The Role of the P2X7 Receptor in Ocular Stresses: A Potential Therapeutic Target

The P2X7 receptor is expressed in both anterior and posterior segments of the eyeball. In the ocular surface, the P2X7 receptor is activated in case of external aggressions: preservatives and surfactants induce the activation of P2X7 receptors, leading to either apoptosis, inflammation, or cell proliferation. In the retina, the key endogenous actors of age-related macular degeneration, diabetic retinopathy, and glaucoma act through P2X7 receptors’ activation and/or upregulation of P2X7 receptors’ expression. Different therapeutic strategies aimed at the P2X7 receptor exist. P2X7 receptor antagonists, such as divalent cations and Brilliant Blue G (BBG) could be used to target either the ocular surface or the retina, as long as polyunsaturated fatty acids may exert their effects through the disruption of plasma membrane lipid rafts or saffron that reduces the response evoked by P2X7 receptor stimulation. Treatments against P2X7 receptor activation are proposed by using either eye drops or food supplements.

P2X7 receptors show complex gating behavior: several seconds of ATP exposure induces P2X7 receptors' dilatation from a channel that allows for the passage of small cations to a pore that allows for permeation of larger cations and dyes such as YO-PRO-1 (see YO-PRO-1 staining protocol in [25]). P2X7 receptor activation triggers numerous cellular effects from oxidative stress to apoptosis, including inflammation. As previously described, P2X7 receptors activate apoptotic caspases 3, 8, and 9 [26,27], and are involved in actin reorganization and plasma membrane blebs formation through p38, Mitogen-Activated Protein Kinases (MAPK), and Rho-GTPases [28]. The activation of P2X7 receptors can also induce an inflammatory response through the formation of the inflammasome complex, which leads to proinflammatory cytokine release [26,[29][30][31][32][33][34][35][36]. All these toxic cellular events occur during the pathogenesis of degenerative disorders, which highlights the pivotal role of P2X7 receptors in these diseases. It is worth mentioning that P2X7 receptors are also involved in life signals such as cell proliferation [37,38]. This effect on proliferation has been associated to wound healing when P2X7 receptor activation is induced [39] but also to cancer when P2X7 receptor expression is increased, allowing cancerous cell survival and proliferation [40,41]. The proliferative effects triggered by P2X7 receptor activation may be elicited at basal or low ATP concentrations [42], and/or depends on isoform expression [43].
P2X7 receptors are of great interest to toxicologists because as membrane receptors, they can be easily targeted by therapeutic formulations to block the toxic mechanisms they trigger. This review will be dedicated to the implication of P2X7 receptors in ocular toxic stresses and the existing modulators that are or could be used in ophthalmology.

Exogenous Stresses: Chemical and Mechanical Injuries
The ocular surface is vulnerable to potential environmental stresses by the nature of its function and anatomic location. This section will focus on the role of P2X7 receptors in the main exogenous stresses impacting the ocular surface.

Preservatives
Despite their well-known toxicity for the ocular surface, preservatives are still present in numerous topical ocular medications. The quaternary ammonium benzalkonium chloride (BAC) would be the most frequently used preservative for preparations, such as multidose eye drops, that require the inclusion of an antimicrobial preservative. Preserved multidose eye drops are generally used for long-term treatments such as glaucoma or in case of ocular infections that need long-term antibiotic treatment and repeated treatments (sometimes more than eight times a day). It has now been clearly demonstrated that BAC can induce ocular discomfort, dry eye, itching, or foreign body sensation [44,45]. Those symptoms have been linked, inter alia, to the apoptosis of corneal and conjunctival cells [46][47][48]. We have demonstrated that BAC, at the same concentrations as those used in eye drops (0.0025-0.01%), activates the P2X7 receptor as a consequence of ATP release, leading to the death of corneal and conjunctival cells [11,49,50]. Preserved latanoprost, travoprost, and bimatoprost solutions, all of them being antiglaucoma prostaglandin analog eye drops, induced the activation of P2X7 receptors (+130% to +400%) in conjunctival cells [50] that could partially contribute to inflammatory stimulation throughout the ocular surface in glaucoma patients under treatment. Preserved ofloxacin fluoroquinolone eye drops induced high activation of P2X7 receptors (+900% to +5500%) on ocular surface cells [49]. In that case, the P2X7 receptor acts as a P2Z/P2X7 cytolytic receptor, which may explain the corneal perforations observed after repeated treatment with preserved fluoroquinolone eye drops [51].
In the field of contactology, preservatives are widely used to clean contact lenses using multipurpose solutions. Polidronium chloride (a quaternary ammonium) and polyhexamethylene biguanides (PHMB) are the most commonly used preservatives in contactology. Despite the very low concentrations of preservatives present in multipurpose solutions (polidronium chloride around 0.001%, and PHMB between 0.00005% and 0.0001%), P2X7 receptors were activated on ocular surface cells after a short incubation time [52,53]. Apoptosis induced by multipurpose solutions can lead to contact lens intolerance over time; that is why eye care professionals recommend the use of a supplemental rinse step of contact lenses with unpreserved saline solutions [54-56].

Surfactants
Surfactants may be included in ophthalmic suspensions to disperse the drug effectively during manufacturing and product use. They are also used to stabilize emulsions that are generally prepared by dissolving or dispersing lipophilic active ingredients into an oil phase by adding suitable emulsifying agents and mixing with water to form oil-in-water emulsions. Nevertheless, excessive amounts can lead to irritation in the eye [57,58]. Sodium lauryl sulfate (SLS) is an anionic surfactant used in cosmetics (toothpastes, shampoos, shaving foams, etc.), and yet it is classified as an irritant product. Pauloin et al., indicated that SLS activates P2X7 receptors using an in vitro corneal cell model and that these P2X7 toxic effects can be inhibited by high-molecular weight hyaluronan [59]. Polysorbates are used in lubricant eye drops to help reduce tear evaporation by stabilizing the lipid layer. They are also widely used, as well as caprylocaproyl polyoxylglycerides (Labrasol™) and polyethoxylated castor oil (Cremophor™ EL), in nanoemulsions for ophthalmic preparations to stabilize lipophilic active ingredients such as ciclosporin A [60]. Nevertheless, the cytotoxic effects they exert on ocular surface cells [61,62] may limit their use. We showed that castor oil, caprylocaproyl polyoxylglyceride, and polysorbate 85 all induced P2X7 receptor activation in conjunctival cells [63,64].

Trauma
Corneal abrasion is one of the most common eye injuries. The causes are as numerous as they are different: tree branches, makeup brushes, a finger, a pet, workplace debris, sports equipment, sand, dust, etc. The main consequences of corneal abrasions are significant discomfort, red eyes, and photophobia [65], and complications can be severe and may lead to blindness if not treated correctly. Corneal abrasions lead to the loss of corneal cells, which makes the eye more susceptible to infection. Therefore, wound healing in the cornea is essential for maintaining the health of the ocular tissue and preventing pathologies. The early response after injury is critical as it initiates essential signaling pathways required for proper wound healing. The P2X7 receptor is necessary for the healing of abrasion wounds by promoting epithelial cell adhesion to the basement membrane coordinating Ca 2+ mobilization, cytoskeletal rearrangement, and normal stromal collagen structure [12,13]. Interestingly, in P2X7 −/− mice, the downward trend in the rate of epithelial wound repair was associated to deleterious morphologic changes at the leading edge of the wound, compared to control mice. Both P2X7 and P2Y2 receptors are necessary for proper corneal wound repair, with P2X7 receptors coordinating signals along the wound margin and P2Y2 coordinating signals back from the wound. Mankus et al. observed that the P2X7 receptor in the corneal epithelium is expressed in both full-length and variant forms and displays different functions [66]. The variant P2X7 receptor may be responsible for corneal tissue flexibility: it is mainly expressed in corneal cells that migrate and become stratified during wound healing. More recently, it has been shown that P2Y2 receptor stimulation supplemented with a P2X7 agonist such as BzATP can improve corneal wound healing outcomes [67].

Endogenous Stresses: Biological Stresses
An endogenous stress takes its origin from the inside of the organism. The P2X7 receptor is related to numerous biological endogenous stresses that occur in the eye, especially in the retina. Among these stresses, we can highlight the main actors of age-related macular degeneration (AMD) such as amyloid β and oxysterols, high glucose inducing diabetic retinopathy, and increased hydrostatic pressure leading to glaucoma.

AMD
AMD is one of the most common causes of severe vision loss worldwide. Proteolipidic deposits called drusen, formed in the retina, characterize AMD. Amyloid β, a highly toxic peptide, is found in drusen [68,69]. We demonstrated that the P2X7 receptor plays a key role in amyloid β-induced degeneration of retinal cells [70]. Amyloid β lead to apoptosis, a hallmark of retinal degeneration [71], via P2X7 receptor activation in human retinal Müller glial cells. The P2X7 receptor is also involved in microglia activation and alterations induced by amyloid β [72][73][74].
Oxysterols, oxidized derivatives of cholesterol, are known for their involvement in degenerative diseases [75,76]. In AMD, oxysterols accumulate in drusen and have been associated with the retinal degeneration process [77][78][79]. We recently highlighted the key role of P2X7 receptor activation in oxysterol-induced retinal degeneration in human retinal pigmented epithelial cells [80].

Diabetic Retinopathy
A high level of serum glucose triggers diabetes and ophthalmic complications such as diabetic retinopathy, which is characterized by alterations of retinal blood vessels. A high glucose concentration induced ATP-mediated apoptosis through P2X7 receptor activation in human fibroblasts [81]. In retinal neurons and microglia, intracellular calcium increased after purinergic stimulation [82]. In the case of diabetes, retinal microvessels are more sensitive to P2X7 receptor activation, meaning that for the same activation of P2X7 receptors, cellular effects and particularly apoptosis were increased in retinal microvessels exposed to high glucose concentrations [83].

Glaucoma
Glaucoma is a major cause of blindness worldwide. An increase in intraocular pressure alters the optic nerve, leading to vision loss. Acute elevation of intraocular pressure leads to an increase in P2X7 receptor expression in retinal ganglion cells [84]. Besides, neuronal mechanical deformations that occur after changes in intraocular pressure induced ATP release in retinal ganglion cells, triggering P2X7 receptor stimulation [85]. Stimulation of the P2X7 receptor then mediated retinal ganglion cell death, which plays a role in ischemia-induced neurodegeneration in the human retina [86]. More generally, ATP-induced activation of P2X7 receptors contributes to the pathogenesis of glaucoma [87].

Anti-P2X7 Strategies in Ophthalmology
The P2X7 receptor has received particular attention as a potential therapeutic target because of its widespread involvement in numerous ocular diseases. It appears to be a key regulatory element of apoptosis, inflammation, and cell death in general. Several P2X7 receptor antagonists have been evaluated in ophthalmology, and both topical and oral administrations have been considered.

Topical Administration
Topical administration, mostly in the form of eye drops, is employed to treat diseases of the anterior segment, usually the cornea and the conjunctiva. Hyaluronan is a natural polysaccharide used in ophthalmology in artificial tears for the treatment of dry eye syndrome [88][89][90][91], due to its lubricant and viscoelastic properties. Hyaluronan has also been considered as a potent P2X7 receptor modulator: a pretreatment with hyaluronan before BAC or SLS incubation was able to significantly decrease SLS-induced P2X7 activation in corneal and conjunctival cells [59, 92,93]. One possible mechanism is that hyaluronan physically coats the cell membrane via strong links with CD44 receptors. At the same time, it masks P2X7 receptors, preventing their activation.
The P2X7 receptor is potently inhibited by divalent cations such as calcium, magnesium, zinc, and copper, that on the one hand alter the affinity of ATP binding to the P2X7 receptor in an allosteric manner, and on the other hand directly interact with the P2X7 receptor [94][95][96][97][98]. It has been demonstrated that some marine solutions containing Ca 2+ , Mg 2+ , and Zn 2+ inhibited basal activation of P2X7 receptors in ocular surface cell lines [11]. Such solutions that are rich in divalent cations could be easily used as P2X7 receptor modulators or to potentiate P2X7 receptor antagonists.

Oral Administration
The oral route of drug administration to target the eye may not be the most efficient delivery system due to absorption from the gastrointestinal tract and high systemic clearance rates. Nevertheless, oral antioxidants are prescribed to dry AMD patients since no truly effective treatment is currently available for patients with advanced disease. A proof-of-principle clinical trial in AMD patients confirmed the positive effects of antioxidant saffron administration in neurodegenerative diseases and its persistence over time [99,100]. Recent data showed that saffron may exert its protective role in neurodegeneration by reducing the intracellular calcium response evoked by P2X7 receptor stimulation [101].
Omega-3 polyunsaturated fatty acids were included in the formulation used in the Second Age-Related Eye Disease Study (AREDS2) to evaluate their effects on slowing the progression of AMD [102]. The results of AREDS2 showed that omega-3 fatty acid supplementation did not yield a statistically significant reduction in the progression of AMD [103]. However, previous observational studies suggested a link between high dietary consumption of omega-3 fatty acids and decreased risk of developing advanced AMD [104,105]. We demonstrated that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) represent efficient modulators of amyloid β toxicity in retinal cells with the condition that these omega-3 fatty acids are brought as triglycerides in fish oils, and not as ethyl esters like in AREDS2 [70]. The preventive effects of fish EPA and DHA against amyloid β-induced apoptosis relies on the inhibition of P2X7 receptors through lipid raft disruption. In combination with Brilliant Blue G (BBG), a specific P2X7 receptor inhibitor, they fully prevented amyloid β cytotoxic effects. It was then concluded that marine oils rich in EPA and DHA plus BBG in food supplements could be proposed to prevent AMD. Other studies determined the promising role of BBG as a therapeutic agent to inhibit AMD expansion, as it might prevent retinal pigmented epithelium and photoreceptor cell death [106][107][108]. Cornea section + Mouse [12] Corneolimbal epithelial cells (telomerase immortalized cells) + + Human [66,109] Conjunctiva Conjunctival epithelial cells + Human [109] Conjunctival epithelial cells (Wong-Kilbourne derivative of Chang conjunctiva (WKD) cell line) + Human [11] Goblet cells + Rat [14] Lens Lens fiber cells + + Rat [21] Lens epithelial cells + Human [11] Retina Müller glial cells + + Human [18,70] Retina section + + Rat [110] Retinal ganglion cells + Rat [111] Retinal pigmented epithelial cells (Acute Retinal Pigment Epithelial-19 (ARPE-19) cell line) + Human [11] Retinal pigmented epithelial cells (primary culture) + + Human [112] Retinal pigmented epithelial cells + + Mouse [113] Photoreceptors + + Rat [114] Ciliary body Ciliary epithelial cells + Bovine [115]

Conclusions
Excessive ATP release is implicated in numerous ocular stresses. As the P2X7 receptor is expressed in the whole eye, it plays a key role in ATP-induced mechanisms, whether they are triggered in the anterior segment or the posterior segment, and whether they are induced by exogenous chemicals such as preservatives or by endogenous agents such as amyloid β. Consequently, P2X7 receptor inhibition is considered as a potent therapeutic strategy and several P2X7 receptor antagonists could be used in the ophthalmology field, but further clinical studies are necessary to clearly identify efficient and bioavailable ophthalmic formulations.