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Am J Physiol Heart Circ Physiol 294: H750-H763, 2008. First published November 30, 2007; doi:10.1152/ajpheart.00161.2007
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Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Tomoko Ichiki,1 Michihisa Jougasaki,1 Manabu Setoguchi,1 Junichi Imamura,1 Hitoshi Nakashima,1 Tatsuru Matsuoka,1 Masahiro Sonoda,1 Kazuhiko Nakamura,1 Shinichi Minagoe,1 and Chuwa Tei2

1Institute for Clinical Research, National Hospital Organization Kagoshima Medical Center, and 2Department of Cardiovascular, Respiratory, and Metabolic Medicine, Graduate School of Medicine, Kagoshima University, Kagoshima, Japan

Submitted 8 February 2007 ; accepted in final form 28 November 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-{kappa}B DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-{kappa}B pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.

atherosclerosis; vascular inflammation

A CARDIAC HYPERTROPHIC FACTOR, cardiotrophin-1 (CT-1), was uniquely discovered in mouse embryoid bodies differentiated from embryonic stem cells via the expression cloning technique that is used to monitor myocyte hypertrophic activity (24). CT-1 is a 21.5-kDa protein member of the IL-6 cytokine family and one of the endogenous ligands for glycoprotein 130 (gp130) (25). CT-1 increases cardiac myocyte length but has little or no effect on width, promotes cardiac myocyte survival, and protects human myocardium against ischemic injury (11, 26). CT-1-mediated cardiac myocyte hypertrophy resembles volume-overload hypertrophy in human congestive heart failure (42). CT-1 is also associated with the pathophysiology of heart diseases, including hypertension (15), myocardial infarction (9), valvular heart disease (34), and congestive heart failure (17, 18). CT-1 exerts its biological actions through its receptor complex, gp130 and leukemia inhibitory factor receptor (LIFR). Heterodimerization of gp130 and LIFR activates signal transducing pathways, such as Ras-dependent MAP kinase and Janus kinase (Jak)-signal transducers and activators of transcription (STAT) cascade (19, 20, 44). In addition to the MAP kinase and Jak-STAT signaling system, CT-1 activates phosphatidylinositol 3-kinase (PI3-kinase) in cardiac myocytes (21) and enhances transcription factor NF-{kappa}B DNA-binding activities (5, 10).

Monocyte-endothelial cell interactions are thought to be critical for the initiation and progression of various inflammatory vascular diseases and atherosclerosis. An early event in the development of atherosclerosis is recruitment of monocytes into the subendothelial regions of the artery, which ultimately leads to formation of foam cells (6, 29). Cell adhesion molecule is inducible on the vascular endothelial cell surface and supports adhesion of various leukocytes, including monocytes, to the vascular endothelium. Intercellular adhesion molecule-1 (ICAM-1) is expressed in human atherosclerotic plaques by an immunohistochemical method, and ICAM-1 may be involved in mediating monocyte adhesion to the vascular endothelium in the early step of atherosclerosis (4, 27, 28). Another important proatherogenic molecule is monocyte chemoattractant protein-1 (MCP-1), which recruits monocytes, precursors of foam cells, into the arterial wall (30). In mouse models of atherosclerosis, deficiency of MCP-1 or its receptor CCR2 leads to the reduction of atherosclerotic lesion size, and overexpression of MCP-1 accelerates progression of atherosclerosis (1, 3, 12, 14), suggesting that MCP-1 plays an important pathophysiological role in atherosclerosis.

In a previous report, we showed that CT-1 was highly expressed in the vascular endothelial cells (16); however, the physiological and pathophysiological role of CT-1 in the vascular endothelium remains unknown. Specifically, the role of CT-1 in the pathogenesis of atherosclerosis and the cross talk between CT-1 and ICAM-1/MCP-1 have not been clarified. The present study was designed to investigate the localization of CT-1 in atherosclerotic lesions obtained from carotid endarterectomy (CEA) and to examine the actions of CT-1 on the monocyte-endothelial cell interaction, as well as on the gene and protein expressions of ICAM-1 and MCP-1, in human aortic endothelial cells (HAECs). We further clarified the mechanisms involved in the induction of ICAM-1 and MCP-1 by CT-1 in HAECs.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Carotid specimens and immunohistochemistry for atherosclerotic plaque. We studied five atherosclerotic lesions obtained from five patients (68.2 ± 2.8 yr old, 4 men and 1 woman) who underwent CEA for the treatment of internal carotid artery stenosis. The patients had no inflammatory or neoplastic diseases and had not been treated with anti-inflammatory drugs. The atherosclerotic plaque obtained from CEA was immediately fixed with 4% formaldehyde, embedded in paraffin, and used for immunohistological staining. In addition, CEA tissues were immediately frozen for Western immunoblot analysis. The indirect immunoperoxidase method was used for immunohistochemical analysis, as described previously (18). The primary antibodies against CT-1, gp130, and LIFR were monoclonal anti-human CT-1 antibody (1:200 dilution; Research Diagnostics, Flanders, NJ), polyclonal anti-human gp130 antibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal anti-human LIFR antibody (1:200 dilution; Santa Cruz Biotechnology), respectively. The primary antibodies, such as monoclonal anti-human von Willebrand factor (vWF) antibody (DakoCytomation, Carpinteria, CA), monoclonal anti-human CD68 antibody (DakoCytomation), and monoclonal anti-human {alpha}-smooth muscle actin antibody (DakoCytomation) were used for identification of endothelial cells, macrophages, and smooth muscle cells, respectively. Adsorption tests were performed to examine the immunohistochemical specificity of the reaction between each antibody and the tissues. The specificity was further confirmed by substitution of nonimmune goat serum (NGS; R & D Systems, Minneapolis, MN) or PBS for primary antibody. This study was approved by the Institutional Review Committee at our hospital, and written informed consent was obtained from each patient before the operation.

Cell culture and reagents. HAECs (Clonetics, San Diego, CA) were seeded in plastic plates precoated with type I collagen (Asahi Techno Glass) and maintained in medium 199 (GIBCO Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FCS (Trace Scientific), 100 µg/ml heparin (ICN Biochemicals, Aurora, OH), 20 µg/ml endothelial cell growth supplement (Wako Pure Chemical), 10 µg/ml human epidermal growth factor (PeproTech, Rocky Hill, NJ), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO Invitrogen). HAECs were cultured at 37°C in 5% CO2-95% air in a humidified atmosphere. At confluence, HAECs appeared as a typical "cobblestone"-patterned monolayer. HAECs at passages 5–8 were used in the present experiments. Human monocytic THP-1 cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 medium (Asahi Techno Glass) containing 10% heat-inactivated FCS and 5 x 10–5 mol/l β-mercaptoethanol (Sigma, St. Louis, MO). Pharmacological inhibitors, such as PD-98059, SB-203580, and LY-294002, were obtained from Wako Pure Chemical, and parthenolide was purchased from MP Biochemicals (Solon, OH). Human CT-1, TNF-{alpha}, and human IL-1β were purchased from PeproTech. No endotoxin was detected in the medium, and CT-1-enhanced gene and protein expressions of ICAM-1 and MCP-1 were not affected by treatment with polymixin B.

Immunocytochemistry for HAECs. Cultured HAECs were immediately fixed with 1% buffered paraformaldehyde (Wako Pure Chemical) for 20 min. The indirect immunoperoxidase method was used for immunocytochemical analysis, as described previously (16). The primary antibodies against ICAM-1 and MCP-1 were monoclonal anti-human ICAM-1 antibody (1:100 dilution; DakoCytomation) and monoclonal anti-human MCP-1 antibody (1:100 dilution; R & D Systems), respectively. Adsorption tests were performed to examine the immunocytochemical specificity of the reaction between each antibody and the cells. The specificity was further confirmed by substitution of NGS or PBS for primary antibody.

Adhesion assay. HAECs were grown to confluence on six-well culture plates precoated with type I collagen, incubated with CT-1 in the presence or absence of monoclonal anti-human ICAM-1 antibody, anti-human gp130 antibody, anti-human LIFR antibody, and pharmacological inhibitors, such as PD-98059, SB-203580, LY-294002, and parthenolide, for 24 h before the adhesion assays, and washed with Hanks' balanced salt solution (HBSS: 2 mmol/l Ca2+, 2 mmol/l Mg2+, and 20 mmol/l HEPES; GIBCO Invitrogen). Human monocytic THP-1 cells (2 x 106) were added to each well containing HAECs. THP-1 cells were allowed to incubate with the endothelial monolayer at room temperature for 30 min on a rocking platform. Each well was turned 90° at 15 min to allow uniform distribution of the THP-1 cells across the endothelial monolayer. Nonadherent mononuclear cells were carefully removed by two washes with HBSS, and the adherent cells were fixed with 2% glutaraldehyde (Wako Pure Chemical) in HBSS. Adherent cells were counted by microscopy. Twenty fields were counted for each well, and the mean number of adhering cells per field was calculated.

Chemotaxis assay. Chemotactic activity was determined using a microchemotaxis chamber with a polyvinylpyrrolidone-free polycarbonate filter (5-µm pore size). HAECs were treated with CT-1 in the presence or absence of anti-human MCP-1 antibody, anti-human gp130 antibody, anti-human LIFR antibody, and pharmacological inhibitors, such as PD-98059, SB-203580, LY-294002, and parthenolide; then the culture medium was removed and transferred to the lower chamber of ChemoTx microplates (Neuro Probe, Gaithersburg, MD). An aliquot of THP-1 monocytic cell suspension (2 x 106 cells/ml) was washed, resuspended in medium 199, and added to the upper compartment, and the cells were allowed to transmigrate for 90 min at 37°C. After migration, the surface of the filter facing the THP-1 cell suspension was scraped off, and those migrating to the lower chamber and adhering to the underside of the filter were fixed, stained with Diff-Quick (American Scientific Products, McGaw Park, IL), and counted (5 fields/filter). The number of cells that migrated to the lower chamber was counted with a hemocytometer.

Total RNA preparation and RNase protection assay. Total RNA was extracted from HAECs with use of the Pure Link Micro-to-Midi total RNA extraction kit (Invitrogen). A biotin-labeled antisense RNA probe cocktail was transcribed from a set of custom-designed cDNA templates (BD Biosciences Pharmingen, San Jose, CA) with use of the MAXIscript in vitro transcription kit (Ambion, Austin, TX). Full-length probe sizes for ICAM-1, MCP-1, and GAPDH were 284, 232, and 124 bp, respectively, and protected fragment sizes were 255, 203, and 96 bp, respectively. The biotin-labeled antisense probes were hybridized to 5 µg of total RNA and subjected to RNase digestion with the RPAIII kit (Ambion). The RNase-protected fragments were purified, resolved on 6% denaturing tribromoethanol-urea polyacrylamide gels (Invitrogen), and transferred to nylon membranes (Invitrogen). The protected fragments were visualized by incubation of the membranes with an alkaline phosphate-streptavidin solution with chemiluminescence reagent (BrightStar BioDetect, Ambion). The intensities of the blots of ICAM-1 and MCP-1 mRNA were quantified using an LAS-3000 Lumino image analyzer (Fuji Film) and normalized to GAPDH mRNA. Yeast RNA served as a negative control.

Western immunoblot analysis. For Western immunoblot analysis, HAECs were serum deprived and stimulated with CT-1 in the presence or absence of pharmacological inhibitors for the indicated time. The cells were washed with cold PBS and immediately harvested in ice-cold cell lysis buffer (Cell Signaling Technology, Beverly, MA) together with 1 mmol/l phenylmethylsulfonyl fluoride (PMSF; Roche Diagnostics) and protease inhibitor cocktail (Complete Mini, Roche Diagnostics). Frozen carotid atherosclerotic plaques were homogenized and immediately harvested in a similar fashion. After centrifugation, the supernatants were recovered, and protein concentration was assessed using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Aliquots of 50 µg of proteins were resuspended in SDS sample buffer [6% SDS, 30% glycerol, 0.03% bromphenol blue, 187.5 mmol/l Tris·HCl (pH 6.8), and 1.25 mol/l DTT], sonicated, boiled for 5 min, and separated by 4–12% NuPAGE Bis-Tris gel (Invitrogen). The proteins were transferred to a polyvinylidene difluoride membrane (Invitrogen) by electrotransfer for 1 h. The membrane was soaked in blocking buffer (5% nonfat dry milk in 0.1% Tween-TBS) and then incubated with the primary phospho-specific antibodies against ERK1/2 MAP kinase, p38 MAP kinase, and Akt (1:1,000 dilution; Cell Signaling Technology) and the primary antibodies against ICAM-1 and MCP-1 (1:200 dilution) in 5% BSA and 0.1% Tween-TBS overnight at 4°C. After the membrane was washed in 0.1% Tween-TBS, it was incubated with alkaline phosphate-conjugated secondary antibodies (Cell Signaling Technology) for 1 h. The protein bands were detected with CDP-Star chemiluminescent substrate (Cell Signaling Technology). The intensities of the blots were quantified using the LAS-3000 Lumino image analyzer. The blots were stripped and reprobed with total antibodies against ERK1/2 MAP kinase, p38 MAP kinase, and Akt (Cell Signaling Technology) or primary antibody against β-actin (Santa Cruz Biotechnology).

Detection of ICAM-1 protein expression in HAECs. Protein expressions of ICAM-1 were determined using a commercially available cell ELISA kit according to the manufacturer's instruction (Protein Detector ELISA kit, KPL, Gaithersburg, MD). Briefly, HAECs were cultured in 96-well microplates precoated with type I collagen. After incubation, HAECs were washed with warm PBS and immediately fixed with 1% buffered paraformaldehyde for 20 min. The monolayers of HAECs were washed with PBS and incubated with BSA blocking solution for 10 min. Then the cells were incubated with monoclonal anti-human ICAM-1 antibody (1:500 dilution) for 1 h. After they were washed, HAECs were incubated with secondary antibody solution for 1 h. Color formation with substrate solution was measured at 405 nm with a microplate reader (model 680, Bio-Rad, Hercules, CA).

Measurement of MCP-1 protein release from HAECs. Protein concentrations of MCP-1 were determined using a commercially available ELISA kit (Quantikine, R & D Systems) according to the manufacturer's instruction. Briefly, after incubation, the culture medium was removed. Samples and standards were added to each well of the microtiter plate, which was precoated with anti-human MCP-1 monoclonal antibody, and incubated for 2 h. Each well was washed with washing buffer and incubated with the enzyme-linked polyclonal antibody specific for human MCP-1 for 1 h. The wells were washed to remove unbound antibody-enzyme reagent, and substrate solution was added to each well. After incubation for 20 min at room temperature, the enzyme reaction was stopped. MCP-1 concentration was determined by comparison of the optical density results with the standard curves. Intra- and interassay variations were 5% and 6%, respectively.

Preparation of nuclear extracts and EMSA. HAECs were washed with cold PBS and scraped, and cell pellets were collected by centrifugation. A protein extraction kit (NucBuster, EMD Biosciences) was used to extract nuclear protein from HAECs. Protein concentrations were assessed by a bicinchoninic acid protein assay kit. EMSA was performed using an EMSA gel-shift kit (Panomics, Redwood, CA) by incubation of a biotin-labeled NF-{kappa}B consensus probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') with a nuclear extract. Briefly, nuclear extracts (5 µg) were incubated with 10 ng of the biotin-labeled probe at room temperature for 30 min. The protein-DNA complexes were separated by electrophoresis on a 6% DNA retardation gel (Invitrogen) and electronically transferred to a nylon membrane. For chemiluminescence band detection, the membrane was incubated with an alkaline phosphate-streptavidin solution and chemiluminescence reagent. The intensities of the blots were quantified using the LAS-3000 Lumino image analyzer.

Statistical analysis. Results of quantitative studies are expressed as means ± SE. Each data point represents the average of three to six experiments. Statistical comparisons were performed by analysis of variance for repeated measures followed by Fisher's least significant difference test when appropriate. Comparisons between groups were performed by Student's unpaired t-test. Statistical significance was accepted for P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Immunohistochemical staining for CT-1, gp130, and LIFR in human atherosclerotic tissue obtained from CEA. Figure 1 illustrates typical immunohistochemical staining for CT-1, gp130, and LIFR in CEA tissue. Immunoreactive CT-1 was detected in endothelial cells, macrophages, and smooth muscle cells in the intima and media. Immunoreactivites for gp130 and LIFR were colocalized with CT-1 in endothelial cells, macrophages, and smooth muscle cells in the intima and media. Immunohistochemical localizations of CT-1, gp130, and LIFR in the endothelial cells were judged by comparison with staining for vWF. The specimens treated with preabsorbed antibody, NGS, or PBS, instead of each primary antibody, demonstrated little or no immunoreactivity.


Figure 1
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  		Fig. 1. Immunohistochemical staining for cardiotrophin-1 (CT-1), glycoprotein 130 (gp130), leukemia inhibitory factor receptor (LIFR), and von Willebrand factor (vWF) in human atherosclerotic tissue from carotid endarterectomy (CEA). Left and middle panels: CEA tissue from right internal carotid artery of a 59-yr-old man with transient ischemic attack. Right panels: CEA tissue from right internal carotid artery of a 71-yr-old man with cerebral infarction. Nonimmune goat serum (NGS) served as a negative control. Original magnification: x100 (left panels) and x400 (middle and right panels).

 
Immunocytochemical staining and Western immunoblot analysis for CT-1, gp130, LIFR, ICAM-1, and MCP-1. Positive immunostaining for CT-1, gp130, and LIFR was observed in the cultured HAECs (Fig. 2A). These immunocytochemical findings were supported by the Western immunoblot analysis showing that each band for CT-1, gp130, and LIFR was detected in the protein extracts from HAECs (Fig. 2B). Immunoreactivities for ICAM-1 and MCP-1 were also detected in HAECs (Fig. 2C). Intensities of immunocytochemical staining for ICAM-1 and MCP-1 were increased in cultured HAECs treated with 10–8 mol/l CT-1 for 24 h compared with the untreated cells (Fig. 2C). The cells treated with preabsorbed antibody, NGS, or PBS, instead of each primary antibody, demonstrated little or no immunoreactivity. Western immunoblot analysis revealed that CT-1 significantly enhanced protein expression of ICAM-1 (Fig. 2D) and MCP-1 (Fig. 2E) in HAECs.


Figure 2
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  		Fig. 2. Immunocytochemical staining and Western immunoblot analysis for CT-1, gp130, LIFR, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1). A: immunocytochemical staining for CT-1, gp130, and LIFR in cultured human aortic endothelial cells (HAECs). NGS served as a negative control. Original magnification x400. B: Western immunoblot analysis of protein expressions for CT-1, gp130, and LIFR in HAECs. C: intensities of immunocytochemical staining for ICAM-1 and MCP-1 were increased in cultured HAECs treated with 10–8 mol/l CT-1 for 24 h. NGS served as a negative control. Original magnification x400. D and E: Western immunoblot analysis revealed that protein expressions for ICAM-1 (D) and MCP-1 (E) were increased in cultured HAECs treated with 10–8 mol/l CT-1 for 24 h. Bars represent densitometric analyses of each expression signal after normalization to expression of β-actin and relative to culture medium (CM). Values are means ± SE of 3 independent experiments. *P < 0.05 vs. CM.

 
CT-1-induced THP-1 monocyte adhesion. Adhesion of human monocytic THP-1 cells to HAECs was increased by treatment of HAECs with 10–8 mol/l CT-1 for 24 h compared with untreated cells (Fig. 3A). The enhanced adhesion was reduced to below the basal level by treatment with mouse monoclonal anti-human ICAM-1 antibody, suggesting that ICAM-1 is involved in the mechanism of CT-1-enhanced THP-1 monocyte adhesion. Anti-gp130 antibody, anti-LIFR antibody, ERK1/2 MAP kinase inhibitor (PD-98059), p38 MAP kinase inhibitor (SB-203580), PI3-kinase inhibitor (LY-294002), and NF-{kappa}B inhibitor (parthenolide) also significantly inhibited CT-1-enhanced monocyte adhesion (Fig. 3A).


Figure 3
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  		Fig. 3. CT-1-induced THP-1 monocyte adhesion and migration. A: monocytes were added to HAECs that had been treated with culture medium (CM) and CT-1 (10–8 mol/l) in the presence or absence of anti-ICAM-1 antibody (10 µg/ml), anti-gp130 antibody (1 µg/ml), anti-LIFR antibody (1 µg/ml), ERK1/2 MAP kinase inhibitor [PD-98059 (PD), 30 µmol/l], p38 MAP kinase inhibitor [SB-203580 (SB), 10 µmol/l], phosphatidylinositol 3-kinase (PI3-kinase) inhibitor [LY-294002 (LY), 30 µmol/l], and NF-{kappa}B inhibitor [parthenolide (Par), 10 µmol/l]. Relative adhesion is the ratio of adhering THP-1 cells to HAECs treated with CT-1 to those adhering to untreated cells. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1. B: monocyte chemotaxis was promoted in response to CM treated with CT-1 and preincubation of CM from CT-1-treated HAECs with neutralizing anti-MCP-1 antibody (80 µg/ml), anti-gp130 antibody, anti-LIFR antibody, PD-98059, SB-203580, LY-294002, and parthenolide resulted in an inhibition of chemotaxis. Relative migration is the ratio of migrating THP-1 cells toward CM from HAECs treated with CT-1 to those from untreated cells. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 
CT-1-induced THP-1 monocyte chemotaxis. Chemotaxis assay revealed that culture medium from CT-1-treated HAECs promoted chemotaxis in human monocytic THP-1 cells compared with the culture medium from untreated cells (Fig. 3B). Enhanced migration of THP-1 cells was significantly suppressed by preincubation of the culture medium with anti-human MCP-1 antibody, suggesting the involvement of MCP-1 in CT-1-induced enhancement of THP-1 monocyte migration. Anti-gp130 antibody, anti-LIFR antibody, PD-98059, SB-203580, LY-294002, and parthenolide also significantly reduced CT-1-induced monocyte migration (Fig. 3B).

CT-1 stimulates phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt in HAECs. Western immunoblot analysis revealed that CT-1 stimulated phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt, with a peak at 10 min and a decline to the basal level by 120 min (Fig. 4, A–C). CT-1 dose dependently stimulated phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt (Fig. 4, D–F).


Figure 4
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  		Fig. 4. Western immunoblot analysis showing CT-1-stimulated phosphorylation of ERK1/2 MAP kinase (A and D), p38 MAP kinase (B and E), and Akt (C and F) in HAECs. A–C: HAECs were treated with CT-1 (10–8 mol/l) for 0–120 min. D–F: HAECs were treated with 10–8–10–12 mol/l CT-1 for 10 min. Bars represent results from densitometric analysis of each phosphorylation signal after normalization to total protein and relative to each phosphorylation of 0 min (A–C) or CM (D–F). Values are means ± SE of 3 (A–C) or 6 (D–F) independent experiments. *P < 0.05 vs. 0 min (A–C) or CM (D–F).

 
Effects of pharmacological inhibitors on CT-1-stimulated phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt. To confirm the specificity of the phosphorylation of each kinase, HAECs were pretreated with ERK1/2 MAP kinase inhibitor (PD-98059, 30 µmol/l), p38 MAP kinase inhibitor (SB-203580, 10 µmol/l), or PI3-kinase inhibitor (LY-294002, 30 µmol/l) for 1 h and then incubated with CT-1 (10–8 mol/l) for 10 min. CT-1-induced phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt was suppressed by pretreatment with PD-98059, SB-203580, and LY-294002, respectively (Fig. 5). However, CT-1-induced phosphorylation of p38 MAP kinase was increased by pretreatment with LY-294002 (Fig. 5B).


Figure 5
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  		Fig. 5. Effects of pharmacological inhibitors on CT-1-stimulated phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt. HAECs were pretreated with ERK1/2 MAP kinase inhibitor (PD, 30 µmol/l), p38 MAP kinase inhibitor (SB, 10 µmol/l), and PI3-kinase inhibitor (LY, 30 µmol/l) for 1 h and then incubated with CT-1 (10–8 mol/l) for 10 min. CT-1-induced phosphorylation of ERK1/2 MAP kinase (A), p38 MAP kinase (B), and Akt (C) was suppressed by the pharmacological inhibitors. Results from densitometric analysis are means ± SE of 3 independent experiments. *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 
CT-1 increases gene expression of ICAM-1 and MCP-1 in HAECs. RNase protection assay revealed that CT-1 increased ICAM-1 and MCP-1 mRNA. ICAM-1 mRNA was activated at 1 and 2 h of treatment, and MCP-1 mRNA was increased at 1 h after treatment with 10–8 mol/l CT-1 (Fig. 6, A and C). Treatment with CT-1 dose dependently induced ICAM-1 and MCP-1 mRNA; incubation with CT-1 at 10–9–10–8 mol/l and 10–10–10–8 mol/l significantly increased ICAM-1 and MCP-1 mRNA, respectively (Fig. 6, B and D).


Figure 6
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  		Fig. 6. CT-1-stimulated gene expression of ICAM-1 mRNA and MCP-1 mRNA in HAECs. CT-1 stimulated gene expressions of ICAM-1 mRNA during 1 and 2 h of incubation (A) and MCP-1 mRNA at 1 h of incubation (C). CT-1 stimulated gene expressions of ICAM-1 mRNA at 10–9–10–8 mol/l (B) and MCP-1 mRNA at 10–10–10–8 mol/l (D). Results from densitometric analysis are presented as density of ICAM-1 or MCP-1 mRNA relative to GAPDH mRNA and relative to 0 h (A and C) or CM (B and D). Values are means ± SE of 3 independent experiments. *P < 0.05 vs. 0 h (A and C) or CM (B and D).

 
Effects of PD-98059, SB-203580, and LY-294002 on CT-1-stimulated gene expression of ICAM-1 and MCP-1. HAECs were incubated with pharmacological inhibitors of ERK1/2 MAP kinase (PD-98059, 30 µmol/l), p38 MAP kinase (SB-203580, 10 µmol/l), and PI3-kinase (LY-294002, 30 µmol/l) for 1 h and then treated with CT-1 (10–8 mol/l). RNase protection assay demonstrated that CT-1-induced augmentation of ICAM-1 and MCP-1 gene expression was attenuated in HAECs pretreated with PD-98059, SB-203580, and LY-294002 (Fig. 7).


Figure 7
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  		Fig. 7. Effects of pharmacological inhibitors on CT-1-stimulated gene expression of ICAM-1 and MCP-1 mRNA. CT-1-induced gene expression of ICAM-1 (A) and MCP-1 (B) mRNA was suppressed by the pharmacological inhibitors. HAECs were pretreated with ERK1/2 MAP kinase inhibitor (PD, 30 µmol/l), p38 MAP kinase inhibitor (SB, 10 µmol/l), and PI3-kinase inhibitor (LY, 30 µmol/l) for 1 h and then incubated with CT-1 (10–8 mol/l) for 1 h. Results from densitometric analysis are presented as density of ICAM-1 or MCP-1 mRNA relative to GAPDH mRNA and relative to CM. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 
CT-1 increases ICAM-1 protein expression and MCP-1 protein secretion in HAECs. ICAM-1 protein expression was significantly increased in a time-dependent manner by treatment with 10–8 mol/l CT-1 (Fig. 8A). CT-1 at 10–11–10–8 mol/l increased ICAM-1 protein expression in HAECs (Fig. 8B). On the other hand, HAECs spontaneously secreted MCP-1 protein into the culture medium, and CT-1 stimulated MCP-1 protein secretion from HAECs in a time-dependent manner (Fig. 8C). Incubation of HAECs with 10–9–10–8 mol/l CT-1 increased MCP-1 protein secretion from HAECs (Fig. 8D).


Figure 8
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  		Fig. 8. CT-1-stimulated ICAM-1 protein expression and MCP-1 protein secretion in HAECs. CT-1 stimulated ICAM-1 protein expression (A) and MCP-1 protein secretion (C) in a time-dependent manner in HAECs. Spontaneous secretion of MCP-1 without CT-1 is also shown in C (open bars). CT-1 stimulated ICAM-1 protein expression (B) and MCP-1 secretion (D) in a dose-dependent manner in HAECs. Bars represent expression relative to 0 min (A) or CM (B) in ICAM-1 and protein release per 105 cells in MCP-1 (C and D). Values are means ± SE (n = 6). *P < 0.05 vs. 0 h (A) or CM (B and D). {dagger}P < 0.05 vs. CM at the same time (C).

 
Effects of TNF-{alpha} and IL-1β on ICAM-1 and MCP-1 expression. The effects of CT-1 on ICAM-1 and MCP-1 expression were compared with the effects of other inflammatory cytokines, such as TNF-{alpha} and IL-1β. ICAM-1 (Fig. 9A) and MCP-1 (Fig. 9C) were stimulated at lower concentrations by TNF-{alpha} (10–12 and 10–13 mol/l TNF-{alpha} for ICAM-1 and MCP-1, respectively) than by CT-1 (10–8 mol/l). Similarly, IL-1β induced ICAM-1 (Fig. 9B) and MCP-1 (Fig. 9D) at much lower concentrations (10–13 and 10–14 mol/l IL-1β for ICAM-1 and MCP-1, respectively).


Figure 9
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  		Fig. 9. Effects of TNF-{alpha} and IL-1β on ICAM-1 and MCP-1 expression. HAECs were treated with TNF-{alpha}, IL-1β, and CT-1 (10–8 mol/l) for 24 h (ICAM-1) or 4 h (MCP-1). TNF-{alpha} stimulated ICAM-1 protein expression (A) and MCP-1 protein secretion (C) in a dose-dependent manner in HAECs. IL-1β also stimulated ICAM-1 protein expression (B) and MCP-1 secretion (D) in a dose-dependent manner in HAECs. Results represent amount relative to CM in ICAM-1 (A and C) and amount of protein release per 105 cells in MCP-1 (B and D). Values are means ± SE (n = 6). *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 
Effects of PD-98059, SB-203580, and LY-294002 on CT-1-induced ICAM-1 protein expression and MCP-1 protein secretion. HAECs were incubated for 1 h with pharmacological inhibitors of ERK1/2 MAP kinase (PD-98059, 3–30 µmol/l), p38 MAP kinase (SB-203580, 0.1–10 µmol/l), and PI3-kinase (LY-294002, 3–30 µmol/l) before treatment with CT-1. Pretreatment of HAECs with 3–30 µmol/l PD-98059, 0.1–10 µmol/l SB-203580, and 30 µmol/l LY-294002 inhibited CT-1-stimulated ICAM-1 protein expression in HAECs (Fig. 10, A–C). Similarly, augmented secretion of MCP-1 protein by CT-1 was suppressed by pretreatment of HAECs with 3–30 µmol/l PD-98059, 0.1–10 µmol/l SB-203580, and 3–30 µmol/l LY-294002 in a dose-dependent manner (Fig. 10, D–F).


Figure 10
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  		Fig. 10. Effects of pharmacological inhibitors on CT-1-stimulated ICAM-1 protein expression and MCP-1 protein secretion. CT-1-induced ICAM-1 protein expression (A–C) and MCP-1 protein release (D–F) were suppressed by the pharmacological inhibitors. HAECs were treated with CT-1 (10–8 mol/l) for 24 h (ICAM-1) or 4 h (MCP-1) with or without pretreatment with specific inhibitors [PD-98059 (3–30 µmol/l, A and D), SB-203580 (0.1–10 µmol/l, B and E), and LY-294002 (3–30 µmol/l, C and F)]. Results represent amount relative to CM in ICAM-1 and amount of protein release per 105 cells in MCP-1. Values are means ± SE (n = 6). *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 
NF-{kappa}B DNA-binding activity and effects of pharmacological inhibitor of NF-{kappa}B on gene and protein expressions of ICAM-1 and MCP-1. To further examine the involvement of the NF-{kappa}B pathway, EMSA was performed using nuclear extracts and an oligonucleotide probe containing consensus NF-{kappa}B DNA-binding sequences. EMSA revealed that CT-1 increased the amount of DNA-protein complex with the NF-{kappa}B probe (Fig. 11A). The CT-1-induced increase in the DNA-protein complex was suppressed by the addition of an excess amount of cold probe (Fig. 11A). Additionally, CT-1-induced activations of ICAM-1 and MCP-1 expression were examined in the presence and absence of parthenolide, an inhibitor of the NF-{kappa}B pathway. Parthenolide inhibited CT-1-stimulated ICAM-1 (Fig. 11B) and MCP-1 (Fig. 11C) mRNA. Similarly, pretreatment with 1–10 µmol/l parthenolide inhibited CT-1-enhanced ICAM-1 protein expression (Fig. 11D) and MCP-1 protein secretion (Fig. 11E).


Figure 11
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  		Fig. 11. NF-{kappa}B DNA-binding activity and effects of parthenolide on gene and protein expression of ICAM-1 and MCP-1. A: CT-1 increased NF-{kappa}B DNA-binding activity. HAECs were treated with or without 10–8 mol/l CT-1 for 30 min. Unlabeled probe (cold excess) was used for competition to verify that the bands were NF-{kappa}B specific. B and C: CT-1-induced gene expression of ICAM-1 mRNA (B) and MCP-1 mRNA (C) were suppressed by the NF-{kappa}B inhibitor parthenolide (10 µmol/l). HAECs were pretreated with parthenolide for 1 h and then incubated with 10–8 mol/l CT-1 for 1 h. D and E: CT-1-induced ICAM-1 protein expression (D) and MCP-1 protein secretion (E) were suppressed by parthenolide. HAECs were treated with 10–8 mol/l CT-1 for 24 h (ICAM-1) or 4 h (MCP-1) with or without pretreatment with parthenolide (1, 5, and 10 µmol/l). Results represent amount relative to CM in ICAM-1 and amount of protein release per 105 cells in MCP-1. Values are means ± SE (n = 6). *P < 0.05 vs. CM. {dagger}P < 0.05 vs. CT-1.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
The present study has demonstrated that CT-1, a member of the IL-6 cytokine family, has properties that could be considered proatherogenic. 1) CT-1 and its receptor system, gp130 and LIFR, were colocalized in atherosclerotic human tissue. 2) Immunocytochemical examination demonstrated that CT-1 and its receptor system, as well as ICAM-1 and MCP-1, were present in HAECs and that CT-1 increased the immunoreactive intensity of ICAM-1 and MCP-1 in HAECs. 3) CT-1 enhanced human monocytic THP-1 cell adhesion to HAECs and promoted migration of THP-1 cells. 4) CT-1 stimulated phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and PI3-kinase in HAECs. 5) EMSA revealed that CT-1 increased NF-{kappa}B DNA-binding activity. 6) CT-1 increased gene and protein expression of ICAM-1 and MCP-1, which were blocked by cotreatment with the pharmacological inhibitors of ERK1/2 MAP kinase, p38 MAP kinase, PI3-kinase, and NF-{kappa}B.

Monocyte adhesion to the vascular endothelium and migration into the subendothelial areas of the artery are key initial steps in the development of atherosclerosis (6, 29). It has been reported that ICAM-1 was expressed in human atherosclerotic plaques, suggesting that ICAM-1 plays a role in atherosclerosis (4, 27, 28). On the other hand, MCP-1 is a member of the C-C chemokines, which recruit monocytes, the precursors of foam cells, into the arterial wall (30); therefore, MCP-1 also plays a pathophysiological role in atherosclerosis (1, 3, 12, 14). CT-1, a member of the IL-6 cytokine family, is one of the endogenous ligands for gp130 (24, 25), and IL-6 has been reported to play an important pathophysiological role in atherosclerosis. IL-6 mRNA was highly expressed in human atherosclerotic lesions (32). Recruitment of macrophages and leukocytes was reduced in apolipoprotein E and IL-6 double-knockout compared with apolipoprotein E-knockout mice (31). IL-6 and soluble ICAM-1 were predictors of progressive peripheral atherosclerosis (39). In the present study, CT-1 and its receptor component gp130 and LIFR were colocalized in human atherosclerotic plaque. In addition, ICAM-1 and MCP-1, together with CT-1 and its receptor system, were present in the vascular endothelial cells, and CT-1 stimulated gene expression and protein expression of ICAM-1 and MCP-1. Therefore, it is possible that CT-1 indirectly enhances monocyte adhesion to the vascular endothelium and promotes monocyte migration into the subendothelial area through activation of ICAM-1 and MCP-1. This idea is supported by the observations that CT-1-induced accelerated monocyte adhesion and migration were suppressed by anti-ICAM-1 antibody and anti-MCP-1 antibody, respectively. In addition, CT-1-enhanced monocyte adhesion and migration were also suppressed by anti-gp130 antibody, anti-LIFR antibody, and pharmacological inhibitors, such as PD-98059, SB-203580, LY-294002, and parthenolide. Therefore, the present study raises the possibility that CT-1 could be a novel atherogenic factor through the mechanisms that involve its receptor system, ERK1/2 MAP kinase, p38 MAP kinase, PI3-kinase, and the NF-{kappa}B pathway.

The present study has demonstrated that expressions of ICAM-1 and MCP-1 are upregulated by CT-1 in HAECs. To better understand the pharmacological activities of CT-1, we compared the effect of CT-1 on ICAM-1 and MCP-1 expressions with those of other inflammatory cytokines, such as TNF-{alpha} and IL-1β. The pharmacological actions of CT-1 to increase protein expressions of ICAM-1 and MCP-1 were much less significant than those of TNF-{alpha} and IL-1β. Since CT-1 is considered to act as an autocrine and/or paracrine factor (22), locally induced CT-1 may activate ICAM-1 and MCP-1 in vascular endothelium in an autocrine and/or paracrine manner. The present finding of colocalization of CT-1 with ICAM-1 and MCP-1 supports this idea. On the other hand, previous reports have demonstrated that CT-1 has a cytoprotective effect in cardiac myocytes (5, 33). Freed and co-workers (8) reported that CT-1 has the ability to repair the infarcted heart. Toh and co-workers (36) reported that transplantation of CT-1-expressing skeletal myoblasts could be useful in the treatment of heart failure, because CT-1 has protective effects against ventricular remodeling. These findings suggest that CT-1 may be used in the treatment of human cardiac diseases in the future. General use of CT-1 in the treatment of cardiac diseases must be considered carefully, because CT-1 has extracardiac actions, such as inflammatory and atherogenic properties.

The maximum increases in ICAM-1 and MCP-1 mRNA in HAECs were observed at 1–2 h of treatment and 1 h after treatment with CT-1, respectively. Fritzenwanger and co-workers (10) reported that CT-1-induced augmentation of MCP-1 mRNA reached its peak 6 h after CT-1 treatment in human umbilical vein endothelial cells. Although the reason for the discrepancy in the time course of MCP-1 mRNA upregulation between the present study and the report of Fritzenwanger and co-workers is unknown, it could be due to different cell types, the presence or absence of serum deprivation, or a different assay procedure.

We are interested in the signaling pathways by which CT-1 upregulates gene and protein expressions of ICAM-1 and MCP-1 in HAECs. First, we investigated the involvement of MAP kinase. CT-1-induced upregulations of gene and protein expressions of ICAM-1 and MCP-1 were inhibited by PD-98059 (Fig. 7, A and B, and Fig. 10, A and D), and PD-98059 completely blocked CT-1-stimulated phosphorylation of ERK1/2 MAP kinase (Fig. 5A). These results suggest that CT-1-induced expression of ICAM-1 and MCP-1 involved the ERK1/2 MAP kinase pathway. Similarly, CT-1-stimulated gene and protein expression of ICAM-1 and MCP-1 were inhibited by SB-203580 (Fig. 7, A and B, and Fig. 10, B and E), and SB-203580 attenuated CT-1-stimulated phosphorylation of p38 MAP kinase (Fig. 5B). These phosphorylation data are supported by previous reports that SB-203580 attenuated phosphorylation of p38 MAP kinase (23, 41). However, other investigators reported that SB-203580 inhibits p38 MAP kinase activity by blocking the binding of ATP to the active site of p38 but has no effect on tyrosine phosphorylation of p38 MAP kinase (37, 43). Although the influence of SB-203580 on the phosphorylation of p38 MAP kinase needs further evaluation, our results suggest that CT-1-induced upregulation of ICAM-1 and MCP-1 involves the p38 MAP kinase pathway. Second, we elucidated the role of PI3-kinase and examined the effect of LY-294002 on the gene and protein expression of ICAM-1 and MCP-1 in HAECs. Pretreatment of HAECs with LY-294002 inhibited CT-1-induced gene and protein expressions of ICAM-1 and MCP-1 (Fig. 7, A and B, and Fig. 10, C and F). LY-294002 selectively and completely blocked phosphorylation of Akt in HAECs (Fig. 5C). These results indicate that CT-1-mediated activation of PI3-kinase is required for upregulation of ICAM-1 and MCP-1.

Interestingly, CT-1-mediated phosphorylation of p38 MAP kinase was further stimulated by the PI3-kinase inhibitor LY-294002 (Fig. 5B). This finding suggests that there is cross talk between the p38 MAP kinase pathway and the PI3-kinase pathway and that PI3-kinase may suppress the activity of p38 MAP kinase. Indeed, this idea is supported by a previous report that LY-294002 induces activation of p38 MAP kinase as well as its upstream activators MAP kinase kinases 3 and 6 (13). Thus, if the p38 MAP kinase system alone is the major signaling pathway of ICAM-1 and MCP-1 upregulation, one would expect ICAM-1 and MCP-1 expression levels to be increased upon treatment with LY-294002. However, in the present study, LY-294002 inhibited gene and protein expression of ICAM-1 and MCP-1 (Fig. 7, A and B, and Fig. 10, C and F). These findings suggest that PI3-kinase-inhibiting actions of LY-294002 surpass its p38 MAP kinase-activating effects in inhibiting expression of ICAM-1 and MCP-1. Therefore, further studies using not only the methods of transfection of small interfering RNA or dominant-negative gene, but also overexpression techniques, are needed to elucidate the relationship between the PI3-kinase pathway and the p38 MAP kinase pathway in CT-1-mediated upregulation of ICAM-1 and MCP-1.

In cardiac myocytes, the cytoprotective effect of CT-1 acts through MAP kinase and the PI3-kinase pathway (5, 33), and the hypertrophic effect of CT-1 is mediated by STAT3 and negatively regulated by ERK1/2 MAP kinase (35). CT-1-induced collagen and DNA synthesis of cardiac fibroblasts are mediated through the activation of Jak/STAT, ERK1/2 MAP kinase, PI3-kinase, and tyrosine kinase (9, 38). Although, in human umbilical vein endothelial cells, CT-1 stimulated MCP-1 through the Jak/STAT pathway (10), it is unknown whether the MAP kinase or PI3-kinase pathway is involved. In the present study, HAECs were treated with PD-98059, a specific ERK1/2 MAP kinase inhibitor, SB-203580, a p38 MAP kinase inhibitor, or LY-294002, a PI3-kinase inhibitor, before incubation with CT-1. As shown in Figs. 7 and 10, PD-98059, SB-203580, and LY-294002 blocked gene and protein expression of ICAM-1 and attenuated gene expression and protein secretion of MCP-1 in HAECs, indicating that the ERK1/2 MAP kinase, p38 MAP kinase, and PI3-kinase pathways are required for the induction of ICAM-1 and MCP-1 by CT-1.

Several reports have described CT-1-mediated activation of transcription factors. Craig and co-workers (5) reported that CT-1 had cytoprotective actions through the activating transcription factor NF-{kappa}B in cardiac myocytes. Fritzenwanger and co-workers (10) reported that CT-1 induced MCP-1 synthesis through the activation of NF-{kappa}B in human umbilical vein endothelial cells. Human ICAM-1 gene included binding sites for the transcription factors SP-1, AP-1, and NF-{kappa}B (40), and human MCP-1 gene also included binding sites for the transcription factors SP-1, AP-1, and NF-{kappa}B (2, 7). In the present study, CT-1 enhanced NF-{kappa}B DNA-binding activity, and the NF-{kappa}B inhibitor parthenolide inhibited CT-1-stimulated monocyte adhesion and migration, as well as gene and protein expression of ICAM-1 and MCP-1. Therefore, this study confirmed that CT-1-induced ICAM-1/MCP-1 upregulation is mediated at least by the NF-{kappa}B pathway. Further studies are needed to elucidate CT-1-activated transcriptional factors.

In conclusion, this study demonstrates that CT-1 and its receptor system are colocalized in human atherosclerotic plaques and that CT-1 enhances monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, PI3-kinase, and NF-{kappa}B systems. These findings raise the possibility that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.


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 ABSTRACT
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The present study was supported by a grant from National Hospital Organization Collaborative Clinical Research.


   ACKNOWLEDGMENTS
 
We thank Yoshiko Kojima for secretarial work and Yoko Takenoshita for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Jougasaki, National Hospital Organization Kagoshima Medical Center, 8-1 Shiroyama-cho, Kagoshima 892-0853, Japan (e-mail: michi{at}qjun.hosp.go.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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