Endotoxin Induces Toll-Like Receptor 4 Expression in Vascular Smooth Muscle Cells via NADPH Oxidase Activation and Mitogen-Activated Protein Kinase Signaling Pathways
Objective— Toll-like receptor 4 (TLR4) plays a major role mediating endotoxin-induced cellular inflammation and regulates vascular smooth muscle cell (VSMC) proliferation, which is related to atherogenesis and restenosis. This study was conducted to investigate the mechanisms involved in lipopolysaccharide (LPS)-induced TLR4 expression in VSMCs.
Methods and Results— Stimulation of human aortic smooth muscle cells (HASMCs) with LPS significantly increased TLR4 expression. The increase was regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (including the activation of subunits p47phox and Rac1), which mediates the production of reactive oxygen species and the activation of intracellular mitogen-activated protein kinase signaling pathways. Treatment with polyethylene-glycol-conjugated superoxide dismutase, N-acetylcysteine (NAC), diphenylene iodonium (DPI), or apocynin significantly decreased LPS-induced TLR4 expression. An actinomycin D chase experiment showed that LPS increased the half-life of TLR4 mRNA. Inhibition of NADPH oxidase activity by DPI, apocynin, or NAC significantly decreased TLR4 mRNA stability, as did the knock-down of RAC1 gene expression by RNA interference. We also demonstrated in an animal model that LPS administration led to a significant elevation of balloon-injury–induced neointimal hyperplasia, and of TLR4 expression, in rabbit aorta.
Conclusion— These findings suggest that NADPH oxidase activation, mRNA stabilization, and MAPK signaling pathways play critical roles in LPS-enhanced TLR4 expression in HASMCs, which contributes to vascular inflammation and cardiovascular disorders.
Toll-like receptors (TLRs) are type I transmembrane receptors that expressed on the cell membrane after LPS stimulation.1 More than 10 human TLRs have been identified,2 but only the functions of TLR2 and TLR4 have been established.3 TLRs are critical for the induction of downstream signals in atherogenesis during endotoxin-mediated vascular disturbances. Epidemiological research suggests that endotoxins, such as LPS, are strong risk factors for cardiovascular disorders.4,5 Failure of coronary artery bridge graft or restenosis is caused by constitutive chronic infection or inflammation.6 Previous studies have demonstrated that TLR4 is abundantly expressed in failing myocardium,7 and in macrophages infiltrating lipid-rich atherosclerotic lesions.8 Moreover, an association between the functional expression of TLR4 and the subsequent augmentation of intimal hyperplasia has recently been described.9 The first vascular cells that come into contactwith circulating LPS may be endothelial cells; however, vascular smooth muscle cells (VSMCs) may also contact
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circulating LPS, with the loss of the endothelial integrity. TLR4 mediates and regulates LPS-induced proinflammatory activation in VSMCs.10 LPS-induced systemic inflammatory responses could increase neointima formation after balloon injury and stent implantation, with the resulting proliferation of smooth muscle cells playing a key role in atherogenesis.11
TLR4 exists in VSMCs, may play a major role in restenosis, and therefore may make a fundamentally significant contribution to the crucial pathophysiological relationship between inflammation and cardiovascular disorders.12 TLR4 expression under LPS stimulation is controlled by transcriptional and posttranscriptional mechanisms,13 which could be induced by reactive oxygen species (ROS) in mammalian cells.14 Many studies have shown that the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is associated with atherosclerosis, hypertension, and heart failure.15,16 NADPH oxidase, an important determinant of the redox states of vascular cells,17 is composed of several kinds of components, including Nox1, Nox4, p22phox, p47phox and Rac protein in VSMCs.18 However, the mechanisms by which LPS affects TLR4 expression in VSMCs are still unclear.
The aim of this study was to explore the cellular events and the underlying mechanisms involved in LPS-induced TLR4 expression in VSMCs, and we also examined whether LPS increased neointimal hyperplasia and TLR4 expression in balloon-injured rabbit aorta. Our findings suggest that NADPH oxidase activation, mRNA stabilization, and MAPK signaling pathways play critical roles in LPS-enhanced TLR4 expression in VSMCs, which contributes to vascular inflammation and cardiovascular disorders.
Materials and Methods
Human aortic smooth muscle cells (HASMCs; Cascade Biologics, Portland, Ore) were grown and passaged as described previously.19 Cells were used at passages 3 to 8. Before treatment or stimulation with reagents, the cells were serum starved for 24 hours.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using a TRIzol reagent kit (Invitrogen), according to the manufacturer’s instructions. The detailed Methods are published in the supplemental Methods, available online at http://atvb.ahajournals.org.
Membrane Fraction and Total Cell Lysate Preparation
Membrane fractions and total cell lysate were prepared as described in the supplemental Methods.
Western Blot Analysis
Western blot analysis was used to determine the changes in total and cell-surface levels of TLR4 and NADPH oxidase component p47phox, and the cytosolic activation of p38 MAPK, ERK1/2, and SAPK/JNK in HASMCs stimulated with LPS (please see the supplemental Methods).
NADPH Oxidase Activity Assay
NADPH oxidase activity was determined with superoxide-dependent lucigenin chemiluminescence (please see the supplemental Methods).
Pull-Down Assay for Rac1 Activity
Rac1 activation was measured using a glutathione S-transferase–(p21-activated kinase)–p21 binding domain (GST–[PAK]–PBD) fusion protein, which binds activated Rac1 (please see the supplemental Methods).
Knock-Down Gene Expression With Interference RNA
Intracellular Rac1 expression was knocked down by transfection with interference RNA (siRNA). The detailed method is published in the supplemental Methods.
Actinomycin D Chase Experiments Evaluate mRNA Stability
Actinomycin D (20 μg/mL for 1 hour) was added to HASMCs after treatments under various experimental conditions. Total RNA was extracted at 0, 15, 30, 60, 120, 180, or 240 minutes after the addition of actinomycin D, and quantitative real-time polymerase chain reaction (PCR) was performed. The mRNA decay curves were constructed and half-lives (t1/2s) were calculated from the curves.
Animal Balloon-Injury Experiment
The Zealand white rabbits (purchased from the animal center of National Yang-Ming University, Taipei, Taiwan, ROC) were randomly divided into four groups. Group 1 was the control; group 2 (LPS) received intravenous injections of LPS (110 ng/kg) through the ear vein at the end of weeks 2 and 3; group 3 (endothelium-denudation [ED]) and group 4 (ED+LPS) received the balloon-injury treatment of the abdominal aorta (please see the supplemental Methods) at the end of week 2; group 4 also received intravenous injections of LPS immediately (the end of week 2) and one week (the end of week 3) after the balloon injury. The animals were euthanized at the end of week 5, and the vessels were collected for immunohistochemical or immunofluorescent staining.
Measurement of Erythrocyte Sedimentation Rate and Serum C-Reactive Protein
Erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) levels were analyzed to evaluate the LPS-induced and balloon-injury-induced systemic inflammatory responses (please see the supplemental Methods).
Immunohistochemical staining was performed on serial 5-μm-thick paraffin-embedded sections from rabbit abdominal aortas using anti-TLR4 and anti–α-actin antibodies (please see the supplemental Methods).
Values were expressed as means±SEM. Statistical evaluation was performed using Student t test and one- or two-way ANOVA followed by Dunnett test. A probability value of <0.05 was considered significant.
LPS Induces TLR4 mRNA and Protein Expression in HASMCs
Total RNA was extracted and reverse transcribed for PCR from HASMCs after stimulation with 25 ng/mL LPS for 0.5, 1, 2, or 4 hours. LPS significantly induced TLR4 mRNA expression. The expression of TLR4 mRNA was elevated and reached a maximum at 2 hours (198.6±14.5% of the control) after stimulation with LPS (Figure 1A). The addition of cycloheximide (a protein synthesis inhibitor) or actinomycin D (an RNA polymerase inhibitor) significantly reduced TLR4 expression in HASMCs treated with LPS (Figure 1B and 1C), suggesting that the LPS-induced expression of TLR4 requires de novo RNA and protein synthesis, and that short-half-life proteins may be involved.20
LPS-Induced TLR4 Expression Is Mediated by NADPH Oxidase Activation and ROS Generation
HASMCs were treated with 25 ng/mL LPS for 20, 40, or 60 minutes, and then the membrane fraction was assayed for NADPH oxidase activity. LPS treatment resulted in a time-dependent increase in enzyme activity. Diphenylene iodonium (DPI) was used to determine NADPH oxidase activity (Figure 2A). HASMCs were treated with 25 ng/mL LPS for 10, 20, 40, 60, or 120 minutes. The membrane fraction was prepared for the measurement of p47phox by Western blot and the total cell lysate for the measurement of Rac1 activity by a GST-(PAK)-PBD fusion protein pull-down assay, respectively. LPS treatment rapidly induced p47phox translocation to the cell membrane and the activation of Rac1 in a time-dependent manner (Figure 2B). Activation of these proteins reached a maximum at 60 minutes after LPS stimulation. LPS or H2O2 significantly induced TLR4 mRNA expression, which was significantly blocked by 100 U/mL polyethylene-glycol-conjugated superoxide dismutase (polyethylene glycol (PEG)-SOD, a permeable antioxidant enzyme), 10 mmol/L N-acetylcysteine (NAC, a glutathione precursor), 30 μmol/L DPI, or 100 μmol/L apocynin (a specific NADPH oxidase inhibitor) (Figure 2C).
LPS-Induced TLR4 Expression Is Mediated by Rac1 Activation
HASMCs were pretreated with C3 transferase from Clostridium botulinum (C3 TCB, a Rho A/B/C inhibitor), C difficile toxin B-1470 (CTB-1470, a Rac/cdc42 inhibitor), or Y27632 (a nonspecific Rho protein inhibitor) for 1 hour before LPS treatment and TLR4 mRNA expression was analyzed by real-time PCR. Pretreatment of HASMCs with Y27632 or CTB-1470, but not with C3 TCB, significantly inhibited LPS-induced TLR4 mRNA expression, indicating that Rac/cdc42 but not RhoA/B/C mediates TLR4 mRNA expression (Figure 2D). LPS-induced TLR4 mRNA expression was completely blocked by Rac1, when an RNA silencing technique was used (Figure 2E), but not by the negative control siRNA.
Intracellular MAPK Signaling in LPS-Induced TLR4 Expression
LPS markedly induced the phosphorylation of MAPKs, including p38 MAPK, ERK1/2, and SAPK/JNK (Figure 3A), after exposure to 25 ng/mL LPS for 15 minutes. DPI and apocynin significantly decreased MAPK (p38 MAPK, ERK1/2, and SAPK/JNK) activation in LPS-stimulated HASMCs (Figure 3B), suggesting that NADPH oxidase–derived ROS are involved in the activation of MAPKs.
Real-time PCR demonstrated that LPS-induced TLR4 mRNA expression was reduced by SP600125 (an SAPK/JNK inhibitor) or PD98059 (an ERK1/2 inhibitor), but not by SB203580 (a p38 MAPK inhibitor) (Figure 3C). LPS-induced TLR4 protein expression was significantly reduced by SB203580, SP600125, and PD98059 (Figure 3D and 3E) suggesting that SAPK/JNK and ERK1/2 play more significant roles than that of p38 MAPK in the transcriptional regulatory signaling pathway. Interestingly, total and membrane TLR4 protein expression was inhibited by SB203580, indicating that p38 MAPK might be involved in the posttranscriptional regulation of TLR4.
NADPH Oxidase–Mediated ROS Are Involved in LPS-Enhanced TLR4 mRNA Stability
To determine whether LPS affects the steady-state dynamic balance between the rate of transcription and the message stability of TLR4 mRNA, an actinomycin D chase experiment13 was conducted. The half-life (t1/2) of mRNA, deduced under various conditions according to a specific formula, indicated that LPS stimulation rapidly increased the stability of TLR4 mRNA in HASMCs (LPS group, 320.3±39.1 minutes versus control group, 59.3±7.9 minutes; Figure 4A). Pretreated with 10 mmol/L NAC, 30 μmol/L DPI, 100 μmol/L apocynin, or transfected with Rac1 siRNA significantly decrease the LPS-induced extension of the TLR4 mRNA half-life (122.9±9.5 minutes, 94.3±10.8 minutes, 74.6±10.6 minutes, and 110.2±11.3 minutes, respectively; Figure 4B).
TLR4 Protein Expression in Endothelium-Denuded Abdominal Aortas of Rabbits With Systemic Inflammation
To examine whether LPS administration affects the expression of TLR4 in vivo, New Zealand White rabbits were injected with LPS after endothelial denudation of the abdominal aorta. During the experimental period, ESR and CRP levels in the different groups were measured and the results are shown in supplemental Table I (available online at http://atvb.ahajournals.org). The neointima was markedly thickened in the ED+LPS group (intima/media area ratio, 1.61±0.12) compared with that of the control group (intima/media area ratio, 0.64±0.05), whereas only slightly thickened neointima was observed in the LPS group (intima/media area ratio, 0.92±0.07) and the ED group (ratio of intima/media area, 1.12±0.05; Figure 5). Immunohistochemical staining showed that strong TLR4 staining was observed on the luminal surfaces in the LPS group and on the thickened neointima in the ED+LPS group, as compared with the TLR4 staining in the control and ED groups. TLR4 was predominantly observed in the smooth muscle cells (α-actin) of the neointima in the LPS and ED+LPS groups.
This study revealed that p47phox- and Rac1-dependent NADPH oxidase activation, ROS production, and MAPK signaling pathways play critical roles in LPS-enhanced TLR4 expression and TLR4 mRNA stabilization in HASMCs. We also demonstrated that LPS increased neointimal hyperplasia, as well as TLR4 expression, in balloon-injured rabbit aortas. These data provide evidence for the direct involvement of VSMCs in LPS-mediated inflammatory activation, which may contribute to the progression of cardiovascular disorders.
LPS is considered to be a strong stimulator of the pathogenesis of atherosclerosis.4 There is evidence that low concentrations of LPS induce potent inflammatory activation in intact human blood vessels.5 The present study shows that, like cardiomyocytes and microvascular endothelial cells,7 HASMCs exhibit lower expression of TLR4 under basal conditions, compared with monocytic cells/THP-1 cells (data not shown). LPS treatment upregulated TLR4 expression, and promoted a proinflammatory phenotype in VSMCs, which may potentially play an active role in vascular inflammation.12
NADPH oxidase is composed of gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac proteins.21 Several homologues of p47phox (such as NoxO1), p67phox (such as NoxA1), and gp91phox (Nox1–5 and Duox1–2) have recently been identified.22–24 ROS generated by NADPH oxidase are considered to be second messengers in the inflammatory response.25,26 NADPH oxidase is predominantly composed of p22phox, p47phox, and Rac1 in VSMCs.18 The activation of NADPH oxidase mediates the proliferation of VSMCs and the inhibition of NADPH oxidase attenuates ROS production in response to platelet-derived growth factor (PDGF).27 The expression and activity of membrane-bound Rac1 increase in infected VSMCs, and Rac1-mediated functional NADPH oxidase is required for vascular inflammation.28 In the present study, we firstly demonstrated that LPS increases TLR4 expression via the translocation of p47phox and the activation of Rac1 and NADPH oxidase in HASMCs. We are aware that p47phox and Rac1 may be important modulators of LPS-induced TLR4 expression in HASMCs, although how the components of activated NADPH oxidase interact directly with TLR4 is far from clear.
MAPK signaling pathways are associated with the vascular inflammation that is modulated by ROS.18 Activation of MAPK proteins, including p38 MAPK, JNK/SAPK, and ERK1/2, is critical in the cellular responses associated with inflammatory stimuli (such as LPS) and oxidative stress.29 In vascular cells, H2O2 activates p38 MAPK, JNK/SAPK, and ERK1/2.30,31 DPI, NAC, p22phox siRNA, or catalase may inhibit the activation of p38 MAPK and JNK/SAPK mediated by Rac1-dependent H2O2 production.30,32,33 Furthermore, H2O2 and shear stress also activate ERK1/2, and antioxidants and mutation of the Rac1 gene inhibit ROS-induced ERK1/2 phosphorylation.34 According to this evidence, p38 MAPK, JNK/SAPK, and ERK1/2 are redox sensitive and contribute to inflammation, suggesting that these signaling pathways might also be redox-sensitive targets.18 ROS mediate the activation of inflammatory signaling and promote the expression of gene products that play critical roles in vascular diseases. Clarification of these mechanisms will allow us to develop appropriate therapeutic strategies for redox-associated vascular disturbances.
Actinomycin D (a transcription inhibitor) prevents the transcription of new mRNA. Cycloheximide is commonly assumed to inhibit cytoplasmic protein synthesis in eukaryotes. Our results show that LPS-induced TLR4 upregulation was blocked by actinomycin D and cycloheximide, suggesting that the regulation of TLR4 expression might be mediated by transcriptional and posttranscriptional mechanisms. Similar to the expression of TLR4 mRNA, the expression of total TLR4 protein was inhibited by an SAPK/JNK inhibitor (SP600125) and decreased to below the baseline value for unstimulated cells. Previous studies have demonstrated that TLR4 mediates the proinflammatory phenotype of VSMCs and that LPS treatment upregulates TLR4 expression, which potentially plays an active role in vascular inflammation.12 In this study, HASMCs exhibited constitutive low-level expression of TLR4 mRNA and protein under basal conditions. These results suggest that SAPK/JNK plays a major role in the signaling pathway of TLR4 mRNA expression in HASMCs. The proximal sequence of the TLR4 gene contains the promoter of activator protein-1 (AP-1),35 and MAPKs are important intracellular signaling regulators that modulate the activation of transcription factors associated with TLR4 expression.12 Therefore, it is reasonable that the SAPK/JNK inhibitor SP600125 diminished TLR4 gene expression in HASMCs. ERK1/2 inhibitors also reduced TLR4 gene expression in HASMCs, suggesting that ERK1/2-related downstream signaling may be involved in TLR4 expression. LPS-induced TLR4 mRNA expression was not reduced by a p38 MAPK inhibitor. However, Western blot analysis showed that total and membrane TLR4 protein expression was significantly inhibited by SB203580 in LPS-stimulated HASMCs. These results suggest that p38 MAPK is involved in LPS-induced TLR4 expression in HASMCs via posttranscriptional mechanisms.
The rabbit abdominal aorta denudation model, with LPS administration, which was modified from a previous report,11 was used in the present study. Rabbits were injected with 110 ng/kg body weight LPS, which represents an endotoxin level of less than 1 ng/mL of plasma. The combined results of ESR and serum CRP measurements are a useful indicator of inflammation. The heart rate, rectal body temperature, and respiratory rate of the rabbits were kept in the normal range after LPS was administered. Elevated CRP and ESR, but the maintenance of normal vital signs, indicated that the dose of LPS was sufficient to produce inflammation in the animals, which remained in a nonseptic state. Recent findings have demonstrated that a repertoire of TLR4 is associated with atherosclerotic lesions and that the expression of TLR4 is upregulated in macrophages and endothelial cells in lesions.36 Functional TLR4 may be present in VSMCs because these cells respond to LPS,10 and an intravenous bolus of LPS increases TLR4 expression.37 In the LPS group, the intensity of TLR4 immunostaining was elevated along the inner wall of the aorta and the slightly thickened intima, suggesting that LPS-induced systemic inflammation deforms the function of the endothelium and provides the inflammatory cells and VSMCs lining the subendothelial space. Several lines of evidence show that TLR4 signaling promotes the release of chemokines and proinflammatory factors,38,39 and that LPS upregulates TLR4 expression, which is important for the subsequent production of cytokines. Moreover, nonspecific systemic stimulation of the innate immune response, which is induced by endotoxin, is associated with intimal hyperplasia in balloon-injured rabbits.11 LPS-induced cytokines in endothelium-denuded-rabbit plasma or HASMC culture medium may also influence TLR4 expression. Further studies are required to clarify the interaction between LPS, cytokine production, and TLR4 expression.
In summary, the present study demonstrates that LPS-enhanced TLR4 expression and mRNA stabilization in HASMCs is mediated by NADPH oxidase–related ROS production and MAPK signaling pathways in vitro. We also showed that LPS enhances neointima formation, as well as TLR4 expression, in balloon-injured vessels in vivo. Our findings suggest that endotoxemia is associated with cardiovascular diseases and that therapy involving antiinflammatory agents is a promising way of preventing atherosclerosis and restenosis.
Sources of Funding
This work was supported in part by the C. Y. Foundation for Advancement of Education, Sciences and Medicine, National Science Council of Taiwan (NSC 93-314-B-010-004, NSC 94-2314-B-010-052), and Taipei Veterans General Hospital (VGH 94-207, V95CT-099, and V95ER2-001).
C.Y.L., Y.L.C., and S.J.L. contributed equally to this study.
Original received April 24, 2006; final version accepted September 7, 2006.
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