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Cardiovascular Research 2007 75(3):555-565; doi:10.1016/j.cardiores.2007.04.027
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Copyright © 2007, European Society of Cardiology

Fc{gamma}RIIa mediates C-reactive protein-induced inflammatory responses of human vascular smooth muscle cells by activating NADPH oxidase 4

Jewon Ryu, Cheol Whan Lee, Jin-Ae Shin, Chan-Sik Park, Jae Joong Kim, Seung-Jung Park and Ki Hoon Han*

Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea

* Corresponding author. Asan Medical Center, 388-1 Pungnap-2 dong Songpa-gu 138-736, Seoul, South Korea. Tel.: +82 2 3010 3150; fax: +82 2 486 5918. steadyhan{at}amc.seoul.kr

Received 13 June 2006; revised 12 April 2007; accepted 20 April 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives We investigated the mechanism by which C-reactive protein (CRP) affects pro-inflammatory activities of vascular smooth muscle cells (VSMCs).

Methods and results RT-PCR, flow cytometry, and immunoblotting assays consistently showed the expression of Fc{gamma}RIIa by cultured VSMCs isolated from human coronary arteries. Immunofluorescence staining of human coronary artery plaque showed the co-localization of Fc{gamma}RIIa with {alpha}-actin(+) VSMCs in atheromatous regions. Confocal microscopic image analysis of H2DCFDA-labeled cells showed that CRP induced intracellular reactive oxygen species (ROS) generation by Fc{gamma}RIIa(+) HEK293T cells. Moreover, CRP time- and dose-dependently generated ROS in VSMCs through Fc{gamma}RIIa activation. VSMCs mainly express NADPH oxidase 4 isoform (Nox4), the suppression of which using a specific siRNA completely abolished CRP-induced ROS generation by VSMCs. The downregulation of p22phox, a component of the active Nox4 complex, by transfecting with specific decoy oligomers and functional blocking of Fc{gamma}RIIa not only inhibited the CRP-induced ROS generation but also reduced the degree of AP-1 and NF-{kappa}B activation, the production of MCP-1, IL-6, and ET-1, and the apoptotic changes of VSMCs in response to CRP.

Conclusions CRP-induced ROS generation by VSMCs, which requires functional activation of Fc{gamma}RIIa and NADPH oxidase 4, orchestrates pro-inflammatory activities of VSMCs and may eventually promote atherogenesis and plaque rupture.

KEYWORDS C-reactive protein; Atherosclerosis; Vascular smooth muscle cells; Receptors (Fc{gamma}RIIa); Cytokines; Apoptosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
C-reactive protein (CRP), a prototype acute phase reactant synthesized primarily in the liver, is generated in response to cytokines, most notably IL-6 and TNF-{alpha}. Elevated serum concentration of high-sensitivity CRP is regarded as an independent risk factor for atherosclerotic disease [1]. In addition to its role as an inflammatory marker, CRP may directly trigger inflammatory responses during the process of atherosclerosis. Numerous studies showed that pentameric form of CRP binds to Fc{gamma} receptors (Fc{gamma}Rs), such as Fc{gamma}RI (CD64) [2] and Fc{gamma}RII (CD32) [3], which triggers pro-inflammatory activities by monocytes/macrophages. Moreover, a recent study reported that Fc{gamma}RI and Fc{gamma}RII are also expressed in human aortic endothelial cells (HAECs) and mediate CRP-induced production of inflammatory molecules [4].

The migration of human vascular smooth muscle cells (VSMCs) into the intima results in plaque growth. VSMCs in atheromatous lesions are not quiescent; rather, they actively proliferate and produce cytokines [5]. Moreover, apoptotic changes of VSMCs weaken the fibrous cap of plaques, which eventually make plaques vulnerable to rupture [6]. Several studies provided evidence that VSMCs are also activated by CRP to generate reactive oxygen species (ROS) [7], produce pro-inflammatory cytokines [8] and develop apoptotic changes [9], however, the detailed mechanism by which CRP modulates such a wide variety of pro-inflammatory properties of VSMCs has not been studied.

The present study identifies the specific CRP receptor expressed on human VSMCs, i.e. Fc{gamma}RIIa, a main isoform of CD32. Moreover, we proved that CRP-induced Fc{gamma}RIIa activation triggers NADPH oxidase 4 activation and subsequent ROS generation, which induces pro-inflammatory activities of human VSMCs and their apoptotic changes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture and transfection
Human vascular smooth muscle cells (VSMCs) isolated from coronary arteries (American Type Culture Collection, Manassas, VA) and the HEK293T cell line (Invitrogen, San Diego, CA) were maintained in F-12 K medium (Kaighn's modification of Ham's F-12 medium) and DMEM with high glucose, respectively, in the presence of 10% FBS with antibiotics, and we used lower passages (less than 6) of cells in the present study. The conformation of human recombinant CRP (Calbiochem, San Diego, CA) was confirmed before adding it to cell cultures [10], and all experiments using CRP were performed in the presence of 25 µg/mL polymyxin B (Sigma Chemical Co. St. Louis, MO), an endotoxin chelator. We confirmed that the tested human recombinant CRP preparation is little contaminated by endotoxin (<1 ng/mL, as determined by a timed-gel endotoxin kit assay (Sigma)) or human immunoglobulin (<0.3 nM) as described [10].

The complete cDNA sequences of human Fc{gamma}RIIa and CRP were subcloned into pcDNA3.1 vector (Invitrogen) after PCR amplification (Table 1). In order to downregulate p22phox, specific double-stranded dumbbell-type phosphothioate-modified decoy oligodinucleotides (ODNs) (5'-GATCTGCCCCATGGTGAGGACC-3') and mismatched ODNs (5'-TAGCATAGCCCTCCGCTGGGG-3') were designed. Complete ring shape formation after annealing (80 °C to 25 °C over 2 h) and ligation step (T4 DNA ligase; Roche Diagnostic GmbH, Annheim Germany, 16 °C for 24 h) was confirmed on a 20% denaturing polyacrylamide gel. pSUPER vectors containing sequences of 19 nucleotides specific to human Nox1, Nox2, or Nox4 for siRNA [11], respectively, were kind gifts from Dr. Bae YS (Ewha Womans University, Seoul, Korea).


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  		Table 1 Primers used for polymerase chain reactions

 
One µg CRP-pcDNA3.1 or Fc{gamma}RIIa-pcDNA3.1, 50 nM decoy ODNs, or 500 ng Nox-pSUPER vectors was incubated for 6 h with 106 VSMCs or HEK293T cells in Opti-MEM (Gibco-BRL, Grand Island, NY) medium in the presence of Superfect (Qiagen, Valencia, CA). We confirmed using a propidium iodide staining that the transfection procedure was not cytotoxic and the viability of control cells at the time of experiments was >90%. Confocal microscopic image analysis of HEK293T cells labeled with a specific antibody showed that transfection efficiency of Fc{gamma}RIIa was >90%.

2.2 Analysis of mRNA expression
The mRNA expression levels of CRP, cytokines (MCP-1, ET-1, and IL-6), GADD153, p22phox, Nox isoforms, and Fc{gamma}Rs were estimated by both semi-quantitative and real-time PCR using specific primer pairs (Table 1). In order to estimate the gene expression, Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) of each gene was obtained by real-time PCR (Roche, Germany) and {Delta}Ct value (the Ct value for cytokine gene expression corrected by the Ct value for the GAPDH housekeeping gene) was calculated.

2.3 Immunoblotting
Proteins of total cell lysate or the cytosolic or mitochondrial fraction were prepared [12], and immunoblotting was performed as described [13] using specific antibodies against Fc{gamma}RIIa (Ancell Corporation, MN), β-actin, or cytochrome c (BD Biosciences). HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:20,000, Jackson ImmunoResearch Lab Inc.) was used as secondary antibody, and the blots were developed using an ECL-kit (Amersham, Piscataway, NJ). Protein was quantified by scanning photodensitometry using a MULTI-IMAGE analysis system and Quantitation software (Bio-Rad Laboratories Inc., Hercules, CA).

2.4 Immunoprecipitation
VSMCs lysed in IMP buffer (50 mM Tris pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 0.3 M NaCl, 1 mg/mL BSA, 2% Igepal CA-630 (v/v), 0.02% NaN3, 1 mM Na3VO4, 10 µg/mL aprotinin, and 10 µg/mL leupeptin) were incubated with 5 µg/mL human recombinant CRP for 2 h and then overnight with 2 µg monoclonal anti-human CRP mouse IgG and 20 µL protein A+G agarose beads (Santa Cruz Biotechnology Inc. CA). Immune complexes bound to beads were washed in IMP-washing buffer (IMP buffer containing 0.1% sodium dodecyl sulfate) and boiled in SDS sample buffer (60 mM Tris, pH 6.8, 2.3% SDS, 10% glycerol, 0.01% bromophenol blue, and 1% 2-mercaptoethanol) for 5 min. Fc{gamma}RIIa protein was detected by immunoblotting as described above.

2.5 Flow cytometry
VSMCs were incubated with Fab fragments of mouse anti-Fc{gamma}RII IgG (1 µg/mL; BD Biosciences) for 30 min at 4 °C and then with FITC-conjugated F (ab')2 fragments of goat anti-mouse IgG (1 µg/mL) for 30 min at 4 °C. As a control, cells were incubated with Fab fragments of nonspecific mouse IgG. Data were analyzed by FACScan using CELLQUEST software (BD Biosciences).

2.6 Detection of Fc{gamma}RIIa in atheroma
Human coronary artery plaque was obtained from a 65-year-old male subject with unstable angina using guided directional coronary atherectomy (DCA) and immersed in isopentan solution at -70 °C and frozen in Jung Tissue Freezing Medium (Leica Instruments GmbH, Nussloch, Germany). Five µm-thick slices mounted onto glass slides were treated with 5% normal swine serum (Vector Laboratories, Burlingame, CA) for 30 min at room temperature to block non-specific binding of antibody, and then incubated with anti-Fc{gamma}RIIa IgG (1:50; Ancell), anti-alpha smooth muscle actin IgG (1:100; LAB VISION Corporation, CA), or anti-CD68 IgG (1:100; Santa Cruz). The antibody bound to tissue was detected by avidin biotin horseradish peroxidase (HRP) and alkaline phosphatase systems, which use 3,3'-diaminobenzidine (DAB) and Vector blue as substrates (Vector Laboratories Burlingame, CA). All sections were lightly counterstained with Mayer's hematoxylin. Alternatively, cell-bound antibodies were detected using Rhodamine (TRITC)-conjugated goat anti-mouse IgG (1:300) and Fluorescein (FITC)-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Lab.) to analyze the co-localization.

2.7 CRP binding assay
Briefly, monolayers of Fc{gamma}RIIa-transfected or mock-transfected HEK293T cells were prepared and treated with 25 µg/mL CRP at room temperature for 30 min. After washing unbound CRP, the monolayers were incubated with mouse monoclonal anti-human CRP IgG (clone CRP-8, Sigma; 1:1000) and FITC-labeled secondary antibody consecutively, and cell-associated fluorescence in cell lysate was measured using a fluoremeter.

2.8 Detection of GTP-bound Rac
The active form of GTP-bound Rac was detected using a pull down assay kit (PIERCE, IL). Briefly, 400 µg protein of cell lysate was incubated with an equal amount of GST-human Pak1-PBD (Rac binding domain of Pak1) to precipitate GTP-bound Rac. The amount of GTP-bound Rac was estimated by immunoblotting as described above, using a mouse anti-Rac monoclonal IgG (PIERCE).

2.9 Confocal microscopic image analysis
Reactive oxygen species (ROS) in VSMCs and HEK293T cells were detected using H2DCFDA (5 µM; Molecular Probes) as described [12]. The spontaneous photo-oxidation of cell-associated H2DCFDA was minimized by collecting the fluorescent image with a single rapid scan. Moreover, the fluorescence reading of each time-point was subtracted from that of unstimulated VSMCs in order to eliminate the effect of the photo-oxidation. For functional inhibition of Fc{gamma}RIIa, cells were incubated with 15 µg/mL anti-human Fc{gamma}RII IgG (Ancell Corp. USA) at 37 °C for 30 min.

2.10 Electrophoretic mobility shift assay (EMSA)
Nuclear protein isolation and EMSA were performed as described [13], using oligonucleotide sequences corresponding to the AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') and NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') binding sites (Santa Cruz). Specificity of each of the developed bands was tested by performing a simultaneous reaction in the presence of a 25-fold excess of unlabeled oligonucleotides.

2.11 Detection of apoptosis
VSMCs undergoing apoptosis were detected by TUNEL staining assay using TdT-FragELTM Kit (Oncogene Research Product, San Diego, CA). Briefly, VSMC monolayers on a 4-well culture plate were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Fixed cells were treated with proteinase K (20 µg/mL, 5 min at room temperature), terminal deoxynucleotidyl transferase (TdT), and streptavidin-horseradish peroxidase (HRP) conjugate and diaminobenzidine, mounted and analyzed by confocal microscopy using a TCS-SP2 system (Leica Microsystems).

2.12 Statistical analysis
SPSS package program was used to perform statistical analysis. Values were expressed as mean±S.D. Differences between two groups were determined by unpaired Student t test. Differences between multiple groups were determined by two-way analysis of variance (ANOVA), where appropriate. Differences were considered significant at p<0.05. Dose responsiveness of CRP on ROS production was analyzed using non-linear regression model, provided by PRISM software 3.0.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Fc{gamma}RIIa is expressed in cultured human VSMCs
RT-PCR showed that human VSMCs isolated from coronary artery express Fc{gamma}RIIa mRNA, meanwhile mRNA expressions of other Fc{gamma}Rs including Fc{gamma}RI, Fc{gamma}RIII were undetectable within 30 cycles of amplification (Fig. 1A). Flow cytometry confirmed surface expression of Fc{gamma}RIIa protein on cultured human VSMCs (Fig. 1B), excluding the possibility that the Fc{gamma}RIIa signal may be derived from contaminating cells. Interestingly, real-time PCR and immunoblotting assay consistently showed that C-reactive protein (CRP) significantly upregulated mRNA and protein levels of Fc{gamma}RIIa by 3 and 4 folds, respectively (p<0.01; Fig. 1A and C). To test whether Fc{gamma}RIIa interacts with CRP, VSMCs treated with CRP were lysed and immunoprecipitated by a specific anti-CRP antibody, and Fc{gamma}RIIa protein was detected using immunoblotting assay. Fc{gamma}RIIa protein was detected in the cell lysate immunoprecipitated by a specific anti-CRP antibody, not by isotype nonspecific IgG (Fig. 1D), confirming Fc{gamma}RIIa expressed in VSMCs recognizes CRP.


Figure 1
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  		Fig. 1 Expression of Fc{gamma}RIIa by human coronary artery VSMCs. Human coronary artery VSMCs were cultured in the presence (‘CRP’; up to 25 µg/mL/24 h) or absence (‘Untreated’) of CRP. (A) mRNA expression levels of Fc{gamma}RI, Fc{gamma}RIIa, and Fc{gamma}RIII were estimated by RT-PCR and real-time PCR. Gel electrophoresis shown represents three independent experiments. Fold changes of Fc{gamma}RIIa mRNA, as determined by real-time PCR, are means±S.D. values. Three independent experiments were performed in duplicate. p<0.01 value was obtained by unpaired Student t test. (B) The surface expression of Fc{gamma}RIIa protein in untreated- (‘Untreated’; dotted line) or CRP-treated VSMCs (‘CRP’; bold line) was measured by performing flow cytometry. Data represent three independent experiments. Non-specific fluorescence was obtained using nonspecific isotype IgG instead of anti-Fc{gamma}RII antibody (‘Cont IgG’; fine line). (C) Fc{gamma}RIIa protein expression level of CRP-treated or CRP-transfected (‘tCRP’) VSMCs were measured by immunoblotting. Bar graphs are mean±S.D. values of band intensities representing five independent experiments. The treatment of VSMCs with CRP significantly increased Fc{gamma}RIIa protein expression level (p<0.01, by two-way ANOVA). NS (statistically non-significant), p<0.001 and p<0.01 in the graph were obtained by unpaired Student t test. (D) The cell lysate of CRP-treated VSMCs was also immunoprecipitated (IP) with anti-CRP antibody or nonspecific isotype IgG, and Fc{gamma}RIIa protein was detected by immunoblotting. Fc{gamma}RIIa protein in total cell lysate without immunoprecipitation was simultaneously detected as a positive control. Immunoblots shown represents three independent experiments.

 
3.2 Fc{gamma}RIIa expression is detected by VSMCs in human atherosclerotic coronary artery
Atheromatous region of the human atherosclerotic coronary artery contained numerous {alpha} smooth muscle actin ({alpha}SMA)(+) VSMCs and CD68(+) macrophages. The area of Fc{gamma}RIIa(+) staining was largely overlapped with both the {alpha}SMA(+) and CD68(+) areas (Fig. 2A). Double staining of specimen with anti-Fc{gamma}RIIa antibody and anti-{alpha}SMA or -CD68 antibody clearly showed the co-localization of Fc{gamma}RIIa with {alpha}SMA(+) VSMCs and CD68(+) macrophages, indicating that both VSMCs and macrophages in atheromatous lesion express Fc{gamma}RIIa (Fig. 2B).


Figure 2
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  		Fig. 2 Localization of Fc{gamma}RIIa in human atherosclerotic plaque. Human atheromatous plaques obtained by directional coronary atherectomy (DCA) were stained with antibodies detecting {alpha} smooth muscle actin ({alpha}SMA) and CD68 to localize VSMCs and macrophages, respectively. The tissue was also stained with a specific antibody to Fc{gamma}RIIa (A). Double immunofluorescence staining was also performed (B). {alpha}SMA or CD68 is shown in green, and Fc{gamma}RIIa is shown in red. Nuclei were also stained with TOPRO3. Figures shown represent three independent experiments.

 
3.3 CRP-induced Fc{gamma}RIIa activation generates intracellular ROS by VSMCs
The basal level of intracellular reactive oxygen species (ROS), as measured by confocal microscopic images after H2DCFDA labeling, was minimal in VSMCs and HEK293T cells. The treatment with 1 µg/ml or higher CRP rapidly developed intracellular ROS in VSMCs in a dose and time dependent manner (p<0.01 by two-way ANOVA), and the degree of which reached maximum in 30 min. Such a response of VSMCs to show CRP-induced ROS generation (25 µg/mL/30 min) was blocked by functional inhibition of Fc{gamma}RIIa (p<0.01; Fig. 3A). As expected, Fc{gamma}RIIa-transfected HEK293T cells showed specific CRP binding (p<0.01; Fig. 3B), and produced intracellular ROS in response to CRP (25 µg/mL/30 min) (p<0.01; Fig. 3C).


Figure 3
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  		Fig. 3 Involvement of Fc{gamma}RIIa in CRP-induced ROS generation. (A) Human VSMCs were labeled with H2DCFDA and stimulated with CRP (0.1 to 25 µg/mL) for up to 60 min and the intensity of cell-associated fluorescence was measured using a plate reader. The fluorescence reading of each time-point was subtracted from that of unstimulated VSMCs in order to eliminate the effect of the photo-oxidation. Data for each time point represent mean±S.D. of triplicate experiments. The treatment with 1 µg/ml or higher CRP significantly developed intracellular ROS in VSMCs in a dose and time dependent manner (p<0.01 by two-way ANOVA). The graph shows dose-responsiveness of CRP on ROS production in 30 min, which was analyzed by Prism 3.0 soft ware and the curve represents nonlinear regression (R square=0.95). NS (statistically non-significant) and p<0.01 values were determined by unpaired Student t test. (B and C) Cultured HEK293T cells were transfected with pcDNA3.1 vector (MOCK) or with same vector containing human Fc{gamma}RIIa cDNA (tFc{gamma}RIIa). In section B, cells were treated with CRP (25 µg/mL) for 30 min and the amount of cell-bound CRP was estimated as described in Methods. The results shown are means±S.D. of cell-associated fluorescence. Three independent experiments were performed in triplicate. p<0.01 value was obtained by unpaired Student t test. In section C, Mock-(MOCK) or Fc{gamma}RIIa-transfected (tFc{gamma}RIIa) HEK293T cells were also labeled with H2DCFDA and stimulated with 25 µg/mL CRP for 30 min and the degree of ROS generation was estimated as described in section A. The results shown are means±S.D. of cell-associated fluorescence from triplicate experiments. p<0.01 value was obtained by unpaired Student t test. Confocal microscopic images, which have been converted to the multi-colored computerized digital images, represent the degree of intracellular ROS amounts (red>yellow>green>white>blue).

 
3.4 NADPH oxidase 4 activation is involved in CRP-induced ROS generation by VSMCs
A pull down assay showed that the stimulation of VSMCs with CRP immediately generated GTP-bound active Rac, a main component of NADPH oxidase (Nox) complex (Fig. 4A). Among Nox isozymes, real-time PCR confirmed that Nox4 expression level was at least 5 folds higher than those of Nox1 and Nox2 (data not shown), and mRNA expression of either Nox1, 2, or 4 isoform was depleted using a specific siRNA, respectively. Alternatively, we tried to downregulate p22phox, another essential component of Nox complex, by transfecting p22phox-specific ODN. The specificity of siRNA and ODN was confirmed by parallel experiments transfecting empty vector or mismatched ODN. We also confirmed the downregulation of mRNA expression levels of Nox isoforms, most notably Nox4, and p22phox by transfected VSMCs, as determined by 30 cycles of PCR (Fig. 4B). Confocal microscopic images of H2DCFDA-labeled VSMCs clearly showed that the magnitude of ROS generated by CRP was abrogated by depleting either Nox4 or p22phox (p<0.01), while transfection of either Nox1 or 2-specific siRNA showed little effect (Fig. 4C). The functional blocking of Fc{gamma}RIIa using a specific antibody also inhibited CRP-induced ROS generation, too (p<0.01).


Figure 4
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  		Fig. 4 Role of Fc{gamma}RIIa and NADPH oxidase on CRP-induced ROS generation by human VSMCs. (A) VSMCs were treated with 25 µg/mL CRP for 10 min and the amount of the GTP-bound active form of Rac (GTP-Rac) was estimated by pull down assay. Excess GDP or GTP{gamma}S was added during the reaction as negative and positive controls, respectively. (B) VSMCs were transfected with pSUPER vector containing sequences of siRNA specific for either Nox1, Nox2, or Nox4, or transfected with p22phox-specific ODNs and cultured for 24 h. Changes of mRNA expression levels of Noxs and p22phox was measured by RT-PCR with 30 cycles of amplification. Nonsense silent siRNA (Mock) and mismatched ODN were used as negative controls. (C) VSMCs were prepared as described in (B), or pretreated with 15 µg/ml neutralizing antibody against Fc{gamma}RII (anti-Fc{gamma}RII Ab; 37 °C for 30 min) were labeled with H2DCFDA, stimulated with 25 µg/mL CRP for 30 min (CRP), and the amount of intracellular ROS was measured as described in Fig. 3C. Data represent five independent experiments. NS (statistically non-significant) and p<0.01 were obtained by unpaired Student t test.

 
3.5 CRP-induced pro-inflammatory activities and apoptotic changes of VSMCs are dependent on NADPH oxidase activation
EMSA showed that CRP-induced activation of AP-1 and NF-{kappa}B (25 µg/mL/24 h) by VSMCs was found to be inhibited by either suppression of NADPH oxidase activities using a p22phox-specific ODN or functional blocking of Fc{gamma}RIIa (Fig. 5A). Moreover, both RT- and real-time PCR showed CRP-induced mRNA expressions of monocyte chemoattractant protein-1 (MCP-1), endothelin-1 (ET-1) and interleukin (IL)-6, and that all of these were attenuated by the functional inhibition of NADPH oxidase or blocking of Fc{gamma}RIIa (p<0.01; Fig. 5B), too.


Figure 5
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  		Fig. 5 Regulatory role of Fc{gamma}RIIa activation and intracellular ROS on CRP-induced AP-1 and NF-{kappa}B activation and cytokine production by human VSMCs. (A) VSMCs were transfected with mismatched ODN or p22phox-specific ODNs (p22phox decoy), or pretreated with 15 µg/ml neutralizing antibody against Fc{gamma}RII (anti-Fc{gamma}RII Ab) or isotype control IgG at 37 °C for 30 min, and treated with 25 µg/mL CRP for 24 h, and AP-1 and NF-{kappa}B activities were measured by EMSA as described in Methods. Where indicated, specificity of each of the developed bands was tested by performing a simultaneous reaction in the presence of a 25-fold excess of unlabeled oligonucleotides as a negative control (unlabeled oligo). Data represent three independent experiments. (B) VSMCs were prepared as described in (A), and the levels of expression of MCP-1, ET-1, IL-6, p22phox and GAPDH mRNAs were estimated by both real-time and semi-quantitative RT-PCR. The results of agarose gel electrophoresis shown in the figures are representative of three independent experiments. Numbers in the table are mean±S.D. values of {Delta}Ct representing six independent experiments. ND; non-detectable in 40 cycles of amplification. p values were obtained by unpaired Student t test.

 
Prolonged treatment of VSMCs with CRP (25 µg/mL/72 h) induced significant morphological changes together with positive for TUNEL staining, indicating that CRP develops apoptosis of VSMCs. The number of TUNEL(+) VSMCs undergoing apoptosis after CRP treatment was markedly reduced by p22phox-specific ODN transfection (Fig. 6A). Immunoblotting analysis showed that the treatment of VSMCs with CRP induced the release of cytochrome c into the cytosol, indicating significant mitochondrial damage. Under identical conditions, expression of the GADD153 gene, an oxidative stress-induced transcriptional factor, was upregulated as described earlier [9]. The transfection of p22phox-specific ODN significantly blocked the CRP-stimulated expression of GADD153 (Fig. 6B).


Figure 6
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  		Fig. 6 Regulatory role of intracellular ROS on CRP-induced apoptotic changes of human VSMCs. VSMCs were transfected with mismatched ODN or p22phox-specific ODNs (p22phox decoy), and treated with 25 µg/mL CRP for 72 h. Figures shown are representative of three independent experiments. (A) Apoptotic changes of VSMCs were analyzed by TUNEL staining as described in Methods. Black dots in the figures represent nuclei of cells undergoing apoptosis. (B) In upper panel, the appearance of cytochrome c in the cytosol (cyt C cytosol) after CRP treatment (25 µg/mL for 72 h; CRP) or CRP overexpression (tCRP) was assayed by immunoblotting. Cytochrome c in the mitochondria (cyt C mitochondria) was simultaneously analyzed as a positive control. Lower panel shows expression of GADD153 mRNA, as measured by RT-PCR, by VSMCs.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Fc{gamma}RIIa has unique signaling motifs, i.e. the immunoreceptor tyrosine-based activation motifs (ITAM), and mediates pro-inflammatory responses by macrophages and neutrophils [14] and aggregation of platelets [15]. The subgroup analysis of Rotterdam study showed that the presence of H131 allele in Fc{gamma}RIIa, which has a lower binding affinity than wild-type Fc{gamma}RIIa, was found to be negatively associated with the severity of atherosclerosis [16]. A previous study showed that Fc{gamma}RII is distributed primarily in cell-rich atheromatous lesions of human carotid and coronary arteries [17]. The present study further clarified that human VSMCs express Fc{gamma}RIIa in vitro and in vivo. We proved that, in addition to Fc{gamma}RIIa expression by cultured human coronary artery VSMCs, the majority of Fc{gamma}RIIa-positive cells in the human atheroma was found to be VSMCs as well as macrophages, suggesting pro-inflammatory activities of VSMCs in atheroma can be mediated through Fc{gamma}RIIa activation.

CRP in atherosclerotic lesion is derived from blood circulation [18] or is built up through de novo synthesis in atheroma, mostly by VSMCs in response to pro-inflammatory cytokines [7]. In the present study, we confirmed that CRP molecules bind and associate with Fc{gamma}RIIa. Moreover, our findings to show CRP-stimulated Fc{gamma}RIIa expression by VSMCs, as proven by immunoblotting assay and FACS analysis, further suggest that more potent Fc{gamma}RIIa activation signals may be generated by VSMCs in response to CRP under atherogenic conditions.

The activation of Fc{gamma}Rs generates oxygen radicals by phagocytes and immune cells [19]. A previous study had described that VSMCs produce ROS when stimulated with >100 µg/mL CRP through unclear mechanism [20]. The present study provides clear evidence that Fc{gamma}RIIa exclusively mediates CRP-induced intracellular ROS generation by human VSMCs. Our results to show potent generation of intracellular ROS by Fc{gamma}RIIa-transfected HEK293T cells in response to CRP indicate that Fc{gamma}RIIa activation is sufficient for CRP to induce ROS generation. In addition, we observed that the minimal effective dose of CRP to induce ROS generation (2–5 µg/mL, equivalent to 19–48 nM) was similar to what had been reported as the Kd value of CRP binding to Fc{gamma}RIIa (38–88 nM) [4,21] and, more importantly, CRP-induced ROS generation by human VSMCs was found to be completely inhibited by anti-Fc{gamma}RIIa antibody.

Human VSMCs express all essential components for NADPH oxidase (Nox) complex [22]. Several previous results to show the co-localization of CRP, Nox4 and p22phox on intra-plaque VSMCs [20,23] support our findings that Nox4 activation follows CRP-induced Fc{gamma}RIIa activation and induces ROS generation by human VSMCs. Our real-time PCR confirmed that, among Nox isoforms, Nox4 is highly expressed in human VSMCs as described earlier [24]. Since Nox4 requires p22phox for its activity [25], we therefore tried to suppress Nox4 activities by transfecting p22phox-specific decoy, as well as transfecting pSUPER vector encoding Nox4-specific siRNA, and both approaches consistently resulted in profound inhibition of CRP-induced ROS generation. Although the functional characteristics of Nox4 have not been thoroughly studied yet, several recent reports claim Nox4 to be regulated. Similar to our results with CRP, Nox4 was found to be Rac1-dependently activated upon stimulation with angiotensin II/arachidonic acid or insulin [26]. In another report, Nox4 mediated LPS-induced ROS generation through direct interaction with TLR4 [11].

Taken together, the present study clearly demonstrates the novel mechanism by which CRP induces ROS generation by human coronary artery VSMCs; the activation of Fc{gamma}RIIa and subsequent assembly of Nox4 complex. The present study further proves that the generated intracellular ROS orchestrates major pro-inflammatory responses of human VSMCs. It is well known that most representative redox-responsive transcription factors, i.e. NF-{kappa}B and AP-1 [7], are activated by CRP. The present study clearly showed that both suppression of ROS generation and functional blocking of Fc{gamma}RIIa reduced the magnitude of NF-{kappa}B and AP-1 activities in CRP-treated VSMCs. As expected, the CRP-stimulated upregulation of NF-{kappa}B-dependent MCP-1 and IL-6 [7], and AP-1-dependent ET-1 [27] were also largely attenuated by Nox inhibition and Fc{gamma}RIIa blocking as well. Therefore, the present study strongly suggests that Fc{gamma}RIIa activation and subsequent ROS generation are prerequisite for CRP-induced activation of NF-{kappa}B and AP-1 and cytokine production.

VSMCs also undergo apoptotic changes when exposed to excessive degree of oxidative stress [28], such as prolonged stimulation with CRP [9]. The present study results to show the inhibition of CRP-induced apoptosis by transfection of a p22phox-specific decoy suggest that CRP-induced Nox4 activation is one of primary triggers of apoptotic signals in VSMCs. A previous study described that upregulation and activation of the growth arrest- and DNA damage-inducible gene GADD153 is involved in the process of CRP-induced apoptotic changes [9]. The present study additionally showed that CRP-stimulated expression of GADD153 expression was also largely dependent on intracellular ROS. A previous study demonstrated that GADD153 promoter has sequences for AP-1 binding element [29], therefore, the activation of AP-1 after CRP treatment may be in part responsible for GADD153 upregulation and subsequent apoptotic changes of VSMCs.

As previously described by Pepys et al. [30], the present study also provides background to suggest that the functional inhibition of CRP can be an effective therapeutic strategy to suppress inflammatory responses. Pepys et al. developed a novel inhibitor of human CRP, i.e. 1,6-bis (phosphocholine)-hexane, which preoccupies complement-binding site on the surface of CRP pentamer, and the injection of which abrogated effects of human CRP to increase infarct size of rats [30]. Taken together with the present study, it is becoming evident that the specific CRP receptors, i.e. Fc{gamma}Rs, are widely distributed among cell types found in human atheroma in vivo. In addition to Fc{gamma}RIIa expression by VSMCs as proven in the present study, human macrophages and aortic endothelial cells (HAECs) express Fc{gamma}RI, II, and III [14], and, Fc{gamma}RI and II [4], respectively. The implication of Fc{gamma}Rs on the progression of atherosclerosis has been proved in a previous animal study by Hernandez-Vargas et al., which showed that apoE knockout mice deficient in Fc{gamma}Rs developed less severe inflammation and atherosclerotic changes in the aortic wall [31]. Therefore, the inhibition of CRP-induced Fc{gamma}R activation, as well as the inhibition of CRP-induced complement activation, may also be an effective way to suppress inflammatory activities of HAECs, VSMCs and macrophages generated by CRP. Similar to our results to show upregulation of Fc{gamma}RIIa by CRP, Hernandez-Vargas et al. also showed that mouse VSMCs isolated from the aorta responded to high concentration of immune complex (150 µg/ml), i.e. another ligand for Fc{gamma}Rs, to induce expressions of Fc{gamma}Rs and production of inflammatory chemokines [31], suggesting the Fc{gamma}R-mediated activation of VSMCs may become significant in hypercholesterolemic mice. Unfortunately, animal experiments using mice provide little information to suggest the role of Fc{gamma}RIIa to trigger VSMCs activities due to the fact that rodents generally lack of Fc{gamma}RIIa while Fc{gamma}RIIb expression is intact [14]. Fc{gamma}RIIb has immunoreceptor tyrosine-based inhibition motifs (ITIM) signaling motifs and the activation of which suppresses the progression of inflammation on the contrary to Fc{gamma}RIIa [14]. Therefore, such a different profile of Fc{gamma}R distribution may in part result in the underestimation of pro-inflammatory properties of CRP in mouse model [32]. In order to prove CRP-induced Fc{gamma}RIIa activation in VSMCs is critical to promote the process of atherosclerosis, future in vivo experiments are anticipated with Fc{gamma}RIIa(+) mice, which had shown to aggravate rheumatoid arthritis and systemic lupus erythromatosus [14].

In summary, the present study clearly describes clear mechanism by which CRP provokes pro-inflammatory activity on human VSMCs. We found novel evidences that Fc{gamma}RIIa is a main CRP receptor expressed in human VSMC, and that CRP-induced Fc{gamma}RIIa activation modulates activation of AP-1 and NF-{kappa}B, production of cytokines, and, the development of apoptosis largely through Nox4-dependent ROS generation. Similar to our results, intracellular ROS have been shown to regulate angiotensin receptor type 1-mediated inflammatory responses by VSMCs [33]. Therefore, generation of intracellular ROS may be a step by which various signaling pathways converge in the development of pro-inflammatory responses by human VSMCs. Our findings to show the expression of Fc{gamma}RIIa in VSMCs in vivo suggests a possibility that CRP may induce VSMCs activation in plaques, which may eventually promote the growth and rupture of the plaque.


   Acknowledgements
 
This study was supported by grants 2007-288 from the Asian Institute for Life Sciences, by grant number FPR02A624110 from the Korean Ministry of Science, and by Cardiovascular Research Foundation in Korea.


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

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