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Shaoqing He, Yong H Park, Thomas Yorio, Raghu R Krishnamoorthy; Endothelins’ effects on gene expression in rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1918.
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A growing body of evidence suggests that endothelin (ET), a family of 21-amino-acid vasoactive peptides, and their receptors (ETA and ETB receptors) are contributors to the neuronal damage in glaucoma. However, ET’s actions in retinal ganglion cells (RGCs) are not fully understood. The aim of this study is to reveal the roles of ETs and their receptors in primary rat retinal ganglion cells.
Primary RGCs were isolated from postnatal day 4-6 rats by panning with Thy-1 antibody. After 7 days culture, isolated RGCs were treated with 100nM of ET-1, ET-2 or ET-3 for 24 hours. Total RNA was extracted using Qiagen RNeasy Mini kit followed by cDNA synthesis and a gene microarray analyses. Affymetrix Rat Genome 230 2.0 Microarray was used to analyze the gene expression in RGCs in response to 100nM ET-1, ET-2 and ET-3 treatments. Realtime PCR to detect gene expression was used to validate the result of Microarray, and immunocytochemical staining was used to confirm the protein expression of regulated genes in RGCs in the same treatment conditions.
31100 gene transcripts and variants from over 28000 rat genes were detected using the probe chip. There was a more than 2.5 fold up-regulation of 1897, 2459 or 2259 genes and down-regulation of 561, 409 or 343 genes with ET-1, ET-2 or ET-3 treatment respectively, compared with control. In real-time PCR validation, there was no appreciable change for Bax, Caspase-2, Caspase-8 and c-Jun. Gene expression of 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. Immunostaining showed a significant increase in ETA, ETB, GAP-43, phosphorylated c-Jun, c-Jun and C/EBPβ with ET-1 treatment, and a slight increase in Bax, Bim and Bcl-XL.
Endothelins induced profound expression alteration in varieties of genes including cytokines, structural proteins, signaling pathways, transcription factors and matrix molecules in RGCs, and also triggered significant changes in neuronal gene expression. The exploration of endothelins’ roles will help understanding on molecular mechanisms underlying glaucomatous changes with ocular hypertension.
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