oxLDL-induced Cell Death Research Articles

480-P: Synergistic Effects of Hyperglycemia and Dyslipidemia on Diabetic Neuropathy—High Glucose Aggravates Oxidized LDL-Induced Schwann Cell Death via Hyperactivation of TLR4

Diabetic neuropathy is a progressive complication of diabetes mellitus, with persistent pain and loss of sensory function. Oxidized LDL (oxLDL), an oxidized form of LDL, has been shown to play a significant role in the development of neuronal dysfunction. Circulating oxLDL is elevated in type 2 diabetes, leading to higher cardiovascular risk. However, the combined effects of hyperglycemia and oxLDL on the progression of diabetic neuropathy remain unknown. Here, we investigated the synergistic effects of high glucose on oxLDL-induced schwann cell death and its detailed mechanism. Immortalized adult mouse Schwann (IMS32) cells were treated with oxLDL (0, 150, 300 μg/mL) under normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) conditions for 24 hours. Following treatment, we evaluated cell viability, gene, and protein expression of Toll-like receptor 4 (TLR4), a receptor for oxLDL. Under NG conditions, 300 μg/mL oxLDL, but not 150 μg/mL reduced the cell viability. Moreover, under HG conditions, both 150 μg/mL and 300 μg/mL oxLDL decreased the cell viability. Likewise, the gene and protein expression of TLR4 was synergistically upregulated by the combination of oxLDL and HG. We further evaluated the effects of TLR4 inhibition on the cell viability and apoptotic caspase-3 activity. The cells were pretreated with TAK-242, a TLR4 inhibitor, for 2 hours prior to 150 μg/mL of oxLDL treatment under NG or HG conditions. TAK-242 pretreatment attenuated the oxLDL-induced cell death in HG conditions. The caspase-3 activity was increased by oxLDL in HG conditions, but not in NG. Furthermore, the increased caspase-3 activity by oxLDL and HG was abolished by TAK-242. These results indicate that high glucose and oxLDL induce activation of TLR4, leading to exacerbated schwann cell apoptosis. The dysregulation of TLR4 signal in diabetes complicated with dyslipidemia may contribute to the pathogenesis of diabetic neuropathy. Disclosure W.Nihei: None. A.Kato: None. T.Himeno: None. M.Kondo: None. J.Nakamura: None. H.Kamiya: Research Support; Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck & Co., Inc., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Japan Tobacco Inc., Novo Nordisk, Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Sanofi, Novo Nordisk, Sumitomo Dainippon Pharma Co., Ltd., Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Daiichi Sankyo, Ono Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Novartis Pharma K.K., Merck & Co., Inc., Sanwa Kagaku Kenkyusho, Otsuka Pharmaceutical Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Astellas Pharma Inc., Terumo Corporation, Fukuda Denshi, LifeScan Diabetes Institute, EA Pharma Co., Ltd, KAKEN PHARMACEUTICAL CO., LTD, AstraZeneca. K.Sango: None. K.Kato: None.

Mulberry supplementation reduces lipid deposition and protects hamster retina from oxLDL damage

• Mulberry reduce lipid deposition and oxLDL to ameliorate retinopathy in hamster fed with high fat diet. • Mulberry protected RPE from oxLDL-caused cell death. • Mulberry inhibited oxLDL to enter cell via regulating the oxLDL receptor. Patients with age-related macular degeneration typically exhibit an accumulation of oxidized low-density lipoprotein (oxLDL) in the retinal Bruch’s membrane. Retinal pigment epithelial (RPE) cells were found to engulf oxLDL, triggering apoptosis. Mulberry and its anthocyanin might reduce oxLDL and lipid deposition in individuals consuming high-cholesterol diet. To investigate the relationship between mulberry water extract (MWE) intake and oxLDL in eye protection, Syrian hamsters fed a high fat diet (HFD) were used herein. After 8 weeks of treatment, MWE was found to reduce lipid deposition in hamsters’ retinas. MWE also reduced the level of oxLDL and lipid peroxidation in eye specimens. Mulberry protected RPE cells from oxLDL-induced cell death and could inhibit the entry of oxLDL into cells by reducing the oxLDL receptor. Taken together, our results indicate that both in HFD fed hamsters and in oxLDL-treated ARPE cells, mulberry can reduce oxLDL accumulation to protect the retina from oxLDL-induced damage.

Oxidised low-density lipoprotein (OXLDL) causes calcium influx and activates mitochondrial permeability transition (MPT) in human macrophages

Oxidized low-density lipoprotein (oxLDL) plays an important role in atherosclerosis development. Studies have implicated the involvement of macrophages and the presence of oxLDL in atherosclerotic plaque. OxLDL is cytotoxic to human macrophages, causing a rapid and concentration and time-dependent cell death. This study aimed to investigate the mechanism of cell death induced by oxLDL in human macrophages. Human macrophages were treated with different concentrations of tetramethylrhodamine methyl ester (TMRM), ruthenium red (RuR) or cyclosporin A (CsA) for 30 minutes followed by addition of median lethal concentration (LC50) of oxLDL and were incubated at 37oC. Fluorescence levels were measured using flow cytometry for TMRM and dihydroethidium (DHE) after a designated incubation time. Cell viability was determined using propidium iodide (PI) via flow cytometry after 24 hours. TMRM results showed that oxLDL caused a time-dependent mitochondrial membrane potential (∆Ψm) loss. Cells treated with RuR and CsA were protected from oxLDL toxicity, possibly by reducing reactive oxygen species (ROS) production and preventing MPT pores opening, respectively. Our findings showed that blocking calcium uptake into the mitochondria may reduce ROS generation and prevents MPT activation. This indicates that oxLDL insult to the mitochondria might play a vital role in oxLDL-induced cell death.

The immunomodulatory effects of barettin and involvement of the kinases CAMK1α and RIPK2

Background: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development.Methods: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQueous One Solution. The kinase inhibition assays were performed using a radioactive (33P-ATP) filter binding assay at the University of Dundee, UK.Results: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α.Conclusions: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.

PPARδ modulates oxLDL-induced apoptosis of vascular smooth muscle cells through a TGF-β/FAK signaling axis

The peroxisome proliferator-activated receptor delta (PPARδ) has been implicated in the modulation of vascular homeostasis. However, its roles in the apoptotic cell death of vascular smooth muscle cells (VSMCs) are poorly understood. Here, we demonstrate that PPARδ modulates oxidized low-density lipoprotein (oxLDL)-induced apoptosis of VSMCs through the transforming growth factor-β (TGF-β) and focal adhesion kinase (FAK) signaling pathways. Activation of PPARδ by GW501516, which is a specific ligand, significantly inhibited oxLDL-induced cell death and generation of reactive oxygen species in VSMCs. These inhibitory effects were significantly reversed in the presence of small interfering (si)RNA against PPARδ, or by blockade of the TGF-β or FAK signaling pathways. Furthermore, PPARδ-mediated recovery of FAK phosphorylation suppressed by oxLDL was reversed by SB431542, a specific ALK5 receptor inhibitor, indicating that a TGF-β/FAK signaling axis is involved in the action of PPARδ. Among the protein kinases activated by oxLDL, p38 mitogen-activated protein kinase was suppressed by ligand-activated PPARδ. In addition, oxLDL-induced expression and translocation of pro-apoptotic or anti-apoptotic factors were markedly affected in the presence of GW501516. Those effects were reversed by PPARδ siRNA, or inhibitors of TGF-β or FAK, which also suggests that PPARδ exerts its anti-apoptotic effect via a TGF-β/FAK signaling axis. Taken together, these findings indicate that PPARδ plays an important role in the pathophysiology of disease associated with apoptosis of VSMC, such as atherosclerosis and restanosis.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways.

Apolipoprotein E receptor-2 deficiency enhances macrophage susceptibility to lipid accumulation and cell death to augment atherosclerotic plaque progression and necrosis

Genome-wide association studies have linked LRP8 polymorphisms to premature coronary artery disease and myocardial infarction in humans. However, the mechanisms by which dysfunctions of apolipoprotein E receptor-2 (apoER2), the protein encoded by LRP8 gene, influence atherosclerosis have not been elucidated completely. The current study focused on the role of apoER2 in macrophages, a cell type that plays an important role in atherosclerosis. Results showed that apoER2-deficient mouse macrophages accumulated more lipids and were more susceptible to oxidized LDL (oxLDL)-induced death compared to control cells. Consistent with these findings, apoER2 deficient macrophages also displayed defective serum-induced Akt activation and higher levels of the pro-apoptotic protein phosphorylated p53. Furthermore, the expression and activation of peroxisome proliferator-activated receptor γ (PPARγ) were increased in apoER2-deficient macrophages. Deficiency of apoER2 in hypercholesterolemic LDL receptor-null mice (Lrp8−/−Ldlr−/− mice) also resulted in accelerated atherosclerosis with more complex lesions and extensive lesion necrosis compared to Lrp8+/+Ldlr−/− mice. The atherosclerotic plaques of Lrp8−/−Ldlr−/− mice displayed significantly higher levels of p53-positive macrophages, indicating that the apoER2-deficient macrophages contribute to the accelerated atherosclerotic lesion necrosis observed in these animals. Taken together, this study indicates that apoER2 in macrophages limits PPARγ expression and protects against oxLDL-induced cell death. Thus, abnormal apoER2 functions in macrophages may at least in part contribute to the premature coronary artery disease and myocardial infarction in humans with LRP8 polymorphisms. Moreover, the elevated PPARγ expression in apoER2-deficient macrophages suggests that LRP8 polymorphism may be a genetic modifier of cardiovascular risk with PPARγ therapy.

Platycodon grandiflorum root attenuates vascular endothelial cell injury by oxidized low-density lipoprotein and prevents high-fat diet–induced dyslipidemia in mice by up-regulating antioxidant proteins

We hypothesized that a Platycodon grandiflorum root (PG) ethyl acetate extract (PGEA) would help reduce the vascular cell injury caused by oxidized low-density lipoprotein (oxLDL) and prevent high-fat (HF) diet-induced dyslipidemia and oxidative stress by up-regulating antioxidant proteins. We investigated the protective effects of PGEA against vascular endothelial cell injury induced by oxLDL and dyslipidemia induced by an HF diet, and the mechanisms underlying these effects were studied. The protective effects of PGEA were investigated with respect to calf pulmonary arterial endothelial (CPAE) cell viability and the lactate dehydrogenase release during oxLDL treatment. The in vivo effects of PGEA were examined using C57BL/6 mice, which were fed an HF diet for 9 weeks. The HF diet was supplemented with 0, 25, or 75 mg/kg PGEA during the last 4 weeks of the experimental period. Histologic analyses of hepatic lipid accumulation were performed. The changes in antioxidant protein levels induced by PGEA, which protects against HF diet-induced oxidative stress, were measured using a proteomics approach. We found that PGEA exhibited antioxidant activity. In CPAE cells, PGEA inhibited both oxLDL-induced cell death and lactate dehydrogenase release. In the HF diet-induced obese mice that received PGEA, we observed significantly reduced plasma and hepatic lipid levels, demonstrating that PGEA has beneficial effects on hyperlipidemia. In addition, we found that PGEA caused the up-regulation of antioxidant proteins. These findings suggest that the antioxidant effects of PGEA may protect against oxidative stress-related diseases.

Lymphocyte cytotoxicity of oxLDL in patients with atherosclerosis.

Atherosclerosis, a chronic inflammatory disease of the vessel wall, is characterized by local and systemic immune responses to a variety of antigens. Oxidized low-density lipoprotein (oxLDL) is considered as an important determining factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the degree of peripheral blood mononuclear cells (PBMC) vulnerability to in vitro oxLDL-induced cytotoxicity from atherosclerotic patients in comparison to healthy individuals. Thirty patients with atherosclerotic lesions, confirmed by angiography, and 30 matched healthy individuals were investigated. PBMC was prepared from individuals' blood samples which were further stimulated with low dose (1 μg/mL) and high dose (50 μg/mL) of extensively oxidized LDL. MTT assay was utilized to measure cell viability and proliferation. Stimulation index (SI) was calculated as mean ratio of optical density (OD) of the stimulated cells divided by OD of untreated cells. Low dose oxLDL treatment caused no significant proliferative or cytotoxic effect in the control group; however, similar treatment caused significant cytotoxic effect in the patients compared to the controls (p=0.026). High dose oxLDL treatment induced more significant cytotoxicity in the patients compared to the controls (p=0.006). Comparison of the SI between the two groups of patients and controls showed significantly lower index by either the low (p=0.03) or the high dose (p<0.001) oxLDL in the patients compared to the controls. PBMC from patients with atherosclerosis showed increased susceptibility to oxLDL-induced cytotoxicity. Our results imply that prolonged exposure to elevated levels of circulating oxLDL could weaken the cellular defense mechanisms by progressive depletion of the pool of antiapoptotic proteins, rendering the cells more vulnerable to oxLDL-induced cell death.

Abstract 5472: Nox4 is a Novel Inducible Source of Intracellular Reactive Oxygen Species in Human Monocyte-Derived Macrophages and Mediates Oxidized LDL-induced Macrophage Death

Nox2/gp91 phox is the large subunit of the integral membrane component of the phagocytic NADPH oxidase (Nox) complex and was believed to be the sole Nox isoform present in monocytes and macrophages. Previously, we reported that increased intracellular ROS formation is required for OxLDL-induced cell death in human monocyte-derived macrophage (HMDM), but the source of these intracellular ROS was unknown (Asmis, Circ.Res. 2003). We now identified Nox4, not Nox2, as the source of OxLDL-induced ROS generation. Nox4 mRNA was detected in human blood monocytes and HMDM. Using a novel rabbit monoclonal antibody that detects Nox4 as a single 66 kDa band, we localized Nox4 to the ER, the nucleus and focal adhesion sites of HMDM, suggesting that Nox4 may play a role in macrophage redox signaling. After stimulation of HMDM with OxLDL, but not native LDL, Nox4 mRNA and protein expression were up-regulated in a dose- and time-dependent manner. Nox4 protein expression was maximal after 6 h and coincided with both increased p22 expression and increased intracellular ROS formation, suggesting that the Nox4/p22 complex is responsible for OxLDL-induced ROS formation in HMDM. Inhibition of MEK1 and ERK1/2 phosphorylation blocked both the induction of Nox4 expression and intracellular ROS formation induced by OxLDL, and protected HMDM from OxLDL-mediated cytotoxicity. In contrast, inhibitors of p38 MAPK or JNK affect neither Nox4 induction nor ROS formation, and showed no cytoprotection. Knockdown of Nox4 with adenovirus-delivered siRNA decreased OxLDL-induced ROS formation and protected macrophages from OxLDL-induced cell injury. In summary, we identified Nox4 as a novel, inducible source of intracellular ROS in human monocytes and macrophages. Nox4 is likely to play important roles in redox signaling and macrophage function. However, if overinduced Nox4-derived ROS promote macrophage death, a mechansim which may have important implications for the pathogenesis of atherosclerosis. This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma &amp; Texas).

Oxidized low‐density lipoprotein (oxLDL)‐induced cell death in dorsal root gangion cell cultures depends not on the lectin‐like oxLDL receptor‐1 but on the toll‐like receptor‐4

DRG cells have been found to undergo apoptosis and necrosis after oxidized low-density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL-induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and toll-like receptor-4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL-treated DRG cell cultures, the increase of cleaved caspase-3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL-treated DRG cell cultures expressed LOX-1 and TLR4 at similar levels. The LOX-1 expression remained unchanged after receptor blockade. However, the inhibition of LOX-1 caused a significant increase of cleaved caspase-3 and a decrease of TLR4 levels. The TLR4-inhibited DRG cell cultures lacked changes in LOX-1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N-terminal kinase (JNK) and a significant decrease of cleaved caspase-3 but did not change the TLR4 level. We conclude that LOX-1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL-induced cell death in DRG cell cultures does not depend on the LOX-1 but on the TLR4.

Sodium ferulate modified gene expression profile of oxidized low‐density lipoproteins‐treated endothelial cells

Oxidized low density lipoprotein (ox‐LDL) is shown to activate endothelial cells to induce inflammatory gene expressions. Sodium ferulate, an effective component extracted from Chinese medicines, is known to have a variety of beneficial pharmacological activities. In this study, we investigate the effects of sodium ferulate on gene expression profile of ox‐LDL‐treated endothelial cells. Cultured human umbilical vein endothelial cells were serum starved for 24 hours, then treated with ox‐LDL (50µg/ml) in the absence or presence of sodium ferulate (5ìM). Sodium ferulate's effects o on gene expression profile and ox‐LDL induced cell death were assessed by using human oligonucleotide microarray gene expression chips and cell viability assay, respectively. Sodium ferulate significantly protected cells against the death induced by ox‐LDL. Meanwhile, a total of 32 genes, including members from families of chemokine, inflammatory factor, and growth factor, up‐regulated by ox‐LDL were significantly down‐regulated to the control level by sodium ferulate administration. These data provided an overview of the gene expression profile of endothelial cells in response to ox‐LDL and sodium ferulate, and demonstrated that sodium ferulate could regulate the expressions of inflammation‐related genes in endothelial cells to ameliorate endothelium function related with atherosclerosis that merit further study.

Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: Role of glutathione reductase and glutaredoxin

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein- S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein- S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein- S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.

OxLDL induced cell death is inhibited by the macrophage synthesised pterin, 7,8-dihydroneopterin, in U937 cells but not THP-1 cells

The atherosclerotic plaque is an inflammatory site where macrophage cells are exposed to cytotoxic oxidised low density lipoprotein (oxLDL). Interferon-γ released from T-cells results in macrophage synthesis of 7,8-dihydroneopterin which has antioxidant and cytoprotective activity. Using the human derived monocyte-like U937 and THP-1 cell lines, we examined whether 7,8-dihydroneopterin could inhibit the cytotoxic effect of oxLDL. In U937 cells, oxLDL caused a dramatic loss of cellular glutathione and caspase independent cell death associated with phosphatidylserine exposure on the plasma membrane. 7,8-Dihydroneopterin completely blocked the cytotoxic effect of oxLDL. In contrast, oxLDL initiated THP-1 cell apoptosis with reduction in cellular thiols, caspase-3 activation and plasma membrane phosphatidylserine exposure. 7,8-Dihydroneopterin was unable to alter these processes or restore the THP-1 cellular thiol content. 7,8-Dihydroneopterin did provide some protection to both THP-1 cells and U937 cells from AAPH derived peroxyl radicals. The preincubation of oxLDL with 7,8-dihydroneopterin did not reduce cytotoxicity, suggesting that 7,8-dihydroneopterin may be acting in U937 cells by scavenging intracellular oxidants generated by the oxLDL. The data show that μM levels of 7,8-dihydroneopterin may prevent oxLDL mediated cellular death within atherosclerotic plaques.

REDD2 gene is upregulated by modified LDL or hypoxia and mediates human macrophage cell death.

Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.

Variations in transmembrane Ca2+ gradient and apoptosis of macrophages induced by oxidized low density lipoprotein.

While Ca2+ has been proposed to be a messenger in OxLDL-induced cell death, few studies have addressed the possibility that it may influence the occurrence of apoptosis and necrosis of macrophages induced by OxLDL in virtue of change of transmembrane Ca2+ gradient including that across plasma membrane and intracellular organelle membranes. In this paper, various lipophilic Ca2+ fluorescent indicators and specific organelle markers were used to study the relationship between the changes of the transmembrane Ca2+ gradients and the OxLDL induced apoptosis of macrophages. Our results showed that following exposure of low dose OxLDL to macrophages, the transmembrane Ca2+ gradient across the plasma membrane, as well as the membrane-proximal Ca2+ gradient, the transnuclear, and the transmitochondrial membrane Ca2+ gradient were all changed significantly. These data suggested that changes in transmembrane Ca2+ gradients might be involved in the apoptosis of macrophages induced by OxLDL.

Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells.

Oxidized low-density lipoprotein (oxLDL) is a potent inducer of apoptosis for vascular cells. In the present study, we demonstrate that the expression of death mediators, including p53, Fas, and Fas ligand (FasL) was substantially upregulated by oxLDL in cultured vascular smooth muscle cells (SMCs). The induction of these death mediators was time dependent and was accompanied by an increase in apoptotic death of SMCs following oxLDL treatment. Two oxysterols, 7beta-hydroxycholesterol and 25-hydroxycholesterol, were also effective to induce the expression of death mediators and apoptosis. alpha-Tocopherol and deferoxamine significantly attenuated the induction of death mediators and cell death induced by oxLDL and oxysterols, suggesting that reactive oxygen species are involved in triggering the apoptotic event. Incubation of cells with FasL-neutralizing antibody inhibited the oxLDL-induced cell death up to 50%. Furthermore, caspase 8 and caspase 3 activities were induced time dependently in SMCs following oxLDL treatment. Collectively, these data suggest that the Fas/FasL death pathway is activated and responsible for, at least in part, the apoptotic death in vascular SMCs upon exposure to oxLDL.

Oxidation-dependent effects of oxidized LDL: proliferation or cell death.

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.

Detection of Cell Death of Cultured Mouse Mesangial Cells Induced by Oxidized Low-Density Lipoprotein

The objectives of the present study using cultured mouse mesangial cells (MMC) were (1) to evaluate the type of cytotoxicity induced by oxidized (ox) LDL, i.e. apoptosis, necrosis and types of other cell death and (2) to investigate the pathway of cell death under incubation with antioxidants or scavenger receptor (SR) antagonists. LDH release and a morphological examination were used in this study. Trypan blue staining of MMC was performed to detect dead cells in culture. Cytotoxicity of ox-LDL in MMC was found to be dose- and time-dependent. In the morphological study of electron microscopy, three different types of cell death in ox-LDL-treated MMC were identified. In the morphological study with semithin sections, these three types of dead cells were identified at different dosages of ox-LDL. Type 1 or type 2 dead cells were observed in low dose ox-LDL or in middle-dose ox-LDL-treated MMC, respectively. Type 3 dead cells were marked in high dose ox-LDL-treated MMC. It appears that the cells were apoptotic (type 1), necrotic (type 3) and other types (type 2). The cytotoxicity of ox-LDL was not mediated by cellular internalization of ox-LDL via SRs. On the other hand, the cytotoxicity of ox-LDL was inhibited by antioxidants such as α-tocopherol, probucol, N-acetyl-cysteine or glutathione ethyl ester. It is indicated that the pathways of ox-LDL induced cell death were distinct from the pathway via SRs.

Oxidized LDL activates fas-mediated endothelial cell apoptosis.

Oxidized low density lipoproteins (OxLDL) promote chronic inflammatory responses in the vasculature that give rise to atherosclerotic plaques. Fas ligand (FasL) is naturally expressed on the vascular endothelium where it can induce apoptosis in Fas-expressing immune cells as they enter the vessel wall. Although vascular endothelial cells are normally resistant to Fas-mediated cell death, OxLDL were shown to induce apoptosis in cultured endothelial cells and endothelium of arterial explants by a process that could be inhibited with Fas L neutralizing antibodies. OxLDL-induced cell death was also reduced in the aortic endothelium cultured from gld (FasL-/-) and lpr (Fas-/-) mice as compared with wild-type mice. OxLDL acted by sensitizing endothelial cells to death signals from the Fas receptor. Thus, the ability of OxLDL to promote Fas-mediated endothelial cell suicide may be a feature that contributes to their atherogenicity.