ET-1 Mediated Extra Cellular Matrix - Collagen Regulation in Human Lamina Cribrosa Cells

Abstract

Purpose:: 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.

Methods:: human lamina cribrosa cells were treated with 1nM, 10nM and 100nM of ET-1 for 24 hrs. Total RNA was isolated and cDNA synthesized. ET_A and ET_B 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 ET_A and ET_B receptors, collagen VI were determined by Western blot using anti ET_A ,anti ET_B 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 ET_B selective antagonist and 1uM BQ123 an ET_A 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.

Results:: Expression of both message and protein of ETA and ET_B receptor, collagen I, collagen VI were detected in human lamina cribrosa cells. The message and protein levels of ET_A receptor were down regulated following treatment with ET-1. An upregulation of message and protein levels of ET_B 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 ET_Aspecific inhibitor BQ123 but not by ET_B 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.

Conclusions:: Human lamina cribrosa cells express functional ET_A and ET_B 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.

Keywords: lamina cribrosa • receptors: pharmacology/physiology • pathobiology