Abstract
Purpose: :
Accelerated loss of human corneal endothelial (HCE) cells can occur in disease or following surgery. Transient sub–lethal hypoxia preconditioning can protect against cell death by inducing expression of cell survival genes. To investigate this protective potential in HCE cells, we established an in vitro model of cell loss using a HCE cell line and two cyto–toxins (tertiary butyl hydroperoxide [tBHP] and paraquat [PQ]).
Methods: :
Exactly 16 hours after tBHP (20–100 µM) and PQ (100–500 µM) treatments, tBHP treated cells were collected; whereas PQ was removed, replaced with fresh medium and cells were allowed to recover for 48 hr before being collected. Cell death was assessed using flow cytometric detection of Annexin–V (An) and propidium iodide (PI) double staining. Mitochondrial membrane potential (MMP) was measured using flow cytometric analysis of JC–1 staining. Cellular oligonucleosome levels were measured using a Cell Death Detection ELISAPLUS kit. For hypoxia samples, plates were placed in a hypoxia chamber (0.6±0.1% O2) at fixed time points (4–24 hours) before or after chemical treatments.
Results: :
tBHP (60 µM) and PQ (500 µM) induced 41.8% and 31.6% An+/PI–/+ cells and MMP decreased 66% and 49%, respectively. Hypoxia pre–treatment (8 hr), significantly reduced An+/PI–/+ cells caused by 60 µM tBHP to 15.3% and a similar reduction was also detected following 18 and 24 hour hypoxia. Hypoxia had no effect on the decrease of MMP caused by 60 µM tBHP. Hypoxia pre–treatments (24 hr) also reduced An+/PI–/+ cells caused by 500 µM PQ to 25.2%. MMP reduced by PQ was also completely prevented by hypoxia. Furthermore, the tBHP and PQ induced cell death and hypoxia cell protection were also confirmed by measuring cellular oligonucleosome levels. Hypoxia post–treatment (24 hr) decreased An+/PI–/+ cells caused by 500 µM PQ to 20.4%, although it failed to prevent the induced MMP collapse.
Conclusions: :
Our results suggest that hypoxia can sufficiently protect cells against cell death caused by tBHP and PQ although no direct link between hypoxia cell protection and MMP preservation was observed.
Keywords: apoptosis/cell death • cornea: endothelium • hypoxia