Lipid peroxidation product 4-hydroxy-trans-2-nonenal causes endothelial activation by inducing endoplasmic reticulum stress.

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.

Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids.

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1ß, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.

Aldose reductase protects against early atherosclerotic lesion formation in apolipoprotein E-null mice.

RATIONALE: Atherosclerotic lesion formation is associated with the accumulation of oxidized lipids. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion; however, their contribution to atherosclerotic lesion formation remains unclear. OBJECTIVE: To test the hypothesis that inhibition of aldehyde removal by aldose reductase (AR), which metabolizes both free and phospholipid aldehydes, exacerbates atherosclerotic lesion formation. METHODS AND RESULTS: In atherosclerotic lesions of apolipoprotein (apo)E-null mice, AR protein was located in macrophage-rich regions and its abundance increased with lesion progression. Treatment of apoE-null mice with AR inhibitors sorbinil or tolrestat increased early lesion formation but did not affect the formation of advanced lesions. Early lesions of AR(-/-)/apoE(-/-) mice maintained on high-fat diet were significantly larger when compared with age-matched AR(+/+)/apoE(-/-) mice. The increase in lesion area attributable to deletion of the AR gene was seen in both male and female mice. Pharmacological inhibition or genetic ablation of AR also increased the lesion formation in male mice made diabetic by streptozotocin treatment. Lesions in AR(-/-)/apoE(-/-) mice exhibited increased collagen and macrophage content and a decrease in smooth muscle cells. AR(-/-)/apoE(-/-) mice displayed a greater accumulation of the AR substrate 4-hydroxy trans-2-nonenal (HNE) in the plasma and protein-HNE adducts in arterial lesions than AR(+/+)/apoE(-/-) mice. CONCLUSIONS: These observations indicate that AR is upregulated in atherosclerotic lesions and it protects against early stages of atherogenesis by removing toxic aldehydes generated in oxidized lipids.

Exposure to acrolein by inhalation causes platelet activation.

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5ppm for 6h) or sub-chronic (1ppm, 6h/day for 4days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

Acrolein consumption induces systemic dyslipidemia and lipoprotein modification.

Aldehydes such as acrolein are ubiquitous pollutants present in automobile exhaust, cigarette, wood, and coal smoke. Such aldehydes are also constituents of several food substances and are present in drinking water, irrigation canals, and effluents from manufacturing plants. Oral intake represents the most significant source of exposure to acrolein and related aldehydes. To study the effects of short-term oral exposure to acrolein on lipoprotein levels and metabolism, adult mice were gavage-fed 0.1 to 5 mg acrolein/kg bwt and changes in plasma lipoproteins were assessed. Changes in hepatic gene expression related to lipid metabolism and cytokines were examined by qRT-PCR analysis. Acrolein feeding did not affect body weight, blood urea nitrogen, plasma creatinine, electrolytes, cytokines or liver enzymes, but increased plasma cholesterol and triglycerides. Similar results were obtained with apoE-null mice. Plasma lipoproteins from acrolein-fed mice showed altered electrophoretic mobility on agarose gels. Chromatographic analysis revealed elevated VLDL cholesterol, phospholipids, and triglycerides levels with little change in LDL or HDL. NMR analysis indicated shifts from small to large VLDL and from large to medium-small LDL with no change in the size of HDL particles. Increased plasma VLDL was associated with a significant decrease in post-heparin plasma hepatic lipase activity and a decrease in hepatic expression of hepatic lipase. These observations suggest that oral exposure to acrolein could induce or exacerbate systemic dyslipidemia and thereby contribute to cardiovascular disease risk.

Myocardial ischaemia inhibits mitochondrial metabolism of 4-hydroxy-trans-2-nonenal.

Myocardial ischaemia is associated with the generation of lipid peroxidation products such as HNE (4-hydroxy-trans-2-nonenal); however, the processes that predispose the ischaemic heart to toxicity by HNE and related species are not well understood. In the present study, we examined HNE metabolism in isolated aerobic and ischaemic rat hearts. In aerobic hearts, the reagent [(3)H]HNE was glutathiolated, oxidized to [(3)H]4-hydroxynonenoic acid, and reduced to [(3)H]1,4-dihydroxynonene. In ischaemic hearts, [(3)H]4-hydroxynonenoic acid formation was inhibited and higher levels of [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE (glutathione conjugate of HNE) were generated. Metabolism of [(3)H]HNE to [(3)H]4-hydroxynonenoic acid was restored upon reperfusion. Reperfused hearts were more efficient at metabolizing HNE than non-ischaemic hearts. Ischaemia increased the myocardial levels of endogenous HNE and 1,4-dihydroxynonene, but not 4-hydroxynonenoic acid. Isolated cardiac mitochondria metabolized [(3)H]HNE primarily to [(3)H]4-hydroxynonenoic acid and minimally to [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE. Moreover, [(3)H]4-hydroxynonenoic acid was extruded from mitochondria, whereas other [(3)H]HNE metabolites were retained in the matrix. Mitochondria isolated from ischaemic hearts were found to contain 2-fold higher levels of protein-bound HNE than the cytosol, as well as increased [(3)H]GS-HNE and [(3)H]1,4-dihydroxynonene, but not [(3)H]4-hydroxynonenoic acid. Mitochondrial HNE oxidation was inhibited at an NAD(+)/NADH ratio of 0.4 (equivalent to the ischaemic heart) and restored at an NAD(+)/NADH ratio of 8.6 (equivalent to the reperfused heart). These results suggest that HNE metabolism is inhibited during myocardial ischaemia owing to NAD(+) depletion. This decrease in mitochondrial metabolism of lipid peroxidation products and the inability of the mitochondria to extrude HNE metabolites could contribute to myocardial ischaemia/reperfusion injury.

Role of endoplasmic reticulum stress in acrolein-induced endothelial activation.

Acrolein is a ubiquitous environmental pollutant and an endogenous product of lipid peroxidation. It is also generated during the metabolism of several drugs and amino acids. In this study, we examined the effects of acrolein on endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with 2 to 10 microM acrolein led to an increase in the phosphorylation of eIF-2alpha within 10 to 30 min of exposure. This was followed by alternate splicing of XBP-1 mRNA and an increase in the expression of the endoplasmic reticulum (ER) chaperone genes Grp78 and Herp. Within 2-4 h of treatment, acrolein also increased the abundance and the nuclear transport of the transcription factors ATF3, AFT4, and CHOP. Acrolein-induced increase in ATF3 was prevented by treating the cells with the chemical chaperone - phenylbutyric acid (PBA). Treatment with acrolein increased phosphorylation of ERK1/2, p38, and JNK. The increase in JNK phosphorylation was prevented by PBA. Acrolein treatment led to activation and nuclear translocation of the transcription factor NF-kappaB and an increase in TNF-alpha, IL-6 and IL-8, but not MCP-1, mRNA. Increased expression of cytokine genes and NF-kappaB activation were not observed in cells treated with PBA. These findings suggest that exposure to acrolein induces ER stress and triggers the unfolded protein response and that NF-kappaB activation and stimulation of cytokine production by acrolein could be attributed, in part, to ER stress. Chemical chaperones of protein-folding may be useful in treating toxicological and pathological states associated with excessive acrolein exposure or production.

Arsenic exacerbates atherosclerotic lesion formation and inflammation in ApoE-/- mice.

Exposure to arsenic-contaminated water has been shown to be associated with cardiovascular disease, especially atherosclerosis. We examined the effect of arsenic exposure on atherosclerotic lesion formation, lesion composition and nature in ApoE-/- mice. Early post-natal exposure (3-week-old mice exposed to 49 ppm arsenic as NaAsO(2) in drinking water for 7 weeks) increased the atherosclerotic lesion formation by 3- to 5-fold in the aortic valve and the aortic arch, without affecting plasma cholesterol. Exposure to arsenic for 13 weeks (3-week-old mice exposed to 1, 4.9 and 49 ppm arsenic as NaAsO(2) in drinking water) increased the lesion formation and macrophage accumulation in a dose-dependent manner. Temporal studies showed that continuous arsenic exposure significantly exacerbated the lesion formation throughout the aortic tree at 16 and 36 weeks of age. Withdrawal of arsenic for 12 weeks after an initial exposure for 21 weeks (to 3-week-old mice) significantly decreased lesion formation as compared with mice continuously exposed to arsenic. Similarly, adult exposure to 49 ppm arsenic for 24 weeks, starting at 12 weeks of age increased lesion formation by 2- to 3.6-fold in the aortic valve, the aortic arch and the abdominal aorta. Lesions of arsenic-exposed mice displayed a 1.8-fold increase in macrophage accumulation whereas smooth muscle cell and T-lymphocyte contents were not changed. Expression of pro-inflammatory chemokine MCP-1 and cytokine IL-6 and markers of oxidative stress, protein-HNE and protein-MDA adducts were markedly increased in lesions of arsenic-exposed mice. Plasma concentrations of MCP-1, IL-6 and MDA were also significantly elevated in arsenic-exposed mice. These data suggest that arsenic exposure increases oxidative stress, inflammation and atherosclerotic lesion formation.

Oral exposure to acrolein exacerbates atherosclerosis in apoE-null mice.

BACKGROUND: Acrolein is a dietary aldehyde that is present in high concentrations in alcoholic beverages and foods including cheese, donuts and coffee. It is also abundant in tobacco smoke, automobile exhaust and industrial waste and is generated in vivo during inflammation and oxidative stress. OBJECTIVES: The goal of this study was to examine the effects of dietary acrolein on atherosclerosis. METHODS: Eight-week-old male apoE-null mice were gavage-fed acrolein (2.5mg/kg/day) for 8 weeks. Atherosclerotic lesion formation and composition and plasma lipids and platelet factor 4 (PF4) levels were measured. Effects of acrolein and PF4 on endothelial cell function was measured in vitro. RESULTS: Acrolein feeding increased the concentration of cholesterol in the plasma. NMR analysis of the lipoproteins showed that acrolein feeding increased the abundance of small and medium VLDL particles. Acrolein feeding also increased atherosclerotic lesion formation in the aortic valve and the aortic arch. Immunohistochemical analysis showed increased macrophage accumulation in the lesions of acrolein-fed mice. Plasma PF4 levels and accumulation of PF4 in atherosclerotic lesions was increased in the acrolein-fed mice. Incubation of endothelial cells with the plasma of acrolein-fed mice augmented transmigration of monocytic cells, which was abolished by anti-PF4 antibody treatment. CONCLUSIONS: Dietary exposure to acrolein exacerbates atherosclerosis in apoE-null mice. Consumption of foods and beverages rich in unsaturated aldehydes such as acrolein may be a contributing factor to the progression of atherosclerotic lesions.

Aldose reductase decreases endoplasmic reticulum stress in ischemic hearts.

Aldose reductase (AR) is a multi-functional AKR (AKR1B1) that catalyzes the reduction of a wide range of endogenous and xenobiotic aldehydes and their glutathione conjugates with high efficiency. Previous studies from our laboratory show that AR protects against myocardial ischemia-reperfusion injury, however, the mechanisms by which it confers cardioprotection remain unknown. Because AR metabolizes aldehydes generated from lipid peroxidation, we tested the hypothesis that it protects against ischemic injury by preventing ER stress induced by excessive accumulation of aldehyde-modified proteins in the ischemic heart. In cell culture experiments, exposure to model lipid peroxidation aldehydes-4-hydroxy-trans-2-nonenal (HNE), 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC) or acrolein led to an increase in the phosphorylation of ER stress markers PERK and eIF2-alpha and an increase in ATF3. The reduced metabolite of POVPC 1-palmitoyl-2-hydroxyvaleroyl phosphatidylcholine (PHVPC) was unable to stimulate JNK phosphorylation. No increase in phospho-eIF2-alpha, ATF3 or phospho-PERK was observed in cells treated with the reduced HNE metabolite 1,4-dihydroxynonenol (DHN). Lysates prepared from isolated perfused mouse hearts subjected to 15 min of global ischemia followed by 30 min of reperfusion ex vivo showed greater phosphorylation of PERK and eIF2-alpha than hearts subjected to aerobic perfusion alone. Ischemia-induced increases in phospho-PERK and phospho-eIF2-alpha were diminished in the hearts of cardiomyocyte-specific transgenic mice overexpressing the AR transgene. These observations support the notion that by removing aldehydic products of lipid peroxidation, AR decreases ischemia-reperfusion injury by diminishing ER stress.