**4. Discussion**

The present study shows that damage observed in human retinal pigmented epithelium (RPE) and retinal endothelium cells under oxidative and inflammatory conditions were restored by the addition of VITD to the media. More specifically, induced inflammatory cytokine levels, early and late apoptosis, and oxidative stress markers were reduced back to control levels. This result suggests that VITD could be a useful candidate in modulating the chronic low-grade inflammation and oxidative stress responsible for the complications in retinal pathologies involving RPE and endothelial cells.

It is well established that glycemic control is an effective managemen<sup>t</sup> to lower the incidence of complications such as DR. However, even an intensive glycemic control is not sufficient to prevent diabetic microvascular pathologies in all patients [4], and hyperglycemia on its own is not sufficient to trigger widespread diabetic microvascular pathologies in all patients [29–31]. Diabetic patients with an initial poor glycemic control have persistent higher incidence of diabetic complications after glucose normalization, a phenomenon described as metabolic memory, suggesting that oxidative stress, non-enzymatic glycation of proteins, epigenetic changes, and chronic inflammation may play a major role in the development and progression of diabetic microvascular complications [32,33] such as diabetic kidney disease [34–36], diabetic polyneuropathy [37,38], and DR [9–11,14]. VITD deficiency is related to a higher risk of DR in type 1 and 2 diabetes mellitus [24,39]. Apart from its role in tissues related to calcium homeostasis [40], high levels of VDR are also present in inflammatory cells such as dendritic cells, macrophages, T-cells, and B-cells, thus supporting the fact that VITD may have a role in inflammatory and immune responses [41].

25-Hydroxylase (encoded by the *CYP27A1* and *CYP2R1* genes), a cytochrome P450 enzyme, catalyzes the formation of vitamin D3 to 25-hydroxyvitamin D3 (25(OH)D3), the main circulating VITD metabolite, and then 25(OH)D3 is converted to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), the most active form by the enzyme 1 alpha-hydroxylase (encoded by the *CYP27B1* gene). 1,25[OH]2D3 is inactivated by 24-hydroxylase (encoded by the *CYP24A1* gene) [42]. Megalin and cubilin, endocytic receptors in the cell membrane, allow the internalization of 25(OH)D3 and 1,25[OH]2D3 into the cell [43]. We demonstrated that the ARPE-19 cell line highly expressed *CYP2R1*, *CYP27B*, and *CYP24A* genes and showed a low expression of *CYP27A* and *cubilin* genes; however, they did not express the megalin gene. The HREC cell line highly expressed the *CYP2R1* and *CYP27B* genes. HREC showed a lower expression of *VDR*, *CYP27A*, *cubilin*, and *megalin* genes and did not express the *CYP24A* gene. This is the first time that the machinery for VITD internalization and metabolization is reported in HREC cells. A recent study reported that VITD treatment enhanced VDR expression in ARPE-19 cells treated with H2O2 [27].

The antioxidant role of vitamin D has been demonstrated in many other micro-environments in the body, especially in the context of diabetes or obesity, including the liver [44], the kidney filtration [45], the heart [46], the hippocampus [47], and the adipose tissue [48], among others. Recently, it has been discovered that vitamin D is not only produced systemically at the renal level, but that extrarenal production is also important. In the eye, there are several tissues in which local production of 1.25(OH)2D3 has been demonstrated, including the sclera, corneal endothelium, ciliary body epithelium, and pigment epithelium, with the corneal endothelium being the eye tissue with the highest conversion rate [49]. We did not find studies focused on the permeability of the retinal blood barrier to VITD; however, it has been demonstrated that the retinal blood barrier has a high permeability to lipophilic substances [50] and also, oral supplemented vitamin D increases the concentration in aqueous humor and tears [49]. To better understand the effect of VITD in the retina, ARPE-19 and HREC cells were subjected to oxidative stress and inflammatory conditions that provoked alterations in tight junctions and also apoptotic signs. Some studies have suggested that VITD can protect against the deleterious effects of reactive oxygen species (ROS), free radicals generated during physiological energy production in the mitochondria [51], therefore improving cell viability in ARPE-19 cells [27] and various tissues [16,52–54]. In line with these observations, we demonstrated that H2O2 and LPS induced the alteration and partial loss of tight junction protein organization (i.e., tortuosity and cytosol localization) in ARPE-19 cells and were restored in oxidative and inflammatory conditions by the addition of VITD to the media. However, results observed in protein expression did not show significant differences in the amount of ZO-1. This phenomenon has been also described by other authors, observing that ZO-1 remains localized in junction despite loss of tight junction protein organization by oxidative stress [55], as observed also in our immunofluorescence images. Inflammatory conditions showed a similar behavior on tight junctions, and their expression was not modified but the organization was altered. VITD addition restored morphological alterations observed in immunofluorescence. The increased early and late apoptosis under oxidative stress and inflammatory conditions was also restored by addition of VITD in ARPE-19 and HREC cells, in concordance with other studies after H2O2 [27] and after high-glucose-induced oxidative stress and inflammation [17].

A recent study showed that VITD treatment also upregulated the expression of antioxidant genes (*catalase*, *CAT*; *superoxide dismutase SOD1* and *SOD2*; *Glutathione peroxidase GPX2* and *GPX3*) in ARPE-19 cells under similar stress conditions [27]. Accordingly, we found that H2O2-treated ARPE-19 cells had significantly increased oxidative stress, and VITD exposure counteracted this 8-OHdG production under oxidative-induced conditions. Other authors showed that increased ROS production and lipid peroxidation downregulated expression of antioxidant genes, and decreased activities of SOD and catalase induced in high glucose-treated ARPE-19 cells was counteracted by VITD exposure [17].

In addition to oxidative stress, the levels of proinflammatory proteins such as MCP-1, IL-1β, IL-6, IL-8, and TNF-α influence the development of DR [56,57], and increased aqueous concentration of those molecules in eyes with severe non-proliferative DR suggests that inflammatory changes

precede the development of neovascularization [58,59]. Endothelial damage is also linked to increased leukocyte adhesion that is explained by the overexpression of endothelial adhesion molecules such as Intercellular adhesion molecule (ICAM)-1, Vascular cell adhesion molecule (VCAM)-1, Platelet endothelial cell adhesion molecule (PECAM)-1, and P-selectin [59,60]. Vascular endothelial growth factor (VEGF) also alters adherens and tight junctional proteins between the endothelial cells [61,62], favoring the infiltration of leukocytes into the retina. This complex of inflammatory events leads to blood–retinal barrier breakdown and with it some of the vision threatening complications such as macular edema. Directly or indirectly VITD regulate over 200 genes involved in cellular proliferation, di fferentiation, apoptosis, angiogenesis, and inflammation [63]. VITD have shown to prevent, slow the progression of, ameliorate inflammation markers of, or decrease the severity of many immune-related disorders such diabetes mellitus [64,65]. In our study, all the inflammatory cytokines investigated were upregulated under inflammatory conditions. TNFα did not result in a statistically significant elevation after LPS induction in HREC cells, probably due to the dispersion observed in control samples. Herein, we observed that the addition of VITD to the media downregulated levels of IL-6, IL-8, MCP-1, IFN-γ, and TNFα in retinal epithelial cells, as also shown by other authors in ARPE-19 cells [66,67]. These results sugges<sup>t</sup> that VITD can control the broad inflammatory spectrum studied that is present in non-proliferative DR, indicating a clear anti-inflammatory response. In the case of retinal endothelial cells, similar results were observed, with the exception of TNFα that were unmodified and IL-18 and IL-12p70 that were also downregulated. Elevated levels of IL1-β and IL18 have been demonstrated in streptozotocin (STZ)-induced diabetic rats [68]. Similarly, serum IL-18 levels have also been reported to be elevated in type 1 diabetic patients, half of which had a form of DR [68]. IL-1β, IL-18, and IL-1 α have pro-inflammatory actions, and in the case of IL-18, a role in angiogenesis [69]. The inflammasome is an oligomer protein complex that triggers the secretion of IL-1β and IL-18 into the extracellular space [70]. Interestingly, the inflammasome has been particularly related to the neovascular pathology occurring in proliferative DR (PDR) [71,72]. While the major pro-inflammatory cytokines such as IL-6, TNFα, and IFN-γ could be detected both in non-PDR and in PDR eyes, inflammasome-related cytokine, IL-18, and caspase-1 were particularly increased in the eyes of PDR patients [72]. In our study, IL-18 levels were e ffectively reduced by VITD in endothelial cells, but not in RPE cells, suggesting a main antiangiogenic role under inflammatory conditions. Moreover, the primary leukocyte populations found in the retina during disease are microglia and macrophages, and it is well known that the activation of the inflammasome is an important mechanism by which these cells cause damage in retinal degenerations. VITD could help to reduce macrophage recruiting by reducing MCP1 levels.

Analyzing the secretome in ARPE-19 cells, researchers found that adding TNFα to the media regulated di fferent proteins secreted by the RPE, which play a critical role in extracellular matrix remodeling, complement network, and angiogenesis [73]. Thus, VITD supplementation could contribute to reduce those e ffects. A mixture of IFN-γ, TNFα, and IL-1β has been shown to decrease the expression of specific genes that play an important role in processes, such as visual cycle, epithelial morphology, melanogenesis, and phagocytosis, in cultured ARPE-19 cells [74]. Therefore, downregulation of those levels by VITD may potentially contribute to restore the RPE dysfunction implicated in retinal diseases, including DR [75,76]. It has been demonstrated that a higher secretion of IL-10 would be a protective factor against the development of proliferative DR (PDR) when proinflammatory cytokines, such as IL-1β, are elevated, as shown in vitreous of PDR patients [77]. In our study, VITD maintained high IL-10 levels, suggesting a possible contribution to an anti-inflammatory environment that must be investigated deeply. Surprisingly, in contrast to other authors [17], IL-1β was not reduced by VITD addition. The bioactive IL12p70 molecule is primarily produced by monocytes, macrophages, dendritic cells, and B-cells. The main functions of IL-12 include the promotion of IFN-γ production from natural killer in cell-mediated immunity [78]. IL-10 is a major inhibitor of IL-12 production by decreasing Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) activation and the association of IL-12p40 promoter with RNA polymerase [78].

Consistent with the anti-inflammation and anti-oxidation role, our results suggested that VITD effectively downregulated in vitro the production of targeted cytokines in DR-related stimuli, suggesting that VITD could block retinal inflammation and oxidative stress associated with DR. Retinal pathologies such as DR are complex diseases with several and different processes involved. We evaluated the effect of vitamin D as an option to restore or to help with the damage provoked by oxidative stress and inflammation. However, different therapies could target multiple steps of oxidative stress for the prevention of this multifactorial blinding complication of diabetes. Santos et al. revised therapies with vitamins and supplements used to treat diabetic retinopathy, and there are various strategies in order to prevent superoxide accumulation, maintain mitochondrial homeostasis, or protect against DNA damage [79]. Other molecules could also be beneficial, even in combination with vitamin D. Although promising, one of the main limitations of this research is the use of an immortalized cell line in which some transcription alterations are produced. Therefore, the results obtained need to be confirmed in primary cells and in experimental in vivo models of retinopathy. Whether these results can be replicated in in vivo models of DR and clinical trials remains to be elucidated.
