Vascular smooth muscle cell activation and growth by 4-hydroxynonenal

The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.     

4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.     

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.     

Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway

4-Hydroxynonenal (HNE) is one of the most abundant aldehyde components of ox-LDL and it exerts various effects on intracellular and extracellular signaling cascades. In this mini-review, a brief synopsis of HNE-modulated signaling pathways will be presented mainly focused on cell death, including recent studies from our laboratory. The results of a number of studies demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. Several signaling pathways have been shown to be modulated by HNE, including MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases and caspases. In order to get insight into the mechanisms of apoptotic response by HNE, MAP kinase and caspase activation pathways have been studied in 3T3 fibroblasts; HNE induced early activation of JNK and p38 proteins but down-regulated the basal activity of ERK-1/2. We have shown that HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with certain molecules such as resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicated a role for JNK-c-Jun/AP-1 pathway. JNK-dependent induction of c-Jun/AP-1 activation data in the literature indicates a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation.     

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.     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Mechanisms of glutamate cysteine ligase (GCL) induction by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end-products of lipid peroxidation and is increased in response to cellular stress and in many chronic and/or inflammatory diseases. HNE can in turn function as a potent signaling molecule to induce the expression of many genes including glutamate cysteine ligase (GCL), the rate-limiting enzyme in de novo glutathione (GSH) biosynthesis. GSH, the most abundant nonprotein thiol in the cell, plays a key role in antioxidant defense. HNE exposure causes an initial depletion of GSH due to formation of conjugates with GSH, followed by a marked increase in GSH resulting from the induction of GCL. GCL is a heterodimeric protein with a catalytic (or heavy, GCLC) subunit and a modulatory (or light, GCLM) subunit. HNE-mediated induction of both GCL subunits and mRNAs has been reported in rat and human cells in vitro; however, the mechanisms or the signaling pathways mediating the induction of Gclc and Gclm mRNAs by HNE differ between rat and human cells. Activation of the ERK pathway is involved in GCL regulation in rat cells while both the ERK and the JNK pathways appear to be involved in human cells. Downstream, MAPK activation leads to increased AP-1 binding, which mediates GCL induction. Some studies suggest a role for the EpRE element as well. As the concentrations of HNE used in all of the studies reviewed are comparable to what may be found in vivo, this makes the findings summarized in this review potentially relevant to GCL regulation in human health and disease.     

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.     

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

The Lipid Peroxidation Product 4-Hydroxynonenal Inhibits Neurite Outgrowth, Disrupts Neuronal Microtubules, and Modifies Cellular Tubulin

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.     

Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition

The effects of the dietary antioxidant N-acetylcysteine (NAC) on alcoholic liver damage were examined in a total enteral nutrition (TEN) model of ethanol toxicity in which liver pathology occurs in the absence of endotoxemia. Ethanol treatment resulted in steatosis, inflammatory infiltrates, occasional foci of necrosis, and elevated ALT in the absence of increased expression of the endotoxin receptor CD 14, a marker of Kupffer cell activation by LPS. In addition, ethanol treatment induced CYP 2 E1 and increased TNFalpha and TGFbeta mRNA expression accompanied by suppressed hepatic IL-4 mRNA expression. Ethanol treatment also resulted in the hepatic accumulation of malondialdehyde (MDA) and hydroxynonenal (HNE) protein adducts, decreased antioxidant capacity, and increased antibody titers toward serum hydroxyethyl radical (HER), MDA, and HNE adducts. NAC treatment increased cytosolic antioxidant capacity, abolished ethanol-induced lipid peroxidation, and inhibited the formation of antibodies toward HNE and HER adducts without interfering with CYP 2 E1 induction. NAC also decreased ethanol-induced ALT release and inflammation and prevented significant loss of hepatic GSH content. However, the improvement in necrosis score and reduction of TNFalpha mRNA elevation did not reach statistical significance. Although a direct correlation was observed among hepatic MDA and HNE adduct content and TNFalpha mRNA expression, inflammation, and necrosis scores, no correlation was observed between oxidative stress markers or TNFalpha and steatosis score. These data suggest that ethanol-induced oxidative stress can contribute to inflammation and liver injury even in the absence of Kupffer cell activation by endotoxemia.     

Cellular lipid peroxidation end-products induce apoptosis in human lens epithelial cells

Hydrogen peroxide (H(2)O(2)), an oxidant present in high concentrations in the aqueous humor of the elderly eyes, is known to impart toxicity to the lens---apoptosis being one of the toxic events. Since H(2)O(2) causes lipid peroxidation leading to the formation of reactive end-products, it is important to investigate whether the end-products of lipid peroxidation are involved in the oxidation-induced apoptosis in the lens. 4-Hydroxynonenal (HNE), a major cytotoxic end product of lipid peroxidation, has been shown to mediate oxidative stress-induced cell death in many cell types. It has been shown that HNE is cataractogenic in micromolar concentrations in vitro, however, the underlying mechanism is not yet clearly understood. In the present study we have demonstrated that H(2)O(2) and the lipid derived aldehydes, HNE and 4-hydroxyhexenal (HHE), can induce dose- and time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases such as caspase-1, -2, -3, and -8 in the cultured human lens epithelial cells. Interestingly, we observed that Z-VAD, a broad range inhibitor of caspases, conferred protection against H(2)O(2)- and HNE-induced apoptosis, suggesting the involvement of caspases in this apoptotic system. Using the cationic dye JC-1, early apoptotic changes were assessed following 5 h of HNE and H(2)O(2) insult. Though HNE exposure resulted in approximately 50% cells to undergo early apoptotic changes, no such changes were observed in H(2)O(2) treated cells during this period. Furthermore, apoptosis, as determined by quantifying the DNA fragmentation, was apparent at a much earlier time period by HNE as opposed to H(2)O(2). Taken together, the results demonstrate the apoptotic potential of the lipid peroxidation end-products and suggest that H(2)O(2)-induced apoptosis may be mediated by these end-products in the lens epithelium.