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.
Project description:Retinal neovascularization is a severe complication of proliferative diabetic retinopathy. We have previously identified that miRNAs is directly involved in the development of retinal neovascularization. Here, we explored the role of miRNAs and its underlying mechanism in modulating angiogenesis.
Project description:Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusive diseases, among others. The use of therapeutics targeting pro-angiogenesis factors such as vascular endothelial growth factor (VEGF) has proven to be highly effective, however considerable side effects exist and serial anti-VEGF treatment has been shown to decrease effectiveness over time. Characterization of additional regulators of neovascularization is needed to further understand neovascular disease and identify possible new therapeutic targets. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxia. EMP2 is highly expressed in human RPE and RPE cell lines. Adult retinal pigment epithelial cell line-19 (ARPE-19) cells were genetically modified to either overexpress EMP2 (OE) or knock down EMP2 (KD) and expression at the RNA and protein level was evaluated using RNA sequencing and western blot respectively. Protein expression was evaluated under both normoxic conditions and conditions of hypoxic stress with 0.5% O2. EMP2 expression was found to positively correlate with expression of the pro-angiogenesis factors hypoxia inducible factor 1-alpha (HIF-1a) and VEGF for both RNA and protein. EMP2 mediated changes in ARPE-19 cells was also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate human umbilical vein endothelial cells (HUVEC) endothelial cell migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potentially important mediator of angiogenesis in the human RPE, a tissue involved in abnormal retinal neovascularization in a number of diseases. EMP2 levels positively correlate with those of the potent pro-angiogenesis mediators HIF-1a and VEGF, however the mechanism of this relationship remains to be clarified. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.
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.
Project description:Background: Retinal neovascularization (RNV) is a leading cause of blindness worldwide. Long non-coding RNA (lncRNA) and competing endogenous RNA (ceRNA) regulatory networks play vital roles in angiogenesis. The RNA-binding protein galectin-1 (Gal-1) participates in pathological RNV in oxygen-induced retinopathy mouse models. However, the molecular associations between Gal-1 and lncRNAs remain unclear. Herein, we aimed to explore the potential mechanism of action of Gal-1 as an RNA-binding protein. Results: A comprehensive network of Gal-1, ceRNAs, and neovascularization-related genes was constructed based on transcriptome chip data and bioinformatics analysis of human retinal microvascular endothelial cells. We also conducted functional enrichment and pathway enrichment analyses. Fourteen lncRNAs, twenty-nine miRNAs, and eleven differentially expressed angiogenic genes were included in the Gal-1/ceRNA network. WT1-AS, LINC01140, and LUCAT1 were situated at the center of the network. Additionally, several key angiogenic genes, such as apelin, angiomotin, and C-X-C motif chemokine ligand 10, were found to potentionally interact with Gal-1 via the ceRNA axis. Furthermore, Gal-1 may be involved in regulating biological processes related to chemotaxis, chemokine-mediated signaling, the immune response, and the inflammatory response. Conclusions: The Gal-1/ceRNA axis identified in this study may play a vital role in RNV. This study provides a foundation for the continued exploration of therapeutic targets and biomarkers associated with RNV.
Project description:Neovascularization contributes to multiple visual disorders including age-related macular degeneration (AMD). Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti-VEGF and concerns of increased risks of developing geographic atrophy following long-term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO/DAND5, a member of the Cerberus-related DAN family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity and AMD. COCO equally abrogated angiogenesis in choroid explants and in a model of choroidal neovascularization. Mechanistically, COCO inhibited the expression of TGFβ and BMP pathwaysand altered ATP production, glucose uptake and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases.
Project description:Background: Retinal neovascularization (RNV) as a result of retinal ischemia, such as in proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), can lead to vitreous hemorrhage, tractional retinal detachment, and irreversible loss of vision if left untreated. Although panretinal laser photocoagulation and anti-vascular endothelial growth factor (VEGF) injections are efficient treatment modalities, a significant number of patients do not respond to either treatment. This clinical finding suggests that other, previously unrecognized mediators and signaling pathways may contribute to RNV development and could represent valuable targets for future treatments. Methods: In search of phylogenetically conserved angiogenic mediators, we examined the transcriptional profile of murine RNV from C57BL/6J mice (n=14) in the oxygen-induced retinopathy (OIR) model as well as human RNV membranes from PDR patients (n=7), who had undergone vitrectomy and compared them with corresponding control tissues (n=13, 10 respectively). Genes that were differentially expressed (DEGs) between RNV and control samples were identified for human and murine samples and their associated gene ontology (GO) clusters analyzed. Lastly, human and murine DEGs were compared to identify phylogenetically conserved factors. Findings: Transcriptional profiles of murine RNV showed that DEGs linked to the activation of the innate immune system (Msn, Cd34), extracellular matrix organisation (Col4a1, Gfap), and regulation of angiogenesis (Col4a2, Fgf2) were significantly upregulated both at the ischemic, preproliferative stage of the disease (OIR p14) as well as at the proliferative stage (OIR p17). While similar GO terms were upregulated in human RNV, only a small overlap in DEGs between both species was detected. Phylogenetically conserved mediators upregulated in both murine and human RNV included ANGPT2, S100A8, MCAM, EDNRA, MRC1, and CCR7. Interpretation: This study identifies phylogenetically conserved inflammatory and pro-angiogenic mediators that are significantly upregulated in both murine and human RNV. Among them, MCAM, ENDRA and MRC1 emerged as the most upregulated, phylogenetically conserved DEGs not yet implicated in human RNV, thus representing potential new treatment targets for ischemic retinal diseases.
Project description:In order to find out the vital genes during retinal neovascularization (RNV), we set up OIR (oxygen-induced retinopathy; induced with 75%±2% oxygen) and wild-type C57BL/6J murine models. We observed the retinal vascular growth process daily both in OIR and wild-type mice through retinal flat-mount, and isolated total retinal RNA at different time points (P8, P9, P12, P13, P30) both in OIR and wild-type mice for gene expression analysis. At least three different retinae were accessed at each time point for observing the retinal vascular growth process. Ten neural retinae from five mice were harvested and pooled into one sample for gene expression analysis. Three biological replicates were used for each time point. Dye-swaps were performed.
Project description:Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we report that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Project description:Choroidal neovascularization (CNV) and the resulting retinal angiogenesis are pathological hallmarks of wet Age-related macular degeneration (AMD). The pathogenesis of CNV is not fully understood, but accumulated evidence has suggested the role of inflammation in the early stage of CNV. To better understand the molecular landscape during the early stage, we performed RNA-Seq and mass spectrometry-based proteomic analysis in the retina of the laser-induced CNV mouse model. Both transcriptomic and proteomic data showed dramatic activation of inflammatory response 3 days post photocoagulation. Integrative analysis suggested a moderate correlation between RNA-Seq and mass spec. Up-regulation of angiogenic factor, basic fibroblast growth factor-2 (Fgf-2), but not vascular endothelial growth factor (Vegf) was observed at both RNA and protein levels, highlighting Fgf-2 as a biomarker and potential therapeutic target during the early stage of CNV. In addition, enrichment analysis indicated a large overlap of inflammation-related genes and pathways at both levels. We also compared our findings with human retinal RNA-Seq data from AMD patients and controls. By using a multi-omics and comparative approach, our findings demonstrate the molecular landscape during the inflammatory stage of mouse CNV and provided new insight into the translation from the mouse model to understanding human AMD and its potential intervention and therapies.