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V. Rao, R. Krishnamoorthy, T. Yorio; ET-1 Mediated Extra Cellular Matrix - Collagen Regulation in Human Lamina Cribrosa Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4190.
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Endothelin -1 (ET-1) has been implicated in glaucoma pathology. A primary site of injury in POAG appears to be at the level of Lamina cribrosa (LC) which demonstrates extensive ECM remodeling. The purpose of this study was to determine the expression of endothelin receptors and ET-1 effects on the regulation of ECM in GFAP negative Lamina cribrosa cells.
human lamina cribrosa cells were treated with 1nM, 10nM and 100nM of ET-1 for 24 hrs. Total RNA was isolated and cDNA synthesized. ETA and ETB receptor, collagen I, collagen VI mRNA was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Total protein lysates of lamina cribrosa cells treated with 1nm, 10nM & 100nM for 24 hrs were SDS PAGE gel and expression of ETA and ETB receptors, collagen VI were determined by Western blot using anti ETA ,anti ETB receptor antibodies and anti collagen VI antibodies respectively. Expression of collagen I and collagen VI following ET-1 treatment was also determined by immunocytochemistry. ET -1 mediated intra-cellular calcium changes in the presence of 1, 10 & 100nM of ET-1, 1uM BQ788 an ETB selective antagonist and 1uM BQ123 an ETA selective antagonist were measured using Fura-2 calcium imaging. ET-1 mediated intra cellular calcium levels was also measured following ET-1(100nM) treatment for 24 hrs.
Expression of both message and protein of ETA and ETB receptor, collagen I, collagen VI were detected in human lamina cribrosa cells. The message and protein levels of ETA receptor were down regulated following treatment with ET-1. An upregulation of message and protein levels of ETB receptor, collagen I, collagen VI was observed following ET-1 treatment. Increase in intracellular calcium was observed in a dose dependent manner with a greater increase at 100nM. The increase in intra cellular calcium was blocked by ETAspecific inhibitor BQ123 but not by ETB specific inhibitor BQ788. The LC cells treated with ET-1 overnight failed to show an increase in intra cellular calcium levels when retreated with ET-1.
Human lamina cribrosa cells express functional ETA and ETB receptors and their expression and function can be altered in response to prolong exposure to ET-1. In LC cells ET-1 also regulates ECM synthesis. The effects on ECM may be important in POAG subjects who have elevated plasma and aqueous humor levels of endothelin-1.
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