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Cardiovascular Research 2004 62(1):185-193; doi:10.1016/j.cardiores.2004.01.002
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Copyright © 2004, European Society of Cardiology

Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells

Sung-Woo Ryooa,b, Dong-Uk Kima, Misun Wona, Kyung-Sook Chunga, Young-Joo Janga, Goo-Taek Oha, Song-Kyu Parka, Pil-Jae Maengb, Hyang-Sook Yooa and Kwang-Lae Hoe*,a

aGenome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon, South Korea
bDepartment of Microbiology, Chungnam National University, Yusong, Daejeon, South Korea

* Corresponding author. Tel.: +82-42-860-4158; fax: +82-42-860-4597. Email address: kwanghoe{at}kribb.re.kr

Received 3 November 2003; revised 13 December 2003; accepted 2 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background and objectives: Endothelial and monocytic cells appear to play a key role in the initiation and progression of atherosclerosis and restenosis via the upregulation of inflammatory cytokines and the formation of oxidized low-density lipoprotein (ox-LDL). However, the role of smooth muscle cells (SMCs) has been underestimated and is not well understood. It was investigated for the first time that native LDL stimulates human SMCs to secrete IL-8. The aim of this study was to investigate the signaling pathway involved in the upregulation of IL-8, induced by LDL in human aortic SMCs. Methods and results: LDL-induced IL-8 expression (mRNA and protein) is specific to SMCs and likely to be regulated at the transcription level in dose- and time-dependent manners, as judged by experiments with actinomycin D and ELISA. Although both p38 and ERK 1/2 MAPKs were activated by LDL, only p38 MAPK is responsible for the LDL effects, as evidenced by a complete blockade of IL-8 upregulation by SB203580. Pretreatment with catalase significantly decreased the extent of IL-8 upregulation, indicating that H2O2 is necessary for the LDL response. Activation of activator protein (AP)-1, but not nuclear factor (NF)-{kappa}B, by p38 or H2O2 appears to be necessary along with the concomitant upregulation of c-fos and c-JUN, as judged by electrophoretic mobility shift and luciferase reporter assays. Conclusions: These data demonstrated that LDL stimulates SMCs to induce IL-8 production in dose- and time-dependent manners at the transcription level and that the LDL signaling in hAoSMCs is conveyed via the generation of H2O2, the phosphorylation of p38 MAPK, the activation of AP-1, and the participation of NF-{kappa}B.

KEYWORDS Atherosclerosis; Interleukins; Lipoproteins; MAP kinase; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The recruitment of mononuclear leukocytes and the migration, growth, and activation of macrophages, lymphocytes, and smooth muscle cells within lesions are critical features of chronic inflammatory response of atherogenesis [1,2]. In this context, vascular cell-derived cytokines are important mediators in the regulation of inflammatory and pathologic situations [2].

The C-X-C chemokine IL-8, originally known as the neutrophil-chemotactic protein or T lymphocyte chemotactic factor, plays a key role in atherosclerosis, as judged by its potential relevance [3–5]. In addition, IL-8 has been reported to possess angiogenic properties [5] and is likely to be involved in the vascular SMC (VSMC) invasion of the intima due to its mitogenic and chemotactic nature [1]. Despite a variety of sources of IL-8 induction, it is well known that the production of IL-8 is specific to reactive oxygen species (ROS) in Hep-G2 cells, A549 epithelial cells, and human skin fibroblasts [6]. At least, four main MAPK members, c-JNK, p38, big MAPK-1, and ERK 1/2 (controversial), have been reported to be activated in SMCs by H2O2 [7]. Expression of the human IL-8 gene can be regulated at both the transcriptional [8,9] and post-transcriptional levels [10], depending on the cell type and inducing agent used. The IL-8 promoter contains consensus binding sites for the inducible transcription factors activator protein (AP)-1, nuclear factor (NF)-{kappa}B, NF-IL-6, and CCAAAT/enhancer-binding protein (C/EBP), which act in concert to synergistically activate the IL-8 promoter [8,11,12].

IL-8 induction is triggered in a number of cell types in response to a number of stimulants. Among them, oxidized low-density lipoprotein (ox-LDL), well known to be a prime risk factor for the development of atherosclerosis and the onset of coronary artery disease [13], is able to induce IL-8 production in all the blood cell types [14,15]. Similarly, other LDL-derivatives, such as electronegative- and enzymatically modified-LDL and oxidized phospholipid, induce IL-8 in human endothelial cells [9,16,17]. In the case of SMCs, previous studies have demonstrated that native LDL (n-LDL) affects vascular homeostasis, such as vasoconstriction and VSMC proliferation [18,19]. The upregulation of IL-8 in SMC is evoked by mechanical stress [12], angiotensin (Ang) II [20], and IL-1 [21]. However, whether native LDL itself could stimulate SMCs to secrete IL-8 has not been examined.

In this study, we report for the first time that IL-8 is upregulated by hVSMCs under hyperlipidemic conditions. In addition, we investigated the signaling pathway by which non-oxidized n-LDL exerts its effects on the upregulation of IL-8 in hAoSMCs, particularly focusing on the potential roles of the MAPKs, ROS, NF-{kappa}B, and AP-1 signaling pathways in this process. The study provides a line of evidence in support of SMC playing a key role in vascular tone through the production of IL-8 in response to high concentrations of LDL.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Reagents
The p38 inhibitor (SB203580), ERK 1/2 inhibitor (PD98059), transcription inhibitor (actinomycin D), tumor necrosis factor (TNF)-{alpha}, and NF-{kappa}B inhibitors (caffeic acid phenethyl ester (CAPE) and parthenolide (PTL)) were purchased from Calbiochem. All reagents, such as H2O2, polyethylene glycol-conjugated superoxide dismutase (SOD), catalase, dimethyl sulfoxide (DMSO), N-acetylcysteine (NAC), diphenylene iodinium (DPI), and lipopolysaccharide (LPS, from E. coli 0111:B4) were purchased from Sigma unless otherwise stated.

2.2. Cell culture
Human aortic SMCs (hAoSMCs) and human coronary smooth muscle cells (HCSMCs) were purchased from Clonetics, and cultured in SmGM-2 Bullet kit medium (Clonetics) at 37 °C in 5% CO2. Twenty-four hours before the experiment, growth medium was removed and replaced with DMEM medium (Gibco) containing 0.1% FBS (Gibco) for 24 h (maintenance medium). Human umbilical vein endothelial cells (HUVECs) were purchased from Angio labs and cultured in medium 199 (Gibco) containing 20% FBS, 2 mM L-glutamine, antibiotics, 30 mg/l endothelial cell growth supplement, and 100 mg/l heparin. Twenty-four hours before the experiment, the growth medium was removed and replaced with heparin- and ECGS-free medium 199 with 4% inactivated human serum (maintenance medium). Human monocytic cell line, THP-1, was purchased from ATCC and cultured in RPMI1640 (Gibco) containing 10% FBS, 2 mM L-glutamine, antibiotics, and 0.05 mM β-mercaptoethanol.

2.3. Isolation of n-LDL
n-LDL (density 1.019–1.063 g/ml) was isolated from the plasma of normocholesterolemic subjects (serum cholesterol <6.2 mM) by differential ultracentrifugation as described by Metzler et al. [22]. During LDL manipulation and storage, particular precautions were taken for the maintenance of n-LDL integrity to prevent LDL from being oxidized. Endotoxin contents were below the detection limit (<1 ng/ml), as measured by the endotoxin assay kit (Pharmingen). Commercial LDL and ox-LDL prepared by reaction with CuSO4 were purchased from Intracel.

2.4. Determination of IL-8 concentration
The amounts of IL-8 released into the media were assayed using a human IL-8 ELISA Kit II (OptEIA, BD Biosciences International) according to the manufacturer's instructions. All experiments were conducted in triplicate.

2.5. Western blot analysis
Cells treated with different chemicals were lysed in SDS sample buffer (62.5 mM Tris pH6.8, 2% SDS, and 10% glycerol). Each sample was resolved on a 10% SDS-PAGE, transferred onto PVDF membranes (Pall), analyzed with antibodies according to the supplier's protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (ECL kit, Amersham). Normalization was performed by a Western blot analysis with the SMC-specific {alpha}-actin antibody (BD Sciences). Polyclonal phospho-p38 MAPK, monoclonal Erk 1/2, and polyclonal c-Jun antibodies were purchased from Cell Signaling Technology. The secondary antibody conjugated with horseradish peroxidase was purchased from Amersham Biosciences.

2.6. Northern blot analysis
Total RNA from hAoSMCs was prepared with the TRIzol Reagent according to the manufacturer's protocol (Gibco). Northern blotting was performed as described previously [23]. Northern probe was prepared by gel-extraction of the PCR-products. PCR-primer sequences of il-8 and c-fos (Bioneer) are as follows: il-8 forward 5'-AGC AGG AAG AAA CCA CCG GAA GG-3', reverse 5'-CAT CTG GCA ACC CTA CAA CAG ACC-3', size of PCR-products, 458 bp; c-fos forward 5'-ACC TAT CTG GGT CCT TCT ATG C-3', reverse 5'-TAA CTA CCA GCT CTC TGC AGT GTC-3', size of PCR-product, 529 bp.

2.7. Electrophoretic mobility shift assay (EMSA)
For the preparation of nuclear extracts, cells were allowed to swell and lysed in hypotonic buffer (10 mM HEPES pH7.9 and 1.5 mM MgCl2) containing a protease inhibitor cocktail solution (Boehringer Mennheim) on ice for 15 min. Nuclei were then harvested by centrifugation at 3000 x g for 5 min at 4 °C. Nuclear lysis was performed in hypertonic buffer (30 mM HEPES pH7.9, 1.5 mM MgCl2, 450 mM KCl, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, and 1 mM PMSF). After centrifugation at 13,000 x g for 20 min, the supernatants were recovered and used in the DNA binding assay. The oligonucleotide sequences for probes (Promega) are as follows; AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3'; NF-{kappa}B, 5'-AGTTGAGGGGACTTTCCCAGGC-3'. EMSA was performed as described previously [23] on a 5% PAGE in 0.5 x TBE buffer (44.5 mM Tris pH 7.5, 44.5 mM boric acid, and 1 mM EDTA).

2.8. Transient transfection and luciferase reporter assay
Sixty percent confluent cells were transfected with pAP-1-Luc or pNF-{kappa}B-Luc plasmids (PathDetect luciferase cis-reporter system containing 7 x AP1 and 5 x NF-{kappa}B enhancer elements, respectively, Stratagene) and pSV-β-Galactosidase control vector for transfection efficiency (Promega) using Superfect reagent (Qiagen). An empty luciferase plasmid, pLuc-MCS, was used as a control. After transfection, the cells were pretreated in serum-free maintenance medium for 16 h, and then treated with LDL for an additional 6 h. The cells were then harvested and the luciferase activities were measured using a Biolumat 9505 luminometer (Berthold) according to manufacturer's instructions (Promega). The luciferase activity was normalized to β-galactosidase activities. All experiments were conducted in triplicate.

2.9. Statistical analysis
All data represents the mean±S.D. of at least three independent experiments. The Unpaired Student's t-test was used to assess the significance of differences between the two groups. A value of P<0.05 was accepted as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. LDL-stimulated IL-8 induction is specific to SMCs
The findings herein show that treatment of human SMCs with n-LDL (100 µg/ml) for 24 h specifically induces IL-8 production (Table 1). To rule out the heterogeneity frequently observed in SMCs, the LDL effects were assessed with two different batches of hAoSMCs and compared with those of HCSMCs as well as those of human monocytic cells (THP-1) and HUVECs. Two different batches of hAoSMCs with the prepared LDL or commercial LDL showed no difference on IL-8 induction and led to a three-fold induction. The LDL effects were more dramatic in HCSMC. In contrast, treatment of THP-1 or HUVECs with LDL failed to induce IL-8 production, as previously reported by other investigators [14,16]. Intriguingly, ox-LDL induced IL-8 production from all the blood cell types, implying that the IL-8 induction mechanisms are different for n-LDL vis-a-vis ox-LDL. These results suggest that the LDL effects are specific to SMCs.


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  		Table 1 Induction levels of IL-8 evoked by various stimuli in blood vessel cells

 
3.2. Treatment of hAoSMCs with LDL induces IL-8 expression in time- and dose-dependent manners
In hAoSMCs, LDL stimulation results in a three-fold increase in IL-8 protein levels. The expression levels of IL-8 reached their maximum level at 10 h after LDL treatment and the three-fold increase was maintained for a period of over 24 h (Fig. 1A). In addition, LDL-stimulated IL-8 production increased steadily in a dose-dependent manner over the range of 5–200 µg/ml after the 24-h LDL treatment (Fig. 1B). These results suggest that treatment of hAoSMCs with LDL induces IL-8 expression in time- and dose-dependent manners.


Figure 1
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  		Fig. 1 Time- and dose-dependent IL-8 induction by LDL. Levels of IL-8 expression were quantified by ELISA analysis at the indicated times after treatment of hAoSMCs with LDL (100 µg/ml) (A) and at the indicated concentrations after a 24-h treatment with LDL (B). n=10, #P<0.05, *P<0.01 versus control. (C) The integrity of the prepared LDL and the stability of LDL (5 µg samples) after a 24-h incubation in culture media were verified by agarose gel electrophroresis using Fat Red 7B staining for lipids. Lane 1, commercial LDL; lane 2, prepared LDL; lanes 3 and 4, prepared LDL after a 12-h (lane 3) or 24-h (lane 4) incubation in culture medium; lane 5, commercial ox-LDL. "0" indicates gel loading slots; "+" and "–" indicate polarity. Representative example of three independent experiments.

 
Since ox-LDL is able to upregulate IL-8 production in all the blood cell types, the integrity of the prepared n-LDL and the stability of n-LDL during experiments were carefully monitored by agarose gel electrophoresis to exclude out the possibility of LDL oxidation (Fig. 1C). In addition, the malondialdehyde concentrations were measured by the thiobarbiturate method, and those of the prepared LDL, commercial LDL, and commercial ox-LDL were undetectable, undetectable, and 7.96±0.61 nmol/10 µg protein, respectively (n=6). The results show that the quality of the prepared LDL is comparable to that of commercial LDL from Intracel and free of oxidation even after a 24-h incubation in culture medium.

3.3. LDL-induced IL-8 expression is regulated at the level of transcription
Expression of the human IL-8 gene is regulated at the transcriptional [8] and post-transcriptional levels [10]. To determine which levels are related with the increases in IL-8 by LDL, the effects of actinomycin D on mRNA synthesis and protein production were first assessed by ELISA (Fig. 2A) and Northern blot analyses (Fig. 2B). Pretreatment of cells with actinomycin D almost inhibited the expression of IL-8 mRNA and protein by LDL. In addition, the effects of LDL on IL-8 mRNA stability was assessed by adding actinomycin D after treatment of LDL or TNF-{alpha} (Fig. 2C). TNF-{alpha} is known to regulate the expression of IL-8 at the transcriptional level [9]. There was no significant difference in the rate of IL-8 mRNA degradation, indicating that increased mRNA levels observed in Fig. 2A are not due to increased mRNA stability and that half-life time for both TNF-{alpha} and LDL is less than 30 min. These results suggest that LDL-induced IL-8 expression is regulated at the level of transcription [9,24,25].


Figure 2
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  		Fig. 2 Regulation of IL-8 expression at the level of transcription. (A and B) hAoSMCs were pretreated with or without actinomycin D (ActD; 10 µg/ml, 1 h), and IL-8 induction by LDL (100 µg/ml, 4 h) was then assessed by ELISA (A) and Northern blot analysis (B). TNF-{alpha} (10 ng/ml, 2 h) and rRNA were used as a positive and normalization controls. n=10, *P<0.01 versus control, #P<0.05 versus LDL; Representative example of three independent experiments. (C) The stability of induced IL-8 mRNA was determined by measuring the message half life. Cells were treated first with LDL for 4 h, and total RNA was extracted at different time intervals after addition of actinomycin D.

 
3.4. Phosphorylation of p38 MAPK is responsible for LDL-stimulated IL-8 induction in hAoSMCs
To investigate the responsible MAPK for the LDL response, the activation of the MAPK members was examined by Western analysis. Treatment of hAoSMCs with LDL induced a time-dependent increase in the phosphorylation of both p38 and Erk 1/2 MAPKs (Fig. 3A). The involvement of JNK was also assessed, but no relationship was detected (data not shown). The maximum phosphorylation of both p38 and Erk 1/2 MAPKs was reached at 5 min after which, p38 and Erk 1/2 phosphorylation both declined slowly and returned to the basal level by 30 and 240 min. To determine which MAPK is responsible for the LDL response, the effects of specific inhibitors of MEK 1/2 (PD98059) and p38 phosphorylation (SB203580) was next assessed. As shown in Fig. 3B, pretreatment of hAoSMCs with SB203580 completely inhibited both p38 phosphorylation (upper panel) and IL-8 induction (lower panel), whereas PD98059 had no detectable effects. These results suggest that the activation of p38 MAPK, but not Erk 1/2 MAPK, is critical for an LDL response.


Figure 3
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  		Fig. 3 Activation of p38 MAPK is essential for LDL-stimulated IL-8 induction. (A) The phosphorylation of both p38 and Erk 1/2 MAPKs was measured by Western blot analysis at the indicated times after treatment of hAoSMCs with LDL (100 µg/ml). (B) hAoSMCs were pretreated with SB203580 (10 µM) or PD98059 (20 µM) for 15 min. Phosphorylation of the MAPK was then measured by Western analysis 5 min after exposure to LDL (upper), whereas IL-8 was quantified by ELISA after a 24-h incubation in the presence or absence of LDL (lower). The {alpha}-actin was used as a normalization control. The MAPK phosphorylation figure represents a typical example of three independent experiments. n=12, *P<0.01 versus control, #P<0.01 versus LDL.

 
3.5 H2O2 participates in the signaling of IL-8 induction by LDL
To determine whether ROS are related to LDL-induced IL-8 production by hAoSMCs, and which, if any, type of ROS is involved, the effects of DPI (a flavin-containing oxidase inhibitor), NAC (a general ROS scavenger), or SOD (an O2– scavenger), DMSO (an OH scavenger), or catalase (an H2O2 scavenger) were assessed. As shown in Fig. 4, LDL-induced IL-8 production was not influenced by pretreatment with DMSO and only mildly affected by pretreatment with DPI, NAC, or SOD. However, LDL effects were severely affected by pretreatment with catalase, but not by heat-inactivated catalase (boiled for 5 min), demonstrating that a nonspecific property of the protein is not causing the inhibition of the LDL response. These results suggest that H2O2, but not O2– and OH, is necessary for the LDL response and that a flavin-containing oxidase, in part, plays a role in the generation of H2O2.


Figure 4
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  		Fig. 4 Participation of H2O2 in the LDL signaling. The indicated ROS-inhibitors were pretreated with hAoSMCs, and their inhibitory effects on IL-8 induction were analyzed by ELISA analysis after an additional 24-h incubation in the presence of LDL (100 µg/ml). The concentrations of inhibitors and treatment time were as follows: DPI (10 µM, 2 h); SOD (500 U/ml, 24 h); NAC (20 mM, 2 h); DMSO (1%, 2 h); catalase (CAT, 500 U/ml, 24 h); heat-inactivated catalase (HI-CAT, 500 U/ml, 24 h). "Q" indicates quiescent cells in the maintenance medium without LDL treatment. n=6–12, *P<0.01 versus control, #P<0.05 versus LDL.

 
3.6. LDL induces activation of AP-1 but not NF-{kappa}B
To determine which transcription factors are responsible for the LDL-induced IL-8 production, the DNA binding activities of AP-1 and NF-{kappa}B were assessed by EMSA. As shown in Fig. 5A, a nuclear extract of LDL-treated hAoSMCs caused a time-dependant increase in AP-1 binding activities, which were comparable to those of TNF-{alpha}-treated cells, used as a positive control. In contrast, LDL had no influence on the binding activities of NF-{kappa}B (Fig. 5B). To determine whether LDL increases transcriptional activities of AP-1 or NF-{kappa}B, hAoSMCs were transiently transfected with AP-1 or NF-{kappa}B promoter linked to a luciferase reporter (Fig. 5C). LDL stimulation of hAoSMCs resulted in a 3.5-fold increase in AP-1 transcriptional activities. In contrast, LDL treatment failed to enhance NF-{kappa}B transcriptional activities. These results suggest that AP-1 activation, but not NF-{kappa}B activation, is necessary for an LDL response.


Figure 5
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  		Fig. 5 LDL induces the activation of AP-1 but not NF-{kappa}B. (A) The increase in AP-1 binding activities in response to LDL (100 µg/ml) was measured by EMSA at the indicated times. The arrows indicate the positions of AP-1 specific complexes. Representative example of three independent experiments. (B) The change in NF-{kappa}B binding activities was measured after a 4-h incubation in the presence or absence of LDL (100 µg/ml). The right-open bracket "{lfloor}" indicates the positions of NF-{kappa}B specific complexes. TNF-{alpha} (10 ng/ml, 1 h) was used as a positive control. For the competition assay, 100 x molar excess of the cold AP-1 or NF-{kappa}B probe was added to the EMSA binding mixture. Representative example of four independent experiments. (C) Cells were transiently co-transfected with plasmids expressing the indicated luciferase cis-reporters and β-galactosidase. Expressed luciferase activities were measured after a 6-h exposure to LDL (100 µg/ml) or TNF-{alpha} (10 ng/ml). n=9, *P<0.05 versus control.

 
3.7. The participation of NF-{kappa}B is necessary for the LDL response
The above results suggest that AP-1 activation, but not NF-{kappa}B activation, is necessary for the LDL response. However, these findings do not necessarily mean that the participation of NF-{kappa}B is not required for the LDL response since the concurrent cooperation of AP-1 and NF-{kappa}B is most common for the transactivation of the IL-8 gene [11,21,26–28]. Therefore, the effects of NF-{kappa}B inhibitors (CAPE and PTL) on LDL-stimulated IL-8 induction were assessed to determine whether the participation of NF-{kappa}B is essential [29–32]. CAPE pretreatment severely reduced the effects of LDL in a dose-dependent manner (Fig. 6A) and PTL pretreatment also showed similar effects. These results indicate that the participation of NF-{kappa}B is also essential.


Figure 6
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  		Fig. 6 Participation of NF-{kappa}B in the LDL signaling. The effects of the NF-{kappa}B inhibitors (PTL, 1 µg/ml, 15 min; CAPE 0, 4, and 15 µM, 15 min) on the LDL-induced IL-8 were assessed after a 24-h incubation in the presence or absence of LDL (100 µg/ml) by ELISA (A and B). n=9, *P<0.01 versus control, #P<0.05 versus LDL.

 
3.8 LDL induces activation of AP-1 via p38 and H2O2 with a concomitant increase in c-JUN and c-fos
To determine the relationship of p38 MAPK and AP-1, the effects of SB203580 on AP-1 activity was assessed. As shown in Fig. 7A, pretreatment of hAoSMCs with SB203580 dramatically blocked LDL-induced AP-1 binding activity, indicating that the activation of p38 MAPK is a prerequisite for the increase in AP-1 activities. In addition, the relationship of H2O2 with AP-1 was assessed. As shown in Fig. 7B, treatment of hAoSMCs with exogenous H2O2 caused a time-dependent increase in AP-1 binding activities, which were comparable to those of LDL-treated cells.


Figure 7
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  		Fig. 7 AP-1 activation via p38 and H2O2 with an increase in c-fos and c-JUN. (A) The inhibitory effects of SB203580 (the p38 inhibitor, 10 µM, 15 min) on AP-1 activation was assessed after a 4-h incubation in the presence or absence of LDL (100 µg/ml). For the competition assay, 100 x molar excess of the cold AP-1 probe was added to the EMSA binding mixture. (B) The stimulatory effects of exogenous H2O2 (10 µM) on AP-1 activation were assessed at the indicated times and compared with those of LDL (100 µg/ml, 4 h). (C) The induced levels of c-fos (upper) and c-JUN (lower) evoked by LDL (100 µg/ml) were assessed at the indicated times by Northern and Western blot analyses, respectively. The EMSA and Northern figures represent typical examples of each three independent experiments.

 
The effects of LDL on the upregulation of AP-1 subunits were next assessed by Northern or Western blot analysis because exogenous H2O2 has been reported to increase expression of c-JUN [33] and c-fos [34]. As shown in Fig. 7C, induction of the c-fos transcript appeared at 20 and 30 min in response to LDL stimulation and returned to basal levels within 120 min, whereas c-Jun protein levels increased steadily over a 24-h time period. The results indicate that the activation of AP-1 evoked by LDL is, at least in part, mediated by de novo c-Fos synthesis. However, it is ambiguous whether an increase in c-JUN is involved in the activation of AP-1, because there is a time difference between the activation of AP-1 and an increase in c-JUN after LDL stimulation (2 and 12 h, respectively). Thus, LDL stimulation elicits H2O2 generation as a major second messenger, which in turn activates AP-1 with a concomitant increase in c-JUN and c-fos via the phosphorylation of p38 MAPK [25].


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Vascular researches have been focused on elucidating the roles of endothelial and monocytic cells. In contrast, the research area of SMCs has been restricted to vascular homeostasis, such as vasoconstriction and proliferation and their roles in vascular dysfunction have been underestimated even though SMCs comprise a major portion of blood vessel cells. As far as atherosclerosis is concerned, ox-LDL has been highlighted since it is capable of recruiting monocytes. In this context, this study is the first to show that human SMCs can induce IL-8 expression in response to n-LDL. The phenomenon was observed in the course of subtraction experiments between quiescent and LDL-treated hAoSMCs. The finding was unexpected because LDL is not able to induce IL-8 in endothelial and monocytic cells, whereas ox-LDL is able to upregulate IL-8 production in all the blood cell types.

Despite the variety of sources of IL-8 induction, it is well known that ROS play key roles in the signaling of IL-8 production [6,35]. The data herein also demonstrate that H2O2 generated by LDL is mainly involved in IL-8 induction in hAoSMCs. The responsible MAPK for IL-8 production appears to be dependent on the types of cells and the stimulus employed. Concerning the LDL effects in hAoSMCs, p38 MAPK, but not Erk 1/2 MAPK is essential (Fig. 3). The present results are consistent with the case of IL-1β-induced IL-8 expression by hVSMCs [21] but inconsistent with the nickel-induced IL-8 expression by epithelial cells [24].

Although coactivation of both AP-1 and NF-{kappa}B is common for transcriptional regulation of IL-8 [8,11], only activation of AP-1 appears to be necessary for the LDL-stimulated IL-8 induction (Fig. 5). Consistent with this finding, our unpublished data showed that fenofibrate (a PPAR{alpha} agonist) pretreatment completely abrogates the LDL effects through a complete deactivation in AP-1 activities with no change in NF-{kappa}B activities. The results point to the physiological significance of AP-1 as a potential link between LDL and PPAR{alpha} modulations in SMCs [36]. The suppression of AP-1 has been reported to attenuate heart diseases in rats [37] and restenosis in human hyperlipidemic condition [38]. Moreover, the increased AP-1 binding activity was accompanied by an increase in c-fos, which is reported to contribute to a high prevalence of atherosclerosis [39]. In contrast, TNF-{alpha} appears to differentially regulate the transcription of IL-8 from LDL, since TNF-{alpha} stimulation results in the activation of both AP-1 and NF-{kappa}B.

The amounts of IL-8 produced in response to LDL are almost same as those produced by HUVECs in response to TNF-{alpha} (14,035 pg/104 cells versus 13,757 pg/104 cells) (Table 1). Taking into account the fact that VSMCs comprise a major portion of blood vessel cells, the total amounts of IL-8 produced by hAoSMCs in response to n-LDL would be much higher than those produced by HUVECs. Moreover, the IL-8 secreted by hAoSMCs can exert its autocrine effects on hAoSMC proliferation through its cognate receptors CXCR1 and CXCR2 [25] as like the case of HCSMCs [5]. This autocrine effects by IL-8 could be more dramatic when HCSMCs are exposed to high concentration of LDL after balloon injury, since HCSMCs show a prominent response to LDL from the undetectable basal level of IL-8 to 1411 pg/104 (Table 1). Additional studies are required to define the growth stimulatory effects of HCSMCs exerted by bFGF, PDGF-BB, and IL-8 (evoked by LDL) to elucidate which is the major cause of intimal hyperplasia after balloon injury.

In conclusion, we show for the first time that LDL stimulates SMCs to induce IL-8 production in dose- and time-dependent manners at the transcription level and that the LDL signaling is conveyed via the generation of H2O2, the phosphorylation of p38 MAPK, the activation of AP-1, and the participation of NF-{kappa}B. These findings suggest that hVSMCs play important roles in the genesis and progression of atherogenesis via the production of IL-8 in hyperlipidemic conditions. Therefore, our results contribute to a better understanding of the possible role of hVSMCs in atherosclerosis under the pathophysiological conditions of hypercholesterolemia.


   Acknowledgements
 
This work was supported by the Molecular and Cellular BioDiscovery Research Program and 21st Century Frontier R&D Program from the Ministry of Science and Technology of Korea and by Korea Research Institute of Bioscience and Biotechnology.

We are grateful to Drs. Jeong T.S., Park S.K., and Kwak J.W. for their helpful advice on the preparation and quality maintenance of n-LDL.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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