Project description:We have performed a proteomics analysis of a human retinal pigment epithelial cell line (ARPE-19), which represents a widely used model for in vitro studies of cellular and molecular mechanisms related to human RPE cells. The project was jointly supervised by <b>Francesco Giorgianni</b> and <b>Sarka Beranova-Giorgianni</b>.
Project description:The purpose of this study is to determine the changes in gene expression by a human retinal pigment epithelium (RPE) cell line (ARPE-19) in response to combination treatment of TGF and TNF, which induces phenotypic changes in vitro that mimic the EMT (Epithelial-to-Mesenchymal Transition). For this purpose, total RNA was extracted from TGF and TNF-treated ARPE-19 cells and differential gene expression between each time point (0, 1, 6, 16, 24, 42, and 60 hours) was determined using genechip arrays (Affymetrix, Human Genome U133). Keywords: time course
Project description:Overwhelming and persistent inflammation of retinal pigment epithelium (RPE) induces destructive changes in retinal environment by invoking immune activation. In this study, we aimed to investigate RPE-specific biological and metabolic response against intense inflammation, and identify molecular characteristics determining pathological progression. Here, we performed quantitative analyses of proteome and phosphoproteome of lipopolysaccharide (LPS)-stimulated human derived RPE cell line ARPE-19 using the latest isobaric TMT labeling approach coupled with high-resolution mass spectrometry.
Project description:Overwhelming and persistent inflammation of retinal pigment epithelium (RPE) induces destructive changes in retinal environment by invoking immune activation. In this study, we aimed to investigate RPE-specific biological and metabolic response against intense inflammation, and identify molecular characteristics determining pathological progression. Here, we performed quantitative analyses of phosphoproteome of lipopolysaccharide (LPS)-stimulated human derived RPE cell line ARPE-19 using the latest isobaric TMT labeling approach coupled with high-resolution mass spectrometry.
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:We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site and network analyses. Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 up-regulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response.
Project description:We assessed the role of prolyl hydroxylase inhibiton using DMOG on angiogenic competance and metabolic reprogramming of endothelial cells.
Project description:The purpose of this study is to determine the changes in gene expression by a human retinal pigment epithelium (RPE) cell line (ARPE-19) in response to combination treatment of TGF and TNF, which induces phenotypic changes in vitro that mimic the EMT (Epithelial-to-Mesenchymal Transition). For this purpose, total RNA was extracted from TGF and TNF-treated ARPE-19 cells and differential gene expression between each time point (0, 1, 6, 16, 24, 42, and 60 hours) was determined using genechip arrays (Affymetrix, Human Genome U133). Experiment Overall Design: ARPE19 cell lines treated with TGF and TNF for 0, 1, 6, 16, 24, 42, and 60 hour. Each experiment were repeated three times. But 1 hour experiment was repeated two times.
Project description:We performed a proteomic analysis of a retinal pigment epithelium cell line (ARPE-19) exposed to long-term sublethal levels of H2O2 to mimic low-level pro-oxidative insult in age-related macular degeneration (AMD). using a wide range of biochemical analyses and cell culture techniques, we highlighted changes in energy metabolism, extracellular matrix organization and inflammation-related processes, which may help in understanding the molecular pathogenesis of AMD.
Project description:Rho-associated protein kinase (ROCK) inhibitors are highly present in different ocular tissues, and upregulation of ROCK pathway has been shown related to the pathogenesis of multiple ocular disorders, including glaucoma, diabetic retinopathy and age-related macular degeneration (AMD). However, the effect of ROCK inhibitor on AMD is still unknown. The protein profile changes in human retinal pigment epithelium (ARPE-19) cells after two days treatment with a ROCK inhibitor were studied using label-free Zeno SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive ARPE-19 proteome library. This project describes the use of Zeno SWATH MS to examine the underlying biological protein changes following ROCK inhibitor treatment of APRE-19 cells. This knowledge may allow more specific anti-AMD drugs to be developed