, Panreac, Barcelona, Spain) for 2 h. To evaluate the protective e ffect of VITD, we added it (1 nM; Sigma-Aldrich, St. Louis, MO, USA) in concomitance 1 h before the end of the induction time. Lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA) was added for 24 h (20 μg/mL for ARPE-19 and 50 μg/mL for HREC cells) to induce an inflammatory response, and then VITD (1 nM; Sigma-Aldrich, St. Louis, MO, USA) was added to the supernatant for 1 h in concomitance.

#### *2.5. Cell Structure and Integrity: Zonula Occludens (ZO)-1 Immunofluorescence and Western Blot*

The e ffect of VITD on intercellular tight junction status was evaluated by zonula occludens-1 (ZO-1) immunofluorescence. One-hundred thousand ARPE-19 cells per well were seeded on laminin-coated polycarbonate membrane cell culture inserts (Corning Life Science, Tewksbury, MA, USA) and were grown in 1% FBS-DMEM for 4 weeks. Immunofluorescence was then performed using a ZO-1 anti-rabbit Alexa Fluor 594 antibody (1:100, 339194, Invitrogen-Life Technologies, Gaithersburg, MD, USA) diluted in blocking bu ffer, following the same protocol described above. DAPI (4-,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA) was used to stain cell nuclei. Images were obtained with a laser scanning confocal imaging system (LSM800, Zeiss, Oberkochen, Germany). H2O2 (1600 μM, Panreac, Barcelona, Spain) over 6 h was used as a positive control for oxidative stress conditions, and LPS (20 μg/mL; L2880 Sigma-Aldrich, St. Louis, MO, USA) was used for 24 h for inflammatory conditions.

A total of 5 μg of ARPE-19 cell homogenates from three passages were mixed with NuPage (4x, Bio-Rad, Hercules, CA, USA), boiled for 5 min, separated on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred onto a nitrocellulose membrane. After we blocked them with 5% skimmed milk ( *w*/*v*; Scharlau, Barcelona, Spain), 0.1% Tween-20 ( *w*/*v*; Sigma-Aldrich, St. Louis, MO, USA) in tris bu ffer saline (TBS) for 1 h at room temperature (RT), membranes were exposed to the mouse monoclonal ZO-1 antibody (1:1000, #33-9100, Invitrogen, Carlsbad, CA, USA) at RT for 1 h, followed by a horseradish peroxidase-conjugated goa<sup>t</sup> anti-mouse antibody (sc-2005; 1:5000, 1 h, RT Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Signal was detected with an enhanced chemoluminescence (ECL) kit (ECL-Select, #RPN2235, GE Healthcare, Fairfield, CT, USA) and images were captured with ImageQuant 400 (GE Healthcare). The relative intensities of the immunoreactive bands were analyzed with ImageQuant TL (GE Healthcare, Fairfield, CT, USA). The loading was verified by Ponceau S red and an anti-β-actin monoclonal antibody (1:10,000, 1 h, RT; Sigma-Aldrich, St. Louis, MO, USA), followed by a goa<sup>t</sup> anti-mouse antibody (sc-2005; 1:10,000, 5% skimmed milk, 1 h, RT; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and signal was detected using ECL-Prime, #RPN2232 (GE Healthcare, Fairfield, CT, USA). Data are presented as absorbance units (AU) ZO-1/β-actin (% vs. saline).

#### *2.6. Assay to Detect Cell Apoptosis*

Apoptosis in ARPE-19 and HREC was performed in cultured plates using an in situ cell death detection kit with TMR Red according to the manufacturer's instructions (#12156792910, Roche, West Sussex, UK) and stored at 4 ◦C until analysis, with the apoptotic cells being labelled with active caspase-3 antibody (1:100, G7481; Promega, Madison, Wisconsin, USA) using the protocol mentioned above and incubated with the secondary fluorescent antibody donkey anti-rabbit 488 (A21206, Invitrogen). Nuclei were labelled with DAPI and images were obtained using a confocal microscope (LSM800, Zeiss, Oberkochen, Germany). H2O2 (600 μM, Panreac, Barcelona, Spain) for 2 h was used as positive control for oxidative stress conditions.

#### *2.7. Viability*/*Toxicity Assay (MTT)*

Cell viability/toxicity in ARPE-19 and HREC cell lines was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), following the manufacturer's instructions. A total of 10,000 ARPE-19 or HREC cells were grown until confluence in DMEM with 10% FBS onto 96-well plates. Then, cells were cultivated for 1 additional week in serum-reduced medium (1% FBS-DMEM), and VITD was added to the culture medium for 24 h at 1, 5, 10, and 50 nM doses.

#### *2.8. Proliferation Assay (Bromodeoxyuridine, BrdU)*

To examine the e ffect of VITD on ARPE-19 and HREC cell proliferation, we seeded 10,000 cells onto 96-well plates. After 24 h, cells were exposed to VITD (1 nM) for 1 h and the Calbiochem BrdU Cell Proliferation Assay (Calbiochem, La Jolla, CA, USA) was performed in accordance with the manufacturer's instructions.

#### *2.9. Measurement of 8-Hydroxidioxiguanosine (8-OHdG) under Oxidative Stress Conditions*

Oxidative damage was measured in ARPE-19 and HREC supernatants subjected to oxidative stress conditions, as described above. To evaluate the e ffect of VITD, we added 1 nM to the media. Supernatants (100 μL) were evaluated by using the Enzyme-Linked ImmunoSorbent Assay (ELISA) kit #ab201734 (Abcam, Cambridge, MA, USA). Data are presented in nanograms per milliliter (ng/mL).

*2.10. Multiplex Cytokine Analysis under Inflammatory and Basal Conditions: Interleukin (IL)-1*β*, IL-6, IL-8, IL-10, IL-12p70, and IL-18; Interferon (IFN)-*γ*; Monocyte Chemoattractant Protein (MCP)-1; and Tumor Necrosis Factor (TNF)-*α

The cytokine analysis for IL-1β, -6, -8, -10, -12p70, and -18; IFN-γ; MCP1; and TNFα was made using FirePlex Firefly (Abcam, Cambridge, MA, USA) particle multiplex immunoassay for Flow Cytometry and Analysis Workbench, a software for multiplex protein expression assays from Abcam Laboratories. Supernatants were used for this purpose and were measured under inflammatory conditions as abovementioned. All cytokines are expressed in pictograms per milliliter (pg/mL), with the exception of MCP-1 and IL-8, which were expressed in ng/mL.
