Effects Of Tumor Necrosis Factor-Ralated Apoptposis-Inducing Ligand And Its Receptor On Atherogensis

BackgroundTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family。It was first discovered in1996and named as Apo-2ligand (Apo-2L). TRAIL is a281-amino acid protein, which is closely related to Fas/Apo-1ligand. TRAIL induces apoptosis in transformed cell lines of diverse origin. So far, five TRAIL receptors have been identified:TRAIL-R1/DR4(death receptor4)、TRAIL-R2/DR5(death receptor5)、TRAIL-R3/DcRl (decoy receptor1)、 TRAIL-R4/DcR2(decoy receptor1) and OPG (osteoprotegerin). Binding of TRAIL to the DR4/5receptors induces apoptosis of tumor cells while leaving normal untransformed cells unaffected.In addition to inducing apoptosis, TRAIL can also promote the survival, migration and proliferation of vascular smooth muscle cells (VSMCs). TRAIL has been implicated in immunosuppressive, immunoregulatory and immune-effector functions. Moreover, TRAIL plays an important role in the biology of stem cells. TRAIL can be expressed by various cells of the immune system, amongst them natural killer cells, T cells, natural killer T cells, dendritic cells and macrophages. In blood vessels, TRAIL receptors are present in both VSMCs and endothelial cells. Therefore, increasing evidence indicates a role of TRAIL in the regulation of vascular functions and cardiovascular disease. Loss of function studies revealed that global deletion of the TRAIL-gene resulted in enhanced atherogenesis in hyperlipidemic mice, implicating a protective role for TRAIL in atherosclerosis. In clinical studies, it was found that serum TRAIL level was significantly lower in patients with coronary artery disease than in those without. Based on these observations, it is proposed that TRAIL-based therapies may have beneficial pharmacological effects in treating cardiovascular diseases such as atherosclerosis.However, the dose-response relationship of the pharmacological effects of TRAIL on atherogenesis has not yet been fully characterized, given that several lines of evidence suggest that excessive levels of TRAIL may also have proatherogenic actions.For example, it has been demonstrated that exogenous TRAIL may induce prominent inflammatory reactions in both vascular endothelial and VSMCs, leading to increased expression of adhesion molecules and proinflammatory chemokines such as E-selectin, intercellular adhesion molecule (ICAM)-1and interleukin (IL)-8. Moreover, the plasma level of TRAIL has been shown to be elevated in patients with inflammatory diseases such as psoriatic arthritis, systemic lupus erythematosus, and viral infection. It is thought that a chronic systemic inflammatory status may increase the risk of atherosclerosis. Although there was one study showing that TRAIL treatment decreased the total plaque area via inducing apoptosis of macrophages, these effects were only observed in late stage plaques. On the other hand, apoptosis of macrophages may lead to plaque destsbilization. Therefore, current evidence for the role of TRAIL in the atherogenesis was not conculsive, especially in the early stage of the process.Aims(1) To examine the effects of rTRAIL (recombinant TRAIL) on the development of atherosclerosis in hyperlipide mice.(2) To further clarify the role of DR5receptor in basal and TRAIL-stimulated AS development using ApoE/DR5DKO mice. (3) To compare the effects of TRAIL on macrophage apoptosis in early and advanced AS plages and explore the underlying mechanisms.Experiment materials and methodsAnimals Male apoE-/-mice at the age of8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). DR5-/-mice were purchased from Mutant Mouse Regional Resource Centers (MMRRC). Mice were sacrificed by anesthetizing with intraperitoneal (i.p.) injection of0.8%pentobarbital sodium (60mg/kg), followed by cervical dislocation. All animal studies were complied with the Animal Management Rules of the Chinese Ministry of Health and approved by the Ethical and Use Committee of the Qilu Hospital of the Shandong University.Treatment protocols with recombinant TRAIL8-wk apoE-/-mice were randomly divided into different groups. While one group of mice received either rTRAIL (3μg per mouse i.p., diluted in200μl of PBS), the other group received an equal volume of vehicle once per week. TRAIL treatment was started at, and maintained throughout, the same time as the normal or high-fat diet treatment till4/8weeks.Treatment protocols with recombinant TRAIL8-wk ApoE-/-or LDLR-/-mice received high-fat diet treatment for8weeks, then was randomly divided into two groups. One group of mice received either rTRAIL (3μg per mouse i.p., diluted in200μl of PBS), the other group received an equal volume of vehicle once per week for another8weeks.Creation of ApoE/DR5DKO mice ApoE-/-and DR5-/-mice were crossed at the age of8-wk, the genotype of the offsprings were DR5+/-ApoE+/-. Then heterogygous mice were mated with each other. We selected DR5-/-ApoE-/-and DR5+/+ApoE-/-mice by genotyping.Tissue Sample CollectionFor the collection of blood samples, animals were fasted overnight, anesthetized with0.8%pentobarbital sodium. Blood was intracardially collected using a syringe though the heart-tips. Followed sacrificing animals, various tissurs, heart and arteries were dissected. For the analysis of mRNA and protein expression, samples were immediately placed in liquid nitrogen and then the kept in-80℃until use. For histological analysis, samples were fixed with4%paraformaldehyde overnight, followed by washing with lx PBS several times.Blood Lipid AnalysisAfter collection of blood samples, plasma from each mouse was prepared and kept at-80℃until use. Total plasma levels of cholesterol (CHO), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automatic biochemical analyzer (Roche, Germany).Histological Staining and ImmunohistochemistryTissue samples were stained with HE, Masson and Oil-Red O using a standard protocol. Tissues prepared from cryosections or paraffin-embedded samples (5μm thickness) were immunohistologically stained with various primary antibodies, followed by further staining with secondary antibodies and hematoxylin staining.Statistical AnalysisAll experiments were repeated at least three times. Data are presented as mean±standard deviation (SD). Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test as appropriate. P<0.05was considered as statistically significant. Results TRAIL treatment increased atherogenesis in hyperlipidemic miceTRAIL treatment for4and8weeks significantly increased both of the en face atherosclerotic lesion area and the cross sectional lesion size at the aortic root. To clarify whether the proatherogenic effects of TRAIL were related to the concomitant high-fat diet feeding, we repeated the experiments in ApoE-/-mice maintained on normal chow. We found that TRAIL similarly increased atherogenesis in chow-fed animals at4week, although the effects at8week did not reach a statistic significance. To determine whether the genetic background of the hyperlipidemic mice affected the effects of TRAIL, we treated LDLR-/-mice with the high-fat diet for16weeks, as the lesion size at8week in LDLR-/-mice was too small to analyze. We showed that rTRAIL also significantly increased both of the en face and cross sectional lesion areas in the aorta.The effects of TRAIL was not associated with changes in monocyte phenotypeSince the proatherogenic effects of TRAIL could be mediated by changes in the circulating immune cells or the resident vascular cells, next we attempted to dissect the cellular mechanisms of TRAIL-induced atherogenesis. To test whether TRAIL had any effects on the phenotype of circulating monocytes, we isolated CD115+peripheral monocytes from apoE-/-mice untreated or treated with TRAIL. We confirmed that～90%of the CD115+cells were also CD11b+. We found that TRAIL had no major effects on the proportions of Ly-6Chigh, CCR2+, CCR5+or CX3CR1+monocyte populations.TRAIL-stimulated atherogenesis was mediated by vascular inflammationTo determine whether TRAIL might induce atherogenesis by modulating vascular inflammation, we performed qPCR assays in the aortas from apoE-/-mice. We demonstrated that in TRAIL-treated mice, the expression levels of MCP-1, RANTES, vascular cell adhesion molecule (VCAM)-1and intercellular adhesion molecule-1(ICAM-1) were all significantly elevated as compared to untreated animals. In contrast, TRAIL had no significant effects on the expression of CX3CL1(fractalkine). TRAIL treatment also significantly upregulated local expressions of TNF-α, E-selectin, and IL-1.Thus, to further determine whether vascular inflammation had a causal role in TRAIL-induced atherogenesis, we tested the effects of blockade of the two chemokine pathways by treating the animals with a combination of neutralizing antibodies against MCP-1and RANTES. We found that, as compared to normal IgQ co-treatment with the neutralizing antibodies significantly blunted the proatherogenic effects of TRAILTRAIL-induced atherogenesis was DR5-dependentTo further confirm that the TRAIL effects were mediated by the DR5receptor in mice, we created ApoE/DR5DKO mice. We demonstrated that TRAIL-induced increase in atherosclerosis was totally abolished in DKO mice. Surprisingly, however, we found that in animals without TRAIL treatment, the lesion size was also significantly smaller in DR5-/-mice than that in DR5+/+mice.ER stress determined the sensitivity of macrophages to TRAIL-induced apoptosisTRAIL can induce macrophage apoptosis in advanced lesions. However, this has not yet been examined in early stage lesions. We showed that TRAIL-induced intraplaque cell apoptosis was more prominent in the16-week group than that in the8-week group.We hypothesized that ER stress might have a role in determining the macrophage sensitivity to TRAIL-induced apoptosis. To test this possibility, we showed that the expression levels of the ER stress markers GRP78and CHOP were significantly higher in the16-week group. We showed that smooth muscle cells were relatively resistant to TRAIL-induced apoptosis as compared to macrophages. To further confirm that ER stress determined the macrophage sensitivity to TRAIL-induced apoptosis, we induced ER stress in cultured human THP-1cells with tunicamycin. We found that TRAIL-triggered apoptosis was significantly increased in cells with ER stress induction. We also confirmed the sensitizing effect of tunicamycin-induced ER stress on TRAIL-triggered apoptosis in RAW264.7cells.Moreover, we performed CHIP assay in THP-1cells and revealed that the ER stress-related transcription factor CHOP directly bound to the DR5promoter, and this interaction was enhanced following tunicamycin treatment.Conclusion1. TRAIL treatment in vivo significantly increased the development of atherosclerosis in hyperlipide mice.2. TRAIL-stimulated atherogenesis was mediated by vascular inflammation but not associated with changes in monocyte phenotype.3. ER stress could upregulated the expression of DR5, which determined the sensitivity of macrophages to TRAIL-induced apoptosis. BackgroundAtherosclerosis (AS) is a condition first develops in the arterial intima where various cells and cytokines accumulated initiated by lots of causes. Animal experiments indicated that, atherosclerotic lesions concerned three processions:(1) The dysfunction of the endothelium and the proliferation of vascular smooth muscle cells (VSMCs), macrophages, lymphocytes which produce inflammatory and chemotactic factors.(2) Accumulation of VSMCs derived collagen fiber and extracellular matrix, give rise to a fibrous plaque.(3) Accumulation of lipids, cholesterol in matrix and foam cells.Ross et.al gave a hypothesis that AS lesions is a chronic inflammatory disease. The study indicated that chronic inflammatory response is a very important factor in the occurrence and development of atherosclerosis. Actually, the process of AS is a protective response to the injury of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). During that process, inflammation is accompanied, but inflammatory overreaction can reduce to atherosclerosis.Chronic inflammatory plays a major role during all stages of atherosclerosis. There are two ways for cytokines promote inflammatory reactions during atherosclerosis:affect the phenotype of circulating immune cells (almost monocytes); induce inflammation of the resident vascular cells. Our former study showed, TRAIL had no major effects on the proportions of monocyte populations, but induced vascular inflammation.TRAIL and its death receptors are expressed in VSMCs of arteries and pulmonary arteries. Sato et.al detected the expression of TRAIL and receptors on VSMCs in in atherosclerotic plaque, got positive results for three death receptors, DR4, DR5, and Fas. Immunohistochemistry showed that DR5was expressed on VSMCs in the plaque cap region, but not on VSMCs in normal carotid artery walls.It has been demonstrated that exogenous TRAIL may induce prominent inflammatory reactions in both vascular endothelial and smooth muscle cells, leading to increased expression of adhesion molecules and proinflammatory chemokines such as E-selectin, intercellular adhesion molecule (ICAM)-1and interleukin (IL)-8. Moreover, the plasma level of TRAIL has been shown to be elevated in patients with inflammatory diseases such as psoriatic arthritis, systemic lupus erythematosus, and viral infection.During atherogenesis, macrophages migrate into the subendothelial space and internalize chemically modified (e.g. oxidized) low-density lipoproteins (LDL), leading to formation of cholesterol-laden foam cells. This process is the central pathophysiological mechanism responsible for the initiation of atherosclerosis. Studies have demonstrated that lipid uptake by macrophages is mediated by various types of scavenger receptors, of which the most functionally important ones include scavenger receptors class A (SR-AI and-All), scavenger receptor-BI (SR-BI), CD36and lectin-like LDL receptor-1(LOX-1). However, whether TRAIL has any effects on expression of macrophage scavenger receptors and lipid uptake by macrophages has not yet been studied.Based on these findings, we proposed that TRAIL might have significant impacts on the inflammatory responses of the resident vascular cells; also, TRAIL might affect the expression of macrophage scavenger receptors and lipid uptake by macrophages.Aims (1) To examine whether and how TRAIL influences the inflammatory responses of the resident vascular cells.(2) To examine whether and how TRAIL affects the expression of macrophage scavenger receptors.(3) To see if TRAIL promotes the lipid uptake by macrophages.(4) To examine whether TRAIL influences the inflammatory responses as an obesity adipokine.Experiment MaterialsCulture of cell lines HUVECs were maintained in ECM medium containing10%fetal bovine serum (FBS),1%penicillin/streptomycin and1%growing factors. Murine macrophage cell line RAW264.7and human monocytic cell line THP-1were maintained in Dulbecco’s Modified Eagle Medium (DMEM) or RPMI1640respectively, containing10%FBS and1%penicillin/streptomycin. Primary aortic VSMCs were isolated from wild type mice using enzymatic digestion and were cultured in DMEM containing10%FBS and1%penicillin/streptomycin.Detection of inflammatory reaction HUVECs, RAW264.7cells and VSMCs were incubated with TRAIL (10ng/ml), TNF-a (20ng/ml) for6,12or24hours, respectively. Following mRNA isolation, the expression of ICAM-1, VCAM-1, MCP-1and E-selectin were measured by RT-PCR.Detection of scavenger receptors in macrophages RAW264.7and THP-1cells were incubated with0.1,1,10or100ng/ml TRAIL for24hours. To examine the time course of TRAIL-stimulated scavenger receptor expression, we treated the cells with TRAIL at one concentration. Following mRNA isolation, the expression of SR-AI, SR-BI, CD36, LOX-1were measured by RT-PCR.Analysis of lipid uptake and foam cell formation THP-1cells were allowed to adhere anf differentiate overnight at37℃in the presence of100nM phorbol mytistate acetate (PMA). RAW264.7or differentiated THP-1cells were cultured on Lab-Tek II chamber slides, after TRAIL incubation, cells were loaded with DiI-Ac-LDL (30μg ml-1) for2,4or8hr. Then cells were fixed, and counterstained with DAPI. Fluorescent images were obtained with a confocal microscope. To assess macrophage transformation into foam cells, we analyzed the accumulation of intracellular lipid droplets using Oil Red O staining.Analysis of the roles of scavenger receptorsTo test the roles of scavenger receptors in TRAIL-induced foam cell formation, we examined the effects of the SR-A inhibitor poly(I:C)(1μM), SR-AI siRNA and the SR-BI inhibitor BLT-1(5μM) on TRAIL-stimulated DiI-Ac-LDL uptake in RAW264.7and THP-1cells.Analysis of the role of DR5receptorTo further clarify the specific role of TRAIL receptors in TRAIL-induced foam cell formation, we isolated wild type and DR5-deficient peritoneal macrophages and measured ox-LDL-induced foam cell formation with and without rTRAIL pretreatment.Activation of MAPKs (mitogen-activated protein kinase)RAW264.7cells and VSMCs were incubated with TRAIL (10ng/ml) or TNF-a (20ng/ml) for5,10,20,30or120minutes, respectively. The levels of ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, FADD and TRAF2were detected using western-blot. Activation of NF-kB was detected using the UPSTATE(?) Non-Radioactive Universal EZ-TFA Transcription Factor Assay.Analysis of the roles of MAPKsTo further clarify the specific roles of MAPK members, we examined the effects of the p38inhibitor SB202190, ERK inhibitor U0126, JNK Inhibitor Ⅱ and the NF-kB inhibitor wogonin on TRAIL-stimulated lipid uptake and inflammation in VSMCs and RAW264.7cells.TRAIL and obesityTo explore the potential relationship of TRAIL and obesity, we examined the expression level of TRAIL in white adipose tissue from ob/ob mice and rats with type II diabetes mellitus. We also measured the plasma level of TRAIL in these obese models. Protein expression of TRAIL in adipose tissue was detected by immunohistochemistry.Statistical analysisAll experiments were repeated at least three times. Data are presented as mean±s.e.m. Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test. P<0.05was considered as statistically significant.ResultsTRAIL increased the expressions of inflammatory cytokines in VSMCs and macrophagesIncubation with lOng/ml TRAIL for12hours increased expressions of ICAM-1and VCAM-1in VSMCs, expression of MCP-1was upregulated after24hour of incubation. Incubation with lOng/ml of TRAIL for6hours increased expressions of MCP-1and VCAM-1in RAW264.7cells. But TRAIL did not affect expressions of inflammatory molecules in HUVECs.TRAIL-stimulated inflammatory reponses requires p38or NF-kB activationTreatment with rTRAIL triggered phosphorylation of ERK1/2, p38and JNK both in VSMCs and macrophages. These effects of TRAIL on MAPK signaling were similar to those induced by TNF-a, which was used as a positive control. The total levels of FADD or TRAF2were not changed. Pretreatment of the cells with the selective p38inhibitor SB202190or the NF-kB inhibitor wogonin, but not U0126(ERK pathway inhibitor) or JNK Inhibitor II, blunted rTRAIL-induced upregulation of SR-AI expression.TRAIL promotes lipid uptake and foam cell formation in macrophagesWe found that pretreatment with rTRAIL (10ng/ml) for24hr significantly increased intracellular DiI-Ac-LDL accumulation in RAW264.7and THP-1cells as measured by fluorescent microscopy. To further confirm that the effects of TRAIL on macrophage lipid uptake were relevant to foam cell formation, we pretreated RAW264.7and THP-1cells with rTRAIL and then loaded the cells with ox-LDL for a longer period of48hr. Oil Red O staining clearly demonstrated that rTRAIL treatment increased the amount of intracellular lipid droplets.The effect of TRAIL on foam cell formation was absent in DR5-deficient macrophagesWe showed that similar to cell lines, primary macrophages of the wild type exhibited increased foam cell formation in response to rTRAIL treatment, whereas this effect of TRAIL was totally abolished in DR5-deficient macrophages.TRAIL upregulated the expression of SR-AI and SR-BI in macrophagesIn RAW264.7cells, incubation with rTRAIL from0.1-100ng/ml for24hr significantly increased expressions of SR-AI and SR-BI in a concentration-dependent manner. The responses were maximum at10ng/ml. In contrast to SR-AI and SR-BI, rTRAIL had no significant effects on expressions of CD36or LOX-1. To examine the time course of TRAIL-stimulated scavenger receptor expression, we treated the cells with rTRAIL at10ng/ml for various time, and demonstrated that rTRAIL induced a gradual response over time with the effects being most significant at24hr.. We also performed western blot to measure the SR-AI protein expression. rTRAIL (10ng/ml for24hr) markedly increased the SR-AI protein expression. To confirm the effects of TRAIL in human cells, we repeated the experiments in THP-1cells. rTRAIL produced very similar responses in scavenger receptor expression as in murine cells. In addition, we demonstrated that rTRAIL-induced effects on SR-AI and SR-BI expression were totally absent in DR5-deficient macrophages as compared to wild type cells TRAIL-stimulated lipid uptake was mediated by SR-ATo test the roles of SR-AI and SR-BI in TRAIL-induced foam cell formation, we examined the effects of the SR-A inhibitor poly(I:C)(1μM) and the SR-BI inhibitor BLT-1(5μM) on TRAIL-stimulated DiI-Ac-LDL uptake in RAW264.7cells. We found that pretreatment with poly(I:C), but not BLT-1, significantly blunted the augmenting effects of TRAIL on DiI-Ac-LDL uptake. To further confirm the specific role of SR-AI, we transfected RAW264.7cells with SR-AI siRNA. Similar to the effects of poly(I:C), SR-AI siRNA significantly suppressed the stimulating effect of TRAIL on DiI-Ac-LDL accumulation. The specific role of SR-AI in mediating TRAIL-induced lipid uptake was also confirmed in human THP-1cells.TRAIL-stimulated SR-AI expression requires p38or NF-kB activationPretreatment of the cells with the selective p38inhibitor SB202190or NF-kB inhibitor wogonin, but not U0126(ERK pathway inhibitor) or JNK Inhibitor II, blunted rTRAIL-induced upregulation of SR-AI expression.TRAIL levels are increased in plasma and white adipose tissue in obesity modelsThe plasma level and mRNA expression of TRAIL in adipose tissue from ob/ob mice were increased as compared with wild type mice. Morever, the mRNA expression of TRAIL in adipose tissue was increased in type II diabetic rats as compare with normal Wistar rats. Immunohistochemistry staining showed that TRAIL was expressed in the stromal cells in the adipose tissue, but not in the adipose cells.Conclusion1. TRAIL increased inflammatory responses in VSMCs and macrophages.2. TRAIL increased the expression of scavenger receptor SR-AI, which mediated lipid uptake of macrophages and foam cell formation.3. TRAIL-stimulated inflammation and SR-AI expression requires p38or NF-kB activation.4. TRAIL levels in the plasma and adipose tissues are elevated in obese animals.