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An In Vitro Hypoxia/Reoxygenation Model of Ischemia/Reperfusion Injury with Glucose Oxidase And Catalase
Author(s) -
Bowman Phillip,
Bynum James A,
McFaul Steve J
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.717.11
Subject(s) - catalase , glucose oxidase , ischemia , umbilical vein , hypoxia (environmental) , chemistry , in vivo , oxygen , reperfusion injury , microbiology and biotechnology , hydrogen peroxide , andrology , in vitro , biochemistry , biology , oxidative stress , enzyme , medicine , organic chemistry
Subjecting cells to reduced oxygen (O 2 ) by purging the atmosphere with nitrogen is a slow process that does not reproduce the rapid local reduction in oxygen that occurs in vivo following decreased oxygen availability due to ischemia. Glucose Oxidase (GOX) rapidly oxidizes glucose and when coupled with Catalase (CAT) to decompose hydrogen peroxide rapidly produces a very low oxygen level (< 0.4 μM within 5 min) as may occur during ischemic injury. To better understand the response of human cells to the rapid reduction in O 2 human umbilical vein endothelial cells (HUVEC) were subjected to 0.4 μM O 2 for up to three hours in complete medium containing GOX and CAT or control medium and total RNA extracted and subjected to gene expression profiling with whole genome microarrays. 368 genes were statistically altered in their expression with a false discovery rate < 10%. Most of these genes experienced down regulation. Prominent among genes down regulated was EGLN3 (hypoxia inducible factor 1A {HIF1A} prolyl hydroxylase) that accounted for the rapid rise in HIF1A detected by western blotting. Only 14 genes were upregulated more than 2‐fold. The GOX‐CAT system reliably reduces O 2 levels in the pericellular media around cultured cells in a short time. This system more closely reproduces the rapid hypoxia induced by ischemia brought on by vascular occlusion or injury.