ABSTRACT
PURPOSE: Alpha-enolase (ENO1), a major glycolytic enzyme, is reported to be over-expressed in various cancer tissues. It has been demonstrated to be regulated by the Hypoxia-inducible factor 1-α (HIF-1α), a crucial transcriptional factor implicated in tumor progression and cancer angiogenesis. Choroidal neovascularization (CNV), which is a leading cause of severe vision loss caused by newly formed blood vessels in the choroid, is also engendered by hypoxic stress. In this report, we investigated the expression of ENO1 and the effects of its down-regulation upon cobalt (II) chloride-induced hypoxia in retinal pigment epithelial cells, identified as the primary source of ocular angiogenic factors. METHODS: HIF-1α-diminished retinal pigment epithelial cells were generated by small interfering RNA (siRNA) technology in ARPE-19 cells, a human retinal pigment epithelial cell line. Both normal and HIF-1α-diminished ARPE-19 cells were then subjected to hypoxic challenge using cobalt (II) chloride (CoCl2) or anaerobic chamber. The relation between ENO1 expression and vascular endothelial growth factor (VEGF) secretion by retinal pigment epithelial cells were examined. Protein levels of HIF-1α and ENO1 were analyzed using Western Blot, while VEGF secretion was essayed by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity after hypoxia was detected by Lactate Dehydrogenase (LDH) Assay. RESULTS: Upon 24 hr of CoCl2-induced hypoxia, the expression levels of ENO1 and VEGF were increased along with HIF-1α in ARPE-19 cells, both of which can in turn be down-regulated by HIF-1α siRNA application. However, knockdown of ENO1 alone or together with HIF-1α did not help suppress VEGF secretion in hypoxic ARPE-19 cells. CONCLUSION: ENO1 was demonstrated to be up-regulated by HIF-1α in retinal pigment epithelial cells in response to hypoxia, without influencing VEGF secretion.
Subject(s)
Biomarkers, Tumor/genetics , Cobalt/pharmacology , DNA-Binding Proteins/genetics , Epithelial Cells/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Oxygen/pharmacology , Phosphopyruvate Hydratase/genetics , Tumor Suppressor Proteins/genetics , Vascular Endothelial Growth Factor A/genetics , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Cell Hypoxia , Cell Line , Cell Survival/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression Regulation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , L-Lactate Dehydrogenase/metabolism , Phosphopyruvate Hydratase/antagonists & inhibitors , Phosphopyruvate Hydratase/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Signal Transduction , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
PURPOSE: Retinal ischemia/reperfusion (I/R) occurs in many ocular diseases and leads to neuronal death. Lutein, a potent antioxidant, is used to prevent severe visual loss in patients with early age-related macular degeneration (AMD), but its effect on I/R insult is unclear. The objective of the present study is to investigate the neuroprotective effect of lutein on retinal neurons after acute I/R injury. METHODS: Unilateral retinal I/R was induced by the blockade of internal carotid artery using intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein or vehicle was administered. The number of viable retinal ganglion cells (RGC) was quantified. Apoptosis was investigated using TUNEL assay. Oxidative stress was elucidated using markers such as nitrotyrosine (NT) and poly(ADP-ribose) (PAR). RESULTS: In vehicle-treated I/R retina, severe cell loss in ganglion cell layer, increased apoptosis as well as increased NT and nuclear PAR immunoreactivity were observed. In lutein-treated I/R retina, significantly less cell loss, decreased number of apoptotic cells, and decreased NT and nuclear PAR immunoreactivity were seen. CONCLUSIONS: The neuroprotective effect of lutein was associated with reduced oxidative stress. Lutein has been hitherto used principally for protection of outer retinal elements in AMD. Our study suggests that it may also be relevant for the protection of inner retina from acute ischemic damage.
