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Amany Tawfik, Shanu Markand, Sylvia Magyerdi, Mohamed Al-Shabrawey, Sylvia Smith; Mechanisms of Retinal Vascular Alteration in Hyperhomocysteinemia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2463.
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Excess homocysteine (Hcy), a sulfur containing non-proteinogenic amino acid, is implicated in human vascular disorders. Recently, we described the retinal phenotype in hyperhomocysteinemic mice lacking/deficient in the gene encoding cystathionine β−synthase (CBS). These mice have marked disruption of retinal neuronal layers, decreased ERG function & altered retinal vasculature suggestive of ischemic retinopathy. In this study we explored the mechanism of Hcy-induced retinopathy specifically screening markers of ER stress, oxidative stress, inflammation & NMDA receptor (NMDAr) activation.
Retinas of cbs+/+ (n=21) & cbs-/- (n=22) mice were harvested at ~3 wks. RNA & protein were isolated & subjected to qPCR & western blotting, respectively to analyze expression of ER stress genes & the proteins they encode including BiP, PERK, pPERK, CHOP, ATF6 & RE1α. Retinal cryosections were subjected to immunofluorescence to evaluate BiP, CHOP, NMDAr, TNFα and the oxidative stress marker DACF-2DA. To investigate whether Hcy altered vascular permeability, human retinal endothelial cells (HREC) were exposed to Hcy-thiolactone (20µM, 50µM, 100µM) in the presence/absence of the NMDAr inhibitor MK801 (25µM) or the ER stress inhibitor phenylbutyric acid, PBA (10mM) followed by incubation with FITC-dextran as an indicator of leakage.
Gene & protein analysis revealed ~40% increase in BiP & PERK expression in cbs-/- retinas compared to cbs+/+; there was no significant change in expression of ATF4, ATF6, CHOP, IRE1α . Immunofluorescence studies in retinal cryosections of cbs-/- mice showed a dramatic increase in BiP & CHOP as well as NMDAr, TNFα & DACF-2A. In vitro studies of Hcy-treated HREC showed a 25% increase in FITC-dextran leakage through the HREC monolayer, which was reduced significantly by MK801 and PBA.
Retinal neurovascular alterations observed in severely hyperhomocysteinemic cbs mice are accompanied by increased levels of ER & oxidative stress as well as activation of NMDAr. Importantly, increased vascular permeability may be associated with Hcy-induced leakiness, which can be attenuated by inhibiting ER stress or NMDAr.
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