Abstract
Purpose: :
Elevated concentration of reactive oxygen species, including nitric oxide and light- induced apoptosis, elicits the expression of erythropoietin in the retina and RPE. It is necessary to investigate pro-angiogenic signaling cascade upon erythropoietin (EPO) expression to understand anti-apoptotic role under hypoxia. Since EPO is a potent angiogenic factor independent of vascular endothelial growth factors (VEGF), we examined the crosstalk between angiogenic and inflammatory proteins along with EPO and EPOR expression in the retina and RPE. Our results will provide the molecular mechanism on angiogenic signaling against oxygen imbalance conditions.
Methods: :
Rat retinal cells and ARPE-19 cells were cultured in vitro. Proteins were analyzed by biochemical methods such as gel electrophoresis, Western blotting, immunoprecipitation, imunocytochemistry and EPO knock-down experiment using small interfering RNA (siRNA). Nitic oxide (NO) production from the cells was measured using chemiluminescence detection and cells were also exposed to exogenous levels of NO using a modified silicone rubber that contained covalently linked S-nitrosothiol.
Results: :
EPO was significantly increased in all types of neuronal cells, including neuron, ganglion and glial cells, under hypoxia. We found the up-regulation of EPO, its receptor and downstream signaling under hypoxic and hyperoxic conditions. Angiotensin I/II and VEGFR levels increased upon addition of EPO to the human RPE and retina. Beclin 1 is down-regulated under oxidative stress condition. In 1% O2 hypoxic condition, the neurite outgrowth increased shown by Map-2 staining that represents more branched neurite of neuronal cells compared to control. Thy-1 is expressed in the cell surface of neuronal cell and ganglion cell body and in dendrites and axon after neuronal maturation. In control, Thy-1 is observed only on the cell body.
Conclusions: :
Endogenous signaling of EPO/EPOR provides retinal angiogenesis by up-regulating several other angiogenic factors. Indeed, the induction of EPO expression under hypoxia and ischemia induced inflammation in the retina and RPE provides insight to understanding the neo-vascularization in retinal degenerative diseases.
Keywords: retinal pigment epithelium • proteomics • nitric oxide