April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Sirt-1 Induces Vascular Endothelial Growth Factor Through The Activation of HIF-2α In Hypoxia
Author Affiliations & Notes
  • Sankarathi Balaiya
    Ophthalmology, Univ of Florida College of Medicine, Jacksonville, Florida
  • Vijay Khetpal
    Ophthalmology, Univ of Florida College of Medicine, Jacksonville, Florida
  • KV Chalam
    Ophthalmology, Univ of Florida College of Medicine, Jacksonville, Florida
  • Footnotes
    Commercial Relationships  Sankarathi Balaiya, None; Vijay Khetpal, None; KV Chalam, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2322. doi:
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    • Get Citation

      Sankarathi Balaiya, Vijay Khetpal, KV Chalam; Sirt-1 Induces Vascular Endothelial Growth Factor Through The Activation of HIF-2α In Hypoxia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2322.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Hypoxia is a critical factor in the patho-physiology of various retinal diseases including age related macular degeneration (AMD). Hypoxia up regulates angiogenic growth factors such as erythropoietin (EPO), vascular endothelial growth factor (VEGF) and promotes neovascularization. Hypoxia alters the cellular redox state and activate class III histone deacetylase (HDACs) sirtulin1 (Sirt1). Sirt 1 activates HIF2α (hypoxia inducible factor) which inturn transactivates VEGF and EPO in hypoxic cells. In this study we investigated the role of Sirt1 in choroidal angiogenesis in vitro.

Methods: : Choroidal endothelial cells (RF/6A) were grown and maintained in a semi-confluent state. Hypoxia was induced by exposing the cells to cobalt chloride for 24 hours and confirmed by flow cytometric analysis of S/G2M ratio. The role of Sirt1 in cell viability was evaluated using WST-1 assay with and without blocking Sirt1 inhibitor, sirtinol. The activation of HIF-2α during hypoxia in presence or absence of Sirt1 was noted using immunoblot analysis. VEGF levels were quantified using enzyme linked immunosorbent assay (ELISA).

Results: : Flow cytometric analysis confirmed the induction of hypoxia by cell cycle arrest at 200 µM concentration of cobalt chloride. The viability of choroidal endothelial cell decreased to 46.2% after blocking sirt 1 activity. However, the viability significantly increased to 55.2% and 52.4% after inducing hypoxia at 100 and 200 µM concentrations respectively, compared to control (p<0.01). After the induction of hypoxia, immunoblot analysis showed a four-fold increase in expression of HIF-2α and the expression was suppressed by blocking sirt 1 activity (p=0.01). VEGF levels further confirmed our results by decreased to control level after blocking sirt1 activity in hypoxic cells.

Conclusions: : Induced hypoxia altered sirt 1 levels in choroidal endothelial cells and promoted release of VEGF through transactivation of HIF-2α

Keywords: age-related macular degeneration • choroid: neovascularization • proliferation 
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