Abstract
Purpose: :
Lipoperoxidation products may be associated with the pathogenesis of diabetic microvascular complications, including retinopathy. Acrolein is a major lipid peroxidation product that is known to be elevated in diabetic patients. Retinal pericyte apoptosis is a pathophysiological hallmark of diabetic retinopathy. In the present study we demonstrate that acrolein induces retinal pericyte apoptosis in vitro and we have begun to unravel the molecular mechanisms through which this occurs.
Methods: :
Cultured bovine retinal pericytes were incubated with acrolein (10, 25 & 50 µM) for 24 h. Pericyte apoptosis was visualised and measured by confocal scanning laser microscopy and FACs analysis respectively, using annexin V or JC–1. Intracellular oxidants were detected using DCFH–DA. Caspase activity was quantified using luminometric assay kits and NF–kB (p65 subunit) activity was determined with a transcription factor assay kit. Acrolein–lysine adduct generation in the presence of antioxidants was measured by competitive ELISA using the monoclonal antibody 5F6.
Results: :
Acrolein caused a dose–dependent increase in cytotoxicity, with >50% apoptosis at the highest concentration compared to non–exposed cells (p<0.01). Acrolein–induced cell death was associated with enhanced generation of reactive oxygen species with levels 1.8 and 2.8 fold above basal values for the two highest acrolein concentrations (p<0.01). Interestingly, the activity of caspase 3/7, 8 and 9 declined as a function of the acrolein level, with values falling below 40% of those measured in control cells at a concentration of 50 µM (p<0.01). The activity of the anti–apoptotic transcription factor, NF–kB also decreased with increasing concentrations of acrolein (p<0.01). The antioxidants ascorbic acid, superoxide dismutase and catalase partially reversed the generation of oxidants by acrolein (p<0.01), but failed to elevate NF–kB activity. None of these agents directly scavenged acrolein.
Conclusions: :
These results suggest that acrolein–induced apoptosis in retinal pericytes appears to be caspase–independent and may, at least in part, involve direct attenuation of NF–kB activity by acrolein. Acrolein generation in vivo may contribute to the pathogenesis of diabetic retinopathy.
Keywords: apoptosis/cell death • vascular cells • oxidation/oxidative or free radical damage