Neuroprotective role of erythropoietin by antiapoptosis in the retina

Abstract

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.

Fig. 1

Light and circadian regulation of EPO/EPOR. Retinal cultures derived from rat or early passages of human RPE cells were exposed to light. Proteins were separated by SDS-PAGE and visualized by Western blot test. A: Rhodopsin was up-regulated in retinal cells after exposure to 5,000 lux light. B: RPE65 was increased after 15 min exposure to 5,000 lux light in the RPE. Truncation of RPE65 to 45 kDa was also seen at 30 min. C: Expression of EPO/EPOR in the 12 hr light/12 hr dark in vivo. Two hours’ exposure to room light (300 lux) rapidly increased the expression of EPO. Continuous exposure to light subsequently decreased EPO. Expression of EPO was recovered after prolonged incubation in the dark.

Fig. 2

Exogenous EPO treatment reduced cell death in H₂O₂-treated retina cells. A: Phase-contrast photomicrographs of sham-washed control or culture treated with 40 µM H₂O₂ for 12 hr. Marked cell death occurred in H₂O₂-treated culture. Scale bar = 30 µm. B: Amount of LDH release was measured from the medium of retinal cultures exposed to the indicated concentrations of H₂O₂ for 3, 6, 12, and 24 hr (mean ± SD, ★P < 0.05, n = 6). All LDH values, after subtraction of background value in sham-washed control cultures, were normalized to the mean maximal value (100) in sister cultures exposed for 24 hr to 5 mM H₂O₂, which causes complete cell death. C: Hoechst 33258 staining of control cells exposed to 40 µM H₂O₂, or cells treated to both H₂O₂ and EPO in retinal cultures (scale bar = 25 µm). D: Bars denote the percentage of LDH release in retinal cell cultures after 3, 6, 12, and 24 hr treatments with 40 µM H₂O₂ alone, or with the addition of EPO 50 U. At 6 hr or later, exogenous EPO showed significant cytoprotective effects of retina cells in oxidative stress. E: Amount of LDH release was measured from the medium of RPE cultures exposed to the indicated concentrations of H₂O₂ for 3, 6, 12, and 24 hr (mean ± SD, ★P < 0.05, n = 6). All LDH values, after subtraction of background value in sham-washed control cultures, were normalized to the mean maximal value (100) in sister cultures exposed for 24 hr to 5 mM H₂O₂, which causes complete cell death.

Fig. 3

Up-regulation of EPO/EPOR expression in the retina exposed to hypoxia (1% O₂; A–C), hyperoxia (50% O₂; D,E), or light (5,000 lux; F,G). A: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells by the cell proliferation assay kit (mean ± SD, ★P < 0.05, n = 6) of retinal cultures exposed to 1% O₂ for 1, 3, and 6 hr, respectively. No significant cell death was observed. B: Immunoblots for EPO and EPOR. As early as 1 hr after exposure to hypoxia, expressions of EPO and EPOR were both increased until 6 hr. Increase of EPOR appeared to be more prominent than EPO up-regulation. C: Immunocytochemistry for cell markers of retina (MAP-2 or GFAP, green) and EPO or EPOR antibody (red) at 3 hr after exposure to hypoxia and sham-washed control. Expression of EPO/EPOR increased after exposure to hypoxia. Scale bar = 30 µm. D: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells (mean ± SD, ★P < 0.05, n = 6) of retinal cultures exposed to 50% O₂ for 1, 3, and 6 hr, respectively. No significant cell death was seen. E: Immunoblots for EPO and EPOR show that 3 hr after exposure to hyperoxia, EPO and EPOR expression increased. F: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells (mean ± SD, ★P < 0.05, n = 6) of retinal cultures exposed to 5,000 lux light for 15, 30, and 60 min. At 15 min or longer exposure, significant cytotoxicity was observed. G: Immunoblots for EPO and EPOR show that EPO expression increased as early as 15 min after exposure to the light, until 60 min. Levels of EPOR also increased.

Fig. 4

Up-regulation of EPO/EPOR expression in the RPE exposed to hypoxia (1% O₂; A,B) hyperoxia (50% O₂; C,D), or light (5,000 lux; E,F). A: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells (mean ± SD, ★P < 0.05, n = 6) of human RPE cell cultures exposed to 1% O₂ for 1, 3, and 6 hr. No significant cell death was seen. B: Immunoblots for EPO and EPOR show that 3 hr after exposure to hypoxia EPO/EPOR expression increased. C: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells (mean ± SD, ★P < 0.05, n = 6) of RPE cell cultures exposed to hyperoxia for 1, 3, and 6 hr. No significant cell death was seen. D: At 3 hr after exposure to hyperoxia, EPO and EPOR expression increased. E: Data represent percentage intensity of DNA fluorescence that is proportional to the number of surviving cells (mean ± SD, ★P < 0.05, n = 6) of human RPE cell cultures exposed to 5,000 lux light for 15, 30, and 60 min. No significant cell death was seen. F: As early as 15 min after exposure to light, EPO/EPOR expression increased until 60 min.

Fig. 5

Upstream regulators of EPO in the retina and the RPE. Retinal cultures derived from rat or early passages of human RPE cells were exposed to 1% O₂, 50% O₂, or 5,000 lux light. Proteins were separated by SDS-PAGE and visualized by Western blot test. A: Increased expression of Hif-1α in the retina under hypoxic and hyperoxic conditions. B: Increased expression of Hif-1α in the RPE at hypoxia or hyperoxic conditions C: Up-regulation of TRX and HO-1 in the RPE exposed to light.

Fig. 6

Downstream regulators of EPO in the retina and the RPE. Retinal cultures derived from rat or early passages of human RPE cells were exposed to 1% O₂, 50% O₂, or 5,000 lux light. Proteins were separated by SDS-PAGE and visualized by Western blot test. A: Expression of p-Jak2 increased in the retina exposed to 1% O₂, 50% O₂, or 5,000 lux light. B: Expression of p-Jak2 increased in the RPE exposed to 1% O₂, 50% O₂, or 5,000 lux light. C: Expression of activated caspase-3 after exposure to light in retinal cells. Until 30 min of light exposure, the level of cleaved caspase-3 in retinal cells was unchanged, indicating that endogenous neuroprotective or antiapoptotic proteins including EPO/EPOR against light stress were up-regulated. At 60 min, increased level of cleaved caspase-3 was compatible with significant cell death. Expression of c-fos and BCL-xl in the retina (D) and RPE cells (E). These antiapoptotic proteins were up-regulated in the light.