Simvastatin prevents oxygen and glucose deprivation/reoxygenation-induced death of cortical neurons by reducing the production and toxicity of 4-hydroxy-2E-nonenal

Lipid membrane peroxidation is highly associated with neuronal death in various neurodegenerative diseases including cerebral stroke. Here, we report that simvastatin decreases oxygen and glucose deprivation (OGD)/reoxygenation-evoked neuronal death by inhibiting the production and cytoxicity of 4-hydroxy-2E-nonenal (HNE), the final product of lipid peroxidation. Simvastatin markedly decreased the OGD/reoxygenation-evoked death of cortical neurons. OGD/reoxygenation increased the intracellular HNE level mostly in neuronal cells, not glial cells. Simvastatin decreased the intracellular level of HNE in neuronal cells exposed to OGD/reoxygenation. We further found that HNE induced the cytotoxicity in neuronal cells and synergistically increased the N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity. Simvastatin largely blocked the NMDA neurotoxicity potentiated by HNE. However, simvastatin did not alter the NMDA-evoked calcium influx in the absence or presence of HNE. HNE inhibited the activity of nuclear factor-kappa B (NF-kappaB), and the cytotoxicity of HNE was in good correlation with inactivation of NF-kappaB. Simvastatin reversed the inhibition of NF-kappaB activity induced by OGD/reoxygenation or HNE. The neuroprotection by simvastatin was significantly attenuated by various NF-kappaB inhibitors, implying that simvastatin inhibits the cytotoxicity of HNE at least in part by maintaining the activity of NF-kappaB. Further understanding of the neuroprotective mechanism of simvastatin may provide a therapeutic strategy for oxidative stress-related neurodegenerative diseases.     

4-Hydroxynonenal induces dysfunction and apoptosis of cultured endothelial cells.

Lipolytic products of triglyceride-rich lipoproteins, i.e., free fatty acids, may cause activation and dysfunction of the vascular endothelium. Mechanisms of these effects may include lipid peroxidation. One of the major and biologically active products of peroxidation of n-6 fatty acids, such as linoleic acid or arachidonic acid, is the aldehyde 4-hydroxynonenal (HNE). To study the hypothesis that HNE may be a critical factor in endothelial cell dysfunction caused by free fatty acids, human umbilical endothelial cells (HUVEC) were treated with up to160 microM of linoleic or arachidonic acid. HNE formation was detected by immunocytochemistry in cells treated for 24 h with either fatty acid, but more markedly with arachidonic acid. To study the cellulareffects of HNE, HUVEC were treated with different concentrations of this aldehyde, and several markers of endothelial cell dysfunction were determined. Exposure to HNE for 6 and 9 h resulted in increased cellular oxidative stress. However, short time treatment with HNE did not cause activation of nuclear factor-kappaB (NF-kappaB). In addition, HUVEC exposure to HNE caused a dose-dependent decrease in production of both interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). On the other hand, HNE exerted prominent cytotoxic effects in cultured HUVEC, manifested by morphological changes, diminished cellular viability, and impaired endothelial barrier function. Furthermore, HNE treatment induced apoptosis of HUVEC. These data provide evidence that HNE does not contribute to NF-kappaB-related mechanisms of the inflammatory response in HUVEC, but rather to endothelial dysfunction, cytotoxicity, and apoptotic cell death.     

4-Hydroxynonenal reduces junctional communication between endothelial cells in culture

The effect of 4-hydroxynonenal (HNE), a lipid peroxidation product, on junctional communication (JC) among cultured vascular endothelial cells was assessed by both study of the transfer of microinjected 6-carboxyfluorescein between neighboring cells and measurement by a "cut-loading and dye transfer" technique. Both methods indicated that at concentrations higher than 10(-9) M and testing times between 6 and 8 h HNE reduces endothelial cell junctional communication. At 10(-8) M, a gradual development of HNE effect appears during 6-8 h of exposure but is followed by a slow recovery completed at 20 h. The reduction in junctional communication is not produced by the inhibition of protein synthesis, as tested by radiolabeled leucine incorporation. The HNE effect might be relevant to pathological processes in which lipid peroxidation is associated with uncontrolled cell proliferation, as in atherogenesis and promotion of carcinogenesis by chronic inflammation.     

Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.     

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

Involvement of aldose reductase in the metabolism of atherogenic aldehydes

Phospholipid peroxidation generates a variety of aldehydes, which includes free saturated and unsaturated aldehydes, and aldehydes that remain esterified to the phosphoglyceride backbone - the so-called 'core' aldehydes. However, little is known in regarding the vascular metabolism of these aldehydes. To identify biochemical pathways that metabolize free aldehydes, we examined the metabolism of 4-hydroxy-trans-2-nonenal in human aortic endothelial cells. Incubation of these cells with [3H]-HNE led to the generation of four main metabolites, i.e. glutathionyl HNE (GS-HNE), glutathionyl dihydroxynonene (GS-DHN), DHN and 4-hydroxynonanoic acid (HNA), which accounted for 5, 50, 6, and 23% of the total HNE metabolized. The conversion of GS-HNE to GS-DHN was inhibited by tolrestat, indicating that it is catalyzed by aldose reductase (AR). The AR was also found to be an efficient catalyst for the reduction of the core aldehyde - 1-palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, which is generated in minimally modified low-density lipoprotein, and activates the endothelium to bind monocytes. As determined by electrospray mass spectrometry, reduction of POVPC (m/z=594) by AR led to the formation of 1-palmitoyl-2- (5)-hydrovaleryl-sn-glycero-3-phosphorylcholine (PHVPC; m/z=596). These observations suggest that due to its ability to catalyze the reduction of lipid-derived aldehydes AR may be involved in preventing inflammation and diminishing oxidative stress during the early phases of atherogenesis.     

Protective effect of rat aldo-keto reductase (AKR1C15) on endothelial cell damage elicited by 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE), a major reactive product of lipid peroxidation, is believed to play a central role in atherogenic actions triggered by oxidized lipoproteins. An aldo-keto reductase (AKR) 1C15 efficiently reduces HNE and is distributed in many rat tissues including endothelial cells. In this study, we investigated whether AKR1C15 acts as a protective factor against endothelial damage elicited by HNE and oxidized lipoproteins. Treatment of rat endothelial cells with HNE provoked apoptosis through reactive oxygen species (ROS) formation, mitochondrial dysfunction and caspase activation in the cells. AKR1C15 converted HNE into less toxic 1,4-dihydroxy-2-nonene, and its overexpression markedly decreased the susceptibility of the cells to HNE. The forced expression of AKR1C15 also significantly suppressed the loss of cell viability caused by oxidized low-density lipoprotein and its lipidic fraction. Furthermore, the treatment of the cells with sublethal concentrations of HNE resulted in up-regulation of AKR1C15, which was partially abrogated by the ROS inhibitors. Collectively, these data indicate an anti-atherogenic function of AKR1C15 through the protection of endothelial cells from damage elicited by toxic lipids such as HNE.     

4-Hydroxynonenal,an end-product of lipid peroxidation,induces apoptosis in human leukemic T- and B-cell lines

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.     

4-Hydroxynonenal, an end-product of lipid peroxidation, induces apoptosis in human leukemic T- and B-cell lines

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.     

Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

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.     

Effects of 4-Hydroxynonenal, A Product of Lipid Peroxidation, on Cell Proliferation and Ornithine Decarboxylase Activity

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10^-5 to 10^-6 M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. ³H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.     

Involvement of aldose reductase in the metabolism of atherogenic aldehydes

Phospholipid peroxidation generates a variety of aldehydes, which includes free saturated and unsaturated aldehydes, and aldehydes that remain esterified to the phosphoglyceride backbone — the so-called ‘core’ aldehydes. However, little is known in regarding the vascular metabolism of these aldehydes. To identify biochemical pathways that metabolize free aldehydes, we examined the metabolism of 4-hydroxy-trans-2-nonenal in human aortic endothelial cells. Incubation of these cells with [³H]-HNE led to the generation of four main metabolites, i.e. glutathionyl HNE (GS-HNE), glutathionyl dihydroxynonene (GS-DHN), DHN and 4-hydroxynonanoic acid (HNA), which accounted for 5, 50, 6, and 23% of the total HNE metabolized. The conversion of GS-HNE to GS-DHN was inhibited by tolrestat, indicating that it is catalyzed by aldose reductase (AR). The AR was also found to be an efficient catalyst for the reduction of the core aldehyde — 1-palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, which is generated in minimally modified low-density lipoprotein, and activates the endothelium to bind monocytes. As determined by electrospray mass spectrometry, reduction of POVPC (m/z=594) by AR led to the formation of 1-palmitoyl-2- (5)-hydrovaleryl-sn-glycero-3-phosphorylcholine (PHVPC; m/z=596). These observations suggest that due to its ability to catalyze the reduction of lipid-derived aldehydes AR may be involved in preventing inflammation and diminishing oxidative stress during the early phases of atherogenesis.     

Effects of 4-Hydroxynonenal,A Product of Lipid Peroxidation,on Cell Proliferation and Ornithine Decarboxylase Activity

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10^-5 to 10^-6 M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. ³H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.     

Interaction Between Neutrophils and 4-Hydroxyalkenals and Consequences on Neutrophil Motility

The lipid peroxidation product 4-hydroxynonenal (HNE) and homologous aldehydes have been found to possess chemotactic activity for rat neutrophil leukocytes in the micromolar to picomolar range, depending on the compound. Such an activity is displayed only in the presence of albumin. The mechanisms by which aldehydes could interact with neutrophils are discussed. II is proposed that albumin acts as a carrier for the aldehyde and releases them to a neutrophil receptor. At concentrations around 10^?4M, 4-hydroxyal-kenals have been found to exert toxic effects on a number of cells, including a strong depression of neutrophil motility. Finally, HNE has been found at chemotactic concentrations in the inflammatory site. The possibility that HNE is involved in the neutrophil influx into the inflammatory site is considered.