Role of oxidative stress in chemical hypoxia induced apoptosis

Proliferative retinopathies are the major cause of blindness in industrialized countries and they are characterized by neuronal depolarization, calcium influx and oxidative stress (OS). This condition evokes an unbalanced release of pro- and anti-angiogenic factors from the retinal pigment epithelium (RPE) towards the pro-angiogenic ones, with a consequent aberrant new vessel formation that, together with neurodegeneration, causes irreversible visual loss. It is well known that hypoxia and increase of reactive oxygen species (ROS) which commonly accompany proliferative retinopathies, alter the balance between pro- and anti-angiogenic factors. In the present study we have evaluated a possible role of OS in the modulation of hypoxia activated (HA) factors that induce apoptosis in human RPE-derived cell line (HRPE). After treatment of HRPE cells with 200mM CoCl2 there was a strong induction of HIF1α, confirming that the experimental approach was an acceptable model for in-vitro hypoxia. The cells showed a significantly reduced viability. These data, together with the increased expression of VEGF confirmed that the HRPE cells respond to the lost of oxygen by increasing cell death (most likely apoptosis as shows by a dramatic increase in caspase-3 expression). These effects seem to be related to OS with an increased level of F2-isoprostane in the cellular medium and intra-cellular 4HNE protein adducts. The responses of the HRPE cells to the increased OS is the induction of NFkB activity and this was confirmed by immunohystochemistry analysis. In addiction the increase of OS in these cells is shown by flow cytometry. These results provide evidence of the possible mechanistic pathway involved in retinopathy, where hypoxic event, which leads to increased OS that via the activation of NFkB stimulates the production of VEGF in HRPE cells. Moreover the increase of cells mortality suggest that OS will induce apoptosis.