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Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery.

ABSTRACT: Gene expression in tree shrew choroid was examined during the development of minus-lens induced myopia (LIM, a GO condition), after completion of minus-lens compensation (a STAY condition), and early in recovery (REC) from induced myopia (a STOP condition). Five groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), one minus-lens group wore a monocular -5 D lens for 2 days (LIM-2), another minus-lens group achieved stable lens compensation while wearing a monocular -5 D lens for 11 days (LIM-11); a recovery group also wore a -5 D lens for 11 days and then received 2 days of recovery starting at 35 DVE (REC-2). Two age-matched normal groups were examined at 26 DVE and 37 DVE. Quantitative PCR was used to measure the relative differences in mRNA levels in the choroid for 77 candidate genes that were selected based on previous studies or because a whole-transcriptome analysis suggested their expression would change during myopia development or recovery. Small myopic changes were observed in the treated eyes of the LIM-2 group (-1.0 ± 0.2 D; mean ± SEM) indicating eyes were early in the process of developing LIM. The LIM-11 group exhibited complete refractive compensation (-5.1 ± 0.2 D) that was stable for five days. The REC-2 group recovered by 1.3 ± 0.3 D from full refractive compensation. Sixty genes showed significant mRNA expression differences during normal development, LIM, or REC conditions. In LIM-2 choroid (GO), 18 genes were significantly down-regulated in the treated eyes relative to the fellow control eyes and 10 genes were significantly up-regulated. In LIM-11 choroid (STAY), 10 genes were significantly down-regulated and 12 genes were significantly up-regulated. Expression patterns in GO and STAY were similar, but not identical. All genes that showed differential expression in GO and STAY were regulated in the same direction in both conditions. In REC-2 choroid (STOP), 4 genes were significantly down-regulated and 18 genes were significantly up-regulated. Thirteen genes showed bi-directional regulation in GO vs. STOP. The pattern of differential gene expression in STOP was very different from that in GO or in STAY. Significant regulation was observed in genes involved in signaling as well as extracellular matrix turnover. These data support an active role for the choroid in the signaling cascade from retina to sclera. Distinctly different treated eye vs. control eye mRNA signatures are present in the choroid in the GO, STAY, and STOP conditions. The STAY signature, present after full compensation has occurred and the GO visual stimulus is no longer present, may participate in maintaining an elongated globe. The 13 genes with bi-directional expression differences in GO and STOP responded in a sign of defocus-dependent manner. Taken together, these data further suggest that a network of choroidal gene expression changes generate the signal that alters scleral fibroblast gene expression and axial elongation rate.

SUBMITTER: He L

PROVIDER: S-EPMC4155741 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery.

He Li L Frost Michael R MR Siegwart John T JT Norton Thomas T TT

Experimental eye research 20140415

Gene expression in tree shrew choroid was examined during the development of minus-lens induced myopia (LIM, a GO condition), after completion of minus-lens compensation (a STAY condition), and early in recovery (REC) from induced myopia (a STOP condition). Five groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), one minus-lens group wore a monocular -5 D lens for 2 days (LIM-2), another minus-lens group achieved stable ...[more]

PMID: 24742494

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Project description:PurposeThe tree shrew model of refractive development is particularly useful because, like humans, tree shrews have a fibrous sclera. Selective changes in some candidate extracellular matrix proteins and mRNAs have been found in the sclera during the development of and recovery from induced myopia. We undertook a more neutral proteomic analysis using two-dimensional gel electrophoresis and mass spectrometry to identify scleral proteins that are differentially expressed during the development of and recovery from lens-induced myopia.MethodsFive tree shrews (Tupaia glis belangeri) wore a monocular -5 D lens for four days, starting 24 days after natural eye opening. At the end of this time, all treated eyes had partially compensated for the lens and were -3.5+/-0.7 D (mean+/-SEM) myopic relative to the untreated fellow control eyes. An additional five animals wore a -5 D lens for 11-13 days followed by four days of recovery without the -5 D lens. The amount of recovery was 1.6+/-0.4 D. Scleral proteins from both groups were then isolated and resolved by two-dimensional gel electrophoresis and spots that were differentially expressed were identified by mass spectrometry.ResultsThe scleral protein profile typically displayed about 700 distinct protein spots within the pH 5-8 range. Comparison of the treated-eye and control-eye scleras of the lens-compensation animals revealed five spots that were significantly and differentially expressed in all five pairs of eyes; all were downregulated 1.2 to 1.7 fold in the treated eye. These proteins were identified as: pigment epithelium-derived factor (PEDF), procollagen Ialpha, procollagen Ialpha2, and thrombospondin I (two spots). In the recovering eyes, the two thrombospondin I spots remained lower in abundance while PEDF and the procollagens were no longer downregulated. In addition, 78 kDa glucose-regulated protein (GRP 78), a member of the heat shock protein 70 family, was slightly upregulated 1.3 fold.ConclusionsWe found consistent results across animals that were of a magnitude consistent with the physiologically small changes to the focal plane of these eyes. Changes in collagen confirm previous findings, but downregulation of thrombospondin I adds detail to our understanding of the chain of signals that regulates scleral creep rate. The differential changes in PEDF and GRP 78 were not expected based on previous studies and demonstrate the utility of the proteomic approach in tree shrew sclera.

Project description:During the development of, and recovery from, negative lens-induced myopia there is regulated remodeling of the scleral extracellular matrix (ECM) that controls the extensibility of the sclera. Difference gel electrophoresis (DIGE) was used to identify and categorize proteins whose levels are altered in this process.Two groups of five tree shrews started monocular lens wear 24 days after eye opening (days of visual experience [VE]). The lens-induced myopia (LIM) group wore a -5 D lens for 4 days. The recovery (REC) group wore a -5 D lens for 11 days and then recovered for 4 days. Two normal groups (28 and 39 days of VE; n = 5 each) were also examined, age-matched to each of the treatment groups. Refractive and A-scan measures confirmed the effect of the treatments. Scleral proteins were isolated and resolved by DIGE. Proteins that differed in abundance were identified by mass spectrometry. Ingenuity pathway analysis was used to investigate potential biological pathway interactions.During normal development (28-39 days of VE), eight proteins decreased and one protein increased in relative abundance. LIM-treated eyes were myopic and longer than control eyes; LIM-control eyes were slightly myopic compared with 28N eyes, indicating a yoking effect. In both the LIM-treated and the LIM-control eyes, there was a general downregulation from normal of proteins involved in transcription, cell adhesion, and protein synthesis. Additional proteins involved in cell adhesion, actin cytoskeleton, transcriptional regulation, and ECM structural proteins differed in the LIM-treated eyes versus normal but did not differ in the control eyes versus normal. REC-treated eyes were recovering from the induced myopia. REC-control eye refractions were not significantly different from the 39N eyes, and few proteins differed from age-matched normal eyes. The balance of protein expression in the REC-treated eyes, compared with normal eyes and REC-control eyes, shifted toward upregulation or a return to normal levels of proteins involved in cell adhesion, cell division, cytoskeleton, and ECM structural proteins, including upregulation of several cytoskeleton-related proteins not affected during myopia development.The DIGE procedure revealed new proteins whose abundance is altered during myopia development and recovery. Many of these are involved in cell-matrix adhesions, cytoskeleton, and transcriptional regulation and extend our understanding of the remodeling that controls the extensibility of the sclera. Reductions in these proteins during minus lens wear may produce the increased scleral viscoelasticity that results in faster axial elongation. Recovery is not a mirror image of lens-induced myopia-many protein levels, decreased during LIM, returned to normal, or slightly above normal, and additional cytoskeleton proteins were upregulated. However, no single protein or pathway appeared to be responsible for the scleral changes during myopia development or recovery.

Project description:We compared gene expression signatures in tree shrew sclera produced by three different visual conditions that all produce ocular elongation and myopia: minus-lens wear, form deprivation, and dark treatment.Six groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), two minus-lens groups wore a monocular -5 diopter (D) lens for 2 days (ML-2) or 4 days (ML-4); two form-deprivation groups wore a monocular translucent diffuser for 2 days (FD-2) or 4 days (FD-4). A dark-treatment (DK) group was placed in continuous darkness for 11 days after experiencing a light/dark environment until 17 DVE. A normal colony-reared group was examined at 28 DVE. Quantitative PCR was used to measure the relative differences in mRNA levels for 55 candidate genes in the sclera that were selected, either because they showed differential expression changes in previous ML studies or because a whole-transcriptome analysis suggested they would change during myopia development.The treated eyes in all groups responded with a significant myopic shift, indicating that the myopia was actively progressing. In the ML-2 group, 27 genes were significantly downregulated in the treated eyes, relative to control eyes. In the treated eyes of the FD-2 group, 16 of the same genes also were significantly downregulated and one was upregulated. The two gene expression patterns were significantly correlated (r(2) = 0.90, P < 0.001). After 4 days of treatment, 31 genes were significantly downregulated in the treated eyes of the ML-4 group and three were upregulated. Twenty-nine of the same genes (26 down- and 3 up-regulated) and six additional genes (all downregulated) were significantly affected in the FD-4 group. The response patterns were highly correlated (r(2) = 0.95, P < 0.001). When the DK group (mean of right and left eyes) was compared to the control eyes of the ML-4 group, the direction and magnitude of the gene expression patterns were similar to those of the ML-4 (r(2) = 0.82, P < 0.001, excluding PENK). Similar patterns also were found when the treated eyes of the ML-4, FD-4, and DK groups were compared to the age-matched normal eyes.The very similar gene expression signatures produced in the sclera by the three different myopiagenic visual conditions at different time points suggests that there is a "scleral remodeling signature" in this mammal, closely related to primates. The scleral genes examined did not distinguish between the specific visual stimuli that initiate the signaling cascade that results in axial elongation and myopia.

Project description: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.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.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).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.

Project description:Pathological neovascularization in choroid, a leading cause of blindness, is a characteristic of many fundus diseases, such as diabetic retinopathy and age-related macular degeneration. The present study aimed to elucidate the key signaling pathways in choroidal neovascularization (CNV) by analyzing the mRNA profiles of choroid and retina in tree shrews with CNV. We induced choroidal angiogenesis by laser photocoagulation in 15 tree shrews and obtained mRNA profiles of their choroids and retinas by high-throughput transcriptome sequencing. Hierarchical cluster analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, hematoxylin and eosin (HE) staining, CD31 immunohistochemistry (IHC), and reverse transcription quantitative PCR (RT-qPCR) were performed. After laser photocoagulation, we obtained a total of 350 differentially expressed genes (DEGs) in the choroid, including 59 genes in Module-FASN ("ME-FASN") module and 28 genes in Module-RPL ("ME-RPL") module. A total of 69 DEGs in retina, including 20 genes in Module-SLC ("ME-SLC") module. Bioinformatics analysis demonstrated that DEGs in choroid were mainly involved in membrane transport; DEGs in "ME-RPL" were prominent in pathways associated with IgA production, antigen presentation, and cell adhesion molecules (CAMs) signaling. DEGs in "ME-FASN" were involved in fatty acid metabolism and PPAR signaling pathway, while DEGs in "ME-SLC" were involved in GABAergic synapse, neuroactive life receptor interaction, cholinergic synapse, and retrograde endocannabinoid signaling pathway. PPI network analysis demonstrated that the ribosomal protein family genes (RPL31, RPL7, RPL26L1, and RPL19) are key factors of "ME-RPL," acyl-CoA superfamily genes (ACACA, ACAT1, ACAA2, and ACACB) and FASN are key factors of "ME-FASN" and superfamily of solid carrier genes (SLC17A6, SLC32A1, SLC12A5, and SLC6A1) and complement genes (C4A, C3, and C2) are key factors of "ME-SLC." In conclusion, the present study discovered the important signal transductions (fatty acid metabolic pathway and CAMs signaling) and genes (ribosomal protein family and the complement system) in tree shrew CNV. We consider that our findings hold implications in unraveling molecular mechanisms that underlie occurrence and development of CNV.

Project description:Hyperopic refractive error is detected by retinal neurons, which generate GO signals through a direct emmetropization signaling cascade: retinal pigment epithelium (RPE) into choroid and then into sclera, thereby increasing axial elongation. To examine signaling early in this cascade, we measured gene expression in the retina and RPE after short exposure to hyperopia produced by minus-lens wear. Gene expression in each tissue was compared with gene expression in combined retina + RPE. Starting 24 days after normal eye opening, three groups of juvenile tree shrews (n = 7 each) wore a monocular -5 D lens. The untreated fellow eye served as a control. The "6h" group wore the lens for 6 h; the "24h" group wore the lens for 24 h; each group provided separate retina and RPE tissues. Group "24hC" wore the lens for 24 h and provided combined retina + RPE tissue. Quantitative PCR was used to measure the relative differences (treated eye vs. control eye) in mRNA levels for 66 candidate genes. In the retina after 6 h, mRNA levels for seven genes were significantly regulated: EGR1 and FOS (early intermediate genes) were down-regulated in the treated eyes. Genes with secreted protein products, BMP2 and CTGF, were down-regulated, whilst FGF10, IL18, and SST were up-regulated. After 24 h the pattern changed; only one of the seven genes still showed differential expression; BMP2 was still down-regulated. Two new genes with secreted protein products, IGF2 and VIP, were up-regulated. In the RPE, consistent with its role in receiving, processing, and transmitting GO signaling, differential expression was found for genes whose protein products are at the cell surface, intracellular, in the nucleus, and are secreted. After 6 h, mRNA levels for 17 genes were down-regulated in the treated eyes, whilst four genes (GJA1, IGF2R, LRP2, and IL18) were up-regulated. After 24 h the pattern was similar; mRNA levels for 14 of the same genes were still down-regulated; only LRP2 remained up-regulated. mRNA levels for six genes no longer showed differential expression, whilst nine genes, not differentially expressed at 6 h, now showed differential expression. In the combined retina + RPE after 24 h, mRNA levels for only seven genes were differentially regulated despite the differential expression of many genes in the RPE. Four genes showed the same expression in combined tissue as in retina alone, including up-regulation of VIP despite significant VIP down-regulation in RPE. Thus, hyperopia-induced GO signaling, as measured by differential gene expression, differs in the retina and the RPE. Retinal gene expression changed between 6 h and 24 h of treatment, suggesting evolution of the retinal response. Gene expression in the RPE was similar at both time points, suggesting sustained signaling. The combined retina + RPE does not accurately represent gene expression in either retina or, especially, RPE. When gene expression signatures were compared with those in choroid and sclera, GO signaling, as encoded by differential gene expression, differs in each compartment of the direct emmetropization signaling cascade.

Dataset Information

Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery.

Publications

Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery.

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