Changes In Retinal Gene Expression Research Articles

OR34-02 Fasting Pancreatic Polypeptide Predicts Incident Microvascular And Macrovascular Complications Of Type 2 Diabetes

Abstract Disclosure: A.H. Sam: None. A.J. Buckley: None. B.Y. Lam: None. P.R. Bech: None. K. Meeran: None. M.T. Barakat: None. S.R. Bloom: None. G.S. Yeo: None. N.G. Lessan: None. K.G. Murphy: None. Background: Pancreatic polypeptide (PP) is elevated in people with Type 2 Diabetes (T2D) or with increased visceral adiposity, risk factors for arterial disease. PP acts at neuropeptide Y (NPY) receptors with effects demonstrated on body composition, arterial tone, and retinal vasculature. Hypothesis: We hypothesised that addition of PP to established factors would enhance estimates of vascular risk in people with T2D. We further hypothesised that PP might have direct effects in glucose sensitive tissues. Methods: Animal study: Continuous subcutaneous PP infusion for four weeks in a high fat diet mouse model; retinal mRNA obtained using Laser Capture Microdissection and submitted for Ingenuity Pathway analysis. Human study: Fasting PP measured at enrolment in 1478 participants (224 normal glucose tolerance, 224 prediabetes, 1030 T2D) recruited at Imperial College London Diabetes Centre Abu Dhabi; microvascular and microvascular complications of diabetes recorded over median 5.5 (IQR 4.9-5.8) years follow-up. Results: Animal study: PP infusion was associated with changes in retinal gene expression potentially influencing neuronal survival including BMP receptors, TWEAK and TNFR1, and inhibition of upstream regulators including DAP3 and ALKBH1, even with unaltered weight, BP and blood glucose compared with pair-fed controls. Human study: In univariate analysis and adjusting for established factors including age, diabetes duration, BMI, sBP, smoking and HbA1c, log2(PP) was significantly associated with incident retinopathy (64 events; univariate: hazard ratio (HR) 1.39 (1.14-1.68) p < 0.001), adjusted: HR 1.26 (1.04-1.53) p=0.021), incident albuminuria (34 events; univariate: HR 1.48 (1.14-1.93) p=0.003, adjusted: HR 1.61 (1.21-2.14) p=0.001, and incident macrovascular disease (74 events; univariate: HR 1.46 (1.22-1.74) p<0.0001, adjusted: HR 1.32 (1.09-1.61), p=0.005), in individuals with T2D. Addition of PP to fully adjusted models resulted in modest improvements in the c-statistic (retinopathy: 0.74 without PP, 0.77 with PP; albuminuria: 0.65 without PP, 0.70 with PP; macrovascular: 0.72 without PP, 0.74 with PP). Adjusting for glycaemic status, BMI, sex, age and family history, PP was associated with increased hazard of progression to T2D in participants not diabetic at enrolment (HR 1.40 (1.08-1.82), p=0.011). Conclusions: Elevated fasting PP predicts incident vascular complications of diabetes, independent of known risk factors. Tonic elevation of PP in the mouse affects retinal gene expression, influencing pathways which may influence neuronal survival. Our results suggest roles for the PP-fold family of peptides in the pathophysiology of diabetic complications and vascular risk stratification. Presentation: Sunday, June 18, 2023

Bioinformatic analysis of retinal gene function and expression in diabetic rats

The aim of the present study was to investigate the changes in retinal gene expression at three time points and assess the underlying molecular mechanisms of diabetic retinopathy (DR) in a streptozotocin (STZ)-induced diabetes rat model using bioinformatics analysis. The gene expression profile of GSE28831 was extracted from the Gene Expression Omnibus database and differentially expressed genes (DEGs) were identified at three different time points (1, 4 and 12 weeks) using the limma package in R language. Gene ontology (GO) enrichment analysis of DEGs was performed followed by a principal component and pathway enrichment analysis of the selected DEGs along with protein-protein interaction network construction at the three time points. A total of 402, 105 and 213 DEGs were screened at 1, 4 and 12 weeks, respectively. In addition, the expression of 8 genes was identified to be significantly different at different time points, including cytochrome P450 2B2 (CYP2B2; downregulated gene; P=0.048; at 1 week), mannan binding lectin-associated serine protease-2 (MASP2; downregulated gene; P=0.044), lecithin retinol acyltransferase (LRAT; downregulated gene; P=0.015), retinal pigment epithelium (RPE)-specific protein 65 kDa (RPE65; downregulated gene; P=0.025), 11-cis-retinoldehydrogenase (RDH5; downregulated gene; P=0.04; at 4 weeks), mitogen-activated protein kinase 13 (MAPK13; upregulated gene; P=0.036), LRAT (downregulated gene; P=0.01) and RPE65 (downregulated gene; P=0.009; at 12 weeks). Furthermore, pathway enrichment and GO enrichment analyses revealed that DEGs at 4 weeks were primarily enriched in retinol metabolism and processes associated with visual functions, including ‘visual perception’ and ‘retinol metabolism’. DEGs, including CYP2B2, MASP2, LRAT, RPE65, RDH5 and MAPK13 may be potential targets for the diagnosis and treatment of DR. Thus, the current study demonstrated that abnormal visual functions occur at 4 weeks in STZ-induced diabetic rats. This may provide a scientific basis for the diagnosis and treatment of DR because DEGs may be used to facilitate the development of novel therapeutic strategies to diagnose and treat DR.

RNA sequencing reveals retinal transcriptome changes in STZ-induced diabetic rats.

The present study aimed to investigate changes in retinal gene expression in streptozotocin (STZ)-induced diabetic rats using next-generation sequencing, utilize transcriptome signatures to investigate the molecular mechanisms of diabetic retinopathy (DR), and identify novel strategies for the treatment of DR. Diabetes was chemically induced in 10-week-old male Sprague-Dawley rats using STZ. Flash-electroretinography (F-ERG) was performed to evaluate the visual function of the rats. The retinas of the rats were removed to perform high throughput RNA sequence (RNA-seq) analysis. The a-wave, b-wave, oscillatory potential 1 (OP1), OP2 and ∑OP amplitudes were significantly reduced in the diabetic group, compared with those of the control group (P<0.05). Furthermore, the implicit b-wave duration 16 weeks post-STZ induction were significantly longer in the diabetic rats, compared with the control rats (P<0.001). A total of 868 genes were identified, of which 565 were upregulated and 303 were downregulated. Among the differentially expressed genes (DEGs), 94 apoptotic genes and apoptosis regulatory genes, and 19 inflammatory genes were detected. The results of the KEGG pathway significant enrichment analysis revealed enrichment in cell adhesion molecules, complement and coagulation cascades, and antigen processing and presentation. Diabetes alters several transcripts in the retina, and RNA-seq provides novel insights into the molecular mechanisms underlying DR.

Progressive morphological changes and impaired retinal function associated with temporal regulation of gene expression after retinal ischemia/reperfusion injury in mice

Retinal ischemia/reperfusion (I/R) injury is an important cause of visual impairment. However, questions remain on the overall I/R mechanisms responsible for progressive damage to the retina. In this study, we used a mouse model of I/R and characterized the pathogenesis by analyzing temporal changes of retinal morphology and function associated with changes in retinal gene expression. Transient ischemia was induced in one eye of C57BL/6 mice by raising intraocular pressure to 120 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. At various time points post I/R, retinal changes were monitored by histological assessment with H&E staining and by SD-OCT scanning. Retinal function was also measured by scotopic ERG. Temporal changes in retinal gene expression were analyzed using cDNA microarrays and real-time RT-PCR. In addition, retinal ganglion cells and gliosis were observed by immunohistochemistry. H&E staining and SD-OCT scanning showed an initial increase followed by a significant reduction of retinal thickness in I/R eyes accompanied with cell loss compared to contralateral control eyes. The greatest reduction in thickness was in the inner plexiform layer (IPL) and inner nuclear layer (INL). Retinal detachment was observed at days 3 and 7 post- I/R injury. Scotopic ERG a- and b-wave amplitudes and implicit times were significantly impaired in I/R eyes compared to contralateral control eyes. Microarray data showed temporal changes in gene expression involving various gene clusters such as molecular chaperones and inflammation. Furthermore, immunohistochemical staining confirmed Müller cell gliosis in the damaged retinas. The time-dependent changes in retinal morphology were significantly associated with functional impairment and altered retinal gene expression. We demonstrated that I/R-mediated morphological changes the retina closely associated with functional impairment as well as temporal changes in retinal gene expression. Our findings will provide further understanding of molecular pathogenesis associated with ischemic injury to the retina.

Inactivation of the microRNA-183/96/182cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice. Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay. Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata. An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.

Protective Gene Expression Changes Elicited by an Inherited Defect in Photoreceptor Structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.

Retinal Gene Expression Changes Related to IOP Exposure and Axonal Loss in DBA/2J Mice

To determine the effects of cumulative IOP exposure and axonal damage on retinal gene expression in DBA/2 mice. DBA/2J, DBA/2J(pe) (pearl), and C57BL/6 mice from 3 to 12 months of age were used. IOP was measured with a rebound tonometer, and optic nerve (ON) damage was determined by grading of ON sections. Retinal RNA was subjected to microarray analysis. Comparisons explored the effects of cumulative IOP exposure (cIOPx) as well as ON damage (ONd) in the DBA/2J animals compared with that in the C57BL/6 and pearl mice. RT-PCR was performed to confirm some of the genes and bioinformatic analysis to identify affected gene networks. Microarrays revealed that an increasing number of genes were up- or downregulated in 9- and 12-month DBA/2J mice with various degrees of ONd. A smaller number of genes were expressed differentially between eyes with different cIOPx at the same age, from 6 months on. Expression of 1385 and 1133 genes differed between DBA/2J animals and C57BL/6 or pearl mice, respectively, and some them were confirmed by RT-PCR. Bioinformatics analysis identified functional gene networks, including members of the complement system, that appeared to be related to cIOPx, ONd, or both. Gene expression changes occur in retinas of DBA/2 mice with various amounts of cIOPx as well as ONd. Genes involved, code for proteins with diverse cellular functions and include among others the complement system. cIOPx and ONd affect common as well as unique gene sets.

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome

BackgroundDiabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.MethodsThe retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.ResultsTranscriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.ConclusionsThese results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

Endogenous Elevation of Homocysteine Induces Retinal Neuron Death in the Cystathionine-β-Synthase Mutant Mouse

To determine the effects of endogenous elevation of homocysteine on the retina using the cystathionine beta-synthase (cbs) mutant mouse. Retinal homocysteine in cbs mutant mice was measured by high-performance liquid chromatography (HPLC). Retinal cryosections from cbs(-/-) mice and cbs(+/-) mice were examined for histologic changes by light and electron microscopy. Morphometric analysis was performed on retinas of cbs(+/-) mice maintained on a high-methionine diet (cbs(+/-) HM). Changes in retinal gene expression were screened by microarray. HPLC analysis revealed an approximate twofold elevation in retinal homocysteine in cbs(+/-) mice and an approximate sevenfold elevation in cbs(-/-) mice. Distinct alterations in the ganglion, inner plexiform, inner nuclear, and epithelial layers were observed in retinas of cbs(-/-) and 1-year-old cbs(+/-) mice. Retinas of cbs(+/-) HM mice demonstrated an approximate 20% decrease in cells of the ganglion cell layer (GCL), which occurred as early as 5-weeks after onset of the HM diet. Microarray analysis revealed alterations in expression of several genes, including increased expression of Aven, Egr1, and Bat3 in retinas of cbs(+/-) HM mice. This study provides the first analysis of morphologic and molecular effects of endogenous elevations of retinal homocysteine in an in vivo model. Increased retinal homocysteine alters inner and outer retinal layers in cbs homozygous mice and older cbs heterozygous mice, and it primarily affects the cells of the GCL in younger heterozygous mice. Elevated retinal homocysteine alters expression of genes involved in endoplasmic reticular stress, N-methyl-d-aspartate (NMDA) receptor activation, cell cycle, and apoptosis.

Changes in Gene Expression in Experimental Glaucoma and Optic Nerve Transection: The Equilibrium between Protective and Detrimental Mechanisms

The authors studied retinal gene expression changes in rats after experimental intraocular pressure elevation and optic nerve transection to elucidate molecular mechanisms of retinal ganglion cell (RGC) death. Translimbal laser photocoagulation was used to induce unilateral IOP elevation in 41 albino Wistar rats. In 38 additional animals, unilateral transection of the optic nerve was performed. Retinas were harvested 1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after each treatment, and total RNA was isolated. Pooled RNA from each time point was analyzed with rat genome arrays. Array results were confirmed by real-time PCR, and localization studies were performed using in situ hybridization for select genes. Genes that were upregulated in glaucoma, but not after transection, included Cyclin D2, Stat1, Stat3, c-Fos, Junb, Anxa1, Anxa 3, and CCAAT/enhancer binding protein (Cebp-delta). In glaucoma and transection models, the upregulation of c-Jun, Activating transcription factor 3, Heat shock protein 27, and Timp1 were observed. Comparisons among microarray databases were performed between our data and reports of retinal and optic nerve injury models in mice, rats, and monkeys. Gene expression changes specific to experimental glaucoma injury were identified. The present analysis supports the importance of neuroinflammation and the participation of the tumor necrosis factor alpha signaling pathway in glaucoma injury. The alterations observed include processes that are both protective of and detrimental to the survival of RGCs.

Form-Deprivation Myopia in Chick Induces Limited Changes in Retinal Gene Expression

Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick. One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription-polymerase chain reaction (qPCR) in independent biological replicates. Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II-related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated. Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development.

Microarray Analysis of Retinal Gene Expression in the DBA/2J Model of Glaucoma

The DBA/2J mouse is a model for secondary angle-closure glaucoma, due to iris atrophy and pigment dispersion, which ultimately lead to increased intraocular pressure (IOP). The study was undertaken to correlate changes in retinal gene expression with IOP elevation by performing microarray analysis of retinal RNA from DBA/2J mice at 3 months before disease onset and at 8 months after IOP elevation. IOP was monitored monthly in DBA/2J animals, and animals with normal (3 months) or elevated IOP (8 months) were identified. RNA was prepared from three individual retinas at each age, and the RNA was amplified and used to generate biotin-labeled probe for high-density mouse gene microarrays (U430.2; Affymetrix, Santa Clara, CA). A subset of genes was selected for confirmation by quantitative RT-PCR, by using independent retina samples from DBA/2J animals at 3, 5, and 8 months of age and compared to retinas from C57BL/6J control animals at 3 and 8 months. There were changes in expression of 68 genes, with 32 genes increasing and 36 genes decreasing at 8 months versus 3 months. Upregulated genes were associated with immune response, glial activation, signaling, and gene expression, whereas downregulated genes included multiple crystallin genes. Significant changes in nine upregulated genes and two downregulated genes were confirmed by quantitative RT-PCR, with some showing changes in expression by 5 months. DBA/2J retina shows evidence of glial activation and an immune-related response after IOP elevation, similar to what has been reported after acute elevation of IOP in other models.

Identification of a unique transcript down-regulated in the retina of rainbow trout ( Oncorhynchus mykiss) at smoltification

Developmental and physiological changes in the retina of salmonid fishes occur during smoltification, a metamorphic event associated with thyroid hormone that prepares salmon for oceanic migration. These changes include loss of ultraviolet-sensitive (UVS) cone photoreceptors, switching of visual pigments, alterations in thyroid hormone regulation, and associated changes in behavior. This model provides an opportunity to study substantial neuronal development within an established retina. Little is known, however, about how higher order neurons are altered or how retinal gene expression changes during this transition. Here, we have used differential display RT-PCR and RACE-PCR to identify a previously uncharacterized gene transcript in Oncorhynchus mykiss under developmental regulation in the retina during smoltification, rtp12.5. This unique cDNA encodes a putative protein 112 amino acids long similar to a hypothetical human open reading frame located on chromosome 14.Q24.2. Differential expression was confirmed by RNA dot blot and in situ hybridization. We also present O. mykiss sep15 cDNA sequence and describe its expression in the vertebrate retina. Considering the expression pattern within retinal tissue observed by in situ hybridization, rtp12.5 may be under TH regulation and involved in neuronal remodeling of the retina during loss of UVS cones.