Vitamin D, the Vitamin D Receptor, Calcitriol Analogues and Their Link with Ocular Diseases

The global prevalence of eye diseases continues to grow, bringing with it a reduction in the activity levels and quality of life of patients, and partial or complete blindness if left untreated. As such, there is considerable interest in identifying more effective therapeutic options and preventive agents. One such agent is vitamin D, known to have a range of anti-cancer, anti-angiogenic, anti-inflammatory and anti-oxidative properties, and whose deficiency is linked to the pathogenesis of a range of cardiovascular, cancer, and inflammatory diseases. This review presents the current stage of knowledge concerning the link between vitamin D and its receptor and the occurrence of eye disease, as well as the influence of analogues of calcitriol, an active metabolite of vitamin D. Generally, patients affected by various ocular disorders have vitamin D deficiency. In addition, previous findings suggest that vitamin D modulates the course of eye diseases and may serve as a marker, and that its supplementation could mitigate some disorders. However, as these studies have some limitations, we recommend further randomized trials to clarify the link between vitamin D and its activity with eye disease.


Introduction
The human eye is a delicate structure that receives optical information from the environment, allowing light perception and vision. However, its operation is inhibited by a range of factors, such as aging, genetic predisposition, excessive light exposure, chronic hyperglycemia, autoimmune diseases, angiogenesis, inflammation and oxidative stress; these may lead to the development of a range of eye diseases such as age-related macular degeneration, diabetic retinopathy or dry eye syndrome. In addition, most ocular disorders result in visual impairment or blindness. Approximately 405 million individual cases of visual impairment were reported in 2015, with 36 million people living with vision loss that year. Moreover, the number of patients with blindness is expected to increase about threefold by the year 2050 [1]. Visual impairment is also related to considerable economic burden for patients, their caregivers and medical care in developed countries [2]. In addition, blindness and visual impairment are two of the strongest risk factors for social isolation, reduced quality of life and decline of functional status. Furthermore, the majority of ocular diseases are diagnosed in advanced stages, which excludes effective therapy [3,4]. As such, prevention is a priority and a significant element of therapeutic approaches.
Numerous studies have demonstrated that a healthy diet limits the development or progression of eye disorders. Indeed, vitamin D deficiency, i.e., a 25-hydroxyvitamin D level lower than 20 ng/mL, is a global health problem [5]. The consumption of fruits and vegetables, which are source of polyphenols and carotenoids, as well as vitamins A, C and E, play a significant role in the prevention and treatment of ocular diseases. These compounds have multiple pro-health properties, mainly comprising anti-inflammatory, anti-oxidant and anti-angiogenic activities [6][7][8]. Nevertheless, other agents believed to In addition, the schematic shows the dietary sources of vitamin D. Sundried mushrooms and fatty fishes, including fish oils, are the sources of ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3), respectively. Both are metabolized to 25-hydroxyvitamin D2/D3 in the liver and, subsequently, to other metabolites. Nevertheless, vitamin D3 is almost twice as potent as ergocalciferol in increasing serum 25-hydroxyvitamin D, and the supplementation of vitamin D2 does not result in as high a blood level of 25-hydroxyvitamin D [14,15].
Vitamin D can also be metabolized locally, including in the eyes (Figure 1). Recent studies indicate that vitamin D hydroxylases are present in ocular structures such as the cornea, ciliary body, sclera and retina, suggesting that vitamin D may be an important intraocular mediator in eye diseases [16,17]. Finally, vitamin D supplementation can be used to maintain adequate concentrations.
The biological action of calcitriol, the active form of vitamin D, is mediated by VDR, a member of the nuclear hormone receptor superfamily. VDR functions as a heterodimer with the retinoid X receptor. This complex interacts with specific DNA sequences belonging to the vitamin D response element (VDRE), resulting in the activation or repression of transcription of genes influencing the function of vitamin D [18,19]. It must be emphasized that VDR is expressed in the structural elements of eye [16,20], and that certain polymorphisms of the VDR gene may be related to the occurrence of eye disease [21,22]. In addition, the schematic shows the dietary sources of vitamin D. Sundried mushrooms and fatty fishes, including fish oils, are the sources of ergocalciferol (vitamin D 2 ) and cholecalciferol (vitamin D 3 ), respectively. Both are metabolized to 25-hydroxyvitamin D 2 /D 3 in the liver and, subsequently, to other metabolites. Nevertheless, vitamin D3 is almost twice as potent as ergocalciferol in increasing serum 25-hydroxyvitamin D, and the supplementation of vitamin D 2 does not result in as high a blood level of 25-hydroxyvitamin D [16,17].

Calcitriol Analogues
The biological action of calcitriol, the active form of vitamin D, is mediated by VDR, a member of the nuclear hormone receptor superfamily. VDR functions as a heterodimer with the retinoid X receptor. This complex interacts with specific DNA sequences belonging to the vitamin D response element (VDRE), resulting in the activation or repression of transcription of genes influencing the function of vitamin D [18,19]. It must be emphasized that VDR is expressed in the structural elements of eye [14,20], and that certain polymorphisms of the VDR gene may be related to the occurrence of eye disease [21,22].

Calcitriol Analogues
As mentioned above, calcitriol takes part in the homeostasis of calcium and phosphate, and seems to be a potent agent in the treatment of many diseases. However, the therapeutic effect of exogenous calcitriol often require it to be present in supraphysiological concentrations resulting in toxic and undesirable effects, primarily hypercalcemia, hypercalciuria, excessive bone resorption and vascular calcification [23,24]. To avoid these side effects, structural analogues of calcitriol have been created, e.g., 22-oxacalcitriol and 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D 3 , and these have been found to have potential efficacy in ocular disorders [25,26]. These compounds deserve attention, and their detailed role in some eye diseases are described in the next section.

Age-Related Macular Degeneration (AMD)
AMD is an acquired eye disease that affects the macula, resulting in visual impairment, and even blindness. Various risk factors have associations with development and progression of AMD, including angiogenesis, the inflammatory response, lipofuscin accumulation in retinal pigment epithelial cells, aging, smoking and sunlight exposure [27,28]. In addition, an important component of the neovascular form of AMD is the formation of new retinal blood vessels, as is apoptosis, pyroptosis and necroptosis of retinal cells, and impaired autophagy. It has been reported that vitamin D modulates programmed death pathways, and that it may influence autophagy and possess anti-angiogenic properties [29]. In addition, vitamin D deficiency is associated with a thinning of the ganglion cell complex and retinal nerve fiber layer [30]. Thus, vitamin D could modulate the course of AMD. Some studies indicate a potential association between 25-hydroxyvitamin D deficiency in serum, possibly associated with low vitamin D intake, and the risk of AMD, including the neovascular form [31][32][33][34]. In addition, some studies suggest that high levels of 25-hydroxyvitamin D can decrease the risk of AMD [35,36]. Nevertheless, most available studies, including meta-analyses, do not indicate that high serum vitamin D levels have a significant protective influence against the occurrence of any stages or subtypes of AMD [37][38][39][40][41][42][43]. In addition, in one study, vitamin D was not found to have any significant overall effect on the incidence and progression of AMD [44]. While it is reasonable to expect discrepancies between studies as a result of heterogeneity in study procedures and lack of longitudinal designs, the literature does not provide any clear evidence of a definitive association between serum 25-hydroxyvitamin D level and AMD

Diabetic Retinopathy (DR)
One common microvascular ocular complication of diabetes mellitus that remains the leading cause of preventable blindness is diabetic retinopathy (DR). As of 2010, this eye disorder affected over 100 million patients worldwide, and this number is expected to almost double by the year 2030 [45,46]. Studies indicate that that patients with DR had lower serum levels of vitamin D compared with those without [47] and that vitamin D (25-hydroxyvitamin D) deficiency is significantly associated with an almost twofold greater risk of DR [48][49][50][51][52][53], especially the proliferative form [54,55]. This dependence has been observed for patients with type 1 and type 2 diabetes mellitus [56][57][58]. Furthermore, it has been proposed that a serum concentration of 25-hydroxyvitamin D ≤ 18.6 ng/mL may serve as a sensitive and specific indicator for the proliferative type among subjects with DR [59]. Interestingly, endogenous vitamin D 3 metabolites, such as 1,25-dihydroxyvitamin D, 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D, could be better for predicting DR than total vitamin D level [60].
These findings regarding vitamin D deficiency in patients with DR suggest that vitamin supplementation may offer a protective effect. Indeed, vitamin D has been found to have neuroprotective properties, and its deficiency led to thinning and reduction of mean retinal nerve fiber layer thickness in early-stage DR subjects [61]. In addition, the protective effect of vitamin D in DR may also result from its ability to reduce cholesterol level, improve HDL cholesterol concentration, inhibit the activity of pro-inflammatory cytokines or pro-angiogenic and fibrotic factors, and downregulate ROS production, which play an important role in the pathogenesis of DR [62][63][64][65].
On the other hand, meta-analyses suggest that some polymorphisms of the VDR gene may also act as susceptibility markers for predicting the risk of DR and for earlier diagnosis of eye disease. Polymorphisms BsmI, ApaI, and FokI of the VDR gene are all significantly associated with DR susceptibility [66,67].

Optic Neuritis (ON)
ON is an inflammatory condition of the optic nerve and is a frequent cause of acute injury in adults and children. Its most common etiology is multiple sclerosis (MS) [68]. As vitamin D insufficiency appears to be a risk factor for the development of MS [69], it is possible that vitamin D may indirectly have an impact on the occurrence of ON and related changes. ON is typically associated with a thinning of the retinal nerve fiber layer (RNFL) caused by declines in axonal and macular volume [70]. Studies have found that vitamin D supplementation does not protect from the loss of RNFL and macula thickness in patients with unilateral ON (i.e., who do not fulfill the McDonald criteria for MS) concomitant with vitamin D insufficiency [70]. Nevertheless, ON may be the first manifestation of MS and occur before a full diagnosis of MS itself. Subjects with acute monosymptomatic ON are often characterized by low 25-hydroxyvitamin D levels in serum; however, these low levels of vitamin D are not correlated with the severity of ON [71]. Nevertheless, in ON patients with a low serum level of vitamin D but without a diagnosis of MS, vitamin D supplementation may delay the occurrence of secondary ON and the subsequent conversion to MS [72]. However, vitamin D levels lower than 30 ng/mL did not appear to be associated with RNFL and macular volume thickness in subjects with MS who were not affected by ON [73]. Therefore, the protective effect of vitamin D in ON is uncertain and requires further clinical trials.

Retinal Vein Occlusion (RVO)
One of the most common second causes of retinal vascular abnormality, and a frequent sight-threatening disorder, is retinal vein occlusion (RVO). This disease is classified as central RVO (CRVO), hemi central RVO (HCRVO) and branch RVO (BRVO) depending on the location of occlusion of the vein [74,75]. As vitamin D deficiency is believed to be associated with endothelial dysfunction and vascular diseases [76], RVO patients could have low levels of vitamin D in their serum. Indeed, significantly lower mean 25-hydroxyvitamin D serum levels have been noted in Indian subjects with RVO compared to controls; however, subjects with CRVO and BRVO did not demonstrate significant differences in mean vitamin D level compared to controls [77]. Interestingly, among CRVO patients, the mean concentration of 25-hydroxyvitamin D can be statistically significant in subjects aged under 75 years [78]; therefore, vitamin D level may be a helpful indicator for the prophylaxis, therapy or diagnosis of CRVO in this group.

Myopia
Myopia is the most frequent eye disorder worldwide and is a refractive anomaly. It is primarily caused by an increase in the axial length of the eyeball and elevates the risk of other ocular diseases. Typically, myopia starts in childhood [79,80]. Data indicate that the prevalence of longer axial length and myopia is significantly higher in children, young adults and adults with vitamin D deficiency compared to those with sufficient levels [81][82][83][84][85][86]. However, the relationship between myopia and vitamin D has been contradicted in some studies [87][88][89][90][91]. This discrepancy may result from other factors affecting vitamin D metabolism, such as the time spent outdoors (ultraviolet radiation exposure). It has been demonstrated that increased time spent outdoors, and ultraviolet B exposure, are effective in preventing the onset of myopia and slowing the myopic shift in refractive error associated with reduced myopia [92,93]. Further longitudinal studies are needed to determine whether higher serum 25-hydroxyvitamin D concentration is protective against myopia.

Retinoblastoma
Retinoblastoma is the most common intraocular cancer in children, and new options and strategies for disease therapy are being sought [94]. Initial studies indicate that retinoblastoma cells have VDR expression [95,96]. Treatment with calcitriol has been found to inhibit Y79 cell growth, inducing apoptosis by increasing the level of Bax protein and decreasing that of Bcl-2 protein [95,96]. Calcitriol was also able to induce cell cycle arrest in the G0/1 phase [96]. Moreover, 1,25-dihydroxyvitamin D significantly limited retinoblastoma growth in athymic mice with subcutaneously-injected cancer cells, as well as tumor angiogenesis in a transgenic retinoblastoma murine model [97,98]. However, calcitriol treatment was found to be toxic, resulting in elevated mortality, hypercalcemia and kidney damage [95,99]. Thus, there is a need for compounds that can act as VDR agonists but with lower calcemic effects. Such candidates include the calcitriol analogs 1,25-dihydroxy-16-ene-23-yne-vitamin D 3 , 1α-hydroxyvitamin D 2 and 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol [95,[99][100][101]. It must be emphasized that low doses of calcitriol could consolidate the effect of chemotherapy, one of the therapeutic options of retinoblastoma, and improve treatment effectiveness. In addition, the combination of cisplatin and calcitriol significantly inhibited tumor growth in athymic mice with subcutaneously injected human Y79 retinoblastoma cells without any increase in mortality and with minimal nephrotoxicity [102].

Uveal Melanoma (UM)
UM is a malignant and common primary intraocular tumor with a significant propensity to metastasize in adults. In almost all cases, patients with liver metastasis die within six months, and the median survival time after diagnosis of metastasis is only 3.6 months [103][104][105]. Currently, effective adjuvant therapy is not available to prevent metastases, and neither is there any successful treatment once metastases have developed [106]. Due to the lack of any efficient therapy, new strategies for UM prevention and control are urgently required. Studies show that low vitamin D status is associated with an increased risk of cancer and poor prognosis [107]. Recent studies indicate that components of the vitamin D metabolism, such as VDR, CYP27B1 and CYP24A1, are present in the UM cells [108]. Moreover, a marked reduction of the VDR expression is inversely correlated with melanin level in UM cells, as well as an aggressive UM profile, which contributes to an increased risk of metastases and poorer prognosis [108]. Interestingly, one of the most common susceptibility factors for UM is the inability to tan [109]; the ultraviolet radiation needed for the synthesis of vitamin D appears to have a protective activity against UM [110]. Furthermore, compounds such as calcitriol and calcidiol, i.e., the biologically-active metabolites of vitamin D and its precursor, have been found to sensitize melanoma cells to radiation used in the therapy of UM [111].
Clearly, there is a possible role for vitamin D and its signaling elements in the treatment of UM. Nevertheless, little is known about the impact of vitamin D on the development and progression of UM, especially considering the detailed mechanisms of its action.

Non-Infectious Uveitis
Uveitis is an intraocular inflammatory condition of the uvea (i.e., the iris, ciliary body, and choroid) which may lead to blindness. Uveitis can be classified depending on the primary localization of the inflammation in the eye (as anterior, intermediate and posterior uveitis) or etiology (as infectious and non-infectious). Uveitis may also be clinically divided into active and inactive forms. It is important to note that non-infectious uveitis is an immune-mediated eye disorder associated with systemic diseases such as sarcoidosis [112]. Studies have revealed an association between hypovitaminosis D and an elevated risk of non-infectious uveitis. For example, vitamin D deficiency contributes to higher risk of uveitis in juvenile idiopathic arthritis [113]. Subjects with normal levels of vitamin D had 21% lower odds of non-infectious uveitis than those with low levels of vitamin D [114]. In addition, significantly decreased blood serum levels of 25-hydroxyvitamin D were detected in patients with non-infectious anterior and non-infectious acute anterior uveitis compared to healthy controls [115,116], as well as in subjects with active non-infectious uveitis compared to inactive non-infectious uveitis patients. Vitamin D supplementation and exposure to the sun were associated with lowered disease activity in patients with vitamin D deficiency [117]. However, every unit (1 ng/mL) increase in vitamin D level was found to be associated with 4% to 5% lower odds of developing non-infectious uveitis depending on the literature source [115,118]. Interestingly, patients with sarcoidosis-related uveitis demonstrated elevated median serum levels of 1,25-dihydroxyvitamin D, compared to those with uveitis due to other causes (132.4 vs. 108.0 pmol/L). In addition, patients with sarcoidosis-induced uveitis had a higher median ratio of 1,25-dihydroxyvitamin D/25hydroxyvitamin D (4.17 vs. 2.56): a ratio higher than 3.5 was found to be associated with the diagnosis of ocular sarcoidosis, with 68% sensitivity and 78% specificity [119].
Hence, vitamin D may play a role in the development and course of non-infectious uveitis; however, no detailed causal relationship has been identified. Further studies are necessary to determine the efficacy of vitamin D supplementation to mitigate and reduce risk of non-infectious uveitis.

Vogt-Koyanagi-Harada Disease (VKHD)
VKHD is a rare granulomatous autoimmune disorder that affects tissues and organs containing melanocytes: primarily the eye, inner ear, skin hair or meninges. It is characterized by panuveitis, which results in severely reduced visual acuity or even blindness if not treated appropriately. It is also accompanied by a varying degree of auditory, neurological and cutaneous manifestations. VKHD most frequently affects people from Asia, Latin America and the Middle East, as well as Native Americans [120]. It is known that vitamin D deficiency may be involved in the development of autoimmune diseases [121]. Patients with active VKHD were found to have lower serum levels of 1,25-dihydroxyvitamin D compared to subjects with inactive VKHD and healthy controls. Moreover, 1,25-dihydroxyvitamin D was found to inhibit the proliferation of peripheral blood mononuclear cells and CD4 + T cells, and to slow the production of interleukin (IL)-17 and interferon gamma (IFN-γ) by these cells [122].
Some polymorphisms in the genes of the vitamin D pathway may increase the occurrence of VKHD. One study did not find any polymorphisms in genes such as VDR, CYP24A1 and CYP27B1 that could contribute to VKHD; however, one polymorphism (c.852G > A; p.284 M > I) detected in CYP2R1 appears to be associated with disease and may be pathogenic [123].
Hence, vitamin D may be involved in the development of VKHD, and one CYP2R1 polymorphism may have a causative role. Nevertheless, studies on a larger cohort of patients are necessary to confirm these observations.

Glaucoma
Glaucoma is a leading cause of irreversible vision loss worldwide. It is an optic neuropathy characterized by progressive degeneration of retinal ganglion cells. It is also associated with slow or difficult outflow of aqueous humor, elevated intraocular pressure (IOP) and damage of the trabecular meshwork [124,125]. Interestingly, 1α,25dihydroxyvitamin D 3 is able to protect against oxidative stress-induced damage to the trabecular meshwork known to occur in glaucoma, suggesting that vitamin D may have therapeutic properties [124]. In addition, the same compound has been found to reduce IOP in non-human primates [25]. Unfortunately, this result has not been confirmed in humans: no difference in IOP was found between healthy subjects with low and high serum 25-hydroxyvitamin D levels. Furthermore, vitamin D supplementation in the patients with low serum concentration of 25-hydroxyvitamin D did not change IOP [126].
Interestingly, except for a group of postmenopausal women, vitamin D deficiency may be considered an independent risk factor for open-angle glaucoma. A reverse Jshaped association was found between serum 25-hydroxyvitamin D levels and the risk of open-angle glaucoma, and patients with glaucoma, including open-angle glaucoma, were characterized by lowered serum 25-hydroxyvitamin D (by about 15% compared to the controls) [127][128][129]. Open-angle patients were also found to have significantly lower serum levels of calcitriol, another metabolite of vitamin D, compared with the agematched controls [130]. Moreover, the serum level of vitamin D could serve as a marker of the severity of primary open-angle glaucoma, as patients with advanced glaucoma had decreased concentrations of 25-hydroxyvitamin D in comparison to early glaucoma and healthy subjects [131].

Cataract
Cataract is a common ophthalmic disorder that is associated with clouding of the lens [132]. Available data show that almost 100 million people worldwide are affected by cataract. The possible risk factors include increasing age, female sex, eyeball trauma, ultraviolet-B-exposure, cigarette smoking, diet with a high glycemic index, malnutrition, and genetic factors [133]. Other diseases such as diabetes mellitus, renal impairment, metabolic syndrome and arterial hypertension increase the risk of cataract [133]. In addition, vitamin D may have an effect on cataract. Patients with age-related cataract dad lower mean serum levels of 25-hydroxyvitamin D compared to controls (7.6 ± 5.5 ng/mL vs. 18.5 ± 9.6 ng/mL), and this change was statistically significant. In addition, among different types of age-related cataract, cases of nuclear cataract were characterized by the lowest level of vitamin D [134]. Moreover, vitamin D deficiency in serum was found to be associated with age-related cataract, including the early form of disease [135,136]. Interestingly, studies indicate that the risk of nuclear cataract is inversely correlated with the serum level of 25-hydroxyvitamin D, with it appearing to have a protective role against nuclear cataract [137,138].
Although posterior subcapsular cataract resembles hypocalcemic cataract, the relationship between low serum vitamin D levels and posterior subcapsular cataract formation remains unclear. Brown et al. (2015) suggest that vitamin D deficiency may contribute to the development of posterior subcapsular cataract, and that some comorbidities and nonophthalmic interventions are associated with posterior subcapsular cataract in the presence of decreased levels of 25-hydroxyvitamin D. Interestingly, some patients with early-stage posterior subcapsular cataract co-existing with vitamin D deficiency had a resolution of changes in the lens following daily supplementation of 5000 IU of 25-hydroxyvitamin D over a 2-year follow-up period [139]. In contrast, no vitamin D deficiency was later observed in patients with posterior subcapsular cataract [140]. In turn, diabetic cataract was characterized by significantly higher levels of 25-hydroxyvitamin D in aqueous humor compared to senile cataract [141].
Vitamin D may well play a role in lens metabolism. However, further extensive trials are needed to explain the correlation between vitamin D concentration and cataract, and to understand the detailed mechanisms of vitamin D activity in this regard.

Scleritis
Scleritis is an infrequent inflammatory disorder of the sclera that may be caused by infectious factors, trauma, drugs or irradiation, and one that frequently accompanies immune-mediated diseases. The immune system appears to play an important role in the pathogenesis of non-infectious scleritis [142]. Therefore, there is an association between scleritis and vitamin D, which is able to suppress the immune response mainly by the modulation of T lymphocyte activity. Multivariate analyses revealed a statistically significant link between non-infectious scleritis and decreased 25-hydroxyviatmin D levels at any time point before or after the onset of scleritis (28.1 ng/mL vs. 34.4 ng/mL in controls, p = 0.009) and any time point before (24.1 ng/mL vs. 34.4 ng/mL in controls, p = 0.033). In addition, the same analysis showed that the odds of developing scleritis were 4% lower for every unit (1 ng/mL) increase in the vitamin D level (OR = 0.96, 95% CI = 0.93-0.99, p = 0.009) [118]. Further studies are necessary to establish the potential role of vitamin D supplementation in the development of scleritis.

Dry Eye Syndrome (DES)
DES is a common ophthalmic disorder affecting the ocular surface, which is marked by persistent symptoms. One of the main mechanisms contributing to the development of disease is the inflammatory reaction [143]. Studies indicate that calcitriol, an active metabolite of vitamin D, is able to inhibit dry eye-related corneal inflammation and apoptosis in in vitro and in vivo experimental models of DES, highlighting the protective role of vitamin D [144][145][146]. DES patients had significantly lower serum levels of 25-hydroxyvitamin D, and this correlated with ocular surface disease index [147][148][149][150]. Furthermore, DES patients also displayed significantly lower concentrations of vitamin D in their tears [151], which may result from the fact that the vitamin D deficiency is associated with tear hyperosmolarity, a dysfunction of tear film and reduction of Schirmer's test value [152,153]. Serum level of 25-hydroxyvitamin D was also found to correlate positively with tear break-up time and tear secretion [154].
In contrast, some studies did not show any association between DES and lowered vitamin D levels [155,156]. Vitamin D (cholecalciferol) supplementation was found to improve tear break-up time, tear secretion, eyelid margin hyperemia and the severity of symptoms in patients with DES refractory to conventional treatment concomitant with vitamin D deficiency [157]. In addition, cholecalciferol supplementation enhanced the efficacy of artificial tears and hyaluronate, which are used in the therapy of DES, and may be a useful adjuvant treatment for subjects with DES refractory to topical lubricants [158]. Furthermore, two single nucleotide polymorphisms (Foklrs2228570 and Apal-rs7975232) in the VDR gene seem to be associated with DES and may potentially be used in the diagnosis of disease [22].

Vernal Keratoconjunctivitis (VKC)
VKC is a chronic, allergic, inflammatory disease of the tarsal and/or bulbar conjunctiva, which occurs seasonally and mainly in the pediatric population [159]. Data show that vitamin D may mitigate allergic diseases [160]. It has been demonstrated that children with VKC have lower serum levels of 25-hydroxyvitamin D in comparison to healthy controls. In addition, a significant correlation has been confirmed between vitamin D level and VKC severity, including VKC objective score and basophils in conjunctival scraping [161]. In contrast, a more recent study confirmed an inverse correlation between serum vitamin D levels and VKC severity; however this difference was not statistically significant [162]. Another study found decreased mean serum concentrations of 25-hydroxyvitamin D in children affected by VKC. In addition, the VKC-children tended to spend less time outdoors during daylight compared to controls (160.7 ± 65.9 vs. 229.5 ± 101.2 min), suggesting that a reduced level of vitamin D may play a role. In addition, a statistically significant correlation was observed between serum 25-hydroxyvitamin D levels and time spent outdoors [163].
Severe VKC is often treated using immunomodulatory drugs, such as cyclosporine or tacrolimus [164]. Furthermore, studies indicate that treatment with 1% cyclosporine or 0.1% tacrolimus as eye drops may contribute to the improvement of VKC signs and symptoms, and that these changes are associated with an increase of 25-hydroxyvitamin D level in serum. This rise was higher in the children with limbal VKC than the tarsal form and could result from the fact that limbal VKC is less symptomatic [161,165]. The above data require confirmation and verification through large-scale clinical trials; however, their findings may provide a greater insight into the pathogenesis of VKC and creation of customized therapy.

Keratoconus
Keratoconus is a common, progressive, ectatic, degenerative disease of the cornea. It manifests as a thin cone resulting in refractive errors, such as irregular astigmatism or myopia, with impairment of visual acuity. Although the detailed pathogenesis of keratoconus formation remains unclear, environmental and genetic factors are believed to have an important role, and proposed mechanisms include inflammatory reaction, oxidative stress and proteolytic degradation in the corneal stroma [166,167].
Recent data suggest that keratoconus may be also associated with disturbances of the immune system, and the systemic inflammatory response related to autoimmune diseases can induce the onset of eye disease [168]. As vitamin D is known to have immunomodulating properties [169], some studies have attempted to identify a relationship between its level and the onset of keratoconus. One study found that patients with keratoconus had significantly decreased serum levels of vitamin D compared to age-and sex-matched controls; however, no statistically significant correlation was found between 25-hydroxyvitamin D concentrations and keratoconus severity, based on Pentacam measurements including anterior curvature [170]. Similarly, Zarei-Ghanavati et al. (2020) reported that although severe keratoconus (Krumeich criteria stage IV) was associated with the lowest level of 25-hydroxyvitamin D, no significant differences were observed among individual groups of patients [171].
Reduced vitamin D levels have been found to significantly increase the probability of non-progressive and progressive keratoconus compared to controls by 1.23 and 1.29 times, respectively. Nevertheless, despite both groups demonstrating significantly lower concentrations of vitamin D in comparison to controls, no significant differences were found between non-progressive and progressive disease [172].
There is clearly a need for further studies investigating the potential relationship between keratoconus and vitamin D and to determine whether vitamin D supplementation may prevent or inhibit the course of disease.

Pterygium
Pterygium is a chronic disease of the anterior segment of the eye characterized by benign, uncontrolled, fibrovascular growth of the bulbar conjunctiva across the cornea, leading to impairment of visual acuity [173,174]. Its pathophysiology is not fully known. Inflammation and angiogenesis are considered important in the course of disease. However, chronic exposure to ultraviolet light is believed to have a significant causative relationship with pterygium. Moreover, a strong link has been evidenced between disease and geographical latitude: the prevalence of pterygium is inversely associated with latitude, indicating that ultraviolet radiation in the development of this eye disorders. This may also indirectly suggest that vitamin D plays a role in the formation of pterygium [175][176][177][178].
This was confirmed by , who found that patients with pterygium had higher serum levels of 25-hydorxyvitamin D even after controlling for sunlight exposure time, and these changes were statistically significant. This positive association was found for both sexes. Nevertheless, it must be emphasized that despite significant difference between the patients with and without pterygium, those with eye disease had vitamin D insufficiency, with a mean serum 25-hydroxyvitamin D level of 20.4 ng/mL [179].
These results were later confirmed by another group, who showed significantly higher serum levels of 25-hydroxyvitamin D to be present in men with pterygium compared to healthy male controls. Importantly, the men with pterygium with more outdoor activity had more elevated concentrations than those with dominant indoor activity. No such relationship was observed in controls, indicating that vitamin D has a potential role in the development of pterygium. However, no such differences were found between female patients and healthy subjects [180]. Another study examined the relationship between pterygium, sun exposure, and serum 25-hydroxyvitamin D level in South Korean adults. The frequency of pterygium was found to be increased in elderly subjects and those who lived at low geographical latitudes. In addition, these patients had higher serum levels of 25-hydroxyvitamin D, suggesting that this may be positively correlated with the prevalence of pterygium [178]. However, a recent study in 2021 did not identify any changes in vitamin D concentration in both serum and tear fluid between the subjects with pterygium and healthy subjects [181]. None of the trials mentioned above evaluated or described the mechanisms underlying the relationship between vitamin D and pterygium. Although vitamin D has pro-health properties, including anti-inflammatory activities, it appears to have an insufficient impact on pterygium to prevent disease. Furthermore, many studies have not compared vitamin D concentration with characteristics of pterygium, such as length or histopathological evaluation. Therefore, further clinical trials are warranted.
Interestingly, a recent study did not find any difference between patients with pterygium and healthy subjects with regard to serum vitamin D level; however, VDR protein expression was found to be elevated in endothelial cells of micro-vessels, subepithelial stromal and intravascular inflammatory cells associated with pterygium compared to adjacent healthy conjunctival tissue [182]. Immunohistochemical assays found VDR localization to differ significantly in the pterygium cells compared to normal conjunctival cells. In healthy conjunctiva, VDR was localized mainly in the cytoplasm, while in pterygium cells, VDR was co-localized in the nucleus and cytoplasm. Hence, the nuclear signaling pathways related to VDR may be engaged in the pathogenesis of pterygium [183]. Further analyses and clinical trials are needed to standardize the role of VDR in pathogenesis and the development of pterygium.

Thyroid Eye Disease (TED)
Graves' disease, the most common cause of thyrotoxicosis, is an autoimmune disease affecting the thyroid gland. It is characterized by the presence of autoantibodies directed against antigens in the thyroid. These may cross-react with orbital antigens leading to TED, known also as Graves orbitopathy or thyroid-related orbitopathy. It is an inflammatoryfibrotic orbitopathy that includes orbital tissues, mainly extraocular muscles and orbital fat, causing diplopia, photophobia, exposure keratopathy and eye pain; if left untreated, the condition can generate compressive optic neuropathy [184,185].
Studies indicate that the vitamin D insufficiency may contribute to disturbances in immune system activity, which is an important factor in the development of autoimmune diseases [121]. Meta-analyses indicates that low vitamin D levels are related to the occurrence of Graves' disease, a lower likelihood of remission and a higher recurrence rate [186,187]. Decreased serum concentrations of 25-hydroxyvitamin D have also been noticed in patients with TED. A retrospective study by Sadaka et al. (2019) found that 20% of TED-patients had vitamin D deficiency (a level below 20 ng/mL), and 31% had insufficiency (a level between 20 and 29 ng/mL). However, these results were obtained using a relatively small sample size, and no correlation was made with clinical disease activity or with an unaffected cohort [188].
Assessment of vitamin D level and its supplementation may play an important role in the early management of Graves' disease by preventing the development of TED. Patients with Graves' disease concomitant with TED have lower serum levels of 25-hydroxyvitamin D compared to those with Graves' disease but without TED [189]. Research efforts are now directed toward identifying characteristics, including gene polymorphisms, which could modify the risk of TED and be associated with its occurrence; these could be used to facilitate early diagnosis. Maciejewski et al. (2020) found a C gene polymorphism rs2228570 (FokI) in VDR to occur more frequently in patients compared to unaffected subjects; this may be a risk factor contributing to the development of TED in patients of Caucasian origin with Graves' disease [190].

Benign Essential Blepharospasm (BEB)
BEB is a cranial dystonia characterized by hyperactivity and sustained, involuntary spasms of muscles around the eyes, such as orbicularis oculi, corrugator and procerus. This ocular disorder affects approximately 1.4-13.3 cases per 100,000 people, mainly women, and the typical onset of the disease occurs between the fifth and seventh decades of life. Although the detailed causes of BEB remain unknown [191][192][193], they may be associated with disturbances in the regulation of intracellular and extracellular ionized calcium (Ca 2+ ), an ion responsible for triggering muscle contraction [194]. Interestingly, patients with BEB demonstrate significantly lower serum calcium levels than healthy subjects [194]. This may indicate decreased vitamin D concentrations, whose deficiency is related to lowered ionized calcium levels and even hypocalcemia [195]. In one study, no significant differences in serum 25-hydroxyvitamin D level were found between BEB-patients and unaffected subjects, and only a moderate negative correlation was found between vitamin D levels and the severity of BEB based on the Jankovic score [194]. However, in another study, BEB patients demonstrated significantly lower 25-hydroxyvitamin D and calcium levels, which may suggest a potential cause of this disorder [196]. Further long-term prospective clinical trials are necessary to determine the role of vitamin D in the involvement of BEB pathophysiology.

Potential Mechanisms of the Vitamin D Action against Ocular Diseases
The ocular diseases described above have various risk factors, etiology and ethiopathogenesis. Inflammation, oxidative stress, angiogenesis, and apoptosis play an important role in the development of most of them [94,143,174]. It was shown that vitamin D was able to mitigate the inflammatory response mainly by inhibition of nuclear factor kappa B (NF-κB) signaling pathway, modulation of the immune cells activity and suppression of pro-inflammatory factor expression, such as cyclooxygenase-2 among others, resulting in a reduction of the prostaglandins level. In addition, vitamin D has been found to modulate apoptosis and downregulate the expression of vascular endothelial growth factor (VEGF) inhibiting angiogenesis [197]. Pro-health effects have also been confirmed for eye disorders. Vitamin D counteracted oxidative stress induced by hydrogen peroxide (H 2 O 2 ) in human retinal pigment cells, and mitigated the inflammation induced by H 2 O 2 through a decrease of the protein expression of interleukin (IL) 1β (IL-1β), IL-8, tumor necrosis factor alpha (TNF-α). Furthermore, the anti-inflammatory effect of vitamin D was confirmed in another in vitro model using lipopolysaccharide (LPS)-stimulated human retinal pigment epithelial cells. These data show that vitamin D may inhibit retinal diseases, such as AMD and DR, limiting inflammation [198,199]. The intraperitoneal administration of calcitriol and 22-oxacalcitriol significantly attenuated lesion volume in laser-induced choroidal neovascularization using a mouse model. Therefore, vitamin D and calcitriol analogues may have potential as an interventional treatment for ophthalmic neovascular indications [26]. In hyperosmotic stress-induced human corneal epithelial cells, calcitriol inhibited the reactive oxygen species (ROS)-NLR family pyrin domain containing 3 (NLRP3)-IL-1β signaling axis via activation of the Nrf2-antioxidant pathway, indicating that it may prevent and mitigate DES-related corneal inflammation and oxidative stress at an early stage [144]. Calcitriol is also able to prevent human corneal epithelial cells from apoptosis via activation of autophagy via the VDR pathway [146]. The potential therapeutic mechanism of calcitriol was also revealed in glaucoma. This compound attenuated oxidative stress-induced damage in human trabecular meshwork cells by inhibiting the transforming growth factor beta (TGF-β)-SMAD family member 3 (SMAD3)-VDR pathway [124]. On the other hand, VDR, including its activation and inhibition, may play an important role in the development and progression of ocular diseases. For example, inhibition of VDR exerted a protective role in high-level glucose-induced damage of retinal ganglion cells by activating the signal transducer and activator of the transcription 3 (STAT3) pathway, indicating the potential role of VDR in DR [200]. Mechanisms of vitamin D action in ocular diseases are complex and not fully known. It seems that cholecalciferol and calcitriol may exert anti-inflammatory, anti-oxidative and anti-angiogenic effects accompanied by a reduction of development and progression of ocular diseases. Nevertheless, further studies are necessary to explain the detailed mechanisms of vitamin D action in eye disorders and their association with the VDR activity.

Summary and Conclusions
Based on the papers included in this review, it appears that vitamin D level may be associated with a range of eye diseases, including DR, ON, RVO, myopia, UM, non-infectious uveitis, VKHD, glaucoma, cataract, scleritis, DES, VKC, keratoconus, pterygium, TED and BEB. Measurement of vitamin D level, primarily 25-hydroxyvitamin D, could be a practical marker of the clinical course and severity of some ocular disorders, or could even be used to predict their risk. The data described in this review indicate that vitamin D has some pro-health properties which can be used against the ocular diseases. Therefore, vitamin D may be an agent supporting the available treatment of eye disorders. For example, it has been suggested that deficiency of the vitamin D occurs in patients with Sjögren's Syndrome-related dry eye, and this may result in a modulation of the clinical course [201]. That is why vitamin D may be considered as a modulator of the clinical course of ocular diseases. In addition, vitamin D may serve as marker of the advancement of the disease, since its level is correlated with severity of the symptoms [148]. Thus, vitamin D may be a tool in the diagnostic and therapeutic process, although further studies are required to confirm this. In addition, some VDR gene polymorphisms may serve as prognostic markers. Interestingly, calcitriol analogues appear to limit the development and progression of retinoblastoma.
The studies a described above have some limitations. First, vitamin D deficiency is becoming an increasing problem worldwide, and was observed in most study participants, even those without apparent disease. It is also not always clear whether vitamin D deficiency is the cause or consequence of a disorder, and the molecular mechanisms of vitamin D action remain poorly understood. Other significant limitations of the studies include the small number of patients, high heterogeneity of selected groups and variation in previous vitamin D supplementation. In addition, considerable variation has occurred in the methods used to measure vitamin D serum concentration, environmental conditions and individual factors (including sunlight exposure, physical activity). Most importantly, vitamin D levels vary seasonally, and this would affect the results depending on when the study was performed. Therefore, further randomized controlled trials are needed to clarify conflicting results.
Selected clinical studies and trials investigating the link between vitamin D, VDR and ocular diseases are summarized in Table 1.  Cross-sectional study 1790

Measurement and comparison serum 25(OH)D levels
Association between DR and prevalence of vitamin D deficiency [48] Population-based cross-sectional study 2113 participants aged ≤ 40 years

Evaluation of blood 25-(OH)D levels and ophthalmic examinations
Inverse association of blood 25(OH)D levels with any DR and proliferative DR only in men [49] Meta-analysis Literature database Identification of the association between serum vitamin D levels and DR Statistically significant association between vitamin D deficiency and DR [50] Retrospective study 3054 Asian Indians with type 2 diabetes mellitus

Evaluation of blood 25-(OH)D levels and ophthalmic examinations
Association between lower serum 25(OH)D and increased severity of DR. Association between vitamin D deficiency and two-fold increased risk for proliferative DR [51] Cross-sectional study 638 patients with type 2 diabetes mellitus

Evaluation of blood 25-(OH)D levels and clinical examinations
Vitamin D deficiency is considered as a risk factor for DR [52] Cross-sectional study 4767 diabetic patients

Evaluation of blood 25-(OH)D levels and ophthalmic examinations
Association between lower serum 25(OH)D and higher prevalence of DR in middle-aged and elderly diabetic adults [53] Clinic-based, cross-sectional study 221subjects Evaluation of blood 25-(OH)D levels and ophthalmic examinations Diabetic subjects, especially those with PDR, have lower 25(OH)D levels than those without diabetes [54] Hospital-based cross-sectional study 889 type 2 diabetic patients with or without DR

Evaluation of blood 25-(OH)D levels and clinical examinations
Vitamin D deficiency is significantly associated with risk of proliferative DR [55] Cross-sectional study 517 subjects aged 8-20 years with type 1 diabetes mellitus

Evaluation of blood 25-(OH)D levels and ophthalmic examinations
Association between the vitamin D deficiency and increased prevalence of DR in young people with type 1 diabetes mellitus [56] Meta-analysis Literature database Identification of the association between serum vitamin D levels and DR Association between the vitamin D deficiency and increased risk of DR patients with type 2 diabetes mellitus [57] Retrospective study 182 with type 1 diabetes mellitus

Evaluation of blood 25-(OH)D levels and ophthalmic examinations
Association between the vitamin D deficiency and increased prevalence of DR in patients with type 1 diabetes mellitus [58]  Evaluation of blood 25-(OH)D, 1,25(OH) 2 D, 24,25(OH) 2 D levels and ophthalmic examinations Association between vitamin D 3 metabolites and DR, whereas lack of these dependence for total vitamin D levels [60] Tertiary care center based cross-sectional study Diabetes mellitus without DR (24), non-proliferative DR (24), proliferative DR (24) and controls (24) Evaluation of blood 25-(OH)D levels and ophthalmic examinations Serum vitamin D levels of ≤ 18.6 ng/mL is marker for proliferative DR [59] Systematic review and meta-analysis Literature database Identification of the association between VDR gene polymorphisms and DR susceptibility There was no substantial association between 25(OH)D levels and spherical equivalent or odds of myopia [89]    Identification of the VDR gene polymorphisms and thyroid-associated orbitopathy susceptibility C allele of rs2228570 VDR gene polymorphism may contribute to the development of thyroid-associated orbitopathy [190] [194] Retrospective case-control study 20 patients with BEB and 20 age-and gender-matched health subjects Evaluation of blood 25-(OH)D levels and clinical examinations Strong negative correlation between disease severity and reduced 25(OH) vitamin D in patients with BEB [196] Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.