FACS-array profiling in retinal endothelial cells from living mouse retinas
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE27238 | GEO | 2011/04/17
SECONDARY ACCESSION(S): PRJNA137491
REPOSITORIES: GEO
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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.
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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.
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