Indian hedgehog signaling from endothelial cells is required for sclera and retinal pigment epithelium development in the mouse eye

The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.     

Human Sclera Maintains Common Characteristics with Cartilage throughout Evolution

Background

The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm.

Methodology/Principal Findings

We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-βs and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human ‘sclera’-derived cells can be considered chondrocytes when cultured ex vivo.

Conclusions/Significance

Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.     

The retinal pigment epithelium of the eye regulates the development of scleral cartilage

The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown.Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation.There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro.RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved.These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.     

ρ1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes

rho(1) GABA(C) receptor antagonists inhibit myopia in chick but the site of this effect is not known. The sclera ultimately determines the shape and size of the globe and thus an untested possibility is that GABA agents have a scleral mechanism. Whether rho(1) GABA(C) receptors are expressed and located in chick sclera is unknown. Real-time PCR, western blot and immunohistochemistry were used to determine whether rho1 GABA(C) receptors are expressed and located in chick fibrous and cartilaginous sclera. Both layers of the chick sclera were positive for rho1 GABA(C) receptor mRNA (PCR) and protein (western blot) expression and labeling was observed in both fibroblasts and chondrocytes of the fibrous and cartilaginous layers (immunohistochemistry). These investigations clearly show that chick sclera possesses rho(1) GABA(C) receptors. The sclera is thus a potential previously unrecognized site for activity of rho(1) GABA(C) agents.     

Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.     

Mitf functions as an in ovo regulator for cell differentiation and proliferation during development of the chick RPE

Mitf has been reported to play a crucial role in regulating the differentiation of pigment cells in homeothermal animals, i.e. the melanocytes and the retinal pigment epithelium (RPE). However, less is known about the functions of Mitf in the developing RPE. To elucidate such functions, we introduced wild-type and dominant-negative Mitf expression vectors into chick optic vesicles by electroporation. Over-expression of wild-type Mitf altered neural retina cells to become RPE-like and repressed the expression of neural retina markers in vivo. In contrast, dominant-negative Mitf inhibited pigmentation in the RPE. The percentage of BrdU-positive cells decreased during normal RPE development, which was followed by Mitf protein expression. The percentage of BrdU-positive cells decreased in the wild-type Mitf-transfected neural retina, but increased in the dominant-negative Mitf-transfected RPE. p27^kip1, one of the cyclin-dependent kinase inhibitors, begins to be expressed in the proximal region of the RPE at stage 16. Transfection of wild-type Mitf induced expression of p27^kip1, while transfection of dominant-negative Mitf inhibited p27^kip1 expression. We found that Mitf was associated with the endogenous p27^kip1 5′ flanking region. These results demonstrate for the first time “in vivo” that Mitf uniquely regulates both differentiation and cell proliferation in the developing RPE.     

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 × 10³, 1 × 10⁴ or 1 × 10⁵ cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 × 10⁴ cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 × 10⁴ cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-α induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.     

BMP-2 Is Involved in Scleral Remodeling in Myopia Development

The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.     

Hydrocaffeic and p-coumaric acids,natural phenolic compounds,inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo

This paper studied the effect on UV-B ocular damage of 10µm hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5µm HCAF+5µm p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m²) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m²). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E₂ (PGE₂) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.     

Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks

A previously characterized chick model of myopia was used to evaluate biochemical changes in the sclera which are associated with ocular enlargement and myopia. Chicks were monocularly occluded for 10 days and the DNA, hydroxyproline, and glycosaminoglycan contents of the sclera were compared between the normal and the myopic eyes. No significant differences could be detected in total DNA or hydroxyproline content. There was, however, a 34% increase in glycosaminoglycans and a 20.7% decrease in cell density within the posterior sclera of myopic eyes. The biosynthesis of scleral proteoglycans was determined by measuring 35SO4 incorporation in the sclera of chicks visually occluded for 5, 10, and 15 days. No differences could be detected in 35SO4 incorporation into the cornea or the anterior sclera. However, 35SO4 incorporation was significantly increased in the posterior sclera of myopic eyes by 64% at Day 5, 39% at Day 10, and 49% at Day 15. When fractionated on Sepharose CL-4B, scleral proteoglycans were resolved into two peaks which were identified by Western blot analysis as aggrecan (cartilage proteoglycan) and decorin. Furthermore, Western blot and dot blot analyses indicated that significantly more aggrecan core protein was present in the sclera of myopic eyes compared with equivalent amounts of sclera from control eyes. These results indicate that increased synthesis and accumulation of aggrecan, which increases the volume of extracellular matrix in the posterior sclera, are responsible for the ocular enlargement observed in this model of myopia.     

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective

To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system.

Methods

Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using ³⁵SO₄²⁻-labelling and the dimethylmethylene blue assay. ³H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured.

Results

The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. ³H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the ³⁵SO₄²⁻ incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts.

Conclusion

The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.     

Scleral Micro-RNA Signatures in Adult and Fetal Eyes

Introduction

In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.

Methods

Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.

Results

Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10¹¹). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.

Conclusion

For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.     

Retinal pigment epithelial cell multinucleation in the aging eye – a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age‐related retinal degenerative disorders particularly age‐related macular degeneration. During aging RPE cells decline in number, suggesting an age‐dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose‐dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted ‘postmitotic’ status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long‐standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age‐related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE‐choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two‐step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell‐extracellular matrix and/or cell–cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E₂ (PGE₂) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE₂ pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE₂ receptors) is essential. We therefore examined the production of PGE₂ in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE₂ on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE₂ receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE₂ synthesis was determined by mass spectrometry, cell proliferation by DNA [³H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE₂ into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE₂-dependent proliferation. Exogenously added PGE₂ stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10^-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE₂ was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE₂ release, which stimulates cell proliferation via the EP1 receptor.     

Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which factors control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming growth factor)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral fibroblasts. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral fibroblasts, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately 15% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye growth.     

The immunoregulatory effects of edeine analogues in mice

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 μg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 μg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1–10 μg/ml but was inhibitory at 100 μg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.