Lipid Peroxidation Product 4-Hydroxy-trans-2-nonenal Causes Endothelial Activation by Inducing Endoplasmic Reticulum Stress

Diabetes and Obesity Center, University of Louisville, Louisville, Kentucky 40202, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2012; 287(14):11398-409. DOI: 10.1074/jbc.M111.320416
Source: PubMed


Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.

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    • "The supernatant was used for LC–MS analysis . The gel plug was then reduced and alkylated using a modification of Jensen's method (Jensen et al., 1997) and as previously described (Vladykovskaya et al., 2012), and digested overnight at 37 °C using 2 μg mass spectrometry grade trypsin (Promega, Madison, WI). "
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    ABSTRACT: High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100-300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein-acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1-5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10-20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. Copyright © 2015. Published by Elsevier Inc.
    Toxicology and Applied Pharmacology 04/2015; 285(2). DOI:10.1016/j.taap.2015.03.029 · 3.71 Impact Factor
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    • "In the negative labeling strategy, cells are exposed to oxidants or insults that promote oxidative stress (Fig. 2). These exposures often lead to mitochondrial damage [6] [32], activation of the autophagic program [6] [33], endoplasmic reticulum (ER) stress [34], and apoptosis [34] [35]. The proteins that could be involved with each of these processes can then be examined by postlabeling with BODIPY–iodoacetamide (BD-IAM), with the result being a decrease in (or negative) labeling under conditions of oxidative stress compared with control conditions. "
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    • "Lipid peroxidation disrupts the normal structure of cellular and subcellular membranes. In addition, the process produces byproducts such as 4-hydroxynonenal (4-HNE) or acrolein, both of which bind to proteins and damage their structure and function [30], [31]. The present results show that GRP78 overexpressing cells suppress lipid peroxidation and may contribute to cell survival following H2O2 treatment. "
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    ABSTRACT: Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.
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