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RAJI RAJESH LENIN, Peter Gergely Nagy, Rajashekhar Gangaraju; Inflammation and hyperglycemia mediate endothelial permeability through ER stress mediated posttranslational modifications. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4595.
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Glycosylation of plasma membrane and secretory proteins as a posttranslational modification (PTM) is a global phenomenon and more than 50% of the proteins in nature are glycosylated. Excess O-glycosylation has been linked to the pathogenesis of several diseases including diabetes and in the pathogenesis of DR. We hypothesize that ER stress under chronic pro-inflammatory and hyperglycemic conditions in retinal endothelial cells would lead to increased O-glycosylation, which in turn affect the tight junction proteins in the plasma membrane leading to permeability defects.
Human retinal microvascular endothelial cells (HREC) were treated with 10 ng/mL TNF-α and 30 mmol/L glucose for 24 hours with and without ER stress inhibitor (10µM TUDCA). Gene and protein expression of ER stress markers were analyzed by Taqman assay and Western blot analysis respectively. Alterations in endothelial junction proteins were evaluated by western blot and immunocytochemistry. GRP-78 translocation was assessed with KDEL silencing. Posttranslational modifications were assessed by O-glycosylation immunoblots. Electrical Impedance Cell Substance (ECIS) was used to assess trans-endothelial resistance (TER) with and without TUDCA.
HREC exposed to TNF-a and high glucose for 24hrs demonstrated ER stress as evidenced by increased gene expression of GRP-78, PERK, IRE-1α, sXBP-1 and CHOP and increased protein expression of GRP78. In addition, a decreased TER in HREC was accompanied by decreased VE-Cadherin and ZO-1 expression. On the other hand, TUDCA treated cells reversed these effects. An anchoring peptide, KDEL knockdown and TNF-a and high glucose treatment significantly increased GRP78 translocation to the plasma membrane and increased O-glycosylation proteins.
Considering the fact that chronic hyperglycemia and pro-inflammation lead to disruption of tight junction proteins, activation of GRP78 and increased PTMs, understanding the underlying ER stress mechanisms may shed new insights into novel therapeutic targets.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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