4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

Beta blockade induces apoptosis in cultured capillary endothelial cells

Continuous beta blockade stimulates deposition of collagen in the pulmonary alveolar interstitium of adult rats. It also causes changes to the capillary endothelial cell compartment reminiscent of programmed cell death. To test whether beta blockade results in endothelial cell apoptosis, cultures of capillary endothelial cells were treated with both a wide-spectrum beta blocker and a beta-2-specific antagonist. Apoptosis was measured in these cultures using both terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and annexin-V assays. Both forms of beta blockade stimulated programmed cell death in these cultures. To test whether the apoptotic effect of beta blockade was related to interstitial collagen deposition, capillary endothelial cells were cocultured with beta-blocked pulmonary fibroblast monolayers. Cocultured endothelial cells were substantially protected from apoptosis after beta blockade; coculture over plain tissue culture plastic or over exogenous collagen films had no effect on programmed cell death in endothelial cells. These results suggest that both pulmonary endothelial and interstitial cells are vulnerable to injury from beta blockade but that paracrine interactions between these cells may protect the peripheral lung from substantive damage.     

Ap4A induces apoptosis in human cultured cells.

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'-P1,P3-triphosphate to diadenosine 5',5'-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.     

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.     

4-Hydroxynonenal induces membrane perturbations and inhibition of basal prostacyclin production in endothelial cells,and migration of monocytes.

Cultured bovine aortic endothelial cells (BAEC) were incubated for 5 days with 10^?5 4-hydroxynonenal (HN). HN treated BAEC and controls were either (i) further incubated with ¹²⁵I-polymyxin B (IPxB) or with radioiodinated, inactivated coagulation factor Xa (IFXai) as markers of membrane phospholipid perturbation, or (ii) assayed for the synthesis of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂). Rabbit blood mononuclear cells enriched in monocytes (MC) were isolated and assayed for chemotactic response to HN. The results showed six - fold increases of IPxB and IFXai binding to BAEC treated with HN, as compared to untreated controls. We also found in HN treated cells a marked inhibition of PGI₂ synthesis, but an unmodified TXA₂ production. In addition, HN in the 10-5-10-10 M range induced oriented migration of MC.     

Glycated high-density lipoprotein induces apoptosis of endothelial cells via a mitochondrial dysfunction

Glycation of plasma proteins may contribute to an excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is nonenzymatically glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelium dysfunction in diabetes. This study set out to clarify whether glucose-modified HDL affects the function of endothelial cells by examining the apoptosis of cultured human aortic endothelial cells (HAECs) exposed to a glycated-oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation of HAECs with 100 microg/ml of gly-ox-HDL for 48 h showed apoptotic features, such as cell shrinkage, membrane blebbing, and concentration and fragmentation of the nucleus, and the degree of apoptosis was dose-dependent on the glucose used in the preparation of gly-ox-HDL. Stimulation of HAECs with gly-ox-HDL elicited a marked increase in caspase 3 activity and the expressions of active caspase 3 and caspase 9, whereas concomitant treatment with a caspase 3 inhibitor significantly blocked gly-ox-HDL-induced apoptosis of HAECs. The release of cytochrome c into cytosols markedly increased in HAECs during the treatment with gly-ox-HDL. The increased expressions of Bax and Bad were detected in HAECs incubated for 24 h with gly-ox-HDL, but gly-ox-HDL failed to interfere with the expression of Bcl-2 and Bcl-x. Moreover, in vitro experiments with HDL (gly-HDL) glycated in the presence of 2 mM EDTA and Cu(2+)-oxidized HDL suggested that the apoptotic effect of gly-ox-HDL on endothelial cells might be due to an additional oxidative modification of gly-HDL. Taken altogether, additional oxidation of HDL under hyperglycemic conditions may induce endothelial apoptosis through a mitochondrial dysfunction, following the deterioration of vascular function.     

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.     

4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Introduction

4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.

Methods

Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[³H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.

Results

Our data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.

Conclusion

Our study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.     

Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets

AimsObesity is associated with hypertriglyceridemia and elevated circulating free fatty acids (FFA), resulting in endothelial dysfunction. Endoplasmic reticulum (ER) stress has been implicated in many of these processes. To determine if ER stress participates in palmitate-induced apoptosis, we investigated the effects of diet-induced obesity and palmitate on mouse aortic endothelial cells (MAEC) in vivo and in vitro.Main methodsMale C57BL/6 mice were fed standard chow diets (SCD) or high-calorie and high-cholesterol diets (HCD) for 3 months. Insulin resistance was detected, and the serum, including proinflammatory indices and markers of endothelial function, was also analyzed. The ultrastructure and apoptosis of the endothelial cells in the thoracic aorta were observed. The primary MAEC were separated and treated with palmitate at different concentrations or different times respectively to observe any changes in cellular proliferation, intracellular reactive oxygen species (ROS) levels and apoptosis. Finally, the ER stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were analyzed.Key findingsHCD-fed obese mice became inflammation-activated and insulin-resistant. Swollen mitochondria, expanded ER and apoptosis in the endothelial cells of the thoracic aorta were observed in HCD-fed mice. Palmitate inhibited cell proliferation, increased production of ROS and induced apoptosis in MAEC. CHOP was overexpressed and shifted into the nucleus (mainly), while the expression of GRP78 was upregulated in the palmitate-treated MAEC.SignificanceOur results indicate that diet-induced obesity results in endothelial dysfunction in vivo, and that oxidative and ER stress may be involved in apoptosis induced by the palmitate in vitro.     

Paraquat induces apoptosis of cultured rat cortical cells

Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a universal herbicide. Although systemic treatment with PQ gives rise to the highest level of the herbicide in the cerebral cortex, our knowledge of its effects in this brain region is very limited. We took advantage of rat cortical cell cultures to analyze how PQ affects cortical neurons. Lactate dehydrogenase (LDH) assay and propidium iodide (PI) staining showed that PQ was cytotoxic to cortical neurons with an IC50 on the third day after treatment of approximately 10 microM. PQ-treated cells had shrunken soma with condensed nuclei and disintegrated dendrites, typical signs of apoptosis. Immunocytochemistry of 8-day in vitro (DIV) cells one day after PQ treatment with anti-phospho-H2AX antibody showed that the average number of punctae per nucleus had increased several-fold, indicating substantial DNA fragmentation. Furthermore, double-staining of 7.5 DIV cultures (50 microM PQ) with PI and an antibody against annexin V (AN), an impermeable plasma protein which specifically binds to phosphatidylserine (PS), showed that the percentages of AN(+)/PI(-) cells had also increased several-fold, pointing to considerable movement of PS from the inner to the outer leaflet of the plasma membrane. Taken together, our data indicate that PQ induces apoptosis in cortical cell cultures.     

IL-4 induces apoptosis of endothelial cells through the caspase-3-dependent pathway

The present study was designed to investigate the hypothesis that interleukin-4 (IL-4) can induce apoptosis of human endothelial cells and to study regulatory pathways of this process. Indeed, DNA ladder assay and flow cytometry study showed that IL-4 can induce apoptosis of endothelial cells in a time- and dose-dependent manner. In addition, IL-4 markedly increased activity of caspase-3, and inhibition of this enzyme suppressed IL-4-induced apoptosis in a dose-dependent manner. These results provide the first evidence that IL-4 can induce apoptosis of human endothelial cells. In addition, the data indicate that the caspase-3-dependent pathway is critically involved in this process.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

4-Hydroxynonenal induces a DNA-binding protein similar to the heat-shock factor.

By using a gel mobility assay, we have shown that treatment of HeLa cells with 4-hydroxynonenal, a major product of the peroxidation of membrane lipids and an inducer of heat-shock proteins, has the same effect as heat shock in causing the appearance of a protein which binds to the sequence of DNA specific for the induction of heat-shock genes. Lipoperoxidation and heat exposure seem to share a common mechanism of specific gene activation.     

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.     

Shear stress induces caveolin-1 translocation in cultured endothelial cells

Considering that vascular endothelial caveolae could be flow sensors converting mechanical stimuli into chemical signals transmitted into the cell, this work studied, in vitro, the change of caveolin-1 expression and distribution of cultured endothelial cells exposed to laminar flows. Experimental results showed that, in control cells, caveolin-1 were primarily localized on the cell surface, and presented some local concentrations. In cells exposed to laminar flows, caveolin-1 distribution showed a time-dependent variation. After 24 h of shear (1.0 Pa), the expression of caveolin-1 increased and a local caveolin-1 concentration was found, in most cells, at the upstream side of the cell body where the hydrostatic pressure and the spatial gradient of shear stress were at a maximum. As a comparison, tumor necrosis factor-a induced a decrease of caveolin-1 in the cells.     

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.     

Acrylamide induces mitochondrial dysfunction and apoptosis in BV-2 microglial cells

Acrylamide (ACR), a potent neurotoxin, can be produced during food processing at high temperature. This study examined the redox-dependent apoptotic and inflammatory responses of ACR in an immortalized mouse microglia cell line BV2. The exposure of BV2 cells to ACR reduced cell viability and induced apoptosis in a concentration-dependent manner. ACR impaired cell energy metabolism by decreasing mitochondrial respiration, anaerobic glycolysis, and lowering expression of the complex I, III, and IV subunits. Mitochondrial dysfunction was associated with a decrease of the mitochondrial membrane potential and the Bcl-2/Bax ratio, thus resulting in activation of the mitochondrion-driven apoptotic signaling. This was accompanied by (a) the modulation of redox-sensitive signaling, suppressed Akt activation and increased JNK and p38 activation, and (b) increased expression of NFκB and downstream inducible nitric oxide synthase (iNOS) and nitric oxide generation, thus supporting indirectly a proinflammatory effect of ACR. Nrf2 expression was also increased but not its translocation to the nucleus. Expectedly, the electrophilic attack of ACR on GSH resulted in substantial loss of GSH with a minor GSSG formation. These changes in the cell׳s redox status elicited by ACR resulted in increased H₂O₂ formation. The changes in mitochondrial functionality and complex subunit expression caused by ACR were reversed by N-acetyl-L-cysteine (NAC). Likewise, NAC restored the cell׳s redox status by increasing GSH levels with concomitant attenuation of H₂O₂ generation; these effects resulted in decreased apoptotic cell death and inflammatory responses. ACR-mediated mitochondrial dysfunction along with a more oxidized redox status seems to be critical events leading to activation of the intrinsic apoptotic pathway and inflammatory responses.