Abstract
Purpose :
Endothelin-1(ET-1) and its receptors are involved in the etiology of glaucoma. Previously, we reported that ET-1 treatment induced reactivation of astrocytes (ASTs) and apoptosis of retinal ganglion cells (RGC). However, ET-mediated activation of ASTs and their effect on RGC survival are largely unknown. This study aimed to studying the roles of ETs in the interaction between RGCs and ASTs.
Methods :
Primary rat RGCs were isolated from rat pup retinas using Thy-1-antibody-panning methods and ASTs from optic nerve of pups. ASTs were treated with 100nM endothelin-1 for 24 hours and subsequent protein detection was made using western blot and immunocytochemistry. The conditional medium collected from ET-1-treated ASTs was carried out a protein array to detect the secreted proteins. ET-1-mediated intracellular calcium was monitored in RGCs, ASTs and a co-culture of RGCs and ASTs using Fura-2 AM calcium imaging. Cell apoptosis and necrosis was detected using Annexin V and propidium iodide staining. Oxidative stress was monitored by reactive oxygen species (ROS) staining using CellRox.
Results :
The treatment of ET-1 induced not only an increase of GFAP staining but also the rearrangement of GFAP filaments. A significant increase in protein levels of several secreted proteins in ASTs was determined using a protein array. In addition, ET-1 rapidly triggered an elevation of intracellular calcium influx in ASTs and administration of verapamil, an L-type calcium channel blocker, inhibited the influx of calcium in ASTs. ET-1 also induced the elevation of intracellular calcium in RGCs; however, calcium response showed a longer latency and was not affected by verapamil. Interestingly, ET-1-induced elevation of [Ca2+]i was significantly attenuated in co-culture of RGCs and ASTs, and accordingly less cell death was also observed in co-culture based on our preliminary data. Moreover, ET-1 treatment triggered the overproduction of ROS in both ASTs and RGCs and several genes associated with ROS metabolism and apoptotic pathways were also upregulated in RGCs treated with ET-1.
Conclusions :
ET-1 induced the activation of astrocytes through a mechanism that may be involved in the control of oxidative stress and calcium-mediated signaling. The results suggest that the reactivation of ASTs initially could be neuroprotective, however, long-term it could lead to dysfunction in the optic nerve and affect RGC survival.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.