Nuclear factor-κB (NF-κB) is a key regulator of systematic inflammation in atherosclerosis (AS). The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, has emerged as an important regulator of chronic inflammation. However, the relationship between mTOR and NF-κB remains poorly defined. The aim of the present study was to investigate the role of mTOR in the pro-inflammatory pathway of human monocytes (HMCs) in patients with coronary artery disease (CAD) and to determine the interaction between mTOR and NF-κB signalling in the inflammatory state. HMCs were isolated from fasting blood samples of 68 patients with CAD and 59 subjects without CAD (non-CAD) to test the activity of NF-κB, p65 nuclear translocation and mTOR phosphorylation, which were all significantly elevated in the CAD group compared with those in the non-CAD group. The concentrations of serum interleukin (IL)-6 and tumour necrosis factor (TNF)-α were higher in the CAD group than in the non-CAD group. In an in vitro experiment, HMCs isolated from non-CAD subjects were used as culture model and were treated with sera extracted from CAD patients (CAD sera) or non-CAD subjects (con sera). CAD sera induced time-dependent phosphorylation of mTOR, aberrant NF-κB activation, as well as up-regulation of inflammatory factors. Moreover, inhibition of mTOR by pharmacological or genetic means abolished the CAD sera-triggered NF-κB activation and pro-inflammatory response. Furthermore, lipid-lowering drug statins partly blocked the CAD sera-activated mTOR and pro-inflammatory response. Our results show that CAD patients are in the pro-inflammatory state with increased NF-κB binding activity and enhanced mTOR phosphorylation. We also found that the activation of mTOR is required for the pro-inflammatory response via NF-κB-dependent pathway in HMCs, which unveils the underlying mechanism of AS and potential strategies to attenuate AS in clinical practice.

CLINICAL PERSPECTIVES

  • The role of mTOR in the pro-inflammatory pathway of HMCs in vivo is still unknown and the relationship between mTOR and NF-κB remains the most heated controversy.

  • Herein, we established the casual link between the mTOR and NF-κB signalling pathways in the HMCs of CAD patients. We found that CAD sera induce p65 nuclear translocation, NF-κB activation and IL-6/TNF-α expression through an mTOR phosphorylation-dependent pathway.

  • Given that statins do not completely block mTOR and NF-κB activation, further investigations are required to identify more effective drugs for CAD treatment.

INTRODUCTION

Atherosclerosis (AS) is a low-grade systemic chronic inflammatory disease that causes myriad serious medical problems worldwide [1,2]. Nuclear factor-κB (NF-κB) is a key regulator of systematic inflammation and aberrant NF-κB activation has been considered a key step in atherosclerotic progression [3,4]. Previous studies have shown that NF-κB binding activity and the nuclear translocation of its p65 subunit are increased in human monocytes (HMCs) of coronary artery disease (CAD) and patients with obesity [5,6].

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, has been reported to play an important role in vascular remodelling and AS, including protein synthesis, lipid metabolism, lysosome biogenesis, cell cycle and apoptosis [7,8]. Previous evidence has highlighted that mTOR inhibitors have pleiotropic anti-atherosclerotic effects and the mTOR inhibitor sirolimus decreases the size and the complexity of aortic atherosclerotic lesions in apoE knockout mice [9]. Clinical data have also shown that implantation of the mTOR inhibitor drug-eluting stent (DES) is effective in treating stable chronic angina or acute coronary syndrome [10]. The mTOR inhibitors are known to prevent the up-regulation of monocyte chemoattractant protein-1 (MCP-1) and macrophage accumulation in the rapamycin- or everolimus-treated animals [1113], but the roles of mTOR in the pro-inflammatory pathway of HMCs in CAD patients remain unknown.

Systematic inflammatory state refers to not only the levels of pro-inflammatory factors in serum but also the status of gene expression in HMCs [5,6,14]. HMCs is a detective model for metabolic diseases such as obesity, diabetes and AS [5,15,16]. Circulating monocytes are crucial cellular mediators of atherosclerotic lesion formation that give rise to lesional macrophages, which take in modified lipoproteins such as ox-LDL (low-density lipoprotein) and then become foam cells that drive atherogenesis [17,18].

Clinical evidence indicates that statin [HMG–CoA (3-hydroxy-3-methylglutaryl–coenzyme A) reductase inhibitor] exerts anti-inflammatory effects on the vascular wall by decreasing the number of inflammatory cells in atherosclerotic plaques [19]. Simvastatin, atorvastatin and lovastatin up-regulate IκB-α protein levels, but down-regulate the activation of transcription factor NF-κB in endothelial cells (ECs) [20]. Previous studies have also demonstrated that atorvastatin not only down-regulates toll-like receptor 4 (TLR4) levels in monocytes from the peripheral blood of stable CAD patients, but also inhibits senescence in endothelial progenitor cells from CAD patients [21,22].

Moreover, the relationship between mTOR and NF-κB remains the most heated controversy. Some believe that mTOR inhibits NF-κB [23,24], whereas others believe that mTOR is an activator of NF-κB [25,26]. In the present study, we investigated whether mTOR was phosphorylated in HMCs from CAD patients and the molecular relationship between mTOR and NF-κB in HMCs.

MATERIALS AND METHODS

Patients and study design

A total of 68 consecutive subjects with CAD (56.7±8.6 years) and 59 subjects without CAD (non-CAD, 55.8±7.9 years) who had received angiography were recruited from the First Affiliated Hospital of Xi’an Jiaotong University. In the CAD group, patients had typical chest pain on effort, accompanied by definite ST-segment changes and/or T-wave inversions and angiographically documented coronary stenosis, but were negative for cardiac markers or troponin I. In the non-CAD group, patients claimed chest discomfort but had normal coronary arteries on angiography. Exclusion criteria were: previous myocardial infarction within 6 months, renal failure, known history of cancer or chronic-immune mediated disorders or current use of immuno-suppressive agents. Written informed consent was obtained from all the participants. The present study complied with the Declaration of Helsinki and the research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University.

Blood samples collection

Fasting blood sample was collected from each participant. The anti-coagulated blood samples (20 ml) and coagulated blood samples (5 ml) were collected for HMCs isolation and serum collection respectively. Serum from each participant (1 ml) was pooled for cell treatment and was stored at −80°C till use.

Isolation of HMCs and cell culture

Peripheral human blood sample was obtained for human primary monocyte isolation as described previously [27]. Using Ficoll-isopaque (Sigma) density gradient, we isolated mononuclear cells by centrifugation. Then the cells were seeded and incubated in six-well plates for 2 h at 37°C in a 5% CO2 incubator. Non-adherent cells were then removed. The isolated HMCs were cultured in RPMI-1640 with 5% FBS for 24 h and then treated with pooled sera from the CAD or non-CAD group (5%). HMCs were pre-treated with ox-LDL (30 μg/ml), rapamycin (Calbiochem, 20 nmol/l), AZD8055 (Selleck Chemicals, 10 nmol/l), everolimus (Sigma, 20 nmol/l), SN-50 (p50 nuclear transport blocker; Sigma, 10 μmol/l), pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor, Sigma, 100 μmol/l), simvastatin (Sigma, 0.1–100 μmol/l), lovastatin (Sigma, 0.1–100 μmol/l) or pravastatin (Sigma, 0.1–100 μmol/l) under different experimental conditions.

siRNA transfection in HMCs

Human mTOR and control siRNA were obtained from Santa Cruz Biotechnology and transient siRNA transfection was carried out according to the protocol of the manufacturer, as described previously [28]. Briefly, siRNAs were dissolved in double distilled water to prepare a 10 μM stock solution. HMCs grown in six-well plates were transfected with siRNA in OptiMEM (Invitrogen) containing RNAiMax (Invitrogen). For each transfection, 250 μl of transfection medium containing 5 μg of siRNA was gently mixed with 250 μl of transfection medium containing 5 μl of transfection reagent. After 20 min incubation at room temperature, the mixture was added to cells in 2 ml of culture medium and cultured for 24 h before treatment.

Serum concentrations of pro-inflammatory mediators

Concentrations of serum interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were measured with ELISA kits according to the manufacturer’s instructions (ExCell Bio), as described previously [9,17]. The lower limits of detection were 0.2 pg/ml for IL-6 and TNF-α. Each analysis was carried out in duplicate by ELISA assay.

NF-κB DNA-binding activity

Nuclear proteins were extracted according to the manufacturer's instructions (Pierce). The NF-κB DNA-binding activity was measured with an NF-κB transcription factor assay kit (Abcam) according to the manufacturer’s instructions. Briefly, a specific double stranded DNA (dsDNA) sequence containing the NF-κB response element is immobilized on to the bottom of wells of a 96-well plate. NF-κB contained in a nuclear extract, binds specifically to the NF-κB response element. NF-κB is detected by addition of specific primary antibody directed against NF-κB. A secondary antibody conjugated to horseradish peroxidase (HRP) is added to provide a sensitive colorimetric read-out at 450 nm using a microtitre plate reader (BioTek Instruments Inc.).

Western blot analysis

Proteins in HMCs were detected using Western blot analysis as described previously [28,29]. Briefly, cellular proteins were extracted according to the manufacturer's instructions (Santa Cruz) and nuclear proteins were extracted according to the manufacturer's instructions (Pierce). Equal amount of proteins were separated by SDS/PAGE (10% gel) and transferred on to a PVDF membrane (Millipore), which was then incubated with primary antibodies against p65 (Santa Cruz), mTOR (Cell Signaling Technology) and p-mTOR (phosphorylated m-TOR; Cell Signaling Technology) respectively and then with HRP-conjugated anti-rabbit or anti-mouse antibody (Abcam). The membrane was developed and protein signals were detected using chemiluminescence. All values were corrected with GAPDH (Santa Cruz) and Histone 1 (Santa Cruz) as cellular and nuclear loading control.

Total RNA isolation and real-time PCR

Total RNA was isolated by TRIzol extraction (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized with RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) and analysed by real-time quantitative PCR with SYBR ExTaq™ II (TaKaRa). Real-time chain reactions were performed on the iQ5™ Multicolor real-time PCR and detection system (BioRad). Relative expressions were determined by normalizing expression of each target to GAPDH.

Statistical analysis

Statistical analysis was performed with SPSS for Windows (version 13.0). Summary values are expressed as mean±S.E.M. and skewed data are reported as median (interquartile range). The Fisher exact test or chi-square test was used for categorical variables. Analyses of the differences between two groups were performed using the t test. Spearman's correlation analysis was used for analysing the correlation between pro-inflammatory parameters and mTOR expression. Statistical significance was assumed at the 5% α-error level (P<0.05).

RESULTS

Clinical characteristics of the study population

Table 1 summarizes the clinical characteristics of all subjects. There were significant differences in high-sensitivity C-reactive protein (hsCRP) between the CAD and non-CAD groups (P<0.05), whereas no significant differences in other parameters, such as gender, age, body mass index (BMI) and risk factors, were observed.

Table 1
Characteristics of the study population

BP, blood pressure; HDL, high-density lipoprotein. Data are reported as means±S.D., median (interquartile range) or number.

non-CAD (n=59)CAD (n=68)P
Sex, M/F (n/n31/28 38/30 1.00 
Age (years) 55.8±7.9 56.7±8.6 0.56 
BMI (kg/m224.3±3.7 24.6±3.8 0.43 
Risk factors    
Hyperlipidaemia 11 18 0.40 
Current smoker 17 17 0.69 
Diabetes 0.99 
Systolic BP (mm Hg) 127±13 128±14 0.78 
Diastolic BP (mm Hg) 76±7 77±8 0.82 
Fasting glucose (mmol/l) 5.46±0.58 5.51±0.51 0.39 
Total cholesterol (mmol/l) 3.89±0.76 3.95±0.87 0.59 
Triacylglycerols (mmol/l) 1.65±1.01 1.68±1.06 0.88 
HDL cholesterol (mmol/l) 0.89±0.26 0.85±0.25 0.13 
LDL cholesterol (mmol/l) 2.19±0.62 2.32±0.73 0.29 
hsCRP (mg/l) 0.91 (0.21, 4.7) 4.05 (1.01, 11.7) 0.01 
non-CAD (n=59)CAD (n=68)P
Sex, M/F (n/n31/28 38/30 1.00 
Age (years) 55.8±7.9 56.7±8.6 0.56 
BMI (kg/m224.3±3.7 24.6±3.8 0.43 
Risk factors    
Hyperlipidaemia 11 18 0.40 
Current smoker 17 17 0.69 
Diabetes 0.99 
Systolic BP (mm Hg) 127±13 128±14 0.78 
Diastolic BP (mm Hg) 76±7 77±8 0.82 
Fasting glucose (mmol/l) 5.46±0.58 5.51±0.51 0.39 
Total cholesterol (mmol/l) 3.89±0.76 3.95±0.87 0.59 
Triacylglycerols (mmol/l) 1.65±1.01 1.68±1.06 0.88 
HDL cholesterol (mmol/l) 0.89±0.26 0.85±0.25 0.13 
LDL cholesterol (mmol/l) 2.19±0.62 2.32±0.73 0.29 
hsCRP (mg/l) 0.91 (0.21, 4.7) 4.05 (1.01, 11.7) 0.01 

CAD patients are in a pro-inflammatory state

We first examined levels of the inflammatory cytokine IL-6 and TNF-α in serum and HMCs. As shown in Figure 1A, both IL-6 and TNF-α serum levels were markedly higher in the CAD group than in the non-CAD (P<0.05) and their mRNA expression in CAD patients was also elevated compared with the non-CAD subjects (P<0.05; Figure 1A), suggesting the increased transcriptional expression of inflammatory cytokines in CAD patients.

The proinflammation, NF-κB and mTOR signalling pathways in HMCs of CAD patients

Figure 1
The proinflammation, NF-κB and mTOR signalling pathways in HMCs of CAD patients

HMCs from CAD patients (n=68) and non-CAD subjects (non-CAD, n=59) were isolated and evaluated. (A) Serum concentrations of IL-6 and TNF-α were elevated and the mRNA expression of IL-6 and TNF-α in HMCs was also increased in CAD patients compared with those in the non-CAD subjects. (B) NF-κB-binding activity, p65 expression in nuclear extracts and p-mTOR and mTOR expression in HMCs. (C) Representative Western blots of p-mTOR, mTOR, nuclear p65, NF-κB-binding activity and the mRNA expression of IL-6 and TNF-α from non-CAD subjects (n=4) and CAD patients (n=3) are shown. In (A) and (B) each box shows the lower and upper quartile values and the line indicates the median value. The error bars show tenth and ninetieth percentiles. *P<0.05 compared with non-CAD group; au, arbitrary units.

Figure 1
The proinflammation, NF-κB and mTOR signalling pathways in HMCs of CAD patients

HMCs from CAD patients (n=68) and non-CAD subjects (non-CAD, n=59) were isolated and evaluated. (A) Serum concentrations of IL-6 and TNF-α were elevated and the mRNA expression of IL-6 and TNF-α in HMCs was also increased in CAD patients compared with those in the non-CAD subjects. (B) NF-κB-binding activity, p65 expression in nuclear extracts and p-mTOR and mTOR expression in HMCs. (C) Representative Western blots of p-mTOR, mTOR, nuclear p65, NF-κB-binding activity and the mRNA expression of IL-6 and TNF-α from non-CAD subjects (n=4) and CAD patients (n=3) are shown. In (A) and (B) each box shows the lower and upper quartile values and the line indicates the median value. The error bars show tenth and ninetieth percentiles. *P<0.05 compared with non-CAD group; au, arbitrary units.

Increased NF-κB binding activity of HMCs in CAD patients

It is known that activated NF-κB translocates to the nucleus, binds specific DNA sequences and promotes the transcription of target genes such as IL-6 and TNF-α [23]. Because both of these factors were activated by NF-κB, the nuclear NF-κB DNA binding activity was investigated to see whether the inflammatory factors were activated by the transcription factor. As shown in Figure 1B, the NF-κB binding activity significantly increased in the CAD group compared with the non-CAD group (P<0.05).

Next, we examined the nuclear translocation of NF-κB subunit p65 in HMCs and the p65 nuclear levels were higher in the CAD group than in the non-CAD group (P<0.05; Figure 1B). These results indicate the transcriptional activation of NF-κB in CAD patients.

Enhanced mTOR phosphorylation at Ser2448 of HMCs in CAD patients

In order to determine the levels of mTOR and its phosphorylation (p-mTOR) at Ser2448, a well characterized activated form of mTOR, Western blots were performed. There was no difference in the total mTOR expression between the two groups, but the level of p-mTOR (Ser2448) was markedly elevated in the CAD group compared with the non-CAD group (Figure 1B), which demonstrated the enhanced mTOR signalling activation in CAD patients.

Activated mTOR in HMCs correlates with the pro-inflammatory state

There were positive correlations between p-mTOR and p65 nuclear translocation. We found that in some samples, the expression of p65 was lower with less p-mTOR signalling (lanes 2 and 4, Figure 1C), and in some other samples, where more p65 nuclear translocations occurred, the mTOR phosphorylation was higher (lanes 5–7, Figure 1C), all of which paralleled with the NF-κB DNA-binding activity and IL-6/TNF-α expressions. Furthermore, Spearman's correlation was used to examine the positive relationships. As shown in Table 2, p-mTOR showed significant positive correlations with NF-κB DNA-binding activity, p65 nuclear translocation and IL-6/TNF-α expression, implying that there should be a cooperation or interaction between the mTOR and NF-κB signalling in the HMCs.

Table 2
Associations between mTOR and inflammatory parameters

NF-κB is presented as NF-κB binding activity; p65 is the nuclear protein level; IL-6 and TNF-α are presented as their mRNA levels; p-mTOR and mTOR are their protein levels. n=66. Spearman's correlation coefficient was used to assess the relationships. *P<0.05; **P<0.01; ***P<0.001.

NF-κBp65IL-6TNF-αp-mTOR
NF-κB –     
p65 0.571*** –    
IL-6 0.197* 0.369*** –   
TNF-α 0.380*** 0.497*** 0.556*** –  
p-mTOR 0.429*** 0.625*** 0.281** 0.508*** – 
mTOR 0.166 0.193* −0.002 0.112 0.121 
NF-κBp65IL-6TNF-αp-mTOR
NF-κB –     
p65 0.571*** –    
IL-6 0.197* 0.369*** –   
TNF-α 0.380*** 0.497*** 0.556*** –  
p-mTOR 0.429*** 0.625*** 0.281** 0.508*** – 
mTOR 0.166 0.193* −0.002 0.112 0.121 

CAD sera, but not control sera, activate mTOR and pro-inflammatory signalling in a time-dependent manner

It has been reported that the sera of CAD patients are atheroprone and pro-inflammatory and can be used for cell culture to mimic pathological conditions [14,30]. Thus, we treated HMCs isolated from the non-CAD group with sera pooled from CAD patients (CAD sera) or non-CAD subjects (Con sera) and monitored mTOR and NF-κB activations. As depicted in Figure 2A, CAD sera significantly increased p-mTOR expression, NF-κB DNA-binding activity, p65 nuclear translocation and IL-6/TNF-α expression in a time-dependent manner. However, no activation was observed using con sera (P>0.05; Figure 2B). These data suggest that atheroprone sera activate mTOR and the pro-inflammatory reaction. Moreover, p-mTOR was significantly elevated 4 h after CAD sera intervention, whereas NF-κB activation, p65 nuclear translocation and IL-6/TNF-α expression were significantly elevated 8 h after CAD sera intervention (P<0.05; Figure 2A), suggesting that the pro-inflammatory response was activated after the mTOR phosphorylation induced by atheroprone sera.

CAD sera induce mTOR phosphorylation and NF-κB activation in HMCs

Figure 2
CAD sera induce mTOR phosphorylation and NF-κB activation in HMCs

HMCs isolated from non-CAD subjects were treated with the pooled CAD sera or con sera for the indicated periods of time. (A) CAD sera-induced mTOR phosphorylation, NF-κB activation, p65 nuclear translocation and IL-6/TNF-α mRNA expression. (B) Effect of con sera on mTOR and NF-κB signalling. Data are means±S.E.M. (n=5); *P<0.05 compared with baseline.

Figure 2
CAD sera induce mTOR phosphorylation and NF-κB activation in HMCs

HMCs isolated from non-CAD subjects were treated with the pooled CAD sera or con sera for the indicated periods of time. (A) CAD sera-induced mTOR phosphorylation, NF-κB activation, p65 nuclear translocation and IL-6/TNF-α mRNA expression. (B) Effect of con sera on mTOR and NF-κB signalling. Data are means±S.E.M. (n=5); *P<0.05 compared with baseline.

Ox-LDL first activates mTOR phosphorylation and then pro-inflammatory signalling

Ox-LDL (oxidized LDL), a well-known atherosclerotic risk factor, has been reported to activate NF-κB and pro-inflammatory signalling in vivo and in vitro [31,32]. In order to examine whether ox-LDL activates mTOR signalling, we tested its effects on an HMCs model at different time points (0–24 h). As depicted in Figure 3A, ox-LDL activated mTOR phosphorylation and pro-inflammatory response in a time-dependent manner.

NF-κB inhibition blocks ox-LDL activated pro-inflammatory response, but not mTOR phosphorylation

Figure 3
NF-κB inhibition blocks ox-LDL activated pro-inflammatory response, but not mTOR phosphorylation

HMCs from non-CAD subjects were treated with ox-LDL for the indicated periods of time. (A) ox-LDL-induced mTOR phosphorylation, NF-κB activation, p65 nuclear translocations and IL-6/TNF-α mRNA expression. (B) NF-κB inhibitor PDTC and p50 nuclear transport blocker SN-50 both inhibited ox-LDL-induced NF-κB activation and pro-inflammatory gene expressions, but had little effect on the phosphorylation of mTOR. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with ox-LDL.

Figure 3
NF-κB inhibition blocks ox-LDL activated pro-inflammatory response, but not mTOR phosphorylation

HMCs from non-CAD subjects were treated with ox-LDL for the indicated periods of time. (A) ox-LDL-induced mTOR phosphorylation, NF-κB activation, p65 nuclear translocations and IL-6/TNF-α mRNA expression. (B) NF-κB inhibitor PDTC and p50 nuclear transport blocker SN-50 both inhibited ox-LDL-induced NF-κB activation and pro-inflammatory gene expressions, but had little effect on the phosphorylation of mTOR. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with ox-LDL.

Phosphorylation of mTOR was significantly elevated at 4 h, whereas NF-κB activation, p65/p50 nuclear translocations and IL-6/TNF-α expression were significantly elevated at 8 h (P<0.05; Figure 3A), suggesting that ox-LDL treatment induced mTOR phosphorylation and then the activated pro-inflammatory response.

Inhibitors of NF-κB block the pro-inflammatory response but not mTOR phosphorylation

We further determined whether NF-κB regulated mTOR phosphorylation and the pro-inflammatory response with PDTC (NF-κB inhibitor) and SN-50. PDTC or SN-50 inhibited ox-LDL-induced NF-κB activation and pro-inflammatory gene expression (P<0.05 for all), but had little effect on the phosphorylation of mTOR (Figure 3B), suggesting that mTOR may functioned as the upstream regulator of NF-κB and the pro-inflammatory response.

Inhibition of mTOR abolishes the CAD sera-induced pro-inflammatory response

According to the results presented above, there may be a cause and effect relationship between mTOR phosphorylation and NF-κB activation. So we used rapamycin, everolimus and AZD8055 (potent mTOR inhibitors) to examine whether mTOR inhibition blocked NF-κB activation and pro-inflammatory gene expression in HMCs. As depicted in Figure 4A, these inhibitors significantly inhibited mTOR phosphorylation, NF-κB activation, p65 nuclear translocations and IL-6/TNF-α expression (P<0.05).

Pharmocological inhibition or knockdown of mTOR suppresses NF-κB activation

Figure 4
Pharmocological inhibition or knockdown of mTOR suppresses NF-κB activation

HMCs isolated from non-CAD subjects were treated with ox-LDL (30 μg/ml) for the indicated periods of time. (A) HMCs were pre-treated with rapamycin (20 nmol/l), everolimus (20 nmol/l) and AZD8055 (10 nmol/l) for 1 h and then treated with CAD sera for 8 h. The cells were harvested for analysis of mTOR and NF-κB signalling. (B) Genetic knockdown of mTOR by siRNA attenuated NF-κB activation and pro-inflammatory gene expression. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with CAD sera only.

Figure 4
Pharmocological inhibition or knockdown of mTOR suppresses NF-κB activation

HMCs isolated from non-CAD subjects were treated with ox-LDL (30 μg/ml) for the indicated periods of time. (A) HMCs were pre-treated with rapamycin (20 nmol/l), everolimus (20 nmol/l) and AZD8055 (10 nmol/l) for 1 h and then treated with CAD sera for 8 h. The cells were harvested for analysis of mTOR and NF-κB signalling. (B) Genetic knockdown of mTOR by siRNA attenuated NF-κB activation and pro-inflammatory gene expression. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with CAD sera only.

Knockdown of mTOR blocks the pro-inflammatory response

We next performed mTOR siRNA knockdown in HMCs (Figure 4B). Knockdown of mTOR by siRNA significantly suppressed CAD sera-induced NF-κB activation and pro-inflammatory gene expression, suggesting that mTOR activation was required for the pro-inflammatory response of HMCs.

Statins partly block mTOR activation and the pro-inflammatory response

Statins are well-known and widely used lipid-lowering medicines that have been proven to provide therapeutic benefit and an anti-inflammatory response in EC, macrophage and smooth muscle cells. Whether statins regulate mTOR and pro-inflammatory pathways in HMCs has not been reported. Thus, we tested the effects of simvastatin, lovastatin and pravastatin. Simvastatin (10 μmol/l), lovastatin (10 μmol/l) and pravastatin (10 μmol/l) partly blocked the CAD sera-induced mTOR phosphorylation and NF-κB activation (44% to 57% of CAD sera only; P<0.05 for all; Figure 5). Furthermore, we tested the dose-dependent effects of statins and the data showed that simvastatin (0.1–100 μmol/l), lovastatin (0.1–100 μmol/l) and pravastatin (0.1–100 μmol/l) partly, but not totally, blocked CAD sera-induced mTOR activation (see Supplementary material). These data indicate that further studies are needed to explore mTOR/NF-κB inhibitors for CAD and AS treatment.

Statins partly block activated mTOR and pro-inflammatory responses

Figure 5
Statins partly block activated mTOR and pro-inflammatory responses

HMCs were pre-treated with simvastatin (10 μmol/l), lovastatin (10 μmol/l) and pravastatin (10 μmol/l) for 1 h and then treated with CAD sera for 8 h. The cells were harvested for analysis of mTOR and NF-κB signalling. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with CAD sera only.

Figure 5
Statins partly block activated mTOR and pro-inflammatory responses

HMCs were pre-treated with simvastatin (10 μmol/l), lovastatin (10 μmol/l) and pravastatin (10 μmol/l) for 1 h and then treated with CAD sera for 8 h. The cells were harvested for analysis of mTOR and NF-κB signalling. Data are means±S.E.M. (n=4); *P<0.05 compared with control; †P<0.05 compared with CAD sera only.

DISCUSSION

In the present study, we unveiled a novel mechanism by which mTOR modulates p65 nuclear accumulation and pro-inflammation in HMCs. Our results suggest that CAD sera and ox-LDL, via mTOR phosphorylation and activation, induce NF-κB activation, p65 nuclear translocation and IL-6/TNF-α expressions, which may provide insight into potential new therapeutic targets for clinical AS treatment.

NF-κB, the first reported inflammatory transcription factor, is composed of p65 dimers and is inactive in the cytoplasm. Once activated, the NF-κB dimers translocate to the nucleus, bind specific DNA sequences and promote the transcription of TNF-α, IL-6 and other pro-inflammatory genes [3,14]. However, how NF-κB is regulated by upstream signalling, especially in human HMCs, is not clear. In the present paper, we demonstrate that p-mTOR mediates NF-κB activation and pro-inflammation in HMCs.

The results obtained in the present study are at variance with previous data. It has been suggested that mTOR inhibits NF-κB p65 signalling or systematic pro-inflammation. Minhajuddin et al. [23] have demonstrated that in the ECs cultured with serum-free medium, rapamycin augments, whereas overexpression of mTOR inhibits thrombin-induced NF-κB activation and intercellular cell adhesion molecule-1 (ICAM-1) expression. Others have found that mTOR overexpression attenuates lipopolysaccharide (LPS)-induced secretion of IL-6 and the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy [24]. NF-κB signalling may act upstream of mTOR in some cancer cell lines. For example, in the oesophagus, in premalignant epithelial cells cultured with serum-free medium, NF-κB signalling is required for bile-acid-induced mTOR activation and cancer cell proliferation [33]. These distinct signalling interaction patterns of mTOR and NF-κB may be due to different cell types and serum-free cell culture and different experimental conditions. All of these cells cannot represent monocytes from the human body.

Several lines of evidence support the key role of mTOR in NF-κB p65 activation and/or systematic pro-inflammation. First, in bone marrow ECs, mTOR activation is essential for cytokine-induced cell-cycle progression, whereas synergized blockade of mTOR and NF-κB pathways effectively inhibits cell proliferation [26]. Secondly, in THP-1 macrophage (a human leukaemic cell line), mTOR phosphorylation is required for ox-LDL-induced TLR4 expression, lipid-efflux impairment and foam cell formation [25]. Müller-Krebs et al. [34] have found that after incubation with podocytes for 48 h, everolimus, a mTOR inhibitor, suppresses NF-κB activation and protein expression of IL-6. Moreover, in the present study, we found a strong correlation between mTOR phosphorylation and NF-κB activation, suggesting that p-mTOR plays an important role in mediating NF-κB activation and pro-inflammation.

Many studies have demonstrated the important roles of statins in regulating endothelium function and anti-inflammation [1922]. Satoh et al. [21] have found that atorvastatin treatment markedly attenuates TLR4-induced pro-inflammation in monocytes of stable CAD patients, which may explain the beneficial clinical effects of statins in CAD and AS treatment. However, it is still unclear whether statins can regulate the signalling pathway of mTOR in HMCs. Herein, we found that statins partly, but not completely, inhibit mTOR phosphorylation and the activated pro-inflammatory response in HMCs. These results may add useful and fundamental information for statins’ anti-inflammatory effect in vivo. Further studies are necessary to explore synergistic mTOR inhibitors for treating CAD and AS.

In conclusion, we firstly analysed the cooperation and interaction between mTOR and NF-κB in HMCs and found that mTOR activation is required for NF-κB activation, p65 nuclear translocation and systemic inflammatory responses in HMCs of CAD patients, thus identifying mTOR as a therapeutic target for AS treatment.

Abbreviations

     
  • AS

    atherosclerosis

  •  
  • BMI

    body mass index

  •  
  • CAD

    coronary artery disease

  •  
  • EC

    endothelial cell

  •  
  • HMC

    human monocyte

  •  
  • HRP

    horseradish peroxidase

  •  
  • hsCRP

    high-sensitivity C-reactive protein

  •  
  • IL-6

    interleukin-6

  •  
  • LDL

    low-density lipoprotein

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NF-κB

    nuclear factor-κB

  •  
  • Ox-LDL

    oxidized LDL

  •  
  • PDTC

    pyrrolidine dithiocarbamate

  •  
  • p-mTOR

    phosphorylated mTOR

  •  
  • SN-50

    p50 nuclear transport blocker

  •  
  • TLR

    toll-like receptor

  •  
  • TNF

    tumour necrosis factor

AUTHOR CONTRIBUTION

Shanshan Gao and Weimin Liu devised and conducted the experiments and wrote the manuscript. Xiaozhen Zhuo, Lijun Wang, Tao Sun, Gang Wang and Yuling Tian participated in cell isolation and Western blots. Junhui Liu and Juan Zhou partially performed clinical data collection and analysis of data. Yue Wu and Zuyi Yuan conceived the project, designed the experiments and wrote the manuscript.

We thank Dr Zhiquan Liu and Dr Min Gong for their helpful suggestions in study design.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 81100209, 81025002, 81400302 and 91339116]; and the 973 Project [grant number 2012CB517804].

References

References
1
Libby
 
P.
Ridker
 
P.M.
Hansson
 
G.K.
 
Progress and challenges in translating the biology of atherosclerosis
Nature
2011
, vol. 
473
 (pg. 
317
-
325
)
[PubMed]
2
Galkina
 
E.
Ley
 
K.
 
Immune and inflammatory mechanisms of atherosclerosis (*)
Annu. Rev. Immunol.
2009
, vol. 
27
 (pg. 
165
-
197
)
[PubMed]
3
Hayden
 
M.S.
Ghosh
 
S.
 
NF-kappaB, the first quarter-century: remarkable progress and outstanding questions
Genes Dev.
2012
, vol. 
26
 (pg. 
203
-
234
)
[PubMed]
4
Tsai
 
A.K.
Steffen
 
B.T.
Ordovas
 
J.M.
Straka
 
R.
Zhou
 
X.
Hanson
 
N.Q.
Arnett
 
D.
Tsai
 
M.Y.
 
Short-term fenofibrate treatment reduces elevated plasma Lp-PLA2 mass and sVCAM-1 levels in a subcohort of hypertriglyceridemic GOLDN participants
Transl. Res.
2011
, vol. 
158
 (pg. 
99
-
105
)
[PubMed]
5
Ghanim
 
H.
Aljada
 
A.
Hofmeyer
 
D.
Syed
 
T.
Mohanty
 
P.
Dandona
 
P.
 
Circulating mononuclear cells in the obese are in a proinflammatory state
Circulation
2004
, vol. 
110
 (pg. 
1564
-
1571
)
[PubMed]
6
Zhao
 
Z.
Wu
 
Y.
Cheng
 
M.
Ji
 
Y.
Yang
 
X.
Liu
 
P.
Jia
 
S.
Yuan
 
Z.
 
Activation of Th17/Th1 and Th1, but not Th17, is associated with the acute cardiac event in patients with acute coronary syndrome
Atherosclerosis
2011
, vol. 
217
 (pg. 
518
-
524
)
[PubMed]
7
Laplante
 
M.
Sabatini
 
D.M.
 
mTOR signaling in growth control and disease
Cell
2012
, vol. 
149
 (pg. 
274
-
293
)
[PubMed]
8
Inoki
 
K.
Kim
 
J.
Guan
 
K.L.
 
AMPK and mTOR in cellular energy homeostasis and drug targets
Annu. Rev. Pharmacol. Toxicol.
2012
, vol. 
52
 (pg. 
381
-
400
)
[PubMed]
9
Elloso
 
M.M.
Azrolan
 
N.
Sehgal
 
S.N.
Hsu
 
P.L.
Phiel
 
K.L.
Kopec
 
C.A.
Basso
 
M.D.
Adelman
 
S.J.
 
Protective effect of the immunosuppressant sirolimus against aortic atherosclerosis in apo E-deficient mice
Am. J. Transplant.
2003
, vol. 
3
 (pg. 
562
-
569
)
[PubMed]
10
Niccoli
 
G.
Sgueglia
 
G.A.
Cosentino
 
N.
Piro
 
M.
Toma
 
A.
Cataneo
 
L.
Fracassi
 
F.
Porto
 
I.
Leone
 
A.M.
Burzotta
 
F.
, et al 
Impact of gender on clinical outcomes after mTOR-inhibitor drug-eluting stent implantation in patients with first manifestation of ischaemic heart disease
Eur. J. Prev. Cardiol.
2012
, vol. 
19
 (pg. 
914
-
926
)
[PubMed]
11
Castro
 
C.
Campistol
 
J.M.
Sancho
 
D.
Sanchez-Madrid
 
F.
Casals
 
E.
Andres
 
V.
 
Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27 Kip1 -independent pathway
Atherosclerosis
2004
, vol. 
172
 (pg. 
31
-
38
)
[PubMed]
12
Pakala
 
R.
Stabile
 
E.
Jang
 
G.J.
Clavijo
 
L.
Waksman
 
R.
 
Rapamycin attenuates atherosclerotic plaque progression in apolipoprotein E knockout mice: inhibitory effect on monocyte chemotaxis
J. Cardiovasc. Pharmacol.
2005
, vol. 
46
 (pg. 
481
-
486
)
[PubMed]
13
Baetta
 
R.
Granata
 
A.
Canavesi
 
M.
Ferri
 
N.
Arnaboldi
 
L.
Bellosta
 
S.
Pfister
 
P.
Corsini
 
A.
 
Everolimus inhibits monocyte/macrophage migration in vitro and their accumulation in carotid lesions of cholesterol-fed rabbits
J. Pharmacol. Exp. Ther.
2009
, vol. 
328
 (pg. 
419
-
425
)
[PubMed]
14
Wu
 
Y.
Zhang
 
W.
Liu
 
W.
Zhuo
 
X.
Zhao
 
Z.
Yuan
 
Z.
 
The double-faced metabolic and inflammatory effects of standard drug therapy in patients after percutaneous treatment with drug-eluting stent
Atherosclerosis
2011
, vol. 
215
 (pg. 
170
-
175
)
[PubMed]
15
Henning
 
R.J.
Dennis
 
S.
Sawmiller
 
D.
Hunter
 
L.
Sanberg
 
P.
Miller
 
L.
 
Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia
Transl. Res.
2012
, vol. 
159
 (pg. 
497
-
506
)
[PubMed]
16
Weber
 
C.
Erl
 
W.
Weber
 
K.S.
Weber
 
P.C.
 
HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia
J. Am. Coll. Cardiol.
1997
, vol. 
30
 (pg. 
1212
-
1217
)
[PubMed]
17
Osterud
 
B.
Bjorklid
 
E.
 
Role of monocytes in atherogenesis
Physiol. Rev.
2003
, vol. 
83
 (pg. 
1069
-
1112
)
[PubMed]
18
Bekkering
 
S.
Quintin
 
J.
Joosten
 
L.A.
van der Meer
 
J.W.
Netea
 
M.G.
Riksen
 
N.P.
 
Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes
Arterioscler. Thromb. Vasc. Biol.
2014
, vol. 
34
 (pg. 
1731
-
1738
)
[PubMed]
19
Vaughan
 
C.J.
Gotto
 
A.J.
Basson
 
C.T.
 
The evolving role of statins in the management of atherosclerosis
J. Am. Coll. Cardiol.
2000
, vol. 
35
 (pg. 
1
-
10
)
[PubMed]
20
Dichtl
 
W.
Dulak
 
J.
Frick
 
M.
Alber
 
H.F.
Schwarzacher
 
S.P.
Ares
 
M.P.
Nilsson
 
J.
Pachinger
 
O.
Weidinger
 
F.
 
HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells
Arterioscler. Thromb. Vasc. Biol.
2003
, vol. 
23
 (pg. 
58
-
63
)
[PubMed]
21
Satoh
 
M.
Tabuchi
 
T.
Minami
 
Y.
Takahashi
 
Y.
Itoh
 
T.
Nakamura
 
M.
 
Expression of let-7i is associated with toll-like receptor 4 signal in coronary artery disease: effect of statins on let-7i and toll-like receptor 4 signal
Immunobiology
2012
, vol. 
217
 (pg. 
533
-
539
)
[PubMed]
22
Tabuchi
 
T.
Satoh
 
M.
Itoh
 
T.
Nakamura
 
M.
 
MicroRNA-34a regulates the longevity-associated protein SIRT1 in coronary artery disease: effect of statins on SIRT1 and microRNA-34a expression
Clin. Sci.
2012
, vol. 
123
 (pg. 
161
-
171
)
[PubMed]
23
Minhajuddin
 
M.
Fazal
 
F.
Bijli
 
K.M.
Amin
 
M.R.
Rahman
 
A.
 
Inhibition of mammalian target of rapamycin potentiates thrombin-induced intercellular adhesion molecule-1 expression by accelerating and stabilizing NF-kappa B activation in endothelial cells
J. Immunol.
2005
, vol. 
174
 (pg. 
5823
-
5829
)
[PubMed]
24
Song
 
X.
Kusakari
 
Y.
Xiao
 
C.Y.
Kinsella
 
S.D.
Rosenberg
 
M.A.
Scherrer-Crosbie
 
M.
Hara
 
K.
Rosenzweig
 
A.
Matsui
 
T.
 
mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy
Am. J. Physiol. Cell Physiol.
2010
, vol. 
299
 (pg. 
C1256
-
C1266
)
[PubMed]
25
Yu
 
M.
Kang
 
X.
Xue
 
H.
Yin
 
H.
 
Toll-like receptor 4 is up-regulated by mTOR activation during THP-1 macrophage foam cells formation
Acta Biochim. Biophys. Sin.
2011
, vol. 
43
 (pg. 
940
-
947
)
26
Costa
 
L.F.
Balcells
 
M.
Edelman
 
E.R.
Nadler
 
L.M.
Cardoso
 
A.A.
 
Proangiogenic stimulation of bone marrow endothelium engages mTOR and is inhibited by simultaneous blockade of mTOR and NF-kappaB
Blood
2006
, vol. 
107
 (pg. 
285
-
292
)
[PubMed]
27
Chen
 
T.
Huang
 
Z.
Wang
 
L.
Wang
 
Y.
Wu
 
F.
Meng
 
S.
Wang
 
C.
 
MicroRNA-125a-5p partly regulates the inflammatory response, lipid uptake, and ORP9 expression in oxLDL-stimulated monocyte/macrophages
Cardiovasc. Res.
2009
, vol. 
83
 (pg. 
131
-
139
)
[PubMed]
28
Zhuo
 
X.Z.
Wu
 
Y.
Ni
 
Y.J.
Liu
 
J.H.
Gong
 
M.
Wang
 
X.H.
Wei
 
F.
Wang
 
T.Z.
Yuan
 
Z.
Ma
 
A.Q.
Song
 
P.
 
Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress
Apoptosis
2013
, vol. 
18
 (pg. 
800
-
810
)
[PubMed]
29
He
 
M.
Liang
 
X.
He
 
L.
Wen
 
W.
Zhao
 
S.
Wen
 
L.
Liu
 
Y.
Shyy
 
J.Y.
Yuan
 
Z.
 
Endothelial dysfunction in rheumatoid arthritis: the role of monocyte chemotactic protein-1-induced protein
Arterioscler. Thromb. Vasc. Biol.
2013
, vol. 
33
 (pg. 
1384
-
1391
)
[PubMed]
30
Chitalia
 
V.C.
Shivanna
 
S.
Martorell
 
J.
Balcells
 
M.
Bosch
 
I.
Kolandaivelu
 
K.
Edelman
 
E.R.
 
Uremic serum and solutes increase post-vascular interventional thrombotic risk through altered stability of smooth muscle cell tissue factor
Circulation
2013
, vol. 
127
 (pg. 
365
-
376
)
[PubMed]
31
Lou
 
Y.
Liu
 
S.
Zhang
 
C.
Zhang
 
G.
Li
 
J.
Ni
 
M.
An
 
G.
Dong
 
M.
Liu
 
X.
Zhu
 
F.
, et al 
Enhanced atherosclerosis in TIPE2-deficient mice is associated with increased macrophage responses to oxidized low-density lipoprotein
J. Immunol.
2013
, vol. 
191
 (pg. 
4849
-
4857
)
[PubMed]
32
Li
 
D.
Saldeen
 
T.
Romeo
 
F.
Mehta
 
J.L.
 
Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB
Circulation
2000
, vol. 
102
 (pg. 
1970
-
1976
)
[PubMed]
33
Yen
 
C.J.
Izzo
 
J.G.
Lee
 
D.F.
Guha
 
S.
Wei
 
Y.
Wu
 
T.T.
Chen
 
C.T.
Kuo
 
H.P.
Hsu
 
J.M.
Sun
 
H.L.
 
Bile acid exposure up-regulates tuberous sclerosis complex 1/mammalian target of rapamycin pathway in Barrett's-associated esophageal adeno-carcinoma
Cancer Res.
2008
, vol. 
68
 (pg. 
2632
-
2640
)
[PubMed]
34
Muller-Krebs
 
S.
Weber
 
L.
Tsobaneli
 
J.
Kihm
 
L.P.
Reiser
 
J.
Zeier
 
M.
Schwenger
 
V.
 
Cellular effects of everolimus and sirolimus on podocytes
PLoS One
2013
, vol. 
8
 pg. 
e80340
 
[PubMed]

Author notes

1

These authors contributed equally to this work.

Declaration: The authors have no financial or personal relationship with organizations that could potentially be perceived as influencing the described research.

Supplementary data