Skip Navigation

Cardiovascular Research 2003 59(3):776-787; doi:10.1016/S0008-6363(03)00459-0
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yang, S.-P.
Right arrow Articles by Lai, J.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, S.-P.
Right arrow Articles by Lai, J.-H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Carvedilol, a new antioxidative β-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-{kappa}B activity

Shih-Ping Yanga,1, Ling-Jun Hob,1, Yi-Ling Linb, Shu-Meng Chenga, Tien-Ping Tsaoa, Deh-Ming Changc, Yu-Lin Hsuc, Chin-Yi Shihc, Ting-Yi Juanc and Jenn-Haung Laic,*

aCardiology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
bInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC
cRheumatology/Immunology and Allergy, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Kung Rd., Neihu 114, Taipei, Taiwan, ROC

haungben{at}tpts5.seed.net.tw

* Corresponding author. Tel.: +886-2-8792-7135; fax: +886-2-8792-7136.

Received 10 February 2003; accepted 8 May 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The activation of T lymphocytes contributes to the inflammatory process of atherosclerosis. Here we examined the effects of carvedilol, a new β-blocker containing an antioxidative property, on the activation of T cells. Methods: Human peripheral blood T cells were negatively selected from whole blood. Cytokines were measured by ELISA. The NF-{kappa}B and related protein activity was determined by electrophoretic mobility shift assays, Western blotting, kinase assays and transfection assays. Results: Carvedilol was nontoxic at concentrations less double equals10 µM, however, higher dosages (≥20 µM) induced T cell apoptosis. We demonstrated that carvedilol inhibited cytokine production from various stimuli-activated T cells. Carvedilol also suppressed the expression of T cell activation markers, including CD25, CD69 and CD71. Molecular investigation indicated that carvedilol specifically downregulated NF-{kappa}B but not activator protein 1 DNA-binding activity in activated T cells. The inhibitory effect was likely due to its antioxidative property. Meanwhile, carvedilol prevented stimuli-induced I{kappa}B{alpha} degradation. Such an effect was mediated through the inhibition of I{kappa}B{alpha} kinase activity. The inhibitory specificity on NF-{kappa}B by carvedilol was also demonstrated in transfection assays. Conclusions: Our results demonstrated a novel therapeutic mechanism of carvedilol in atherosclerosis, namely the inhibition of T cell activation via downregulating NF-{kappa}B activity.

KEYWORDS Carvedilol; Atherosclerosis; T cells; Cytokines; NF-{kappa}B


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
While the formation of atherosclerosis is considered to be a slowly progressive process, many factors like plaque disruption and superimposed thrombosis may result in the nonlinearity of this process and cause symptoms of unstable angina or acute myocardial infarction. Although the exact mechanisms are far from understood, the accumulated evidence indicates that the development of plaque rupture or coronary thrombosis may be unrelated to the severity of coronary plaque, whereas, it may be associated with the inflammatory process of plaque lesions [1]. In patients receiving directional coronary atherectomy, a positive correlation between the extent of initial coronary plaque inflammation and the recurrence of unstable angina is also observed in a long-term follow-up after this procedure [2]. Similarly, inflammation has been shown to affect plaque stability in restenotic coronary lesions that cause unstable coronary syndromes [3].

Besides the relatively clear role of monocytes/macrophages in the pathogenesis of atherosclerosis, the importance of T cells in this process is beginning to be appreciated. Earlier observations by Emeson and Robertson [4] revealed that in young adults and children dying from acute trauma, their aorta sections contain many T cells in those eccentric intima thickenings. The activated T cells can also be found in cryostat sections of carotid endarterectomy specimens [5]. In addition, the percentages of activated T cells in atherectomy specimens correlate well with the severity of the ischemic coronary syndrome [6]. These observations suggest that T cells may participate in initial stages of atherosclerosis formation. When circulating lymphocytes were examined in patients with coronary syndrome, the numbers of activated T cells as well as their secreted cytokines are significantly higher in unstable angina compared to stable angina patients [7,8]. Moreover, Caligiuri et al. [9] demonstrated that the T cell response found in unstable angina patients is driven by a certain antigen which could be readily detected in the culprit coronary atherosclerotic plaques. Collectively, the series of investigations have established the indispensable role of T cell activation as well as the secreted cytokines in the pathogenesis of coronary artery diseases [10].

Carvedilol is a new antihypertensive drug that preserves potent an antioxidative property aside from its β-blocking effects. Besides antihypertension, its uses for reduction of infarct size and for prevention of restenosis after coronary atherectomy [11] as well as for chronic heart failure [12,13] have been extensively reported. Noticeably, all these diseases are tightly correlated with the status of inflammation. Therefore, the therapeutic benefits of carvedilol in these diseases suggest that this drug may possess anti-inflammatory effects. The antioxidative property of carvedilol further supports this possibility. We therefore set out to examine whether carvedilol preserves anti-inflammatory effects that probably involve downregulation of T cell activation. In the present study, we demonstrated that carvedilol effectively inhibited cytokine production from activated T cells and the underlying mechanisms might involve the downregulation of NF-{kappa}B signaling pathways.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Preparation of carvedilol and drug cytotoxicity measurement
The powder of carvedilol, (±)-1-[carbazoloyl-(4)-oxyl]-3-[2-methoxy-phenoxy] ethylamino-2-propanol, was kindly provided by Roche Pharmaceuticals. The drug was dissolved in DMSO to make a 100 mM stock concentration. The rest of other β-blockers such as propranolol, pindolol and labetalol (Sigma, St. Louis, MO, USA) were similarly prepared. For experiments, the required concentrations of each drug were made by further dilution of the concentrated stock solution with culture medium. The culture medium contained RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 1000 U/ml penicillin–streptomycin (Gibco-BRL, Gaithersburg MD, USA). The final concentrations of DMSO were consistently <0.01% that showed no cytotoxicity to T cells.

2.2 Preparation of peripheral blood T cells
Human peripheral blood T cells were negatively selected from whole blood [14]. Briefly, buffy coat from blood bank (Taipei, Taiwan) was mixed with Ficoll-Hypaque, after centrifugation, the layer of mononuclear cells was collected. After lysis of red blood cells, the peripheral blood mononuclear cells were laid on Petri dishes to remove adherent cells and then incubated with antibodies including L243 [anti-DR; American Type Culture Collection (ATCC), Rockville, MD, USA], OKM1 (anti-CD11b; ATCC) and LM2 (anti-Mac1; ATCC) for 30 min at 4°C. The cells were then washed with medium containing 10% fetal bovine serum and incubated with magnetic beads conjugated with goat anti-mouse IgG (R&D, Minneapolis, MN, USA). The antibody-stained cells were then removed with a magnet. Following a repeat of the above procedures, the T cells were obtained with a purity of >98% as determined by the percentage of CD3+ cells in flow cytometry (Beckton Dickinson, Mountain View, CA, USA).

2.3 Cell stimulation and cytokine determination
For cell activation, the following stimuli and concentrations were used: phorbol 12-myristate 13-acetate (PMA, Sigma) at 10 or 50 ng/ml; ionomycin (Sigma) at 1 µM; phytohemagglutinins (PHA, Sigma) at 10 µg/ml; concanavalin A (ConA, Calbiochem, La Jolla, CA, USA) at 10 µg/ml; H2O2 (Sigma) at various doses as indicated in the figure legends; immobilized anti-CD3 monoclonal antibodies (mAbs) (OKT3, ATCC) at 10 µg/ml and soluble anti-CD28 mAbs (clone 9.3, kindly provided by Dr. Carl June, Naval Institute, Bethesda, NIH) at 1 µg/ml concentrations. The cells were incubated with a series of stimuli for variable time points and the cell pellets or supernatants were collected for further analysis. The determination of cytokine concentrations was performed with ELISA as described [14].

2.4 Measurement of cell surface molecule expression
This method has been described in our previous work [14]. Briefly, after washing, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-interleukin (IL)-2 receptor {alpha} (anti-IL-2R{alpha} or anti-CD25), anti-CD69, anti-CD71, or FITC-conjugated isotype-matched mAbs (PharMingen, San Diego, LA, USA) and the expression of these cell surface molecules was determined with a flow cytometer (Becton Dickinson). The determination of phosphatidylserine exposure, an indication of cell apoptosis, was determined by annexin V staining as described [15]. The percentages of each cell surface molecule expression were used to evaluate the drug effects.

2.5 DNA fragmentation assay
The drug-treated or -untreated cells were washed and pelleted and resuspended in hypotonic lysis buffer (1% Triton X-100, 50 mM Tris–HCl, pH 7.9, 10 mM EDTA, and 50 µg/ml RNase A) for 10 min at room temperature. After centrifugation, the supernatant was purified using a DNA Miniprep procedure (Promega, Madison, WI, USA). After sequential washing with ethanol, the DNA was eluted and analyzed on an agarose gel as described [15].

2.6 Nuclear extract preparation
Nuclear extracts were prepared according to our published work [16]. Briefly, the treated cells (2x106 cells) were left at 4°C in 50 µl of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM PMSF, and 3.3 µg/ml aprotinin] for 15 min with occasional gentle vortexing. The swollen cells were centrifuged at 15 000 rpm for 3 min. After removal of the supernatants (cytoplasmic extracts), the pelleted nuclei were washed with 50 µl buffer A and subsequently, the cell pellets were resuspended in 30 µl buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 3.3 µg/ml aprotinin) and incubated at 4°C for 30 min with occasional vigorous vortexing. Then the mixtures were centrifuged at 15 000 rpm for 20 min, and the supernatants were used as nuclear extracts.

2.7 Electrophoretic mobility shift assay (EMSA)
The EMSA was performed as detailed in our previous report [16]. The oligonucleotides containing NF-{kappa}B binding site (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), activator protein 1 (AP-1) binding site (5'-CGC TTG ATG AGT CAG CCG GAA-3') and Oct-1 binding site (5'-TGT CGA ATG CAA ATC ACT AGA A-3') were purchased and used as DNA probes (Promega). The DNA probes were radiolabeled with [{gamma}-32p]ATP using the T4 kinase according to the manufacturer’s instructions (Promega). For the binding reaction, the radiolabeled NF-{kappa}B, AP-1 or Oct-1 probe was incubated with 5 µg of nuclear extracts. The binding buffer contained 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1 mM MgCl2, 4% glycerol, and 2 µg poly(dI–dC). The reaction mixture was left at room temperature for the binding reaction to proceed for 20 min. If unradiolabeled competitive oligonucleotides were added, they were used as 100-fold molar excess and preincubated with nuclear extracts for 10 min before the addition of the radiolabeled probes. The final reaction mixture was analyzed in a 6% nondenaturing polyacrylamide gel with 0.25x Tris–borate–EDTA (TBE) as an electrophoresis buffer.

2.8 Western blotting
ECL Western blotting (Amersham, Arlington Heights, IL, USA) was performed as described previously [15]. Briefly, after an extensive wash, the cells were pelleted and resuspended in lysis buffer. After periodic vortexing, the mixture was centrifuged and the supernatant was collected and the protein concentration measured. Equal amounts of whole cellular extracts were analyzed on 10% SDS–PAGE and transferred to the nitrocellulose filter. For immunoblotting, the nitrocellulose filter was incubated with TBS–T containing 5% nonfat milk (milk buffer) for 2 h, and then blotted with antisera against I{kappa}B{alpha}, IKK{alpha} (Santa Cruz Biotechnology) or β-actin (PharMingen) for overnight at 4°C. After washing with milk buffer twice, the filter was incubated with donkey anti-mouse IgG conjugated to horseradish peroxidase at a concentration of 1:5000 for 30 min. The filter was then incubated with the substrate and exposed to an X-ray film.

2.9 Immunoprecipitation kinase assay
The construction of plasmid pGEX-4T-2–I{kappa}B{alpha} containing sequences of N-terminal 4–54 amino acids of I{kappa}B{alpha} fused in-frame to the expression plasmid pGEX-4T-2 has been detailed [17]. The GST–I{kappa}B{alpha} fusion protein induced in E. coli transfected with pGEX-4T-2–I{kappa}B{alpha} was used as a substrate for I{kappa}B{alpha} kinase (IKK{alpha}). The c-Jun N-terminal kinase (JNK) substrate, GST–c-Jun fusion protein, was a kind gift from Dr. S.-F. Yang (Academia Sinica, Taiwan). Myelin basic protein used as a substrate for extracellular signal regulated protein kinase (ERK) and p38 was purchased from Sigma. The antibodies for kinase assays were either kindly provided by Dr. Tse-Hua Tan (Baylor College of Medicine, TX, USA, for both JNK and p38) or purchased from Santa Cruz Biotechnology (for IKK{alpha} and ERK). To perform immunoprecipitation kinase assay, the whole cellular extract 50–100 µg was incubated with 5 µl of specific antibody in incubation buffer containing 25 mM HEPES (pH 7.7), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton-X-100, 20 mM β-glycerophosphate, 0.1 mM Na3VO4, 2 µM leupeptin and 400 µM PMSF overnight. The mixture was then immunoprecipitated by addition of protein A beads and rotated at 4°C for 2 h. After extensive washing, twice with HEPES washing buffer containing 20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA and 0.05% Triton X-100, twice with LiCl washing buffer containing 500 mM LiCl, 100 mM Tris (pH 7.6), 0.1% Triton X-100 and 1 mM DTT and twice with kinase buffer containing 20 mM MOPS (pH 7.2), 2 mM EDTA, 10 mM MgCl2, 0.1% Triton X-100 and 1 mM DTT, the beads were resuspended in 40 µl kinase buffer with addition of cold ATP (30 µM), substrates and 10 µCi of [{gamma}-32P]ATP. The mixture was incubated at 30°C with occasional gentle mixing for 30 min. The reaction was then terminated by resuspending in 1% SDS solubilizing buffer and boiled for 5 min and analyzed in SDS–PAGE.

2.10 Transfection assays
The transfection assays were performed according to our previous work [16] with some modifications. In order to reduce cell damage, we used the transfection reagent TransFastTM (Promega) to transfect plasmids into cells instead of using electroporation. In brief, 1x106 human T cell line Jurkat were evenly mixed in a well of a 6-well plate with 2 µg of reporter plasmid pNF–{kappa}B-Luc or pAP–1-Luc (Stratagene, La Jolla, CA, USA) and TransFastTM transfectant (6 µl) in triplicate. At 48 h after transfection, the cells (in a well) were equally distributed into five for conditions indicated in Fig. 8 (unstimulated, PMA+ionomycin-stimulated, PMA+ionomycin+ 1, 5 or 10 µM of carvedilol pretreatment). The drug was added 2 h before the addition of PMA (10 ng/ml) and ionomycin (1 µM). After another 24 h, the cell pellets were collected, the total cell lysates prepared and the luciferase activities were determined according to the manufacturer’s instructions (Promega).


Figure 8
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Carvedilol suppressed transcriptional activity of NF-{kappa}B but not AP-1. Human T cell line Jurkat at 1x106/ml were mixed together with pNF–{kappa}B-Luc (labeled as NF-{kappa}B) or pAP–1-Luc (labeled as AP-1) reporter plasmids and the transfection reagent TransFastTM as described in Methods. At 48 h after transfection, the cells were equally divided and pretreated or not with carvedilol at various dosages for 2 h. After stimulation with PMA (10 ng/ml)+ionomycin (1 µM) for another 24 h, cells were collected and the total cell lysates were analyzed for luciferase activities. The representative data out of at least three independent experiments are shown. P, P value, by paired Student’s t test.

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Safety ranges of carvedilol in human peripheral blood T cells
When the drug is administered chronically, carvedilol can attain a plasma concentration of 0.4 µM [18]. However, the tissue concentrations of carvedilol are considered to be higher than the plasma levels [19]. Therefore, for the study of short-term effects of carvedilol, relatively higher concentrations were used both in vitro and in vivo. The analysis with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assays and trypan blue exclusion assays showed that when carvedilol was used at concentrations less double equals10 µM, there was no significant toxicities on examined human peripheral blood T cells (data not shown). However, higher concentrations of carvedilol killed cells (data not shown). We then determined whether the mechanism of carvedilol-induced T cell death was mediated through necrotic or apoptotic process. As illustrated, at concentrations higher than 20 µM, carvedilol treatment of T cells resulted in both the generation of DNA fragmentation (Fig. 1A) and the exposure of phosphatidylserine (Fig. 1B, determined by annexin V binding), characteristics of cellular apoptosis.


Figure 1
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Carvedilol at concentrations higher than 20 µM induced apoptosis of human peripheral blood T cells. Purified human peripheral blood T cells at 5x105/ml were treated with various concentrations of carvedilol for 48 (A) or 24 (B) h. The DNA was extracted from T cells and the fragmentation assays were performed as described in Methods (A). Mr: 100-bp marker. Alternatively, the cells were stained with annexin V and analyzed in a flow cytometer (B). The representative data shown were obtained from more than six different donor T cells with similar results.

 
3.2 Carvedilol inhibited the cytokine production from activated T cells
Although inflammation has been demonstrated to be one of the major factors leading to atherosclerosis, the etiology that initiates this inflammatory process is not exactly clear. We therefore chose a wide range of stimuli including PHA, ConA and anti-CD3+anti-CD28 mAbs to activate human peripheral blood T cells and then measured the effects of carvedilol on stimulated cells. We showed that carvedilol not only inhibited these stimuli-induced production of cytokines from T cells, both T helper 1 (IL-2 and interferon-{gamma}) and T helper 2 (IL-4 and IL-10) cytokines were equally susceptible to the inhibition by carvedilol in a dose-dependent manner (Fig. 2).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of carvedilol on cytokine production from activated T cells. Purified human peripheral blood T cells at 5x105/ml were pretreated in triplicate with various concentrations of carvedilol for 2 h and then stimulated with PHA, ConA, or anti-CD3+anti-CD28 mAbs for another 24 h. The supernatants were collected for IL-2, IL-4, IL-10 and interferon-{gamma} measurements. The representative data (in pg/ml) out of at least three different donor T cells are shown. 3+28=anti-CD3+anti-CD28 mAbs stimulation.

 
3.3 Carvedilol inhibited T cell activation marker expression
Since many T cell cytokines were susceptible to the inhibition by carvedilol, we determined if the expression of T cell activation markers could also be affected by carvedilol. As shown in Fig. 3, after activation with anti-CD3+anti-CD28 mAbs, T cells greatly expressed several cell surface activation markers, including CD25, CD69 and CD71. In the presence of carvedilol, the expression of these activation markers was significantly suppressed.


Figure 3
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Carvedilol inhibits the expression of CD25 (IL-2R{alpha}), CD69 and CD71 on activated T cells. Human peripheral blood T cells at 5x105/ml were treated with various concentrations of carvedilol for 2 h and then stimulated or not with anti-CD3+anti-CD28 mAbs for another 24 h. The expression of CD69, CD71 and CD25 was measured by a flow cytometer. The representative data out of three are shown.

 
3.4 Carvedilol downregulated NF-{kappa}B activity in activated T cells
The extensive inhibition of cytokine production and activation marker expression in T cells by carvedilol raises the possibility that carvedilol may target certain signaling molecules that are commonly involved in T cell activation. We therefore examined the effects of carvedilol on the activation of transcription factors NF-{kappa}B, a family of proteins extensively involved in a variety of signaling pathways. After stimulation with PMA+ionomycin, peripheral blood T cell nuclear extracts contained strong NF-{kappa}B DNA-binding activity. The binding was specific because only wild type but not mutant {kappa}B oligonucleotides could completely compete the binding (Fig. 4A). In the presence of carvedilol, the PMA+ionomycin-induced NF-{kappa}B DNA-binding activity was greatly suppressed (Fig. 4B). As a positive control, aspirin also strongly inhibited NF-{kappa}B DNA-binding activity (Fig. 4B). Remarkably, carvedilol at a concentration as low as 1 µM effectively downregulated CD28-costimulated NF-{kappa}B DNA-binding activity (Fig. 4C, upper panel). Under such circumstances, carvedilol had no effect on AP-1 and Oct-1 DNA-binding activity (Fig. 4C, middle and lower panels). When carvedilol was present before the addition of stimuli, it effectively blocked NF-{kappa}B DNA-binding activity (Fig. 4D). However, the inhibitory effect was totally abolished when carvedilol was added after the addition of stimuli (Fig. 4D). In contrast, while CD28 costimulation strongly induced AP-1 DNA-binding activity, carvedilol added either before or after the addition of stimuli did not show any suppressive effects (Fig. 4D). These results suggest the relative specificity of carvedilol on inhibition of NF-{kappa}B DNA-binding activity.


Figure 4
View larger version (44K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Carvedilol blocks NF-{kappa}B DNA-binding activity. Human peripheral blood T cells at 2x106/ml were pretreated with various concentrations of carvedilol (B and C, 10 µM for D) or 5 mM aspirin (B) for 2 h or various time points (D) and then stimulated or not with PMA+ionomycin for 3 h (A and B) or anti-CD3+anti-CD28 mAbs for 10 h (C and D). The nuclear extracts were prepared and analyzed by EMSA. The 32P-labeled oligonucleotides containing the {kappa}B, AP-1 or Oct-1 site were used as probes. A competition study was done with unradiolabeled wild-type (wt) or mutant (mt) {kappa}B oligonucleotides (A). The competitors were added 10 min before the addition of the radiolabeled {kappa}B probe. After addition of the radiolabeled probe, the whole reaction mixture was incubated for 20 min and then analyzed on a 6% native polyacrylamide gel. In (C), V stands for vehicle with 0.01% DMSO; in (D), – stands for the time point before adding the stimuli and + stands for the time point after adding the stimuli.

 
3.5 Carvedilol blocked NF-{kappa}B DNA-binding activity mainly via its antioxidative effect
Since carvedilol preserves both β-blocking and antioxidative properties, we were interested to determine which of these two effects is responsible for the inhibition of NF-{kappa}B DNA-binding activity. As shown in Fig. 5A, carvedilol but not the other examined β-blockers such as propranolol, pindolol and labetalol could inhibit NF-{kappa}B DNA-binding activity. In addition, in the presence of carvedilol, the oxidant H2O2-induced NF-{kappa}B DNA-binding activity was greatly reduced (Fig. 5B). These results suggest that the inhibition of NF-{kappa}B DNA-binding activity by carvedilol was mainly mediated through its antioxidative rather than β-blocking capacity.


Figure 5
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Carvedilol inhibits NF-{kappa}B DNA-binding activity mainly via its antioxidative effect. Human peripheral blood T cells at 2x106/ml were pretreated with 10 µM of carvedilol or other β-blockers such as propranolol, pindolol and labetalol for 2 h and then stimulated or not with PMA+ionomycin for 3 h. The nuclear extracts were prepared and analyzed by EMSA as described in Fig. 4A. Similarly, after pretreatment with 10 µM of carvedilol, T cells were stimulated with various concentrations of H2O2 and the NF-{kappa}B DNA-binding activity was measured with EMSA (B). The representative data out of two are shown.

 
3.6 Carvedilol blocked I{kappa}B{alpha} degradation through inhibiting I{kappa}B{alpha} kinase (IKK{alpha}) but not mitogen-activated protein (MAP) kinase activity
The inhibition of NF-{kappa}B activity by carvedilol suggests that this drug may have some effects on the NF-{kappa}B-associated protein, I{kappa}B{alpha} that retains NF-{kappa}B in an inactive status in the cytosol of resting T cells. We showed that carvedilol successfully blocked I{kappa}B{alpha} degradation induced by PMA+ionomycin stimulation (Fig. 6). As a comparison, carvedilol had no effects on β-actin levels. Because the degradation of I{kappa}B{alpha} was initiated through the phosphorylation of 32 and 36 serine residues by IKK (or IKK{alpha}, used in this study), we determined whether carvedilol had any effect on IKK{alpha} activities. We demonstrated that carvedilol nearly completely blocked the IKK{alpha} activities (Fig. 7A and B). In contrast to the consistent inhibitory effects on IKK{alpha} activity, carvedilol showed limited effects on MAP kinases such as ERK, p38 and JNK activities (Fig. 7C). Interestingly, in one donor T cell, we did observe mild to moderate inhibition of JNK activity by carvedilol (data not shown). This could be due to the possible effects of donor variations.


Figure 6
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Prevention of I{kappa}B{alpha} degradation by carvedilol. Human peripheral blood T cells at 2x106/ml were pretreated or not with 10 µM of carvedilol for 2 h and then stimulated with PMA (50 ng/ml)+ionomycin (1 µM) for various time points. After washing, cell pellets were collected and the total cell lysates were analyzed for the protein levels of I{kappa}B{alpha} and β-actin in Western blotting assays as described in Methods. The representative data out of three are shown.

 

Figure 7
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Blockade of IKK{alpha} but not MAP kinases activity by carvedilol. To reduce the basal activities of kinases, human peripheral blood T cells at 2x106/ml were maintained in a serum-free medium for 16 h and then were pretreated or not with 10 µM of carvedilol for 2 h and then stimulated with PMA (50 ng/ml)+ionomycin (1 µM) for various time points. After washing, cell pellets were collected and the total cell lysates were immunoprecipitated with anti-IKK{alpha} (A), anti-ERK, anti-p38 or anti-JNK (C) antibodies. After sequential washing, the substrates (GST–I{kappa}B{alpha} for IKK{alpha}, GST–c-Jun for JNK and myelin basic protein for ERK and p38) and 10 µCi of [{gamma}-32P]ATP were added. After kinase reaction, the reaction mixture was analyzed. For determination of the IKK{alpha} protein level, Western blotting assays as described in Fig. 6 were performed. The representative data (A and C) and the statistical analysis of the results from three different donor T cells (B) are shown. The fold induction was presented as a comparison with the intensity determined in medium control.

 
3.6 Carvedilol inhibited NF-{kappa}B transcriptional activity
To further investigate the regulatory effect of carvedilol on NF-{kappa}B, we transiently transfected NF-{kappa}B-luciferase or AP-1-luciferase reporter plasmids into human T cell line Jurkat. At 48 h after transfection, the cells were treated with carvedilol at various dosages and then stimulated with PMA+ionomycin to induce NF-{kappa}B or AP-1 transcriptional activity. Consistently, we demonstrated that carvedilol significantly inhibited the transcriptional activity of NF-{kappa}B but had no suppressive effects on AP-1 transcriptional activity (Fig. 8).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The increase of activated monocytes/macrophages and T cells in coronary artery diseases is observed not only in plaque lesions but also in circulation populations [20,21]. Thus, the determination of cytokine production from peripheral blood T cells can, to a certain extent, reflect the situation in plaque lesions [22]. Importantly, serum cytokines like IL-2 and tumor necrosis factor-{alpha} can deliver negative ionotropic effects on cardiac papillary muscles through nitric oxide-dependent mechanisms [23]. Besides, the increased serum cytokines in ischemic heart disease may also account for the mechanisms associated with the atherosclerosis formation and plaque instability such as deposition and activation of cellular elements on the vessel wall, increased cell adhesiveness, superoxide radical generation and endothelial injury [24–26]. In this context, the inhibition of cytokine production from activated T cells may provide a potential therapeutic benefit in the treatment of coronary artery diseases.

Most studies examining the role of cytokines in coronary artery diseases were mainly conducted to measure a pool of cytokines in serum or plaque lesions and correlate the cytokine levels with the disease severities. Through this approach, several cytokines or mediators have been identified to be associated with the development of myocardial ischemia [5,8,22,24,27]. Our studies thus expanded these earlier observations to examine cell-specific cytokines in individual cell type and also determined the effects of carvedilol on cytokine production. We demonstrated that carvedilol effectively inhibited cytokine production from various stimuli-activated T cells (Fig. 2). The inhibition of T cell activation by carvedilol was also observed in examining the expression of cell surface activation markers (Fig. 3).

Within examined dosages used in this study, carvedilol has been shown to preserve broadspectrum effects in different tissue cells. At concentrations of 1–10 µM, carvedilol inhibits low density lipoprotein oxidation by the human umbilical vein endothelial cells [28]. Carvedilol, at a concentration of 10 µM, also effectively reduces cardiac necrosis size in ischemia–reperfusion-mediated heart injury [29] and epinephrine-induced apoptosis in human coronary artery endothelial cells [30]. In cultured human pulmonary artery vascular smooth muscle cells, carvedilol at 0.1–10 µM, produced a concentration-dependent inhibition of the mitogenesis induced by various stimuli, including platelet-derived growth factor, epidermal growth factor, thrombin, and serum [31]. Carvedilol, with an IC50 value of 3 µM, also caused a concentration-dependent inhibition of vascular smooth muscle cell migration induced by platelet-derived growth factor [31]. Aside from these effects, several other therapeutic mechanisms of carvedilol were also demonstrated which include the preservation of anti-nitric oxide activity [32], the inhibition of free-radical-induced cardiac damage [33] as well as the suppression of reactive oxygen species generation by leukocytes [34]. In the present study, we add several novel observations in the field of carvedilol research and conclude that carvedilol is a strong NF-{kappa}B inhibitor in T cells. These include the suppression of binding of NF-{kappa}B to the {kappa}B site (Fig. 4), the prevention of I{kappa}B{alpha} degradation (Fig. 6), the inhibition of IKK{alpha} activity (Fig. 7) as well as the blockade of NF-{kappa}B-dependent reporter gene transcription (Fig. 8). Furthermore, we demonstrated that the carvedilol-mediated suppression of NF-{kappa}B DNA-binding activity was mainly through its antioxidative rather than its β-blocking property (Fig. 5).

While NF-{kappa}B transcription factors play critical roles in T cell activation and survival, the activation of NF-{kappa}B can also be readily detected in various cardiac damage-associated events. For example, the induction of ischemia–reperfusion activates NF-{kappa}B in rat heart tissues [35]. In pigs fed with hypercholesterolemic diet, the activation of NF-{kappa}B is found in intimal cells of coronary arteries [36]. Convincingly, the activated NF-{kappa}B is detected only in human atherosclerotic plaques but not in normal vessels [37]. The activation of NF-{kappa}B through a toll-like receptor in human adventitial fibroblasts may also relate to the process of atherosclerosis [38,39]. Other indirect supports come from the observations that the genes of adhesion molecules, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, important in atherosclerotic process, are regulated by NF-{kappa}B [40]. The therapeutic effects of low-dose aspirin on vascular inflammation, plaque stability, and atherogenesis also involve the down-regulation of NF-{kappa}B activity in a low-density lipoprotein receptor-deficient mice model [41]. In addition, as a well-known regulator of NF-{kappa}B, the oxidative stress has been consistently observed in many conditions, including hyperlipidemia, hypertension and diabetes mellitus that are highly associated with the development of atherosclerosis (reviewed in [42]). Furthermore, the administration of a synthetic double-stranded DNA ‘decoy’ that binds to NF-{kappa}B transcription factors and blocks their binding to {kappa}B binding site greatly reduces myocardial infarct size in vivo [43]. Given the negative roles of NF-{kappa}B-regulated T cell cytokines like IL-2 and interferon-{gamma} in coronary artery diseases, the inhibition of NF-{kappa}B by carvedilol should provide additional beneficial therapeutic effects. Finally, because NF-{kappa}B plays important roles in many aspects of T cell activation induced by a variety of mitogens, cytokines and pathogens, this group of transcription factors may lead to reasonable targets for therapeutic purposes for atherosclerosis.

Time for primary review 28 days.


   Acknowledgements
 
Supported in part by the National Health Research Institutes (NHRI-EX92-9208SI), the National Science Council (NSC-91-2314-B-016-044), Taiwan, R.O.C. and by the Roche Pharmaceuticals. The kind gifts from Drs. T.-H. Tan, S.-F. Yang and Carl June and Roche Pharmaceuticals are highly appreciated.


   Notes
 
1 S.-P. Yang and L.-J. Ho contributed equally to this work. Back


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

  1. Hansson G.K. Immune mechanisms in atherosclerosis. Arterioscl Thromb Vasc Biol (2001) 21:1876–1890.[Abstract/Free Full Text]
  2. Meuwissen M., Piek J.J., van der Wal A.C., et al. Recurrent unstable angina after directional coronary atherectomy is related to the extent of initial coronary plaque inflammation. J Am Coll Cardiol (2001) 37:1271–1276.[Abstract/Free Full Text]
  3. Piek J.J., van der Wal A.C., Meuwissen M., et al. Plaque inflammation in restenotic coronary lesions of patients with stable or unstable angina. J Am Coll Cardiol (2000) 35:963–967.[Abstract/Free Full Text]
  4. Emeson E.E., Robertson A.L. Jr. T lymphocytes in aortic and coronary intimas. Their potential role in atherogenesis. Am J Pathol (1988) 130:369–376.[Abstract]
  5. Hansson G.K., Holm J., Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol (1989) 135:169–175.[Abstract]
  6. van der Wal A.C., Piek J.J., de Boer O.J., et al. Recent activation of the plaque immune response in coronary lesions underlying acute coronary syndromes. Heart (1998) 80:14–18.[Abstract/Free Full Text]
  7. Neri Serneri G.G., Prisco D., Martini F., et al. Acute T-cell activation is detectable in unstable angina. Circulation (1997) 95:1806–1812.[Abstract/Free Full Text]
  8. Liuzzo G., Kopecky S.L., Frye R.L., et al. Perturbation of the T-cell repertoire in patients with unstable angina. Circulation (1999) 100:2135–2139.[Abstract/Free Full Text]
  9. Caligiuri G., Paulsson G., Nicoletti A., Maseri A., Hansson G.K. Evidence for antigen-driven T-cell response in unstable angina. Circulation (2000) 102:1114–1119.[Abstract/Free Full Text]
  10. Weyand C.M., Goronzy J.J., Liuzzo G., et al. T-cell immunity in acute coronary syndromes. Mayo Clinic Proc (2001) 76:1011–1020.[ISI][Medline]
  11. Serruys P.W., Foley D.P., Hofling B., et al. Carvedilol for prevention of restenosis after directional coronary atherectomy: final results of the European carvedilol atherectomy restenosis (EUROCARE) trial: S.B. 211475, a metabolite of carvedilol, reduces infarct size after myocardial ischemic and reperfusion injury in rabbits. Circulation (2000) 101:1512–1518.[Abstract/Free Full Text]
  12. Yancy C.W., Fowler M.B., Colucci W.S., et al. Race and the response to adrenergic blockade with carvedilol in patients with chronic heart failure. New Engl J Med (2001) 344:1358–1365.[Abstract/Free Full Text]
  13. Nakamura K., Kusano K., Nakamura Y., et al. Carvedilol decreases elevated oxidative stress in human failing myocardium. Circulation (2002) 105:2867–2871.[Abstract/Free Full Text]
  14. Lai J.H., Ho L.J., Kwan C.Y., Chang D.M., Lee T.C. Plant alkaloid tetrandrine and its analog block CD28-costimulated activities of human peripheral blood T cells: potential immunosuppressants in transplantation immunology. Transplantation (1999) 68:1383–1392.[CrossRef][ISI][Medline]
  15. Lai J.H., Ho L.J., Lu K.C., et al. Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction. J Immunol (2001) 166:6914–6924.[Abstract/Free Full Text]
  16. Lai J.H., Horvath G., Subleski J., et al. RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter. Mol Cell Biol (1995) 15:4260–4271.[Abstract]
  17. Lai JH. The role of Rel/NF-{kappa}B and I{kappa}B{alpha} proteins in CD28 signal transduction. Baylor College of Medicine, USA. 1996 (Thesis).
  18. McPhillips J.J., Schwemer G.T., Scott D.I., Zinny M., Patterson D. Effects of carvedilol on blood pressure in patients with mild to moderate hypertension. A dose response study. Drugs (1988) 36:82–91.[Medline]
  19. Yue T.L., Ma X.L., Gu J.L., Ruffolo R.R. Jr., Feuerstein G.Z. Carvedilol inhibits activation of stress-activated protein kinase and reduces reperfusion injury in perfused rabbit heart. Eur J Pharmacol (1998) 345:61–65.[CrossRef][ISI][Medline]
  20. Jude B., Agraou B., McFadden E.P., et al. Evidence for time-dependent activation of monocytes in the systemic circulation in unstable angina but not in acute myocardial infarction or in stable angina. Circulation (1994) 90:1662–1668.[Abstract/Free Full Text]
  21. Lee W.H., Lee Y., Kim J.R., et al. Activation of monocytes, T-lymphocytes and plasma inflammatory markers in angina patients. Exp Mol Med (1999) 31:159–164.[ISI][Medline]
  22. Ikonomidis I., Andreotti F., Economou E., et al. Increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. Circulation (1999) 100:793–798.[Abstract/Free Full Text]
  23. Finkel M.S., Oddis C.V., Jacob T.D., et al. Negative ionotropic effects of cytokines on the heart mediated by nitric oxide. Science (1992) 257:387–389.[Abstract/Free Full Text]
  24. Vaddi K., Nicolini F.A., Mehta P., Mehta J.L. Increased secretion of tumor necrosis factor-{alpha} and interferon-{gamma} by mononuclear leukocytes in patients with ischemic heart disease. Relevance in superoxide anion generation. Circulation (1994) 90:694–699.[Abstract/Free Full Text]
  25. Liuzzo G., Vallejo A.N., Kopecky S.L., et al. Molecular fingerprint of interferon-{gamma} signaling in unstable angina. Circulation (2001) 103:1509–1514.[Abstract/Free Full Text]
  26. Geng Y., Hansson G.K., Holme E. Interferon-{gamma} and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. Circ Res (1992) 71:1268–1276.[Abstract/Free Full Text]
  27. Saitoh T., Kishida H., Tsukada Y., et al. Clinical significance of increased plasma concentration of macrophage colony-stimulating factor in patients with angina pectoris. J Am Coll Cardiol (2000) 35:655–665.[Abstract/Free Full Text]
  28. Yue T.L., Wang X., Gu J.L., Ruffolo R.R. Jr., Feuerstein G.Z. Carvedilol prevents low-density lipoprotein (LDL)-enhanced monocyte adhesion to endothelial cells by inhibition of LDL oxidation. Eur J Pharmacol (1995) 294:585–591.[CrossRef][ISI][Medline]
  29. Yue T.L., Ma X.L., Wang X., et al. Possible involvement of stress-activated protein kinase signaling pathway and Fas receptor expression in prevention of ischemia–reperfusion-induced cardiomyocyte apoptosis by carvedilol. Circ Res (1998) 82:166–174.[Abstract/Free Full Text]
  30. Romeo F., Li D., Shi M., Mehta J.L. Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway. Cardiovasc Res (2000) 45:788–794.[Abstract/Free Full Text]
  31. Ohlstein E.H., Douglas S.A., Sung C.P., et al. Carvedilol, a cardiovascular drug, prevents vascular smooth muscle cell proliferation, migration, and neointimal formation following vascular injury. Proc Natl Acad Sci USA (1993) 90:6189–6193.[Abstract/Free Full Text]
  32. Yoshioka T., Iwamoto N., Tsukahara F., et al. Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells. Br J Pharmacol (2000) 129:1530–1535.[CrossRef][ISI][Medline]
  33. Flesch M., Maack C., Cremers B., et al. Effect of β-blockers on free radical-induced cardiac contractile dysfunction. Circulation (1999) 100:346–353.[Abstract/Free Full Text]
  34. Dandona P., Karne R., Ghanim H., et al. Carvedilol inhibits reactive oxygen species generation by leukocytes and oxidative damage to amino acids. Circulation (2000) 101:122–124.[Abstract/Free Full Text]
  35. Li C., Browder W., Kao R.L. Early activation of transcription factor NF-{kappa}B during ischemia in perfused rat heart. Am J Physiol (1999) 276:H543–552.[ISI][Medline]
  36. Wilson S.H., Caplice N.M., Simari R.D., et al. Activated nuclear factor-{kappa}B is present in the coronary vasculature in experimental hypercholesterolemia. Atherosclerosis (2000) 148:23–30.[CrossRef][ISI][Medline]
  37. Brand K., Page S., Rogler G., et al. Activated transcription factor nuclear factor-{kappa} B is present in the atherosclerotic lesion. J Clin Invest (1996) 97:1715–1722.[ISI][Medline]
  38. Vink A., Schoneveld A.H., van der Meer J.J., et al. In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions. Circulation (2002) 106:1985–1990.[Abstract/Free Full Text]
  39. Edfeldt K., Swedenborg J., Hansson G.K., Yan Z.Q. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation (2002) 105:1158–1161.[Abstract/Free Full Text]
  40. Iiyama K., Hajra L., Iiyama M., 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) 85:199–207.[Abstract/Free Full Text]
  41. Cyrus T., Sung S., Zhao L., et al. Effect of low-dose aspirin on vascular inflammation, plaque stability, and atherogenesis in low-density lipoprotein receptor-deficient mice. Circulation (2002) 106:1282–1287.[Abstract/Free Full Text]
  42. Collins T., Cybulsky M.I. NF-{kappa}B: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest (2001) 107:255–264.[ISI][Medline]
  43. Morishita R., Sugimoto T., Aoki M., et al. In vivo transfection of cis element ‘decoy’ against nuclear factor-{kappa}B binding site prevents myocardial infarction. Nat Med (1997) 3:894–899.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yang, S.-P.
Right arrow Articles by Lai, J.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, S.-P.
Right arrow Articles by Lai, J.-H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?