title: Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells.
abstract: Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O2)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Sergio F
authors.initials: SF
authors.lastname: Martin
authors.forename: Subroto
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Narasimham
authors.initials: N
authors.lastname: Parinandi
authors.forename: B Rita
authors.initials: BR
authors.lastname: Alevriadou
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D018929:Cell Culture Techniques; D002460:Cell Line; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000821:virology; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D016877:Oxidants; C107953:RAC1 protein, human; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein