Dataset Information

Role of bone morphogenetic proteins in form-deprivation myopia sclera.

ABSTRACT:

Purpose

To clarify the role of bone morphogenetic proteins (BMP-2,-4,-5) in sclera remodeling during myopia induction and their effect on sclera fibroblasts in cell culture.

Methods

Reverse transcription and polymerase chain reaction (RT-PCR) as well as immunofluorescence were used to detect the expression of the BMPs in human and guinea pig posterior sclera. In guinea pig form-deprivation myopia (FDM) model, RT-PCR and western blotting were used to investigate changes of BMP expression in the posterior sclera. Human sclera fibroblast (HSF) was primarlly cultured and treated with various doses of BMP-2. Cell proliferation was evaluated by the MTT assay. RT-PCR and western-blot were used to determine the changes of collagen I, aggrecan, and possible activated signal pathway. Cell phenotype and activated signal pathway, especially for α-smooth muscle actin (α-SMA) and phospho-smad1/5/8 were then further investigated by cytoimmunofluorescence staining.

Results

Both human and guinea pig sclera express BMP-2, -4, and -5. In FDM eyes, BMP-2 and BMP-5 expression were reduced in the posterior sclera. Cell proliferation increased significantly (p<0.05) and more cells differentiated into myofibroblast when incubated with 100 ng/ml BMP-2 . The expressions of collangen I, aggrecan, and phospho-smad1/5/8 significantly increased (p<0.05 respectively) as well.

Conclusions

Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, the expressions of BMP-2 and BMP-5 decreased in FDM eyes. BMP-2 might be able to promote HSF proliferation and differentiation, as well as to help extracellular matrix synthesis potentially through classical Smad pathway.

SUBMITTER: Wang Q

PROVIDER: S-EPMC3056124 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Publications

Role of bone morphogenetic proteins in form-deprivation myopia sclera.

Wang Qing Q Zhao Guiqiu G Xing Shichao S Zhang Lina L Yang Xian X

Molecular vision 20110308

<h4>Purpose</h4>To clarify the role of bone morphogenetic proteins (BMP-2,-4,-5) in sclera remodeling during myopia induction and their effect on sclera fibroblasts in cell culture.<h4>Methods</h4>Reverse transcription and polymerase chain reaction (RT-PCR) as well as immunofluorescence were used to detect the expression of the BMPs in human and guinea pig posterior sclera. In guinea pig form-deprivation myopia (FDM) model, RT-PCR and western blotting were used to investigate changes of BMP expr ...[more]

PMID: 21403850

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Project description:PURPOSE: Aquaporins (AQP) form a family of specialized water channels known to transport water across cell membranes and reduce osmotic gradients. The isoform AQP4 is highly expressed in the astroglia of the brain and Müller cells in the retina. In the brain, AQP4 play a role in the control of cerebral edema by shunting excess fluid into blood vessels and by upregulating during conditions of hyperosmolarity. Thus, on the basis of the hyperosmolarity seen across the retina and choroid of hatchling chickens made myopic by form deprivation (FD), we predicted an upregulation of retinal AQP4 expression during induction of myopia. METHODS: Two-day-old hatchling chicks were monocularly form-deprived for 48, 72, or 96 h, and then after biometric assessment, the eyes of these animals and the normal controls of the same age were enucleated. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 protein in the chick retina or for immunolocalization using polyclonal AQP4 antibodies to determine regional distribution and intensity of labeling during the induction of form deprivation myopia (FDM). RESULTS: As expected, ultrasonography demonstrated that all post hatchling eyes showed rapid elongation with occluded eyes elongating faster than fellow eyes or normal controls and becoming progressively more myopic with the duration of visual deprivation. Western blot analyses revealed an approximately 30 kDa band immunoreactive for AQP4 protein and confirmed the presence of AQP4 in chicks. Immunohistochemical staining showed the greatest positive immunoreactivity for antibodies to AQP4 in the inner retina along the vitreoretinal interface, nerve fiber layer, ganglion cell layer, and inner plexiform layer in all animals. The control eyes showed relatively constant levels of AQP4 expression until day 5 after which the level appeared to decrease. By comparison, positive AQP4 immunoreactivity in the nerve fiber layer increased significantly over the first 48 h in form-deprived eyes and in fellow eyes and then decreased over the next 48 h but not to the level of expression in the normal untreated eyes. CONCLUSIONS: This is the first study to demonstrate the presence of AQP4 protein in the chick retina and to associate AQP4 expression in the inner retina with the initiation of form deprivation and the period of fastest axial elongation. This increased expression of AQP4 channels near the vitread border during the time of rapid growth suggests a role for AQP4 as a conduit for movement of retinal fluid into the vitreous in form-deprived chicks.

Project description:Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to the abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. By combining analysis of gene expression, injection of the thymidine analog 5-bromo-2'-deoxyuridine and immunocytochemistry, we show that the retinal periphery in both juvenile rhesus macaques and green monkeys harbors a population of mitotically active neuroprogenitor cells that proliferate when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with the axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. cDNA subtractions, construction of cDNA libraries and differential screening were performed as previously described (Tkatchenko et al, 2000). Two subtractions were carried out resulting in two cDNA libraries. One library was enriched in clones potentially up-regulated in the retina of the closed eye and the other was enriched in cDNAs potentially down-regulated. One thousand clones from each library were analyzed, and 535 differentially-expressed cDNAs were amplified by PCR and spotted onto glass slides coated with poly-L-lysine (five duplicates for each cDNA). The slides were then processed, hybridized with Cy3- and Cy5-labeled complex cDNA probes and washed in 0.2xSSC as described at http://cmgm.stanford.edu/pbrown/protocols/. Two hybridizations with color swap were carried for each monkey. After hybridization, slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). The images were acquired and processed using the GenePix Pro 5.1 software package (Axon Instruments). The data obtained were normalized against GAPDH expression level and mean values (the reported values) and P-values (probability associated with a Student's t-test) were calculated for duplicate experiments (n = 10) with color swap using Microsoft Excel. Genes were defined as differentially regulated if P< 0.05. Cluster analysis was performed using the Genesis software package (provided by Alexander Sturn, Graz University of Technology, Austria).

Dataset Information

Role of bone morphogenetic proteins in form-deprivation myopia sclera.

Purpose

Methods

Results

Conclusions

Publications

Role of bone morphogenetic proteins in form-deprivation myopia sclera.

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