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Shaoqing He, Yong H Park, Thomas Yorio, Raghu R Krishnamoorthy; Endothelin-Mediated Gene Expression in Rat Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4405.
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© ARVO (1962-2015); The Authors (2016-present)
The vasoactive peptides, endothelins (ETs), including ET-1, ET-2 and ET-3, and their receptors (ETA and ETB receptors) are contributors to neuronal damage in glaucoma. However, ETs’ direct actions on retinal ganglion cells (RGCs) are not fully understood. The aim of this study was to investigate the effect of ETs on gene expression in primary rat retinal ganglion cells.
Primary RGCs were isolated from retinas of postnatal 4-6 days rat pups by panning with a Thy-1.1 antibody. After 7 days in culture, isolated RGCs were treated with 100nM of ET-1, ET-2 or ET-3 for 24 hours followed sequentially by a gene microarray, real-time PCR and immunocytochemical analyses. Affymetrix Rat Genome 230 2.0 Microarray was used to analyze gene expression in RGCs. Real-time PCR to detect gene expression was used to validate some of the results of the microarray. Additionally, immunocytochemical staining and immunoblot analysis were carried out to confirm the protein expression of regulated genes in RGCs following the same treatment conditions.
Using microarray, 31100 gene transcripts and variants were detected. There was more than 2.5-fold upregulation of 1848, 2394 or 2109 genes, and downregulation of 423, 299 or 372 genes with ET-1, ET-2 or ET-3 treatment respectively, compared to untreated controls. Real-time PCR showed a uniform increasing trend for most of the gene expression obtained from the microarray, including cdk1, s100 family (A4, A6 and A11), il-6, il-11, and ETB receptor. On the other hand, ETA and Bcl-2 in ET-1 treatment was decreased to 70% of control, whereas ETB increased to 13.6 fold compared to sham control. In real-time PCR validation, there was no appreciable change in expression levels of Bax and c-Jun. Immunostaining showed a significant increase in ETA, ETB, Growth Associated Protein 43 (GAP-43), phosphorylated c-Jun, c-Jun and Bax with ET-1 treatment. Immunoblot analysis confirmed ET-1-mediated upregulation of GAP-43, c-Jun and phosphorylated c-Jun in RGCs.
Bcl-2 family, matrix metalloproteinases (MMPs), c-Jun and ET receptors were the major genes or proteins that were regulated by ET-mediated signaling. Endothelins induced a profound alteration in expression of a variety of genes including cytokines, structural proteins, signaling pathways, transcription factors and matrix molecules in RGCs.
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