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Michael E. Boulton, Li Liu, Zhijuan Chen, Jun Cai, Xiaoping Qi, Maria B. Grant; Targeting The IRE1α/XBP1 Arm Of The Unfolded Protein Response To Inhibit Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1793.
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Growth factors, cytokines, hypoxia and oxidative stress all result in an accumulation of unfolded/misfolded proteins in the endoplasmic reticulum that activate the IRE1 pathway of the unfolded protein response (UPR). The purpose of this study was to determine if inhibition of the IRE1α/XBP1 arm of the UPR could prevent choroidal neovascularization and identify possible mechanisms involved in this inhibition.
In vitro studies utilized retinal and choroidal microvascular endothelial cells that were exposed to varying doses of either putative IRE1α/XBP1 blockers (MG132 and 17-AAG) or siRNAs against IRE1α, XBP1 or α-basic crystallin (CRYAB). In vitro angiogenesis was assessed by the Matrigel tubule formation assay. VEGF degradation was evaluted using the limited proteolysis assay and Western Blot. For in vivo studies, mice received varying concentrations of intravitreal injections of MG132, 17-AAG or siRNAs against IRE1α, XBP1 and CRYAB immmediately following laser induction of CNV. Animals were sacrificed at 14 days post laser and the volume of CNV lesions quantified.
MG132 and 17-AAG showed a marked dose-dependent inhibition of in vitro angiogenesis and choroidal neovascularization in the mouse (p<0.005). The importance of the IRE1α/XBP1 pathway was further confirmed by siRNA knock down of IRE1α and XBP1 which similarly produced a greater than 50% inhibition of angiogenesis both in vitro and in vivo. Activation of the IRE1α/XBP1 pathway leads to increased expression of the chaperone protein CRYAB which is able to bind VEGF and protect it from intracellular degradation. siRNA knockdown of CRYAB inhibited angiogenesis and resulted in increased proteolysis of intracellular VEGF. MG132, 17-AAG and siRNA knock down of IRE1α and XBP1 all significantly increased VEGF proteolysis (p<0.005).
Activation of the unfolded protein response sustains angiogenesis by promoting CRYAB-VEGF binding preventing degradation of intracellular VEGF. The IRE1α/XBP1 pathway of the UPR offers a potential therapeutic target in the treatment of aberrant ocular neovascularization.
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