RNA analysis of the circadian rhythm of choroidal neovascularization
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE253949 | GEO | 2024/10/09
REPOSITORIES: GEO
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Project description:Emerging evidence suggests a link between the circadian clock and retinopathies though the causality has not been established. Circadian clocks in the mammalian retina regulate a diverse range of retinal functions that allow the retina to adapt to the light-dark cycle. We report that clock genes are expressed in the embryonic retina, and the embryonic retina requires light cues to maintain robust circadian expression of the core clock gene, Bmal1. Deletion of Bmal1 and Per2 from the retinal neurons results in retinal angiogenic defects similar to when animals are maintained under constant light conditions. Using two different models to assess pathological neovascularization, we show that neuronal Bmal1 deletion reduces neovascularization with reduced vascular leakage, suggesting that a dysregulated circadian clock primarily drives neovascularization. Chromatin immunoprecipitation sequencing analysis suggests that semaphorin signaling is the dominant pathway regulated by Bmal1. Our data indicate that therapeutic silencing of the retinal clock could be a common approach for the treatment of certain retinopathies like diabetic retinopathy and retinopathy of prematurity.
2022-07-01 | GSE207271 | GEO
Project description:Neovascular age-related macular degeneration represents the most common cause of blindness in the western world. Alterations of the outer Blood-retina barrier integrity and a localized inflammatory microenvironment lead to sprouting of choroidal neovascularization in intimate contact with surrounding myeloid cells and ultimately lead to visual impairment. The discovery of novel targets interfering with angiogenesis and inflammation is vital for the future treatments in AMD patients. To identify novel potential targets in the local phagocytes of the retina, microglia, we performed a comprehensive RNA-seq analysis in the mouse model of laser-induced choroidal neovascularization (mCNV). Here, we identified the angiogenic factor Osteopontin (Opn), also known as "secreted phosphoprotein 1” (Spp1), to be one of the most highly expressed genes in retinal microglia in the course of CNV formation. We could confirm the presence of SPP1 at the lesion site in recruited retinal microglia of Cx3cr1CreER:Rosa26-Tomato reporter mice using immunohistochemistry and in whole retinal tissue lysates by ELISA compared to controls highlighting a massive local production of SPP1. Inhibition of SPP1 by intravitreal injection of anti-SPP1 antibody significantly increased the lesion size compared to IgG-treated control eyes. In line with the results in rodents, we found an increased SPP1 mRNA expression in surgically extracted human choroidal neovascular (hCNV) membranes by the quantitative RNA-seq approach of massive analysis of cDNA ends (MACE) and found numerous IBA1+SPP1+ myeloid cells in human CNV membranes. Taken together, these results highlight the importance of SPP1 in the formation of CNV and potentially offer new opportunities for therapeutic intervention by inhibiting the SPP1 pathway.
2021-02-16 | GSE160011 | GEO
Project description:RNA analysis of the circadian rhythm of choroidal neovascularization
| PRJNA1067979 | ENA
Project description:In order elucidate the key signaling pathways in choroidal neovascularization, we induced choroidal angiogenesis by laser photocoagulation in 12 tree shrews and obtained mRNA profiles of their choroids and retinas by high-throughput transcriptome sequencing. Gene ontology(GO) and kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis, hierarchical cluster analysis, weighted gene co-expression network analysis, protein-protein interaction (PPI) network analysis, and reverse transcription quantitative PCR (RT-qPCR) were performed.
2021-03-21 | E-MTAB-10198 | biostudies-arrayexpress
Project description:PURPOSE Diseases that involve choroidal or retinal endothelial vascular cells are leading causes of vision loss: age-related macular degeneration, retinal ischemic vasculopathies and non-infectious posterior uveitis. Proteins differentially expressed by these endothelial cell populations are potential drug targets. We used deep proteomics to define the molecular phenotype of human choroidal and retinal endothelial cells at the protein level. METHODS Choroidal and retinal endothelial cells were separately isolated from 5 human eye pairs by selection on CD31. Total protein was extracted and digested, and peptide fractions were analysed by reverse-phase liquid chromatography tandem mass spectrometry. Peptide sequences were assigned to fragment ion spectra and proteins were inferred using public protein databases. Protein abundance was determined by spectral counting. Protein expression in choroidal versus retinal endothelial cells was compared using edgeR package in R, and annotation enrichment analysis was performed. RESULTS Human choroidal or retinal endothelial cells expressed 5042 non-redundant proteins. Setting the false discovery rate at 5%, 498 proteins (14.4%) of 3454 quantifiable proteins with minimum mean spectral counts of 2.5 were differentially expressed between cell populations. Choroidal and retinal endothelial cells were enriched in angiogenic proteins, and retinal endothelial cells were also enriched in immunologic proteins. CONCLUSIONS This work demonstrates the protein heterogeneity of human choroidal and retinal vascular endothelial cells and provides multiple candidates for further study as novel treatments or drug targets for posterior eye diseases.
2018-03-26 | PXD005972 | Pride
Project description:Background: As a widespread post-transcriptional RNA modification, N5-methylcytosine (m5C) is implicated in a variety of cellular responses and processes that regulate RNA metabolism. Despite this, a clear understanding of m5C modification’s role and mechanism in angiogenesis is still lacking. Methods: Single-cell RNA sequencing data was analyzed to determine expression of m5C methylase NSUN2. m5C levels were determined by mRNA isolation and anti-m5C dot blot in both hypoxia-induced endothelial cells (ECs) and laser-induced choroidal neovascularization (CNV). In addition, endothelial cell and endothelium‐specific NSUN2‐knockout mouse model were used to investigate the effect of NSUN2 silence on angiogenic phenotype. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5C-methylated RNA immunoprecipitation sequencing (m5C-meRIP-seq). CUT&Tag sequencing was performed to test the histone lactylation signal in the promoter region of NSUN2. Finally, AAV-mediated short hairpin RNAi knockdown of NSUN2 gene expression (AAV-shNSUN2) was constructed to investigate the effect of inhibiting CNV. Results: First, we discovered that m5C methylase NSUN2 expression level and mRNA m5C level were significantly higher in CNV-ECs than in normal ECs. NSUN2 knockdown in ECs inhibited proliferative, migration, and tube formation activities of ECs. Moreover, compared with EC NSUN2flox/flox mice, EC-specific NSUN2-deficient (EC NSUN2-/-) mice displayed less retinal vascular leakage after laser induction. Through multiomics analyses, we subsequently identified A-kinase anchoring protein 2 (AKAP2), a scaffolding protein which isolate Protein kinase A (PKA) to specific subcellular locations through binding to its regulatory subunit, as a downstream candidate target of NSUN2 in ECs. Overexpression of exogenous AKAP2 markedly reversed the inhibitory phenotypes in NSUN2-deficient ECs. Interestingly, laser induced NSUN2 up-regulation was driven by lactate-mediated lactylation on histone H3K18. In CNV models, AAV-mediated repression of NSUN2 modulated highly retinal vascular leakage and choroidal thickness. Conclusion: Overall, our findings indicate that NSUN2 is a novel therapeutic target for choroidal neovascularization.
2024-05-19 | GSE267661 | GEO
Project description:Background: As a widespread post-transcriptional RNA modification, N5-methylcytosine (m5C) is implicated in a variety of cellular responses and processes that regulate RNA metabolism. Despite this, a clear understanding of m5C modification’s role and mechanism in angiogenesis is still lacking. Methods: Single-cell RNA sequencing data was analyzed to determine expression of m5C methylase NSUN2. m5C levels were determined by mRNA isolation and anti-m5C dot blot in both hypoxia-induced endothelial cells (ECs) and laser-induced choroidal neovascularization (CNV). In addition, endothelial cell and endothelium‐specific NSUN2‐knockout mouse model were used to investigate the effect of NSUN2 silence on angiogenic phenotype. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5C-methylated RNA immunoprecipitation sequencing (m5C-meRIP-seq). CUT&Tag sequencing was performed to test the histone lactylation signal in the promoter region of NSUN2. Finally, AAV-mediated short hairpin RNAi knockdown of NSUN2 gene expression (AAV-shNSUN2) was constructed to investigate the effect of inhibiting CNV. Results: First, we discovered that m5C methylase NSUN2 expression level and mRNA m5C level were significantly higher in CNV-ECs than in normal ECs. NSUN2 knockdown in ECs inhibited proliferative, migration, and tube formation activities of ECs. Moreover, compared with EC NSUN2flox/flox mice, EC-specific NSUN2-deficient (EC NSUN2-/-) mice displayed less retinal vascular leakage after laser induction. Through multiomics analyses, we subsequently identified A-kinase anchoring protein 2 (AKAP2), a scaffolding protein which isolate Protein kinase A (PKA) to specific subcellular locations through binding to its regulatory subunit, as a downstream candidate target of NSUN2 in ECs. Overexpression of exogenous AKAP2 markedly reversed the inhibitory phenotypes in NSUN2-deficient ECs. Interestingly, laser induced NSUN2 up-regulation was driven by lactate-mediated lactylation on histone H3K18. In CNV models, AAV-mediated repression of NSUN2 modulated highly retinal vascular leakage and choroidal thickness. Conclusion: Overall, our findings indicate that NSUN2 is a novel therapeutic target for choroidal neovascularization.
2024-05-19 | GSE267662 | GEO
Project description:To investigate the tRF/tiRNA expression pattern in the development of choroidal neovascularization, we built a laser-induced choroidal neovascularization mouse model and performed the tRF/tiRNA sequencing on RPE-choroid-sclera complexes at different time points after model establishment, while the tissues from mice without treatment were used as control group.
2022-08-05 | GSE210099 | GEO
Project description:Choroidal neovascularization (CNV) is a severe complication of the late-stage form of wet age-related macular degeneration. In order to identify lncRNAs and coding genes involved in the pathogenesis of CNV, we performed microarray analysis to identify expression profiles of lncRNAs and mRNAs in laser-induced CNV samples and controls in mice.
2019-04-13 | GSE129743 | GEO
Project description:Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, we hypothesized that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from 6 human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasite:cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8747 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false discovery rate set at 5 %. For selected genes, differences in level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphical descriptive analysis demonstrated strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 of 8,747 transcripts (8.9 %) were differentially represented. Notably, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. Our findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte. Experiment Overall Design: Retinal and choroidal vascular endothelial cells were isolated from both eyes of 6 human cadaver donors. Endothelial cells of each subtype from each donor were separately pooled and cultured. In a first series of experiments (Experiment 1), donor-matched cultures of retinal and choroidal endothelial cells derived from 3 donors were incubated with either T. gondii tachyzoites or medium alone. In a second set of experiments (Experiment 2), cultured retinal and choroidal endothelial cells derived from the other 3 donors were stimulated with LPS or medium alone. Whenever possible, 2 replicate dishes of endothelial cells were subjected to each set of conditions. Subsequent to incubations, mRNA was extracted from a cell lysate prepared from each dish of cells, and cRNA derived from each mRNA preparation was separately hybridized to one oligonucleotide expression array.
2008-06-15 | E-GEOD-7850 | biostudies-arrayexpress