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Don K. Davis, Jr., S Balaiya, KV Chalam; Transcription Regulator Sirt1 Protects Choroidal Endothelial Cells from Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2313.
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© ARVO (1962-2015); The Authors (2016-present)
Age related macular degeneration (AMD) is the leading cause of visual impairment in people over the age of 65, in the Western world. The exudative form of AMD is characterized by choroidal neovascularization (CNV) secondary to VEGF mediated abnormal proliferation of choroidal endothelial cells. Reactive oxygen species/oxidative stress is a major factor associated with AMD. Changes in cellular redox state activate a mammalian silent information regulator sirt1 (Sirtuins). Sirt1, a NAD+ dependent class III histone deacetylase involved in response to molecular damage and oxidative stress, modulates gene silencing, cell division and apoptosis. Activated sirt1 stimulate hypoxia inducible factor (HIF) signaling and in turn augment the transcription of vascular endothelial growth factor A (VEGF-A) and angiogenesis. The purpose of the present study is to identify the role of sirt1 activity in choroidal endothelial cells under oxidative stress.
Monkey choroidal endothelial cells (RF/6A) were maintained in a semi-confluent state in appropriate conditions and serum starved for 24 hrs. Cells were treated subsequently with different concentrations (100 to 700 µM) of hydrogen peroxide (H2O2) to induce oxidative stress. Sirt1 activity was inhibited using a pharmacological inhibitor, sirtinol (100 µM). The role of sirtuin activity was evaluated by measuring the production of reactive oxygen species/oxidative stress after blocking sirt1 activity using dihydrorhodamine 123 (DHR123). After inhibiting sirtiun action, oxidative stress induced cytotoxicity on choroidal endothelial cells was measured by WST-1 assay. Oxidative stress induced HIF signaling was identified using immuno blot analysis.
DHR123 results showed that after inhibiting sirt1 activity, the level of oxidative stress increased 6.6% at 400 µM concentration of H2O2, in a dose dependent manner. Following the inhibition of sirtiun, WST-1 assessment revealed that the cytotoxic effects of oxidative stress on choroidal endothelial cells treated with 400 µM of H2O2 increased 5% compared to control . Further, cytotoxicity increased 35.1%, 62.9%, 71.2% from control with increasing concentration of H2O2 (500, 600 and 700 µM, respectively; p-value<0.01).
Sirtuin protects the choroidal endothelial cell viability at higher concentrations (500, 600 and 700 µM) of oxidative stress and can have a therapeutic role in inhibition of choroidal neovascularisation, a major component of blinding age related macular degeneration.
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