Identification of RALDH2 as a visually regulated retinoic acid synthesizing enzyme in the chick choroid.

PURPOSE: All-trans-retinoic acid (atRA) has been implicated in the local regulation of scleral proteoglycan synthesis in vivo. The purpose of the present study was to identify the enzymes involved in the synthesis of atRA during visually guided ocular growth, the cells involved in modulation of atRA biosynthesis in the choroid, and the effect of choroid-derived atRA on scleral proteoglycan synthesis. METHODS: Myopia was induced in White leghorn chicks by form deprivation for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of atRA synthesizing enzymes was evaluated by semiquantitative qRT-PCR, in situ hybridization, and immunohistochemistry. atRA synthesis was measured in organ cultures of isolated choroids using LC-tandem MS quantification. Scleral proteoglycan synthesis was measured in vitro by the incorporation of (35)SO(4) in CPC-precipitable glycosaminoglycans. RESULTS; RALDH2 was the predominant RALDH transcript in the choroid (> 100-fold that of RALDH3). RALDH2 mRNA was elevated after 12 and 24 hours of recovery (60% and 188%, respectively; P < 0.01). The atRA concentration was significantly higher in cultures of choroids from 24-hour to 15-day recovering eyes than in paired controls (-195%; P < 0.01). Choroid conditioned medium from recovering choroids inhibited proteoglycan synthesis to 43% of controls (P < 0.02, paired t-test; n = 16) and produced a relative inhibition corresponding to a RA concentration of 7.20 × 10(-8) M. CONCLUSIONS: The results of this study suggest that RALDH2 is the major retinal dehydrogenase in the chick choroid and is responsible for increased atRA synthesis in response to myopic defocus.

Increased hyaluronan synthase-2 mRNA expression and hyaluronan accumulation with choroidal thickening: response during recovery from induced myopia.

PURPOSE: Several studies have convincingly shown that in chicks, compensation for imposed focus involves immediate changes in choroid thickness. The molecular events associated with choroidal thickening and the regulation of the choroidal response are largely unknown. METHODS: Form-deprivation myopia was induced in the right eyes of 2-day-old chicks by the application of translucent occluders for 10 days and was followed by unrestricted vision for an additional 1 to 20 days (recovery). Individual choroids were isolated from treated and control eyes and used for reverse transcription-quantitative PCR, hyaluronan (HA) localization with biotinylated hyaluronic acid binding protein (b-HABP), and analyses of HA size and concentration by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS). RESULTS: HAS2 gene expression increased significantly after 6 hours of unrestricted vision (>7-fold) and peaked at 24 hours (>9-fold). In untreated eyes, HA was localized to perivascular sheaths of larger choroidal blood vessels; however, after 4 to 15 days of recovery, intense labeling for HA was detected throughout the thickened choroidal stroma. Analyses of choroidal HA by SEC-MALLS indicated that HA concentration was significantly increased in recovering choroids compared with controls after 4 to 8 days of recovery (≈3.5-fold). CONCLUSIONS: Newly synthesized HA accumulates in the choroidal stroma of recovering eyes and is most likely responsible for the stromal swelling observed during recovery from myopia. This HA accumulation is initiated by a rapid increase in choroidal expression of the HAS2 gene in response to myopic defocus.

Melatonin receptor expression in Xenopus laevis surface corneal epithelium: diurnal rhythm of lateral membrane localization.

PURPOSE: Melatonin receptors are seven-pass G protein-coupled receptors located in many tissues throughout the body, including the corneal epithelium (CE), and relay circadian signals to the target cells. The purpose of this study was to determine more precisely the cellular distribution of the melatonin receptors in the surface cells of the CE of Xenopus laevis, and to examine the relative distribution of melatonin receptor subtype expression at different times during the circadian cycle. METHODS: Cryostat sections and whole corneas of adult Xenopus laevis were processed for immunocytochemistry using antibodies specific for each of the three melatonin receptor subtypes (Mel1a, Mel1b, and Mel1c). For the circadian studies, corneas were obtained from euthanized frogs at 4-h intervals during a 24-h period under a 12 h:12 h light-dark cycle. Double-label immunocytochemistry was performed using a Mel1a antibody in combination with antibodies against Mel1b, Mel1c, or the zonula occludens protein ZO-1. Corneal whole-mount specimens and corneal sections were analyzed by laser-scanning confocal microscopy. RESULTS: All three melatonin receptor subtypes were expressed on the surface and sub-superficial layer of CE cells, but with different sub-cellular distributions. The Mel1a receptor was highly localized to the lateral plasma membrane of the surface CE, but also displayed cytoplasmic localization at some times of day, especially at night. Mel1c showed a similar pattern of labeling to Mel1a, but there were some distinctive differences, insofar as the Mel1c receptors were usually located immediately basal to the Mel1a receptors. The relative degree of membrane and cytoplasmic labeling of the Mel1c receptor also oscillated during the 24-h period, but was out of phase with the changes that occurred in the Mel1a receptor localization. Furthermore, in the late afternoon time point, the Mel1a and Mel1c receptors were highly co-localized, suggestive of heterodimerization, whereas at other time points, the two receptors were distinctly not co-localized. Double-label immunocytochemistry of Mel1a and ZO-1 demonstrated that the Mel1a receptor was located basal to the tight junctions, on the lateral membrane in very close proximity to the ZO-1 protein. CONCLUSIONS: Mel1a, Mel1b, and Mel1c receptor subtypes are expressed in the lateral plasma membrane of the Xenopus surface CE, at a position in close proximity to the tight junctions that form the corneal diffusion barrier. The very close association of the Mel1a receptors to the ZO-1 peripheral membrane tight junction proteins is suggestive of a potential role for melatonin in influencing the rate of tight junction formation or breakdown. The transient co-localization of Mel1a and Mel1c late in the light period is suggestive of formation of heterodimers that may influence receptor responsiveness and/or activity during specific periods of the day. The dynamic daily changes in melatonin receptor subtype expression and localization in the surface CE supports the concept that melatonin signaling may affect circadian activities of the surface epithelium of the cornea.

Inhibition of human scleral fibroblast cell attachment to collagen type I by TGFBIp.

PURPOSE: Transforming growth factor beta-induced protein (TGFBIp; 68 kDa) is a secreted extracellular matrix (ECM) protein that has been demonstrated to regulate cell attachment in a variety of cell types. The sclera synthesizes and secretes TGFBIp, which may function to facilitate scleral ECM remodeling events associated with myopia development. Here the authors report that human scleral fibroblasts (HSFs) express TGFBI and that its protein product, TGFBIp, mediates an effect on cell attachment. METHODS: TGFBI/TGFBIp expression was evaluated by RT-PCR and immunoblot of HSF lysates and culture supernatants. The effect of rTGFBIp (50 microg/mL) on cell attachment to collagen type I was determined with the use of fluid-phase cell attachment assays in HSFs, human foreskin fibroblasts (HFFs), and human corneal stroma fibroblasts (HCFs). Binding assays using biotinylated rTGFBIp were used to assess TGFBIp binding to the HSF surface. Flow cytometry and immunocytochemistry were used to determine both alphavbeta3 and alphavbeta5 expression and localization to the HSF cell surface. RESULTS: HSFs expressed TGFBI and secreted TGFBIp (approximately 833 ng/h). rTGFBIp significantly decreased (25 microg/mL; P

Ocular expression of avian thymic hormone: changes during the recovery from induced myopia.

PURPOSE: Several studies suggest that postnatal ocular growth is under the control of factors within the eye that regulate the rate of scleral extracellular matrix remodeling and the rate of ocular elongation. A microarray analysis was employed to identify some of the factors involved in the regulation of visually guided ocular growth. Gene expression was compared in the retina-retinal pigmented epithelium (RPE)-choroid of chick eyes that were decelerating in the rate of ocular growth ("recovering" from myopia) as compared with contralateral control eyes. METHODS: Form-deprivation myopia was induced in the right eyes of two-day-old chicks by the application of translucent occluders. Following 10 days of deprivation, occluders were removed and chicks were provided unrestricted vision for an additional 1-7 days (recovery). After one and four days of recovery, chicks were sacrificed, retina, RPE, and choroid were isolated, and mRNA was subjected to microarray analysis using a chicken immune system 4000 gene microarray. In addition, whole eyes and isolated ocular tissues (retina and RPE, choroid, sclera, and extraocular muscle) of treated and control eyes were subjected to real-time PCR, immunohistochemistry, and western blot analyses to verify gene expression results. RESULTS: Following one day of recovery, only one gene, avian thymic hormone (ATH) was highly upregulated (+12.3 fold). ATH gene and protein expression were confirmed in the retina and choroid as well as in the sclera and extraocular muscle. A significant increase in ATH protein was detected in choroids from treated eyes following four days of recovery as compared to contralateral controls (p<0.05; Wilcoxon signed-rank test). CONCLUSIONS: ATH is expressed in several ocular tissues and is specifically and rapidly (within one day) upregulated in the choroids of chick eyes recovering from induced myopia. This upregulation corresponds to the onset of choroidal thickening and increased choroidal vascular permeability. The identification of ATH in ocular tissues and its increased protein accumulation in the choroid during recovery from induced myopia suggest a novel role for this protein in the choroidal response to myopic defocus.

Choroidal regulation of scleral glycosaminoglycan synthesis during recovery from induced myopia.

PURPOSE: The present study was undertaken to examine the relationship between choroidal permeability and scleral glycosaminoglycan synthesis rates during the development of and recovery from form deprivation myopia. METHODS: Form deprivation myopia was induced in chicks for 10 days and was followed by a period of unrestricted vision for 0 to 15 days (recovery). Choroidal permeability was quantified by measuring albumin leakage from choroidal blood vessels into suprachoroidal fluid using Evans blue. Scleral sulfated glycosaminoglycan synthesis was assessed on punches of sclera obtained immediately after extraction of suprachoroidal fluid for permeability measurements or after incubation with suprachoroidal fluid by measuring the amount of (35)SO(4) incorporated into glycosaminoglycans over a period of 4 hours at 37 degrees C. Suprachoroidal fluid was subjected to size fractionation and proteinase digestion to characterize the bioactive fractions from recovering and control chick eyes. RESULTS: Recovery from prior form deprivation was associated with a significant increase in choroidal permeability, compared with that of myopic eyes and contralateral control eyes, and was coincident with a significant decrease in scleral sulfated glycosaminoglycan synthesis rates in treated eyes compared with contralateral control eyes. Suprachoroidal fluid isolated from recovering chick eyes significantly inhibited scleral glycosaminoglycan synthesis compared with suprachoroidal fluid from control eyes (-54%; P < 0.01; ANOVA). Preliminary characterization of suprachoroidal fluid suggested that all inhibitory activity in suprachoroidal fluid fractions specific to recovering eyes is present in molecular weight fractions of less than 10 kDa. CONCLUSIONS: The results of this study suggest that increased choroidal permeability coincides with a decrease in the rate of scleral glycosaminoglycan synthesis during recovery from myopia. The authors speculate that increased choroidal permeability may represent a mechanism for controlling the rate of delivery of bioactive factors to the sclera to regulate the rate of glycosaminoglycan synthesis in the posterior sclera.

The sclera and myopia.

Myopia is a very common ocular problem, affecting perhaps one billion people worldwide. Most myopia is produced by lengthening of the vitreous chamber of the ocular globe. High myopia is characterized by scleral thinning and localized ectasia of the posterior sclera. The sclera is a dense, fibrous, viscoelastic connective tissue that forms the outer coat of the eye and consists of irregularly arranged lamellae of collagen fibrils interspersed with proteoglycans and non-collagenous glycoproteins. Scleral fibroblasts are located between scleral lamellae, and are responsible for synthesizing the extracellular matrix in which they reside. Research highlighted in this review clearly demonstrates that the sclera is not a static container of the eye, but rather is a dynamic tissue, capable of altering extracellular matrix composition and its biomechanical properties in response to changes in the visual environment to regulate ocular size and refraction. Based on these studies, a strategy directed at reversing myopia-associated scleral extracellular matrix remodeling events would be warranted, particularly in cases of high myopia in humans.

Melatonin receptors in chick ocular tissues: implications for a role of melatonin in ocular growth regulation.

PURPOSE: The influences of diurnal rhythms involving a variety of ocular parameters are implicated in the development of myopia. The purpose of this study was to determine the expression of the melatonin receptor subtype proteins in chick ocular tissues and to examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia. METHODS: Expression of the Mel(1a), Mel(1b), and Mel(1c) melatonin receptor proteins in ocular tissues was examined by Western blot analyses, slot blot analyses, and immunocytochemistry. For examining the effect of melatonin on ocular growth, chicks were maintained on a 12-hour light-dark cycle and were monocularly form-vision deprived in one eye with a translucent occluder for 5 days. During the 5-day treatment period, chicks were injected systemically during the early dark period with melatonin (0.6 mg) or 2% ethanol vehicle control. Ocular dimensions of normal and deprived eyes were examined by high frequency A-scan ultrasound. RESULTS: Immunocytochemical analysis of chick ocular tissues revealed the cellular distribution of the Mel(1a) receptor subtype in the cornea, choroid, sclera, and retina. Western blot and slot blot analyses demonstrated that all three receptors were present in these tissues and they demonstrated distinct diurnal rhythms of protein expression in the retina-RPE-choroid, with peak levels of Mel(1a) and Mel(1b) occurring during the night and peak levels of Mel(1c) during the day. Systemic administration of melatonin resulted in significant changes in anterior chamber depth, vitreous chamber depth, and choroidal thickness of form-deprived and/or control eyes. CONCLUSIONS: Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth.

Interaction of lumican with aggrecan in the aging human sclera.

PURPOSE: Lumican is a keratan sulfate proteoglycan originally identified in cornea, but present in a variety of connective tissues where it presumably regulates collagen fibril formation and organization. The present study was designed to describe the chemical nature of lumican core protein in the aging human sclera. METHODS: Western blot analyses, immunohistochemistry, and immunoaffinity chromatography were used to detect and purify the lumican core protein from tissue extracts from human donors 6 to 89 years of age. Treatment of lumican with chondroitinase ABC, keratanase-I and -II, and/or endo-beta-galactosidase was used to determine the degree of glycosylation of the lumican core protein. RESULTS: Lumican was present in the human sclera as a 70- to 80-kDa core protein with short unsulfated lactosaminoglycan side chains. In addition, on Western blots, a larger >200-kDa species was apparent that was immunologically related to lumican. This high-molecular-weight material increased in scleral extracts with increasing age. The complex was most abundant in unreduced samples, and approximately two thirds of the 70- to 80-kDa lumican core protein was released from the complex on reduction of the scleral extract. Further characterization of the >200-kDa lumican-immunopurified complex indicated that aggrecan (the cartilage proteoglycan) was covalently associated with lumican. CONCLUSIONS: Reducible and nonreducible lumican-aggrecan interactions occur in the scleral extracellular matrix and result in the formation of high-molecular-weight complexes that increase with age. These results represent the first report demonstrating lumican-aggrecan interactions and suggest they may play a role in age-related scleral extracellular matrix changes.