Abstract
Purpose: :
HtrA2/Omi is a nuclear-encoded mitochondrial serine protease with a pro-apoptotic role in caspase dependent cell death. Given that oxidative stress is one of the most important suspected risk factors for age-related macular degeneration (AMD), we investigated the role of HtrA2/Omi in ARPE-19 cells exposed to hydrogen peroxide-induced oxidative stress and in archived eyes with AMD.
Methods: :
Oxidative stress was induced in ARPE-19 cells by 1mM hydrogen peroxide for 2 hours. Expression of HtrA2/Omi in these cells was evaluated using real-time RT-PCR, immunohistochemistry, and western blot analysis. XIAP, caspase-3 and caspase-9 expression was evaluated by immunohistochemistry or western blot analysis. Cellular expression of HtrA2/Omi, activated caspase-3, and activated caspase-9 were also measured after adding UCF-101, an HtrA2/Omi inhibitor, to ARPE-19 cells under H2O2-induced oxidative stress. Cellular expression of HtrA2/Omi, activated caspase-3 and activated caspase-9 were measured. Cell viability was detected by MTT assay. HtrA2 was localized in the retina of AMD and non-AMD control archived eyes by immunohistochemistry.
Results: :
HtrA2/Omi mRNA was upregulated after 2 hour treated with 1mM H2O2. H2O2-induced oxidative stress led to translocation of HtrA2/Omi from mitochondria to the cytosol, neutralization of XIAP, and increased levels of activated caspase-3 and activated caspase-9 in the cytosol. UCF-101 reduced the upregulation of HtrA2 in cytosol and in turn, exerted significant protective effects including inhibition of caspase-9 and caspase-3 activities and decreased cell death. HtrA2/Omi was found in normal retina and was markedly decreased in both wet and dry AMD lesions, especially in the photoreceptor inner segments, where abundant mitochondria reside.
Conclusions: :
H2O2-induced oxidative damage results in HtrA2/Omi relocation from mitochondria to cytosol, where it induces RPE cell apoptosis via a caspase-mediated pathway, possibly resulting in RPE degeneration. UCF-101, a specific HtrA2/Omi inhibitor, reduces expression and cytosolic translocation of HtrA2/Omi, attenuates caspase-3 and caspase-9 activation, and decreases apoptosis of RPE cells. HtrA2/Omi is highly expressed in normal retina, but is decreased in late-stage lesions from both wet and dry AMD, where RPE and photoreceptor are lost. Therefore, HtrA2/Omi-mediated RPE apoptosis due to oxidative stress might play an important role in AMD pathogenesis.
Keywords: apoptosis/cell death • retinal pigment epithelium • age-related macular degeneration