Abstract
Purpose: :
Retinal stem and progenitor cells (RPCs) populate the stem cell niche and are suggested to proliferate, migrate and differentiate in response to signals including hypoxia and local cytokine production. We asked whether Epo was upregulated in RPCs under low oxygen in vitro, and whether Epo, when added to Epidermal Growth Factor (EGF) could enhance RPC proliferation through independent or convergent signaling pathways. We also asked if Epo could increase cell survival on exposure to hydrogen peroxide (H2O2).
Methods: :
RPCs were isolated from post-natal day 2 retina or week 4 ciliary body as previously described. Hypoxia experiments were performed after 10 days of culture in a defined environment of 1% oxygen, 5% CO2 and balanced nitrogen, for 4, 8 and 24 hours. For proliferation assays, the number of colonies per well were counted after 10 days in the presence of EGF (20ng/ml), or EGF + Epo (10, 100, 300 uM). Wortmannin, PD98059, FTI-277 and rapamycin, were added at three concentrations each to evaluate the influence of the PI3K, MEK, RhebGTPase and mTOR pathways respectively. Due to the limitations of working with primary RPCs, the R28 progenitor cell line (Dr G Seigel) was used to develop an H2O2 kill assay. After 24 hour differentiation on laminin in the presence of cAMP, R28 cells were exposed to increasing concentrations of H2O2, with or without Epo, and harvested at 8 and 16 hours comparing Annexin V and propidium iodide staining by Flow Activated Cell Sorting. Quantitative PCR was used to determine the ratio of Bax/Bcl-2 mRNA, an indicator of pro- versus anti-apoptotic gene expression.
Results: :
RPCs exposed to 1% oxygen upregulated Epo mRNA at least 3-fold over baseline (n=6). Added to EGF, Epo increased RPC proliferation up to 2-fold, in a dose-dependent manner (n=9). Wortmannin, PD98059 and rapamycin reduced Epo-dependent proliferation (p<0.05), while FTI-277 had no significant effect. On exposure to 200uM H2O2, Epo (350uM) increased cell survival, shifting cell towards early over late-stage apoptosis (n=6). Exposure to H2O2 significantly increased the Bax/Bcl-2 ratio up to 17-fold over a 16 hour period. The effect of Epo on this ratio is under evaluation.
Conclusions: :
These data demonstrate that hypoxic RPCs upregulate Epo expression. Addition of Epo to EGF-stimulated RPCs increases their proliferation, and interacts via the canonical PI3k-Akt-mTOR pathway. Epo also reduces cell death in the presence of superoxide toxicity. Taken together, these data suggest that Epo could play an important role in the growth and survival of RPCs in the stem cell niche.
Keywords: growth factors/growth factor receptors • apoptosis/cell death • cell survival