Hemoglobin-mediated oxidative stress induces human endothelial barrier dysfunction

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	FDA/CBER	OXIDSTRESS-2

Authors

• E Blumenthal
• F D'Agnillo

Abstract

Systemic exposure to extracellular hemoglobin (Hb) induces vascular dysfunction and toxicity via mechanisms that are not completely defined. We examined the effect of cell-free Hb on endothelial barrier function, tight junction protein expression (ZO-1 and claudin-5), nuclear translocation of beta-catenin and Nrf2, and heme oxygenase-1 (HO-1) expression. Primary human endothelial cells incubated with Hb and glucose oxidase-generated hydrogen peroxide showed increased monolayer permeability that correlated with reduction in claudin-5 expression. ZO-1 expression was unchanged though notable reorganization occurred. Nuclear accumulation of beta-catenin was observed at 6 and 24 hours. Enhanced nuclear levels of Nrf2 were observed at 6 hours and declined by 24 hours. HO-1 expression increased by 6 hours, peaked at 12 hours, and returned to basal levels at 48 hours. Pretreatment with sulforaphane (SFN), a compound that promotes nuclear accumulation of Nrf2, enhanced the HO-1 response. This correlated with decreased monolayer gap formation suggesting a protective role of Nrf2 and/or HO-1. These findings indicate that Hb-mediated oxidative stress produces endothelial barrier dysfunction characterized by disruption in endothelial tight junctions. Transcriptional manipulation of antioxidant pathways may be useful for treating vascular complications associated with hemolytic disease settings.

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