Hypoxia-Reperfusion Produces Reactive Oxygen Species and Cell Death in the Mitotic Lens Epithelial Cells

Abstract

Purpose: : A highly oxidized lens with opaque brown color and intact lens epithelial cells (LECs) are characteristics of blunescent age-related nuclear cataract. Since the ocular lens is located in a highly hypoxic and low glucose environment, we hypothesized that the depressed blood circulation associated with arteriosclerosis, which develops in a majority of elderly populations, will induce a lack of oxygen and glucose (ischemia). The ischemia activates the unfolded protein response (UPR), the production of reactive oxygen species (ROS), and cell death in LECs. Flavins are known to be sources of brown pigments in this cataract.

Methods: : Human LECs and lenses from rats were cultured in different atmospheric oxygen levels. H₂-DCFH-DA and EthD staining methods detect ROS and cell death, respectively. Protein blot analysis and immunohistochemistry were performed with Abs to UPR-specific proteins. A total glutathione (GSH) quantification kit was used for quantification of free GSH.

Results: : LECs in constant levels of oxygen of < 0.02 %, 1%, 4%, or 20% did not induce the UPR and cell death within 48-72 hrs. LECs in lack of glucose in the presence of 4 or 20% oxygen did not induce cell death within 24 hrs. However, a combination of lack of glucose and oxygen induced the UPR in LECs within 1 hr, and 2.5 hrs is sufficient to trigger the production of ROS and cell death. The UPR is preferentially induced in the proliferative LECs. In the rat lenses, ischemia for 2.5 hrs is also sufficient to induce the production of ROS in the mitotic zone of LECs. The UPR specific proteins were increased in this region of LECs. Flavin adenine dinucleotide (FAD) is an essential co-factor for ER oxidoreductin 1 and GSH-reductase, which were significantly up-regulated in LECs under ER stress. We speculate that the produced ROS and accumulated FDA may be internalized in the lens by an action of the internal micro circulatory system to oxidize the lens.

Conclusions: : Depressed blood circulation induces severe hypoglycemia under hypoxia, which induces the UPR, production of ROS, and cell death in the mitotic LECs.

Keywords: aging • oxygen • hypoxia