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Atorvastatin suppresses lipopolysaccharide-induced inflammation in human coronary artery endothelial cells

Abstract

The present study was designed to examine the effects of atorvastatin on vascular inflammatory responses in human coronary artery endothelial cells(HCAECs), when challenged by lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) ligand. HCAECs were pretreated with atorvastatin and induced by LPS. The expression of TLR4, interleukin -6(IL-6), monocyte chemoattractant protein 1(MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1(ICAM-1), nuclear factor-κB (NF-κB) and p38 mitogen activated protein kinase(p38 MAPK) were evaluated using Real-time polymerase chain reaction, cytokine ELISA assay and Western blotting. The results showed that pretreatment with atorvastatin down-regulated the expression of TLR4 in LPS-activated HCAECs. Atorvastatin also attenuated the LPS-induced expression of interleukin IL-6 and MCP-1, at both the transcription and translation level in HCAECs. LPS-induced endothelial cell adhesion molecules, ICAM-1 and VCAM-1 expression were also reduced by pretreatment with atorvastatin. Furthermore, atorvastatin efficiently suppressed LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings show that atorvastatin suppresses endothelial cell inflammation, suggesting that atorvastatin may be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.

Keywords:
Atorvastatin; TLR4; Lipopolysaccharide; Endothelial cells; Inflammation

INTRODUCTION

The inflammatory response of vascular endothelial cells plays important roles in the development and progression of atherosclerosis (Kinlay, Ganz, 1997Kinlay S and Ganz P. Role of endothelial dysfunction in coronary artery disease and implications for therapy. Am J Cardiol. 1997;80(9A):11-16.; Simon et al., 1999Simon BC, Noll B and Maisch B. Endothelial dysfunction--assessment of current status and approaches to therapy. Herz. 1999;24(1):62-71.; Akira et al., 2006Akira S, Uematsu S and Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006; 124(4):783-801.). When stimulated by pathogenic mediators, endothelial cells trigger critical inflammatory responses, including enhancement of endothelial permeability, secretion of cytokines/ chemokines and recruitment of circulating leukocytes (Raetz, Whitfield, 2002Raetz CR and Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700.).

Toll-like receptors (TLRs) are type-I transmembrane receptors expressed on the cell membrane. TLR4, the first of the TLRs described, has been the focus of particular interest since its recognition as the receptor for lipopolysaccharide (LPS) (Akira et al., 2001Akira S, Takeda K and Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001;2(8):675-80.). LPS, a major structural portion of the outer membrane of Gram-negative bacteria, is a ligand and potent agonist of TLR4 (Wiedermann et al., 1999Wiedermann CJ, Kiechl S, Dunzendorfer S, Schratzberger P, Egger G, Oberhollenzer F, et al. Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study. J Am Coll Cardiol . 1999;34(7):1975-81.). Activation of the TLR4 signal is related to its downstream release of inflammatory cytokines in patients with acute coronary syndrome (Methe et al., 2005Methe H, Kim JO, Kofler S, Weis M, Nabauer M and Koglin J. Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome. Circulation. 2005;111(20):2654-61.). Endothelial cells, upon LPS stimulation, initiate the over-production of inflammatory cytokines including IL-6, chemokines such as MCP-1, ICAM-1 and VCAM-1 (Yamagami et al., 2003Yamagami H, Yamagami S, Inoki T, Amano S and Miyata K. The effects of proinflammatory cytokines on cytokine-chemokine gene expression profiles in the human corneal endothelium. Invest Ophthalmol Vis Sci. 2003;44(2):514-20.; Dauphinee and Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.). Accordingly, several reports have suggested the activation of TLR4 plays an important role in the development and progression of some inflammatory diseases (Schroder, Schumann, 2005Schroder NW and Schumann RR. Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis. 2005;5(3):156-64.; Gribar et al., 2008Gribar SC, Anand RJ, Sodhi CP and Hackam DJ. The role of epithelial Toll-like receptor signaling in the pathogenesis of intestinal inflammation. J Leukoc Biol. 2008;83(3):493-8.). Thus, TLR4 is an excellent therapeutic target for the treatment of inflammatory disease.

Atorvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA coenzyme A reductase, inhibits the biosynthesis of cholesterol and associated precursors, which are the isoprenoid products of mevalonate (Sinensky et al., 1990Sinensky M, Beck LA, Leonard S and Evans R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J Biol Chem . 1990;265(32):19937-41.; Takemoto, Liao, 2001Takemoto M and Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol . 2001;21(11):1712-9.). Besides its cholesterol lowering effects, atorvastatin has anti-inflammatory and immunomodulatory benefits. Atorvastatin inhibits LPS-mediated activation of human peripheral mononuclear cells and endothelial cells (Rice et al., 2003Rice JB, Stoll LL, Li WG, Denning GM, Weydert J, Charipar E, et al. Low-level endotoxin induces potent inflammatory activation of human blood vessels: inhibition by statins. Arterioscler Thromb Vasc Biol . 2003;23(9):1576-82.) and it suppresses vascular inflammation and stabilizes vulnerable plaques (Pleiner et al., 2004Pleiner J, Schaller G, Mittermayer F, Zorn S, Marsik C, Polterauer S, et al. Simvastatin prevents vascular hyporeactivity during inflammation. Circulation . 2004;110(21):3349-54.). Atorvastatin down-regulates the expression of TLR4 and reduces levels of the pro-inflammatory cytokine, the tumor necrosis factor β (TNF-β) and IL-6 (Weis et al., 2001Weis M, Pehlivanli S, Meiser BM and von Scheidt W. Simvastatin treatment is associated with improvement in coronary endothelial function and decreased cytokine activation in patients after heart transplantation. J Am Coll Cardiol. 2001;38(3):814-8.). HCAECs, an endothelial cell line derived from human coronary artery endothelial cells, are commonly used for in vitro experimental models of vascular endothelial cells (Liu et al., 2003Liu YC, Lo YC, Huang CW and Wu SN. Inhibitory action of ICI-182,780, an estrogen receptor antagonist, on BK(Ca) channel activity in cultured endothelial cells of human coronary artery. Biochem Pharmacol. 2003;66(10):2053-63.; Dela Paz et al., 2017Dela Paz NG, Melchior B and Frangos JA. Shear stress induces Galphaq/11 activation independently of G protein-coupled receptor activation in endothelial cells. Am J Physiol Cell Physiol. 2017;312(4):428-37.). However, little is known about mechanisms of atorvastatin response for these anti-inflammatory effects in HCAECs.

In this study, we investigated the effects of atorvastatin on the expression of LPS-induced inflammatory genes (IL-6 and MCP-1) and adhesion molecules such as ICAM-1 and VCAM-1 in HCAECs. We also analyzed the LPS-induced phosphorylation of the signaling molecules, NF-κB and p38 MAPK.

MATERIAL AND METHODS

Material

Atorvastatin was supplied by Calbiochem, China. Human Coronary Artery Endothelial Cells was supplied by Cell Applications, China. LPS (Escherichia coli 055: B5), protease inhibitor cocktail, hydrocortisone, human fibroblast growth factor, vascular endothelial growth factor, insulin-like growth-factor, ascorbic acid, epidermal growth factor, heparin and other chemicals, unless otherwise stated, were purchased from Sigma, China. Monoclonal antibodies against p38, phosphor-p38, ICAM-1, VCAM-1 and β-actin were purchased from Cell Signaling Technology, USA. Antibodies to TLR4 (M300) and NF-κB p65 were purchased from Santa Cruz Biotechnology, USA. ECL® (enhanced chemiluminescence) anti-rabbit IgG, HRP (horseradish peroxidase)-linked whole antibody and ECL® Plus Western Blotting Detection System was obtained from GE Healthcare, USA.

Cell culture

Human coronary artery endothelial cells were cultured in EGMTM-2 Endothelial BulletKitTM (Lonza, USA) including endothelial cell growth medium (EGM-2) and supplements with EGM-2 (5% fetal bovine serum, hydrocortisone, human fibroblast growth factor, vascular endothelial growth factor, insulin-like growth-factor, ascorbic acid, epidermal growth factor, gentamicin/amphotericin-1000 and heparin). For experiments, 1×106/mL cells were seeded in 500 μl complete medium in 24-well plates. After growing to 80-90% confluence, the medium was changed. LPS was diluted in complete cell culture medium and added to cells. For access the time of LPS treatment, cells were exposed to LPS for 0min, 5min, 15min, 30min and 60min at 100 ng/mL. For access the concentration of LPS treatment, cells were exposed to LPS at 0 ng/ mL, 1 ng/mL, 10 ng/mL,100 ng/mL and 1000 ng/mL for 30min.For access the time of atorvastatin treatment, cells were pre-treated with atorvastatin for 0h, 6h, 12h and 24h at 1µmol/l. Then, cells were exposed to LPS (100 ng/mL) for 2 h. Atorvastatin remained in culture medium for the duration for the experiment.

For access the concentration of atorvastatin treatment, cells were exposed to atorvastatin at 0 µmol/l, 0.01 µmol/l, 0.1µmol/l,1µmol/l and 10µmol/l for 12h. Then, cells were exposed to LPS (100 ng/mL) for 2 h. Atorvastatin remained in culture medium for the duration for the experiment.

For access the function of atorvastatin in inflammatory, cells were pre-treated with atorvastatin at 1µmol/l for 12h. Then, cells were exposed to LPS (100 ng/mL) for 2 h. Atorvastatin remained in culture medium for the duration for the experiment.

All the experiments were performed in triplicate, with standard deviations (SD) for each time point.

Cytokine ELISA

Levels of IL-6 and MCP-1 in cell culture supernatants were quantified by ELISA kits (R&D Systems, USA). Recombinant cytokines were used to construct standard curves. Absorbance of standards and samples was determined spectrophotometrically at 450 nm, using a microplate reader (Bio-Rad, USA). Results were plotted against standard curves. The assays were carried out according to the protocols provided by the manufacturer.

Quantitative real-time PCR

HCAECs were seeded into six-well dishes, with a density of 5 × 104 cells per well. Cells were exposed to LPS or atorvastatin of different concentrations and stimulated for different time periods. Total cellular RNA was isolated using Trizol reagent (Invitrogen, USA). cDNAs were synthesized according to manufacturer’s instructions using the Reverse Transcription kit (Promega, USA). Real-time PCR involved use of a LightCycler (Roche Applied Science, USA), following manufacturer’s instruction. The transcript levels of GAPDH was quantified as an internal RNA control. Quantitative values were obtained from the threshold cycle value (Ct), the initial point when a significant increase of fluorescence was detected. Experiments were performed in triplicate, for each data point. The following primer sets were used to amplify specific cDNA fragments: GAPDH (forward: 5’-GGC TCT CCA GAA CAT CAT CC-3’; reverse:5’-TTT CTA GAC GGC AGG TCA GG-3’); TLR4 (forward:5’-AGG ATG AGG ACT GGG TAA GGA -3’; reverse: 5’-CTG GAT GAA GTG CTG GGA CA -3’); IL-6 (forward: 5’-CAT CCA TCT TTT TCA GCC ATC TTT-3’; reverse: 5’-TGA CAA ACA AAT TCG GTA CAT CCT-3’). MCP-1(forward: 5’-CAG CCA GAT GCA ATC AAT GCC-3’; reverse: 5’-TGG AAT CCT GAA CCC ACT TCT-3’). The abundance of each gene product was calculated by relative quantification, with values for the target genes, normalized with GAPDH.

Western blot analysis

Western blotting was used to detect the expression of TLR4, ICAM-1, VCAM-1, phosphorylated NF-κB p65, total NF-κB, phosphorylated p38 MAPK, total p38 MAPK, and β-actin. After treatment, HCAECs were washed three times with cold PBS, and lysed with lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.9, 1 mM EDTA, 0.1% Igepal, and 1× protease inhibitor cocktail) for 20 min on ice. Whole cell protein lysates were separated on 12% SDS-PAGE gels and transferred onto PVDF membranes (Invitrogen) by electro-blotting for 2 h at 60-75 V. Once dissembled, the membranes were blocked for 1 h with 5% BSA in PBST (PBS containing 0.1% Tween 20) and incubated with the appropriate primary antibodies (ICAM-1 and VCAM-1 antibody were diluted 1:200 in PBST, β-actin 1:1000, and all others 1:500) overnight at 4°C. After washing with PBST, membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibodies (1:5000 dilution in PBST containing 5% BSA) at room temperature for 1 h. Signals were developed using ECL and exposed on X-ray films. The protein mass was compared after quantifying the intensity of protein bands using Quantity one software (Bio-Rad, Hercules, CA). Experiments were repeated three times.

Data analysis

Data were analyzed on SPSS 10.0 for Windows software. All values were expressed as means ± SD. Comparisons between groups involved use of the one-way ANOVA with Student’s t test. A value of P <0.05 was considered statistically significant.

RESULTS

LPS induced TLR4 mRNA and protein expression in HCAECs

LPS has been shown previously to up-regulate TLR4 expression selectively, in vivo and in vitro (Dauphinee, Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.) To define the optimal conditions of LPS stimulation, we examined the effects of LPS on the expression of TLR4 in HCAECs. The results showed that LPS activated TLR4 channels in HCAECs in a concentration-dependent manner (Figure 1. B, D and F). These experiments suggested the suitable concentration of LPS was 100 ng/mL.

FIGURE 1
TLR4 mRNA and protein expression in HCAECs after LPS stimulation.

HCAECs showed low levels of TLR4 mRNA expression in the absence of LPS stimulation. However, after stimulation with LPS (100 ng/mL), TLR4 mRNA expression rapidly increased and reached maximum expression at 30 min (Figure 1. A). In contrast, the expression of TLR4 protein showed a slower increase in expression and reached maximum expression 2 h after stimulation with LPS (100 ng/mL) in HCAECs (Figure 1. C and E).

Atorvastatin decreases LPS-induced expression of TLR4 mRNA in HCAECs

Atorvastatin has been reported to inhibit TLR4 signaling in vivo and in vitro (Methe et al., 2005Methe H, Kim JO, Kofler S, Weis M, Nabauer M and Koglin J. Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome. Circulation. 2005;111(20):2654-61.; Wang et al., 2011Wang Y, Zhang MX, Meng X, Liu FQ, Yu GS, Zhang C, et al. Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells. Am J Physiol Heart Circ Physiol. 2011;300(5):1743-52.; Fang et al., 2014Fang D, Yang S, Quan W, Jia H, Quan Z and Qu Z. Atorvastatin suppresses Toll-like receptor 4 expression and NF-kappaB activation in rabbit atherosclerotic plaques. Eur Rev Med Pharmacol Sci. 2014;18(2):242-6.). To clarify whether atorvastatin had the same characteristics in HCAECs, we examined the inhibitory effect of atorvastatin on TLR4 expression in these cells. Pretreatment with atorvastatin (1 µmol/l) before LPS addition (100 ng/ mL) (atorvastatin+LPS) markedly reduced TLR4 mRNA level induced by LPS. In HCAECs pretreated with atorvastatin (0.01-10.0 µmol/l) for 12 h before LPS addition (100 ng/mL), atorvastatin had a dose-dependent attenuating effect on TLR4 mRNA levels (Figure 2A). The maximal inhibition of TLR4 mRNA expression was obtained after 12 h of exposure to atorvastatin (1µmol/l) (Figure 2, A and B).

FIGURE 2
TLR4 mRNA expression in HCAECs after atorvastatin pretreatment.

Atorvastatin inhibits LPS-induced IL-6 and MCP-1 mRNA expression and protein secretion in HCAECs

To clarify whether atorvastatin inhibit the downstream genes of TLR4 signaling in HCAECs, we examined the inhibitory effects of atorvastatin on the production of inflammatory mediators in HCAECs. For these studies, we used concentrations of 1 μM atorvastatin and pretreatments of 12 h for cell culture experiments. Using qPCR, we determined whether atorvastatin exerted inhibitory effects on the expression of the inflammatory genes, IL-6 and MCP-1 in LPS-induced HCAECs. As shown in Figure 3A and B, incubation of HCAECs with 100 ng/mL LPS for 2 h, markedly increased the mRNA expression of IL-6 and MCP-1. However, the increased IL-6 and MCP-1 mRNA levels were blunted by the additional pretreatment of atorvastatin (p <0.01).

To further confirm the inhibitory effects of atorvastatin on IL-6 and MCP-1 expression in HCAECs, these cells were pretreated with atorvastatin, then LPS was added for 2 h, and media collected for the measurement of cytokine release using ELISA. As shown in Figure 3C and D, LPS (100 ng/mL) treatment dramatically increased the levels of all cytokines, whereas pretreatment with atorvastatin significantly reduced their levels. Atorvastatin pretreatment reduced LPS-induced IL-6 levels in the media from 1380 pg/mL to 830 pg/mL, and MCP-1 levels from 920 pg/mL to 380 pg/mL, respectively.

FIGURE 3
The effects of atorvastatin on LPS-induced IL-6 and MCP-1 expression in HCAECs.

(A) IL-6 mRNA levels in HCAECs were analyzed by real-time PCR after treatment with LPS and atorvastatin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. (B) MCP-1 mRNA levels in HCAECs were analyzed by real-time PCR after treatment with LPS and atorvastatin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. (C) Levels of IL-6 peptides released in the medium were assessed by ELISA after treatment with LPS and atorvastatin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. (D) Levels of MCP-1 peptides released in the medium were assessed by ELISA after treatment with LPS and atorvastatin. Statistical analysis was performed on Ato, LPS and Ato+ LPS versus Con group, respectively. Data are expressed as mean value SE of three independent experiments. *P<0.05 indicates significant differences.

Atorvastatin suppresses LPS-induced ICAM-1 and VCAM-1 expression in HCAECs

ICAM-1 and VCAM-1 are adhesion molecules expressed by endothelial cells in response to inflammatory stimuli, and are responsible for monocyte adhesion (Iiyama et al., 1999Iiyama K, Hajra L, Iiyama M, Li H, DiChiara M, Medoff BD, et al. Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res. 1999;85(2):199-207.; Yoon et al., 2010Yoon JJ, Lee YJ, Kim JS, Kang DG and Lee HS. Protective role of betulinic acid on TNF-alpha-induced cell adhesion molecules in vascular endothelial cells. Biochem Biophys Res Commun. 2010;391(1):96-101.). Theeffects of atorvastatin on the expression of ICAM-1 and VCAM-1 were tested using LPS-stimulated HCAECs by Western blot analysis (Figure 4). The results demonstrated that LPS (100 ng/mL) significantly increased ICAM-1 and VCAM-1 expression in HCAECs by 3.5- and 5.2-fold, respectively (Figure4. A and B). However, after cells were pretreated with atorvastatin (1 μM/mL) for 12 h, ICAM-1 and VCAM-1 levels induced by LPS were markedly reduced by 48.2 and 61.7%, respectively (Figure4 A and B).

FIGURE 4
The effects of atorvastatin on LPS-induced ICAM-1 and VCAM-1 expression in HCAECs.

Atorvastatin inhibits LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs

We next examined the effects of atorvastatin on the NF-κB signaling pathway, which is involved in the regulation of downstream pro-inflammatory gene expression in HCAECs (Dauphinee, Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.). The results in Figure 5A showed that LPS (100 ng/mL) treatment for 2 h induced a rapid increase in p- NF-κB protein levels in HCAECs. Pretreatment with atorvastatin at concentrations of 1 μM, reduced the level of p- NF-κB to 61.5% that of the LPS-only group. Some studies have reported the activation of the phosphatidylinositol p38 MAPK pathway and its contribution to the up-regulation of inflammatory cytokines in activated endothelial cells (Griendling et al., 2000Griendling KK, Sorescu D, Lassegue B and Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol . 2000;20(10):2175-83.; Hsu, Wen, 2002Hsu HY and Wen MH. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J Biol Chem. 2002;277(25):22131-9.). Thus, we also analyzed the effects of atorvastatin on LPS-induced p38 MAPK activation in HCAECs. As shown in Figure 5B, levels of p- p38 MAPK in HCAECs was rapidly increased after LPS exposure for 2h. Atorvastatin pretreatment resulted in the significant inhibition of LPS-induced P38 MAPK phosphorylation, in contrast to control groups.

FIGURE 5
The effects of atorvastatin on the phosphorylation of NF-κB and p38 MAPK, induced by LPS in HCAECs.

DISCUSSION

This study examined the anti-inflammatory effects of atorvastatin on vascular endothelial cells, using the HCAEC cell line model. Atorvastatin and potent inhibitors of cholesterol biosynthesis are widely used in the treatment of hypercholesterolemia and the prevention of atherosclerotic disease (Alagona, 2010Alagona P, Jr. Pitavastatin: evidence for its place in treatment of hypercholesterolemia. Core Evid. 2010;5:91-105.; Sezer et al., 2011Sezer ED, Sozmen EY, Nart D and Onat T. Effect of atorvastatin therapy on oxidant-antioxidant status and atherosclerotic plaque formation. Vasc Health Risk Manag. 2011;7:333-43.). Atorvastatin exerted its anti-inflammatory effects by inhibiting TLR4 signaling, which was originally characterized as a novel anti-sepsis agent, capable of inhibiting inflammatory mediator production (Wang et al., 2011Wang Y, Zhang MX, Meng X, Liu FQ, Yu GS, Zhang C, et al. Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells. Am J Physiol Heart Circ Physiol. 2011;300(5):1743-52.). Atorvastatin inhibits TLR4 signaling by attenuating LPS-induced rapid TLR4 mRNA expression, which led to the inhibition of two distinct downstream signaling pathways: the inhibition of NF-κB translocation and the inactivation ERK phosphorylation (Fang et al., 2014Fang D, Yang S, Quan W, Jia H, Quan Z and Qu Z. Atorvastatin suppresses Toll-like receptor 4 expression and NF-kappaB activation in rabbit atherosclerotic plaques. Eur Rev Med Pharmacol Sci. 2014;18(2):242-6.). Atorvastatin has also been reported to have anti-inflammatory properties in LPS-stimulated monocytes and endothelial cells (Yang et al., 2012Yang SS, Li R, Qu X, Fang W and Quan Z. Atorvastatin decreases Toll-like receptor 4 expression and downstream signaling in human monocytic leukemia cells. Cell Immunol. 2012;279(1):96-102.). In this study, we demonstrated that atorvastatin exerted anti-inflammatory effects in HCAECs.

TLRs are important in the innate immune response, and expression levels of these receptors reflect the sensitivity of immune cells to initiate an immune response (Abreu et al., 2001Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET and Arditi M. Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol. 2001;167(3):1609-16.; Zarember, Godowski, 2002Zarember KA and Godowski PJ. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol . 2002;168(2):554-61.). Several reports have indicated the regulation of TLR expression by various cytokines and molecules and have linked these observations to pathogenetic roles of TLRs in several diseases (Faure et al., 2001Faure E, Thomas L, Xu H, Medvedev A, Equils O and Arditi M. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation. J Immunol . 2001;166(3):2018-24.; Xu et al., 2001Xu XH, Shah PK, Faure E, Equils O, Thomas L, Fishbein MC, et al. Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation . 2001;104(25):3103-8.). Here, we demonstrate atorvastatin exerts direct regulatory effects on TLR4 expression in HCAECs that influence cellular activation. Statins reduce TLR4 surface expression on CD14 monocytes in vivo and ex vivo in a dose dependent fashion, causing down-regulation of IRAK-1 kinase activity and the reduced expression of pro-inflammatory cytokines and B7-1. Endothelial innate immune responses are key events in vascular inflammation and the development of atherosclerosis (Tousoulis et al., 2006Tousoulis D, Antoniades C, Koumallos N and Stefanadis C. Pro-inflammatory cytokines in acute coronary syndromes: from bench to bedside. Cytokine Growth Factor Rev. 2006;17(4): 225-33.). Upon LPS stimulation, TLR4 activates NF-κB and MAPK via a signal transduction process, involving MyD88 and IL-1 receptor-associated kinases, essential for pro-inflammatory proteins (IL-6 and IL-8) (Bjorkbacka, 2006Bjorkbacka H. Multiple roles of Toll-like receptor signaling in atherosclerosis. Curr Opin Lipidol. 2006;17(5):527-33.). In vivo and in vitro studies have also shown that high level LPS exposure triggers the activation of endothelial cells, resulting in the secretion of pro-inflammatory cytokines, which further impact on cardiovascular disease processes (Dauphinee, Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.). Thus, up-regulation of TLR4 expression may play active roles in inflammatory disease.

The vascular inflammatory process is the result of interactions between exogenous stimuli and endothelial cells (Raetz, Whitfield, 2002Raetz CR and Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700.; Bains et al., 2010Bains SK, Foresti R, Howard J, Atwal S, Green CJ and Motterlini R. Human sickle cell blood modulates endothelial heme oxygenase activity: effects on vascular adhesion and reactivity. Arterioscler Thromb Vasc Biol. 2010;30(2):305-12.). LPS, which is one of the strongest stimulators targeting the endothelium, could increase cytokine expression via a TLR4-dependent mechanism (Frost et al., 2002Frost RA, Nystrom GJ and Lang CH. Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2002;283(3):698-709.). It was reported that high level LPS exposure triggers the activation of endothelial cells, resulting in the secretion of pro-inflammatory cytokines, which further impact on cardiovascular disease processes (Dauphinee, Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.). LPS acted as a TLR4-activating promoter, triggering pro-inflammatory responses and enhancing pro-inflammatory cytokine production (Boone et al., 2004Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat Immunol . 2004;5(10):1052-60.). Classic pro-inflammatory cytokine genes implicated in the pathogenesis of inflammatory responses in HCAECs include IL-6, MCP-1, ICAM-1 and VCAM-1. IL-6 is an important cytokine which is expressed mainly within the endothelium of atherosclerotic plaques (Dengler et al., 2000Dengler TJ, Raftery MJ, Werle M, Zimmermann R and Schonrich G. Cytomegalovirus infection of vascular cells induces expression of pro-inflammatory adhesion molecules by paracrine action of secreted interleukin-1beta. Transplantation. 2000;69(6):1160-8.; Dewberry et al., 2000Dewberry R, Holden H, Crossman D and Francis S. Interleukin-1 receptor antagonist expression in human endothelial cells and atherosclerosis. Arterioscler Thromb Vasc Biol . 2000;20(11):2394-400.; Shemesh et al., 2012Shemesh S, Kamari Y, Shaish A, Olteanu S, Kandel-Kfir M, Almog T, et al. Interleukin-1 receptor type-1 in non-hematopoietic cells is the target for the pro-atherogenic effects of interleukin-1 in apoE-deficient mice. Atherosclerosis. 2012;222(2):329-36.). Another important cytokine produced by endothelial cells is MCP-1, which is the key player in monocyte recruitment; high MCP-1 expression levels were determined in cardiovascular disease patients (Libby, 2002Libby P. Inflammation in atherosclerosis. Nature. 2002;420(6917):868-74.; Dauphinee, Karsan, 2006Dauphinee SM and Karsan A. Lipopolysaccharide signaling in endothelial cells. Lab Invest. 2006;86(1):9-22.). ICAM-1 and VCAM-1, produced by endothelial cells, also act as key components in inflammatory responses, are important in the recruitment of leukocytes to sites of inflammation, and are thus implicated in the pathogenesis of vascular inflammatory diseases such as atherosclerosis (Fotis et al., 2012Fotis L, Giannakopoulos D, Stamogiannou L and Xatzipsalti M. Intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 in children. Do they play a role in the progression of atherosclerosis? Hormones (Athens). 2012;11(2):140-6.). In the present study, we demonstrated that stimulation with LPS (100 ng/mL) increased IL-6, MCP-1, ICAM-1 and VCAM-1 expression in HCAECs, while pretreatment with atorvastatin suppressed LPS-induced expression of IL-6, MCP-1, ICAM-1 and VCAM-1 in these cells.

Previous studies have implicated the activation of MAPKs and NF-κB in mediating the effects of specific TLRs in various cell types (Wang et al., 2010Wang L, Zhang X, Liu L, Yang R, Cui L and Li M. Atorvastatin protects rat brains against permanent focal ischemia and downregulates HMGB1, HMGB1 receptors (RAGE and TLR4), NF-kappaB expression. Neurosci Lett. 2010;471(3):152-6.; Wang et al., 2011Wang Y, Zhang MX, Meng X, Liu FQ, Yu GS, Zhang C, et al. Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells. Am J Physiol Heart Circ Physiol. 2011;300(5):1743-52.). To elucidate the signaling pathways in the downstream atorvastatin-induced inhibition of TLR4, we studied the role of MAPK activation by assessing atorvastatin +LPS-induced expression of the phosphorylated forms of p38 MAPK, and NF-κB. Our results showed that LPS up-regulated TLR4 and elicited activation of the MAPK and NF-κB signaling cascade in HCAECs, which was consistent with previous studies (Bachar et al., 2004Bachar O, Adner M, Uddman R and Cardell LO. Toll-like receptor stimulation induces airway hyper-responsiveness to bradykinin, an effect mediated by JNK and NF-kappa B signaling pathways. EurJ Immunol . 2004;34(4): 1196-207.). p38 MARK actives the immune response by phosphorylate several cellular targets, including cytosolic phospholipase A2, the microtubule-associated protein Tau, and the transcription factors ATF-1 and -2, MEF2A, Sap-1, Elk-1, NF-κB, Ets-1, and p53 (Ono, Han, 2000Ono K and Han J. The p38 signal transduction pathway: activation and function. Cell Signal. 2000;12(1):1-13.; Kyriakis, Avruch, 2001Kyriakis JM and Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001;81(2):807-69.) (103,143).Several reports have showed that atorvastatin down-regulated the activity of p38 (Asehnoune et al., 2004Asehnoune K, Strassheim D, Mitra S, Kim JY and Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol . 2004;172(4): 2522-9.; Nishida et al., 2005Nishida S, Matsuoka H, Tsubaki M, Tanimori Y, Yanae M, Fujii Y, et al. Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK. Mol Cell Biochem. 2005;269(1-2):109-14.), whereas other studies have reported sustained p38 activation, even with atorvastatin treatment (Fukao, Koyasu, 2003Fukao T and Koyasu S. PI3K and negative regulation of TLR signaling. Trends Immunol. 2003;24(7):358-63.). Our data demonstrated that atorvastatin exerted an inhibitory effect on LPS-induced phosphorylation of p38. Since the activation of both NF-κB and p38 MAPK can be triggered by TLR4 signaling, we believe that the effects of atorvastatin on NF-κB and p38 MAPK phosphorylation in HCAECs, are mediated by its inhibition of TLR4 signaling.

CONCLUSION

Our finding showed that atorvastatin down-regulated TLR4 mRNA expression in LPS-activated HCAECs; it effectively blocked LPS-induced expression of IL-6, MCP-1, ICAM-1, and VCAM-1 in HCAECs; and it significantly reduced LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings suggest that atorvastatin might be suitable for development as an anti-inflammatory agent to suppress vascular inflammation and to prevent cardiovascular disease.

ACKNOWLEDGMENTS

This work was supported by the Open Projects of Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province (KF201707).

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Publication Dates

  • Publication in this collection
    22 Apr 2022
  • Date of issue
    2022

History

  • Received
    19 Dec 2018
  • Accepted
    01 Feb 2019
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br