[Inflammatory metabolites of arahidonic acid in tear fluid in UV-induced corneal damage]. / Vospalitel'nye metabolity arakhidonovoi kisloty v sleznoi zhidkosti pri indutsirovannom ul'trafioletovym izlucheniem povrezhdenii rogovitsy.

The ultraviolet (UV) B-induced damage of the eye surface of experimental animals (rabbits) includes loss of corneal epithelium, apoptosis of keratocytes and stromal edema. These changes are accompanied by clinically and histologically manifested corneal inflammation, neutrophil infiltration, and exudation of the anterior chamber of the eye. According to mass spectrometric analysis, UV-induced corneal damage is associated with pronounced changes in the lipid composition of tears, including a decrease in the amount of arachidonic acid and prostaglandin E2 and an increase in the concentrations of prostaglandin D2 and its derivative 15d-PGJ2. In addition, it is accompanied by an alteration in the levels of hydroxyeicosate tetraenic acid derivatives, namely upregulation of 12-HETE and downregulation of 5-HETE. The revealed changes indicate the activation of metabolic pathways involving 5-lipoxygenase, 12-lipoxygenase, cyclooxygenase 1 and 2, and prostaglandin-D-synthase. These findings contribute to understanding mechanisms of UV-induced keratitis and point on feasibility of selective anti-inflammatory therapy for improving corneal regeneration after iatrogenic UV damage.

Iatrogenic Damage of Eye Tissues: Current Problems and Possible Solutions.

Visual system is at high risk of iatrogenic damage. Laser ocular surgery, the use of powerful illumination devices in diagnostics and surgical treatment of eye diseases, as well as long surgeries under general anesthesia provoke the development of chronic degenerative changes in eye tissues, primarily in the cornea and the retina. Despite the existence of approaches for prevention and treatment of these complications, the efficacy of these approaches is often limited. Here, we review the mechanisms of iatrogenic damage to eye tissues at the cellular and biochemical levels. It is well recognized that oxidative stress is one of the main factors hindering regeneration of eye tissues after injuries and, thereby, aggravating iatrogenic eye disorders. It is accompanied by the downregulation of low-molecular-weight antioxidants and antioxidant enzymes, as well as changes in the expression and redox status of proteins in the damaged tissue. In this regard, antioxidant therapy, in particular, the use of highly effective mitochondria-targeted antioxidants such as SkQ1, is considered as a promising approach to the prevention of iatrogenesis. Recent findings indicate that the most efficient protection of eye tissues from the iatrogenic injury is achieved by preventive use of these antioxidants. In addition to preventing corneal and retinal cell death induced by oxidative stress, SkQ1 contributes to the restoration of innate antioxidant defense of these tissues and suppresses local inflammatory response. Since the timing of routine medical manipulations is usually known in advance, iatrogenic damage to the ocular tissues can be successfully prevented using mitochondria-targeted therapy.

[Mechanisms of perioperative corneal abrasions: alterations in tear film proteome]. / Mekhanizmy razvitiia perioperatsionnykh érozii rogovitsy: izmeneniia proteomnogo sostava sleznoi plenki.

Perioperative corneal abrasion is an ophthalmic complication commonly found in patients underwent general anesthesia. In this study, correlations between development of corneal injury and proteomic changes in tear film during general anesthesia were examined using an animal (rabbit) model. Being started after 1-h anesthesia, the process of accumulation of pathological changes in the cornea unequivocally led clinically significant abrasions following 3-6 h of the narcosis. The corneal damage was associated with alterations in profiles of major proteins of the tear film. Analysis of the tear proteome pointed to depression of lachrymal glands function, and suggested serotransferrin, serum albumin and annexin A1 as potential tear markers of the complication. The tear film alterations included fast drop of total antioxidant activity and activity of superoxide dismutase, and decrease in interleukin-4 and increase in interleukin-6 content indicating development of oxidative and pro-inflammatory responses. These findings suggest antioxidant and anti-inflammatory therapy as prospective approach for prevention/treatment of perioperative corneal abrasions. The observed anesthesia-induced effects should be considered in any study of ocular surface diseases employing anesthetized animals.

Alterations in Tear Biochemistry Associated with Postanesthetic Chronic Dry Eye Syndrome.

Perioperative dry eye syndrome (DES) is a common ocular complication of long-term general anesthesia. Chronic DES can lead to permanent damage to the cornea and disturbance of visual function, up to total loss of vision. Here, a relationship between the duration of general anesthesia and the risk of chronic DES in patients was demonstrated. Using an experimental model of perioperative corneal abrasions in rabbits, it was found that introduction of animals to 3-h general anesthesia resulted in clinically significant chronic damage to the cornea in 50% of cases. The development of the complication was not associated with irreversible or long-term impairment of tear secretion, but it was accompanied by a decrease in tear film stability and growth of the total protein content as well as decrease in total antioxidant activity of the tear induced by low molecular weight antioxidants. In addition, anesthesia-induced changes in activity of tear antioxidant enzymes including superoxide dismutase and enzymes providing homeostasis of reduced glutathione (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) were observed. All these alterations were protracted (up to 1-2 weeks) and therefore might account for transition of the perioperative DES into the chronic form. These findings can be useful in the development of novel approaches for the prevention and treatment of chronic forms of DES in the postanesthetic period.

Preventive and therapeutic effects of SkQ1-containing Visomitin eye drops against light-induced retinal degeneration.

The human retina is constantly affected by light of varying intensity, this being especially true for photoreceptor cells and retinal pigment epithelium. Traditionally, photoinduced damages of the retina are induced by visible light of high intensity in albino rats using the LIRD (light-induced retinal degeneration) model. This model allows study of pathological processes in the retina and the search for retinoprotectors preventing retinal photodamage. In addition, the etiology and mechanisms of retina damage in the LIRD model have much in common with the mechanisms of the development of age-related retinal disorders, in particular, with age-related macular degeneration (AMD). We have studied preventive and therapeutic effects of Visomitin eye drops (based on the mitochondria-targeted antioxidant SkQ1) on albino rat retinas damaged by bright light. In the first series of experiments, rats receiving Visomitin for two weeks prior to illumination demonstrated significantly less expressed atrophic and degenerative changes in the retina compared to animals receiving similar drops with no SkQ1. In the second series, the illuminated rats were treated for two weeks with Visomitin or similar drops without SkQ1. The damaged retinas of the experimental animals were repaired much more effectively than those of the control animals. Therefore, we conclude that Visomitin SkQ1-containing eye drops have pronounced preventive and therapeutic effects on the photodamaged retina and might be recommended as a photoprotector and a pharmaceutical preparation for the treatment of AMD in combination with conventional medicines.

Cancer-retina antigens -- a new group of tumor antigens.

Some photoreceptor proteins normally specific for the eye retina are aberrantly expressed in malignant tumors. These proteins include recoverin, visual rhodopsin, transducin, cGMP-phosphodiesterase 6 (PDE 6), cGMP-dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin. By analogy with cancer-testis antigens, these photoreceptor proteins form the group of cancer-retina antigens. It is shown that an aberrant demethylation of the promoter region of recoverin is involved in the aberrant expression of this protein. The cascade Wnt5a → Frizzled-2 → transducin → PDE 6 is shown to function in skin melanoma cells, and this suggests that these cancer-retina antigens can play a functional role. The events accompanying the signal transduction in this cascade, including those involving calcium ions and cGMP-dependent protein kinase (protein kinase G), are discussed.

Organotypic culture of neural retina as a research model of neurodegeneration of ganglion cells.

Organotypic models deserve special attention among the large variety of methods of vertebrate retina cultivation. The purpose of this study was to make a detailed qualitative and quantitative characterization of a model employing roller organotypic cultivation of the neural retina of rat eye posterior segment, with special attention to morphological and functional characteristics of retinal ganglion cells. The study included morphological analysis of retina histological preparations as well as estimation of RNA synthesis and evaluation of neuron survival by the Brachet and TUNEL methods, respectively. Retina has been shown to display normal morphofunctional characteristics for the first 12 h of cultivation. After 24 h, a substantial number of ganglion cells underwent pyknosis and stopped RNA synthesis. Almost all the cells of the retinal ganglion layer became apoptotic by 3-4 days in vitro. In the course of cultivation, neural retina is detached from the underlying layers of the posterior eye segment and undergoes significant cytoarchitectonic changes. The causes of ganglion cell death during organotypic cultivation of eye posterior segment are discussed. This method can serve as a suitable model for the screening of new retinoprotectors and for research on ganglion cell death resulting from retina degenerative diseases, e.g. glaucoma.

Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca2+/recoverin-dependent inhibition of phosphorylation of rhodopsin.

Antibodies AB(60-72) and AB(80-92) against two immune-dominant epitopes of photoreceptor Ca(2+)-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca(2+)-dependent manner, were obtained. The presence of AB(60-72) or AB(80-92) results in a slight increase in Ca(2+)-affinity of recoverin and does not affect significantly a Ca(2+)-myristoyl switch mechanism of the protein. However in the presence of AB(60-72) or AB(80-92) recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca(2+)-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.

Expression of Ca-binding protein recoverin in normal, surviving, and regenerating retina of Pleurodeles waltl adult triton.

Immunohistochemical study of the expression of recoverin (photoreceptor protein) in the retina of Pleurodeles waltl adult triton was carried out in health, during regeneration after removal, and under conditions of long-lasting detachment. Studies with polyclonal (monospecific) antibodies to recoverin showed that normally it is present in the internal segment, connective cilium, in distal portions of the external segments of cones and rods, and in Landolt clubs of displaced bipolar cells. Detachment of the retina is associated with translocation of recoverin from the photoreceptor processes to perikaryons, and the content of recoverin-positive displaced bipolar cells increases. During regeneration of the retina after its excision via conversion of the pigmented epithelial cells, recoverin is synthesized in the prospective photoreceptor perikaryons and then accumulates in the forming inner segments. Hence, recoverin can serve as a reliable marker in studies of photoreceptor differentiation and functioning during regeneration or survival of the retina.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

New method of in vitro culturing of pigment retinal epithelium in the structure of the posterior eye sector of adult rat.

We propose a new method of organotypic roller 3D-culturing of the posterior sector of the eye. The method allows maintaining tissue viability in vitro for 14 days (which considerably surpasses the capacities of stationary culturing) and studying of the behavior, of pigment retinal epithelial cells and choriocapillary membrane. Using this method we demonstrated phenotypic transformation, migration, and proliferation of pigment retinal epithelial cells under conditions of roller organotypic culture. In the absence of the retina, these cells exhibit properties of scavenger cells (phagocytes) both within and outside the layer. Under conditions of roller culturing in vitro, cells of the pigment retinal epithelium undergo changes similar to those observed in various retinal pathologies in vivo, including age-associated changes. Here we discuss the possibility of using the proposed method for evaluation of the effect of various factors added to the culture medium on the pigment epithelium, for modeling of processes developing in damaged pigment epithelium or under conditions of various pathologies, and for the study of regeneration responses in cells of pigment retinal epithelium in adult vertebrates.

Detection of annexin IV in bovine retinal rods.

The fraction of proteins capable of binding to photoreceptor membranes in a Ca2+-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.

Extracts of lung cancer cells reveal antitumour antibodies in sera of patients with lung cancer.

The objective of the present study was to reveal antitumour antibodies in sera of patients with small cell lung cancer (SCLC). The antibodies in sera of patients with SCLC and other tumours were detected by immunoblotting with whole extracts of SCLC cells as the antigen source. Sera of patients with various pulmonological disorders, irradiated during the liquidation of consequences of the Chernobyl nuclear power plant incident (a high-risk group in lung cancer), were also analysed. The present authors' found that SCLC sera contain a set (pattern) of antitumour antibodies which are rarely detected in sera of patients with cancers different from SCLC and very rarely, if ever, present in sera of healthy individuals. The sensitivity and the specificity of the pattern are equal to 80% and 91%, correspondingly. In the high-risk group in lung cancer, the frequencies of the antibodies are somewhat lower than the corresponding values in SCLC sera, but significantly larger than those in healthy sera. The findings of the present study create a basis for clinical application of the antitumour antibodies described.

Low titre autoantibodies against recoverin in sera of patients with small cell lung cancer but without a loss of vision.

To date, many authors have described the presence of autoantibodies against various neuronal proteins, paraneoplastic antigens (PNA), in a serum of patients with different kinds of malignant tumors located outside the nervous system. These autoantibodies may cross-react with the corresponding PNA or their epitopes present in neurons and thus initiate the development of a variety of neurological disorders, paraneoplastic syndromes (PNS), even though the primary tumor and its metastases have not invaded the nervous system. Cancer-associated retinopathy (CAR) is a rare ocular PNS induced by autoantibodies against several retinal antigens, one of which is a photoreceptor calcium-binding protein, recoverin. Only several CAR patients with a few kinds of cancer (endothelial carcinoma, breast cancer, epithelial ovarian carcinoma) have so far been found to contain autoantibodies against recoverin in their sera. As for lung cancer, the majority of CAR cases mediated by anti-recoverin autoantibodies have been revealed in patients with the most malignant lung cancer, small cell lung carcinoma (SCLC), and only one similar case has been described for a patient with non-small lung carcinoma. The common feature of all these anti-recoverin-positive patients, irrespective of the type of cancer, is the presence of both the CAR syndrome and high titres (as a rule, more than 1:1000) of the underlying autoantibodies in their serum. In this study, we have used recombinant myristoylated recoverin to screen serum samples of 50 patients with SCLC by Western blot and revealed 5 individuals with low titres of anti-recoverin antibodies, who have no manifestation of a loss of vision. To our knowledge, this is the first report on the presence of low titre autoantibodies against recoverin in a serum of patients with cancer, but without visual dysfunction.

Phototransduction and calcium.

In the early 1970s it became clear that an unknown intracellular messenger participated in signal transduction in retinal rod cells. Ca2+ and cGMP had been considered as candidates for such a messenger until 1985 when the cGMP-gated conductance of the plasma membrane was discovered. However, for the last several years, Ca2+ has taken revenge as a signal molecule which contributed to the processes underlying the photoreceptor cell recovery. This review describes the overall picture of phototransduction molecular mechanisms with special emphasis on participation of Ca2+ in visual cell signalling.

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites.

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.

Obtaining and characterization of EF-hand mutants of recoverin.

Several EF-hand recoverin mutants were obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase were determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the microM range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures.

Rhodopsin phosphorylation in bovine rod outer segments is more sensitive to the inhibitory action of recoverin at the low rhodopsin bleaching than it is at the high bleaching.

Recoverin, a calcium-binding protein, is supposed to have rhodopsin kinase as a target in the retinal rod cell. In the present work, we show that efficiency of recoverin as an inhibitor of rhodopsin phosphorylation in bovine rod outer segments is inversely proportional to the level of rhodopsin bleaching. These results, together with the data obtained previously in a reconstituted system (Senin et al. (1997) Biochem. J. 321, 551-555), allow us to hypothesize that recoverin might be responsible for a Ca2(+)-dependent regulation of the kinase in vivo, preventing it from participating in the phosphorylation of unbleached rhodopsin.

Recoverin inhibits the phosphorylation of dark-adapted rhodopsin more than it does that of bleached rhodopsin: a possible mechanism through which rhodopsin kinase is prevented from participation in a side reaction.

In its resting state rhodopsin kinase is present in an inactive from and is activated after interaction with light-activated rhodopsin (Rho*). The activated rhodopsin kinase then phosphorylates Rho* but is also able to catalyse the phosphorylation of dark-adapted rhodopsin. A consequence of the latter behaviour of the activated kinase is that at low levels of bleach a large number of phosphoryl groups are incorporated per mol of Rho*. Recoverin- and Ca2+-dependent inhibition of rhodopsin kinase was found to be inversely related to the extent of bleaching; the lower the fraction of rhodopsin bleached, the greater the inhibition. The IC50 of recoverin is approx. 1 microM at a 0.2% level of bleach and about 5 microM in a fully bleached sample. The inhibitory effect of recoverin was studied separately on the phosphorylation of rhodopsin and Rho*. The formation of phosphorylated rhodopsin was inhibited 4.5-fold more strongly than that of phosphorylated Rho*. These results are interpreted to suggest that one of the roles of the recoverin-dependent regulation of the activity of rhodopsin kinase is to prevent the enzyme from participating in the unwanted phosphorylation of dark-adapted rhodopsin, directing it to fulfil its 'correct' function of quenching the transduction activity of Rho*.