Therapeutic potential of topical administration of siRNAs against HIF-1α for corneal neovascularization.

Given the implications of the problem of neovascularization on ocular health, as well as the growth in the number of cases, the purpose of the present study has been testing the efficacy of siRNAs (small interfering RNA) designed to silence Hypoxia Inducible Factor -1α (HIF-1α) and to demonstrate that their use stops neovascularization in a model of corneal burn. Corneal wounds in the limbic zone were made in the eyes of New Zealand white rabbits. Topical applications of siRNAs were done the next day to the wound for four consecutive days and eyes were examined with a slit lamp. Evaluation of neovascularization progress was done by analyzing images by ImageJTM and to determine the neovascular area in Matlab ® was used. At the same time, a rabbit corneal cell line was used for in vitro study of hypoxia exposure and Western blot analysis of the cell's extracts were done. Under normal cell culture oxygenation, the expression of HIF-1α was lower than that observed under hypoxic conditions. After 2 h of hypoxia, there was a significant increase in the HIF-1α expression, effect that was maintained up to 6 h. The increased in HIF-1α was mimicked by a cell permeable prolyl-4-hydroxylase inhibitor. Cobalt chloride showed no capacity to increase HIF-1α in vitro. The effect of three different siRNA on HIF-1α was tested after 4 h of hypoxia. siRNA#1 was able to silence 80% of HIF-1α expression, siRNA#2 and siRNA#3 reduce the expression in 45% and 40% respectively. In addition, the three siRNA were tested in a corneal model of neovascularization. scrambledsiRNA#2 was the most effective inhibitor of blood vessel production, followed by siRNA#3 and siRNA#1. Compared to the scrambled siRNA (100% of blood vessel generation), siRNA#2 blocked the presence of blood vessels by 83 ± 2%, siRNA#3 inhibited 45 ± 7% and siRNA#1 only inhibited 18 ± 5%. The necessary time to observe the 50% of effect showed values of NV50 of 10.2 ± 2.4 days for the scrambled siRNA, 9.1 ± 1.4 for siRNA#1, 6.5 ± 1.85 for siRNA#2 and 4.8 ± 1.8 days for siRNA#3. In conclusion, the topical application of siRNA towards HIF-1α seems to be an effective and reliable method to stop neovascularization.

Efficacy of Artificial Tears Based on an Extract of Artemia salina Containing Dinucleotides in a Rabbit Dry Eye Model.

(1) Background: Artemia salina is a brine shrimp containing high concentrations of dinucleotides, molecules with properties for dry eye treatment. For this reason, the purpose of the study was to evaluate the effect of the artificial tears based on an extract of Artemia salina in a rabbit dry eye model. (2) Methods: A prospective and randomized study was carried out. Twenty rabbits were divided into 4 groups (n = 5, each group): healthy rabbits, dry eye rabbits, dry eye rabbits treated with hypromellose (HPMC), and dry eye rabbits treated with Artemia salina. Dry eye was induced by the topical instillation of 0.2% benzalkonium chloride. The measurements were performed before and after the treatment for 5 consecutive days. (3) Results: The topical instillation of artificial tears containing Artemia salina showed beneficial effects on tear secretion, tear break-up time, corneal staining, the density of Goblet cells, heigh of mucin cloud secreted by these cells, and mRNA levels of IL-1ß and MMP9 in conjunctival cells. Compared with the HPMC, there was a statistically significant improvement (p < 0.05) with the Artemia salina in all the variables under study, except for the conjunctival hyperemia, density of Goblet cells, and mRNA levels of IL-6. (4) Conclusions: The potential of artificial tears based on Artemia salina as a secretagogue agent for dry eye treatment was confirmed, opening the door for future clinical trials and studies to extrapolate the findings for dry eye patients.

Preclinical Development of Artificial Tears Based on an Extract of Artemia Salina Containing Dinucleotides in Rabbits.

PURPOSE: To evaluate the preclinical efficacy of eye drops based on an extract of Artemia salina on the ocular surface of rabbits. Tear secretion, tear break-up time and corneal staining were measured. MATERIAL AND METHODS: A preclinical and short-term prospective study was performed. Twenty New Zealand white rabbits were divided into five groups, with four rabbits per group, each receiving a different concentration of Artemia salina. In each rabbit, an extract of Artemia salina (2%, 4%, 6%, 8% or 10%) was randomly instilled in one eye and saline solution (negative control) in the other eye. Tear secretion, tear break-up time and corneal staining were measured before and after the instillation of five drops per eye (one drop per hour) on the same day. RESULTS: In tear secretion, there was an increase of 43.88 ± 6.73% with 4% Artemia salina in comparison with its baseline measurement (P = .049). The rest of the groups did not show differences (P ≥ 0.05). For tear break-up time, none of the groups showed differences (P ≥ 0.05), while for corneal staining score, there was an improvement of 0.88 ± 0.83 with 4% Artemia salina (P = .038) and a deterioration of 0.50 ± 0.83 with control solution (P = .008). CONCLUSIONS: Short-term instillation of eye drops with 4% Artemia salina produced both stimulation of tear secretion and a slight improvement of physiological corneal staining. Besides, all the doses of up to 10% Artemia salina did not produce undesirable side effects on the ocular surface. Therefore, these eye drops are presented as a possible new treatment for dry eye due to their secretagogue properties and ocular surface regeneration.

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension.

BACKGROUND: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. CONCLUSIONS: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R-MT1R and D3R-MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Light-induced ATP release from the lens.

The recent discovery of the photoreceptor melanopsin in lens epithelial cells has opened the possibility of modulating this protein by light stimulation. Experiments carried out on New Zealand white rabbits have demonstrated that the release of ATP from the lens to the aqueous humor can be reduced either when a yellow filter or a melanopsin antagonist is used. Compared to control (1.10 ± 0.15 µM ATP), the application of a yellow filter (λ465-480) reduced ATP in the aqueous humor 70%, while the melanopsin antagonist AA92593 reduced the presence of ATP 63% (n = 5), an effect which was also obtained with the PLC inhibitor U73122. These results indicate that when melanopsin is blocked either by the lack of light, a filter, or an antagonist, the extracellular presence of ATP is significantly reduced. This discovery may be relevant, on the one hand, because many ocular physiological processes are controlled by ATP and, on the other hand, because it is possible to stimulate ATP release with just light and without using any added substance.

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist.

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.

Increased Ap4A levels and ecto-nucleotidase activity in glaucomatous mice retina.

The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (Ap4A) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. Ap4A levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exhibited Ap4A values, which changed in stimulated retinas as long as the pathology progressed varying from 0.73 ± 0.04 (3 months) to 0.170 ± 0.05 pmol/mg retina (23 months). Concomitantly, NPP1 expression was significantly increased (82.15%) in the DBA/2J mice at 15 months. Furthermore, immunohistochemical studies showed that NPP1 labeling was stronger in OPL and IPL labeling tangentially in the vitreal part of the retina and was upregulated at 15 months of age. Our findings demonstrate that Ap4A decreased levels may be related with exacerbated activity of NPP1 protein in glaucomatous degeneration and in this way contributing to elucidate different mechanisms involved in retinal impairment in glaucomatous degeneration.

Diquafosol Delivery from Silicone Hydrogel Contact Lenses: Improved Effect on Tear Secretion.

PURPOSE: The aim of this study was to evaluate the ability to uptake and to deliver diquafosol from commercial contact lenses (CLs) and its effect on tear secretion. METHODS: For both in vitro and in vivo experiments, two commercial silicone hydrogel (Si-Hy) CLs (comfilcon A and balafilcon A) were used. The CLs were soaked overnight for 12 h in diquafosol solution and control CLs were soaked in saline solution (NaCl 0.9%). The CLs were introduced into a new well container with 1 mL of saline solution, and aliquots of 100 µL were extracted at different times during a period of 6 h to measure the diquafosol release. For in vivo experiments, nine male New Zealand white rabbits were used. CLs soaked in diquafosol were inserted in the eye and compared with control CLs and diquafosol topical instillation. Schirmer's tests were performed to evaluate tear secretion and diquafosol release at different times during the 6-h period. RESULTS: For in vitro experiments, the largest amount of diquafosol was released during the first 24 h for both CL materials under study, without statistical differences between them (P < 0.05). The topical application showed the maximum release at 1 min after instillation, meanwhile the release from both CL materials was at 30 min of insertion. The effect on tear secretion was higher with CL delivery compared with topical instillation (P < 0.05), being 300 min for both CLs and 90 min for topical application. CONCLUSION: The use of CLs increases the residence time of diquafosol on the ocular surface with a concomitant enhancement in tear secretion during longer periods.

The role and therapeutic potential of melatonin in age-related ocular diseases.

The eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration, and diabetic retinopathy. As the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells, and photoreceptors is postmitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, and thus, oxidative damage becomes irreversible. Other conditions such as low-grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory, and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation, and angiogenesis mechanisms.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved.

Low expression of CD39 and CD73 genes in centenarians compared with octogenarians.

Ageing involves a progressive decline of the body's regulatory systems including immune system. Adenosine regulates immune function by interaction with its receptors, mainly adenosine A2A receptor, present on the surface of immune cells. Furthermore, cellular response to this nucleoside is highly dependent on its extracellular concentration that is regulated by ecto-enzymes such as CD39 and CD73. Therefore, the aim of this study was to investigate the effect of age on adenosine A2A receptor, CD39 and CD73 gene expression. Changes in mRNA were measured by quantitative PCR from peripheral blood of young, middle-aged and older adults as well as centenarians. Centenarians showed a prominent decrease of CD39 and CD73 mRNA in comparison with older adults. Regarding to adenosine A2A receptor, we detected two subgroups of centenarians with high and low level of transcript. Additionally, adenosine A2A receptor mRNA level of centenarians, did not correlate with their cognitive impairment. In summary, our pilot study suggests that unlike of adenosine A2A receptor, the level of CD39 as well as CD73 mRNA could be a hallmark of successful human ageing.

TRPV4 Stimulation Induced Melatonin Secretion by Increasing Arylalkymine N-acetyltransferase (AANAT) Protein Level.

Melatonin is a molecule which has gained a great deal of interest in many areas of science; its synthesis was classically known to be in the pineal gland. However, many organs synthesize melatonin, such as several ocular structures. Melatonin is known to participate in many functions apart from its main action regulating the circadian rhythm. It is synthesized from serotonin in two steps, with a rate-limiting step carried out by arylalkymine N-acetyltransferase (AANAT). In this report, the role of TRPV4 channel present in human ciliary body epithelial cells in AANAT production was studied. Several experiments were undertaken to verify the adequate time to reach the maximal effect by using the TRPV4 agonist GSK1016790A, together with a dose-response study. An increase of 2.4 folds in AANAT was seen after 18 h of incubation with 10 nM of GSK1016790A (p < 0.001, n = 6). This increment was verified by antagonist assays. In summary, AANAT levels and therefore melatonin synthesis change after TRPV4 channel stimulation. Using this cell model together with human ciliary body tissue it is possible to suggest that AANAT plays an important role in pathologies related to intraocular pressure.

Understanding the Presence and Roles of Ap4A (Diadenosine Tetraphosphate) in the Eye.

Diadenosine tetraphosphate abbreviated Ap4A is a naturally occurring dinucleotide, which is present in most of the ocular fluids. Due to its intrinsic resistance to enzyme degradation compared to mononucleotides, this molecule can exhibit profound actions on ocular tissues, including the ocular surface, ciliary body, trabecular meshwork, and probably the retina. The actions of Ap4A are mostly carried out by P2Y2 receptors, but the participation of P2X2 and P2Y6 in processes such as the regulation of intraocular pressure (IOP), together with the P2Y2, is pivotal. Beyond the physiological role, this dinucleotide can present on the ocular surface keeping a right production of tear secretion or regulating IOP. It is important to note that exogenous application of Ap4A to cells or animal models can significantly modify pathophysiological conditions and thus is an attractive therapeutic molecule. The ocular location where Ap4A actions have not been fully elucidated is in the retina. Although some analogues show interesting actions on pathological situations such as retinal detachment, little is known about the real effect of this dinucleotide, this being one of the challenges that require pursuing in the near future.

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells.

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.

Signs and Symptoms of Dry Eye in Keratoconus Patients Before and After Intrastromal Corneal Rings Surgery.

PURPOSE: Based on the relationship between keratoconus and dry eye, the aim of this study was to evaluate changes in signs and symptoms of dry eye in keratoconus patients before and after intrastromal corneal ring surgery. METHODS: Fifteen keratoconus patients were enrolled in Fundación Jiménez-Díaz of Madrid and University Clinic of Optometry of the Universidad Complutense de Madrid (Madrid, Spain). Tear break up time (TBUT), Schirmer test without anesthesia, corneal staining, diadenosine tetraphosphate (Ap4A) concentration, and ocular surface disease index (OSDI) were evaluated. Impression cytology combined with laser confocal microscopy was performed to evaluate goblet cell density, mucin cloud height (MCH), and cell layer thickness (CLT). All measurements were performed before (pre) surgery, 1 month (post) and 6 months after surgery (post6m). RESULTS: We found no statistical differences in time in Schirmer test, TBUT, and corneal staining. OSDI scores were 44.96 ± 8.65, 26.30 ± 6.79, and 19.31 ± 4.28 for (pre), (post), and (post6m) surgery, respectively (p < 0.001). Impression cytology showed a decrease in cell density at (post6m) compared with presurgery (47.36 ± 35.15 cells/mm2 and 84.88 ± 32.08 cells/mm2, respectively, p = 0.04). At post6m, the MCH increased compared with presurgery values (13.97 ± 4.26 µm and 6.77 ± 2.51 µm, respectively, p < 0.001). There was a statistically significant increase in CLT in time. Ap4A tear concentrations were lower post6m than presurgery (1.02 ± 0.65 and 2.56 ± 1.10 µM, respectively, p < 0.001). CONCLUSION: Intrastromal corneal ring surgery induces changes improving dry eye symptoms but no changes were found in signs of dry eye after surgery in keratoconus patients except for the MCH that increases drastically. More studies are needed to clarify the reason of its improvement.

Diadenosine tetraphosphate as a potential therapeutic nucleotide to treat glaucoma.

Glaucoma is a neurodegenerative disease that produces blindness. The main factor associated with this disease is an abnormally elevated intraocular pressure (IOP). To date, some attempts have been made to demonstrate the role of nucleotides modulating IOP, but never in a model of glaucoma. The DBA/2J mouse is an animal that develops the pathology spontaneously, starting from the typical rise in IOP at 9 months of age. Using this animal model, together with a control mouse, C57BL/6J, it has been possible to monitor the elevation in IOP in the glaucomatous mice and to check the ability of the dinucleotide diadenosine tetraphosphate AKA Ap4A to reduce IOP. The topical application of Ap4A when IOP is maximal (9-12 months) reduced IOP 30.6 ± 6.6% in the DBA/2J and 17.9 ± 4.0% in the C57BL/6J mice. Concentration response curves in both animal strains produced similar pD2 values; these being 4.9 ± 0.5 and 5.1 ± 0.4 for the normotensive C57BL/6J and the glaucomatous DBA/2J respectively. Antagonist studies showed differences between the control and the glaucomatous animals. In particular, the main receptor reducing IOP in the control animal was the P2Y1 receptor and in the glaucomatous model the P2Y6, although the participation of other P2 receptors cannot be ruled out. The long-term effect of Ap4A applied three times a week for 3 months showed a clear stop in the elevation of IOP in the glaucomatous model, thus indicating the possibility of using Ap4A as an effective compound for the treatment of glaucoma.

Elevated intraocular pressure increases melatonin levels in the aqueous humour.

PURPOSE: To study the levels of melatonin in the aqueous humour of normotensive and hypertensive intraocular pressure (IOP) patients and to compare them to an animal model of glaucoma. METHODS: A total of 37 eyes of 37 patients who underwent cataract surgery were included in the study and were divided into normotensive patients, with IOP below 21 mmHg (n = 23), and hypertensive patients, with IOP > 21 mmHg (n = 14). Glaucomatous DBA/2J (n = 6) and control C57BL/6J (n = 6) mice presenting 3 and 12 months of age for each strain were also used. Human and mice aqueous humours were aspirated using a 30-gauge Rycroft cannula on a tuberculin syringe and further processed to quantify melatonin by high-performance liquid chromatography analysis. RESULTS: Melatonin levels in normotensive patients (IOP below 21 mmHg) presented values as medians (first quartile; third quartile) of 14.62 (5.38;37.99) ng/ml (n = 23), while hypertensive patients (IOP above 21 mmHg) showed melatonin concentrations of 46.63 (10.28; 167.28) ng/ml (n = 14; p < 0.039). Glaucoma mice presented melatonin values of 0.37 (0.34; 0.59) ng/ml (at 3 months of age, before the pathology starts), which increased to 1.55 (0.94; 1.88) ng/ml (at 12 months of age, when the pathology is fully developed and IOP is maximum; n = 6, p < 0.001). Control mice did not significantly modified melatonin concentrations between 3 and 12 months of age. CONCLUSION: Patients with high IOP present increased concentrations of melatonin in their aqueous humour compared to normotensive patients. This has been confirmed in a glaucomatous animal model in which it has been possible to see a correlation between the development of the pathology, with an increase in IOP, and a concomitant elevation of melatonin in the aqueous humour.

Presence of melanopsin in human crystalline lens epithelial cells and its role in melatonin synthesis.

Melanopsin is a non-image forming photoreceptor known to be present in the retina and it is considered to have light regulated tasks among other functions. In the present work, melanopsin presence in human lens epithelial cells as well as in human lens tissue is described for the first time. Moreover, studying the concentration of melatonin and its synthesising enzyme AANAT proved a clear link between melanopsin activation and the suppression of melatonin synthesis. Melanopsin sensitivity to specific wavelength (465-480 nm, blue) was confirmed after making temporal studies incubating lens epithelial cells under light, red, green, blue and total darkness for 2, 4, 8, 12 h and analysing the concentration of both melatonin and its synthesising enzyme AANAT, discovering that melatonin levels after submitting cells to total darkness are significantly higher to ones submitted to white or specifically blue light (***p < 0.001, n = 6). The involvement of melanopsin in the regulation of melatonin was also determined by using a specific inhibitor AA92593 and by inhibiting melanopsin-induced phospholipase C activation. Under this situation neither AANAT nor melatonin levels changed under light conditions (n = 4, ***p < 0.001). The discovery of melanopsin in the lens opens the possibility of regulating melatonin synthesis with the corresponding implication as an antioxidant substance.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures.

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

The influence of rigid gas permeable lens wear on the concentrations of dinucleotides in tears and the effect on dry eye signs and symptoms in keratoconus.

PURPOSE: To evaluate the signs and symptoms of dry eye and dinucleotide secretion in tears of keratoconus patients (KC) and the potential effect of rigid gas permeable (RGP) contact lens wear. METHODS: Twenty-three KC patients and forty control subjects were enrolled in this study. Signs of dry eye including tear volume, tear stability and corneal staining along with symptoms were assessed using the McMonnies questionnaire. Tears were collected using Schirmer strips, and dinucleotide concentrations in collected tears measured using high pressure liquid chromatography. Values obtained in KC and controls were compared. The effect of contact lens wear in KC was also assessed. RESULTS: KC eyes showed a significantly lower tear volume compared to controls, shorter tear break up time (TBUT), higher corneal staining and higher McMonnies dry eye questionnaire scores (p<0.05). When compared with non-wearers, KC contact lens wearers showed significantly higher symptoms, lower Schirmer and TBUT values (p<0.05). Concentration of Ap4A (0.695±0.304µM vs. 0.185±0.178µM) and Ap5A (0.132±0.128µM vs. 0.045±0.036µM) were higher in KC compared to controls (p<0.001) and only Ap4A was statistically higher in RGP wearers compared to non-wearers (0.794±0.478µM vs. 0.417±0.313µM) (p<0.05). CONCLUSION: Signs and symptoms of dry eye as well as concentrations of Ap4A and Ap5A were markedly increased in KC patients compared to controls. Moreover, Ap4A and symptoms of dry eye were statistically higher in RGP wearers compared to non-wearers. This seems to indicate that factors such as RGP contact lens wear might exacerbate the clinical condition of dry eye.