Abstract
This study shows cilostazol effect to prevent remnant lipoprotein particle (RLP)-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). Upon incubation of HUVECs with RLP (50 μg/ml), adherent monocytes significantly increased by 3.3-fold with increased cell surface expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and monocyte chemoattractant protein-1 (MCP-1). Cilostazol (∼1-100 μM) concentration dependently repressed these variables as did (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085) (10 μM), a specific nuclear factor-κB (NF-κB) inhibitor. Cilostazol effects were significantly antagonized by iberiotoxin (1 μM), a maxi-K channel blocker. RLP significantly increased expression of lectin-like receptor for oxidized low-density lipoprotein (LDL) (LOX-1) receptor protein. Upon transfection with antisense LOX-1 oligodeoxynucleotide (As-LOX-1), LOX-1 receptor expression was reduced, whereas HUVECs with sense LOX-1 oligodeoxynucleotide did express high LOX-1 receptor. RLP-stimulated superoxide and tumor necrosis factor-α levels were significantly lowered with decreased expression of VCAM-1 and MCP-1 by transfection with As-LOX-1 as did polyinosinic acid (10 μg/ml, a LOX-1 receptor inhibitor). RLP significantly degraded inhibitory κBα in the cytoplasm and activated nuclear factor-κB (NF-κB) p65 in the nucleus of HUVECs with increased luciferase activity of NF-κB, all of which were reversed by cilostazol (10 μM), BAY 11-7085, and polyinosinic acid. Together, cilostazol suppresses RLP-stimulated increased monocyte adhesion to HUVECs by suppression of LOX-1 receptor-coupled NF-κB-dependent nuclear transcription via mediation of the maxi-K channel opening.
Atherosclerosis is known as chronic inflammatory processes resulting from interaction between oxidized low-density lipoprotein (Ox-LDL), macrophages, lymphocytes, and other cellular elements of the arterial wall (Ross, 1999). Recent clinical evidence has suggested that endothelial dysfunction elicited by Ox-LDL is critically important in the pathogenesis of atherosclerosis, in that inflammation plays a central role in its development (Ross, 1999; Blake and Ridker, 2001). Since Nakajima et al. (1993) have developed a simple and rapid assay method for determination of remnant lipoprotein particles (RLP)-cholesterol, i.e., chylomicron and VLDL remnants, a number of reports have focused on the role of RLP, derived from VLDL and chylomicrons, as an atherogenic factor (Hodis, 1999). It has been shown that RLP elicit endothelial vasomotor dysfunction in human coronary arteries (Doi et al., 1998) and in isolated rabbit aorta (Kugiyama et al., 1998). Endothelium-derived reactive oxygen species initiate and propagate free radical chain reactions in polyunsaturated fatty acid in RLP (Doi et al., 2000). Ox-LDL elicits endothelial dysfunction by enhancing expression of adhesion molecules on the endothelium, which facilitates leukocyte adhesion to the intima (Mehta et al., 1995). Evidence emerges that expression of lectin-like receptor for oxidized-LDL (LOX-1) gene is up-regulated by ox-LDL, angiotensin II, oxygen free radicals, and TNF-α (Li et al., 2002). Nagase et al. (2001) have emphasized the importance of the redox-sensitive up-regulation of LOX-1 receptor gene expression in vascular endothelium. Evidence accumulates that expression of LOX-1 receptors in the vascular endothelium mediates apoptotic cell death in the endothelial cells (Sawamura et al., 1997; Li and Mehta, 2000). Most recently, Shin et al. (2004) have emphasized the importance of RLP to increase expression of LOX-1 receptor protein, in that NAD(P)H oxidase-dependent superoxide production stimulated by RLP is dependent on the activation of LOX-1 receptors.
In the inflammatory and proliferative responses of the endothelium, some adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin, are expressed by the activated endothelial cells in the atherosclerotic lesions (Reape and Groot, 1999), and chemokines such as monocyte chemoattractant protein-1 (MCP-1) and cytokines such as interleukin-1 and tumor necrosis factor-α (TNF-α) are secreted from the endothelial cells (Adams and Shaw, 1994; Bevilacqua et al., 1994).
Cilostazol [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone] has been introduced to increase the intracellular cyclic AMP level by blocking its hydrolysis by type III phosphodiesterase (Kimura et al., 1985). Its principal actions include inhibition of platelet aggregation (Kimura et al., 1985), and vasodilation via mediation of increased cyclic AMP level (Tanaka et al., 1989). Cilostazol was approved for use in the treatment of intermittent claudication by the Food and Drug Administration (Dawson et al., 1998). Recently, cilostazol has been demonstrated to elicit a property to scavenge the hydroxyl and peroxyl radicals (Kim et al., 2002), and to increase the outward K+ current by activating maxi-K channels (Hong et al., 2003). Given the suppressive effect of cilostazol on the production of superoxide and release of cytokines in association with suppression of monocyte adhesion, it is likely that treatment with cilostazol can provide a potential strategy for prevention of atherosclerosis by reducing formation of adhesive and chemoattractant molecules in the endothelial cells and monocytes.
The present study was designed to 1) determine whether the expressions of adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, and chemokine such as MCP-1 were correlated with adhesion of monocytes to HUVECs in the presence of RLP; and 2) examine whether cilostazol inhibits these variables in the absence and presence of iberiotoxin, a maxi-K channel blocker. Furthermore, to explore the implication of LOX-1 receptors in the actions of RLP, the experiment was conducted in the HUVECs transfected with antisense and sense LOX-1 oligodexoynucleotides (As-LOX-1 and sense-LOX-1) in comparison with the effects of BAY 11-7085, an inhibitor of phosphorylation of IκB, and polyinosinic acid, a LOX-1 receptor inhibitor. The study further ascertained whether transcription factor NF-κB plays a role in the interaction between RLP and cilostazol.
Materials and Methods
Isolation of RLP. EDTA-plasma (1 mg/ml EDTA) was obtained at 5 h after the test meal from 10 hyperlipidemic diabetic subjects (n = 12; mean age, 49.5 ± 7.2 years; body mass index, 24.6 ± 1.2 kg/m2; plasma triglyceride, >150 mg/dl; plasma cholesterol, >200 mg/dl). The subjects had no serious diseases and had taken no cardiovascular medications for 7 days. RLP was routinely prepared with columns packed with immunoaffinity gel containing anti-apoA-1 and anti-apoB-100 monoclonal antibodies (donated by Dr. Katsuyuki Nakajima, Japan Immunoresearch Laboratories, Co. Ltd., Gunma, Japan). The unbound fractions containing apoE-enriched lipoproteins and albumin were eluted with phosphate-buffered saline (138 mmol/l NaCl, 2.7 mmol/l KCl, 8.1 mmol/l Na2HPO4, and 1.1 mmol/l KH2PO4, pH 7.4), and the unbound fractions were ultracentrifuged (d < 1.006) to isolate RLP. According to SDS-polyacrylamide gel electrophoresis, the unbound fraction isolated consisted primarily of VLDL remnants and small amounts of chylomicron remnants (determined by densitometric analysis on SDSpolyacrylamide gel electrophoresis, the ratio of the amount of apoB-48 relative to apoB-100 was 0.13 ± 0.02).
Cell Cultures. HUVECs (endothelial cell line derived from the vein of normal human umbilical cord, cell line of CRL-1730; American Type Culture Collection, Manassas, VA) were cultured in the endothelial cell basal medium-2 (EGM-2) Bullet kit media (Clonetics, BioWhittaker, San Diego, CA). Cells were grown to confluence at 37°C in 5% CO2 on 0.2% gelatin-coated culture dishes and used for experiments at not greater than passage 8. U-937 (CRL-1593.2; American Type Culture Collection) cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum.
Preparation of Antisense (Sense) LOX-1 Oligodeoxynucleotide and DNA Transfection. Antisense phosphorothioate oligodeoxynucleotide was synthesized as 16-mer (eight bases) targeted at the 5′-CAG TTA AAT GAG GCC G-3′ part of the LOX-1 sequence. The corresponding control (sense phosphorothioate oligodeoxynucleotide) was 16-mer (eight bases) targeted at 5′-ACC TAC GTG ACT ACG T-3′. All oligonucleotides were synthesized by Bioneer Corporation (Daejon, Korea). Hereafter, the antisense and sense to LOX-1 receptor mRNA will be referred to as antisense LOX-1 and sense LOX-1 oligodeoxynucleotide, respectively.
HUVECs were seeded for 24 h before transfection in tissue culture dishes. At 50 to 70% confluence, the dishes were washed twice with Opti-MEM medium to remove the fetal bovine serum, and a transfection cocktail containing 10 μg of DNA and 10 μl of LipofectAMINE reagent (Invitrogen, Carlsbad, CA) per 100-mm dish was added. The medium was removed and then EGM-2 was added to each dish.
Adhesion Assays. For the adhesion assays, HUVECs were plated on six-well gelatin-coated dishes at a density of 1.2 × 105 cells/well. On the next day, the cells were pretreated with various concentrations of cilostazol or vehicle for 15 min, after which RLP 50 μg/ml was added for additional 4 h. Thereafter, human monocytoid U937 cells (3 × 105 cells/well; American Type Culture Collection) were added to each monolayer and incubated under rotating conditions (63 rpm) at room temperature. Ten minutes later, nonadhering cells were removed by gentle washing with MCDB131 (Invitrogen), and monolayers were fixed with 1% paraformaldehyde.
Enzyme-Linked Immunosorbent Assay. HUVECs were plated on 96-well gelatin-coated tissue culture plates at a density of 2 × 104 cells/well. After 24 h, culture medium was changed to EGM-2 with 0.5% fetal bovine serum. Then, the cells were pretreated with various concentrations of cilostazol or vehicle for 3 h, after which RLP (50 μg/ml) was added for the indicated period.
After treatment with 2% paraformaldehyde, the cells were washed twice with Hanks' balanced salt solution and then incubated for 1 h with antibodies specific to human VCAM-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and ICAM-1 and E-selectin (BD Biosciences Clontech, Camarillo, CA). The cells were incubated for 1 h with second antibody. After washed the wells four times, the second antibody binding was detected by reaction of tetramethylbenzidine with H2O2 (TMB peroxidase EIA substrate kit; BD Biosciences). The absorbance at 450 nm was measured using ELISA reader (Bio-Tek Instruments, Winooski, VT).
Western Blot Analyses. The confluent cells received EGM-2 with 0.5% fetal bovine serum plus drugs 24 h before stimulation with RLP, and then were exposed to RLP for 24 h. The cells were lysed in lysis buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1% Triton X-100. For preparation of nuclear extracts, cells were lysed in buffer A (10 mM HEPES, 10 mM NaCl, 1.5 mM MgCl2, 0.25% Tween 20, 1 mM dithiothreitol, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 15 μg/ml aprotinin). After 5-min incubation at 4°C, nuclei were collected by centrifugation at 4000 rpm, and the pellets were resuspended in buffer B (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.25% Tween 20, 0.2 mM EDTA, 1 mM dithiothreitol, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 15 μg/ml aprotinin) and incubated on ice for 20 min. After centrifugation at 12,000 rpm, 50 μg of total protein was loaded into 10% SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membrane (Amersham Biosciences Inc., Piscataway, NJ). The blocked membrane was then incubated with the indicated antibodies to NF-κB p65, IκB-α (Santa Cruz Biotechnology, Inc.) and LOX-1 receptor (generously donated by Dr Tatsuya Sawamura, National Cardiovascular Center Research Institute, Suita, Osaka, Japan). The immunoreactive bands were visualized using chemiluminescent reagent as recommended by the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical, Rockford, IL). The signals of the bands were quantified using the GS-710 calibrated imaging densitometer (Bio-Rad, Hercules, CA). Cilostazol was applied 2 h before stimulation with RLP (50 μg/ml). The results were expressed as a relative density.
MCP-1 and TNF-α Assay. For analysis of MCP-1 and TNF-α in supernatants, 1 × 106 HUVECs were plated onto 48-well plates. MCP-1 and TNF-α levels in cell-free supernatants were assayed by using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. MCP-1 and TNF-α-contents were assessed by measuring absorbance at 450 nm using ELISA reader (Bio-Tek Instruments) and extrapolating from a stand curve.
Measurement of Superoxide Anion. Endothelial homogenates (100 μg protein/well) were placed into the Luminometer (Microlumat LB96P; EG & G Berthold, Wildbad, Germany). Immediately before recording chemiluminescence, NADH and NADPH (final concentration, 100 μM each) were added, and dark-adapted lucigenin (bis-N-methylacridinium nitrate, 5 μM) was added via an autodispenser. Each experiment was performed in triplicate.
Transfection and Luciferase Assays. NF-κB activity was examined using a luciferase plasmid DNA, pNFκB-Luc, that contains a specific binding sequence for NF-κB (BD Biosciences Clontech). Transfection was carried out using FuGENE6 reagent (Roche Diagnostics, Indianapolis, IN). When cultured HUVECs reached about 50% confluence, cells were treated with 0.1 μg of DNA/0.2 μl of FuGENE6 complexes in a total volume of normal media with 200 μl for 48 h. Cells received serum-free plus drugs 3 h before stimulation with TNF-α/RLP and then were exposed to TNF-α/RLP for 6 h. Cilostazol or other inhibitors was applied 10 min before experiment. Luciferase activity was measured by a luminometer (GENious; Tecan, Salzburg, Austria). Luciferase activities were normalized with protein concentration per each well.
Drugs. Cilostazol was donated by the Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan) and dissolved in dimethyl sulfoxide as a 10 mM stock solution. Iberiotoxin was from Upstate Biotechnology (Lake Placid, NY). BAY 11-7085 was from the Calbiochem (San Diego, CA). Polyinosinoc acid was from the Sigma-Aldrich (St. Louis, MO).
Statistical Analyses. The results are expressed as mean ± S.E.M. Student's t test was used for analyzing the values between vehicle- and agent-treated groups. A value of P < 0.05 was considered to be significant.
Results
RLP-Stimulated Adhesion of Monocytes. Upon incubation of HUVECs with RLP (1, 5, 10, 50, and 100 μg/ml, for 4 h), adherent monocytes increased concentration dependently and reached a plateau at 50 μg/ml RLP, in which the level of adherent monocytes (basal levels, 11.5 ± 2.7 cells/mm2) was 37.9 ± 2.0 cells/mm2 (3.3-fold). The increased monocyte adhesion to HUVECs was concentration dependently suppressed by cilostazol (1, 10, and 100 μM) (Fig. 1, A and B), which was significantly antagonized by iberiotoxin (1 μM). Furthermore, to predict whether RLP-induced increased adhesion of monocytes are dependent on activation of the nuclear transcription NF-κB, BAY 11-7085 (10 μM), a specific NF-κB inhibitor, was used. Monocyte adhesion to HUVECs significantly decreased to 17.6 ± 2.8 cells/mm2 (P < 0.001) by BAY 11-7085 (10 μM) (Fig. 1).
RLP-Stimulated VCAM-1, ICAM-1, and E-Selectin Expressions. When HUVECs were incubated with increasing concentration of RLP (∼1-100 μg) for 36 h, cell surface VCAM-1 levels concentration dependently increased and reached a maximum at 50 to 100 μg/ml RLP by ∼110-120% (data not shown). RLP (50 μg/ml)-stimulated increased VCAM-1 expression was concentration dependently repressed by cilostazol (1, 10, and 100 μM) as well as by BAY 11-7085 (10 μM), indicating implication of activation of NF-κB. Similarly, RLP (50 μg/ml) significantly increased ICAM-1 and E-selectin expression by 102.5% (P < 0.01) and 73.3% (P < 0.01), respectively, both of which were concentration dependently suppressed by cilostazol as well as by BAY 11-7085 (10 μM). Cilostazol (10 μM)-induced suppression of VCAM-1, ICAM-1, and E-selectin expression was all blocked by iberiotoxin (1 μM) (Fig. 2). BAY 11-7085-induced suppression of protein expression was not affected by iberiotoxin (data not shown).
RLP-Stimulated MCP-1 Expression. Upon incubation of HUVECs with RLP (50 μg/ml), MCP-1 expression significantly increased by approximately 170%, which was concentration dependently suppressed by cilostazol (by 35.5% at 10 μM, P < 0.01) and by BAY 11-7085 (by 30% at 10 μM, P < 0.05). Cilostazol-induced decrease in MCP-1 expression was antagonized by iberiotoxin (1 μM) (Fig. 3).
Inhibition of LOX-1 Receptor Expression by As-LOX-1. Incubation of HUVECs with RLP (∼3-100 μg/ml) significantly increased LOX-1 protein expression concentration dependently (Fig. 4A). After transfection with As-LOX-1 for 8 h, basal expression of receptor protein was little affected by RLP (∼10-100 μM), whereas HUVECs transfected with sense-LOX-1 did well respond to RLP with a concentration-dependent increase in LOX-1 receptor expression (10, 50, and 100 μM) (Fig. 4B). As-LOX-1 or sense-LOX-1 alone did not injure the HUVECs when determined as LDH release (data not shown).
Blockade of Monocyte Adhesion to HUVECs and Repression of VCAM-1 and MCP-1 Levels by As-LOX-1. Markedly increased monocyte adhesion to HUVECs stimulated by RLP was significantly reduced by treatment with As-LOX-1 as well as by polyinosinic acid (100 μg/ml). In the line with these results, RLP-induced increases in VCAM-1 and MCP-1 expression were also reduced by transfection with As-LOX-1 as well as polyinosinic acid (100 μg/ml). In contrast, pretreatment with sense-LOX-1 showed no effect (Fig. 5).
Superoxide and TNF-α Production after Transfection with As-LOX-1. When measured superoxide by recording chemiluminescence and TNF-α by ELISA in HUVECs, both superoxide and TNF-α production significantly increased in response to RLP by 87.4% (P < 0.01) and 390% (P < 0.001), respectively. In the HUVECs transfected with As-LOX-1, RLP-stimulated superoxide and TNF-α were significantly lowered, as contrasted to the HUVECs transfected with sense-LOX-1 (Fig. 6).
RLP-Stimulated Degradation of IκBα and Activation of NF-κB. To predict whether expression of adhesion molecules and MCP-1 were regulated by NF-κB, a transcription factor, degradation of IκBα in the cytoplasm and activation of NF-κB in the nuclear extracts were assessed by Western blot assay. Treatment of HUVECs with RLP (50 μg/ml) significantly degraded IκBα (P < 0.01) and markedly activated NF-κB p65 (P < 0.001). Both RLP-induced IκBα degradation and NF-κB p65 activation were significantly reversed by cilostazol (10 μM) and BAY 11-7085, a potent inhibitor of phosphorylation of IκB (Pierce et al., 1997), and polyinosinic acid, a LOX-1 receptor inhibitor (Moriwaki et al., 1998), respectively (Fig. 7).
Likewise, both TNF-α (50 ng/ml) and RLP (50 μg/ml) significantly activated the luciferase activity of NF-κB in HUVECs. Those activations were concentration dependently and significantly reduced by cilostazol (1, 10, and 100 μM) as well as by BAY 11-7085 (10 μM), suggesting that the enhanced nuclear DNA binding activity of the NF-κB transcription factor by TNF-α or RLP was strongly inhibited by cilostazol (Fig. 8).
Discussion
The present study shows that RLP increased the expression of adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, and chemokine such as MCP-1 in the HUVECs in concert with increase in monocyte adhesion to the HUVECs, and these RLP-stimulated variables were significantly suppressed by cilostazol. Adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, belong to the immunoglobulin superfamily of cell adhesion molecules regulating attachment and transendothelial migration of leukocytes (Adams and Shaw, 1994). Evidence accumulates that VCAM-1 plays a dominant role in the initiation of atherosclerosis (Cybulsky et al., 2001), and MCP-1 expression that is up-regulated in the atherosclerotic lesions importantly implicates in the monocyte adhesion to the endothelium (Gu et al., 1998). In the present study, RLP markedly stimulated monocyte adhesion to the HUVECs in association with increased expression of adhesion molecules and chemokine in the HUVECs, which were significantly suppressed by cilostazol. The inhibitory effects of cilostazol on the expression of adhesion molecules were further supported by other reports. Attenuation by cilostazol of the TNF-α-induced MCP-1 production (Nishio et al., 1997) and of the TNF-α-induced transcriptional activity of VCAM-1 promoter (Otsuki et al., 2001) in the HUVECs was demonstrated. Recently, Omi et al. (2004) demonstrated inhibition by cilostazol of expression of adhesion molecules and neutrophil adhesion induced by high glucose through increase in nitric oxide production.
To confirm whether RLP effect is mediated via activation of LOX-1 receptors in the endothelial cells, their effects were assessed in the HUVECs transfected with As-LOX-1. RLP (∼3-100 μM) concentration dependently increased the LOX-1 receptor protein expression in the wild-type and HUVECs transfected with sense LOX-1, but not in the HUVECs with As-LOX-1. The findings showing that RLP-induced increased monocyte adhesion to HUVECs in concert with increased VCAM-1 and MCP-1 expression was significantly reduced by As-LOX-1 transfection as well as by polyinosinic acid (Moriwaki et al., 1998), suggest that expression of LOX-1 receptor is a key factor in RLP-mediated monocyte adhesion to HUVECs and in the expression of adhesion molecules and chemokines. It is explained that polyinosinic acid may antagonize RLP effect by competitive inhibition of RLP binding to LOX-1 receptors considering the report of Moriwaki et al. (1998), in that polyinosinic acid reduced 125I-ox-LDL binding to LOX-1 receptors by sharing the common binding sites on the LOX-1 receptor molecule. It is well known that reactive oxygen species and TNF-α are critically implicated in the development and progression of atherosclerotic lesions in humans (Meyer et al., 1999) and in the induction of endothelial apoptosis (Dimmeler et al., 1998). Our results showed that both superoxide and TNF-α production significantly increased in response to RLP in the wild-type HUVECs as well as in the HUVECs transfected with sense LOX-1, but not in the cells with As-LOX-1. These results are consistent with the report of Shin et al. (2004), in that treatment of HUVECs with monoclonal antibody for LOX-1 receptor attenuated RLP-mediated production of superoxide production and release of cytokines (TNF-α and interleukin-1β) in HUVECs. When we examined effect of RLP on the NF-κB p65 activation in the HUVECs transfected with As- and sense-LOX-1 in comparison with wild-type HUVECs (WT) (data not shown), RLP-stimulated NF-κB p65 activation was significantly lowered in the HUVECs transfected with As-LOX-1 as contrasted with the HUVECs transfected with sense-LOX-1. These results strongly support the hypothesis that LOX-1 receptor activation importantly implicates in the action of RLP as did oxidized LDL (Li et al., 2002).
It is established that NF-κB, a redox-sensitive transcription factor, regulates the transcription of a variety of cellular genes, including injury response (Baeuerle, 1991). Evidence accumulates that NF-κB is activated in the advanced atherosclerosis (Hernandez-Presa et al., 1997). NF-κB is present in the cytosol as a heterodimer composed of NF-κB1(p50) and Rel (p65) subunits bound to the inhibitor protein IκBα/β. Upon activation, NF-κB translocates from the cytosol to the nucleus of the cell, binds to specific DNA sequences, and initiates transcription (Baeuerle and Baltimore, 1998). Considering that NF-κB is an oxidative stress-responsive transcription factor (Brand et al., 1997), it is likely predictable that NF-κB nuclear transcription critically implicates in the cascade of RLP/LOX-1 receptor/oxidative stress (cytokine formation)/monocyte adhesion. The present results showed significantly degraded IκBα in the cytoplasm and activated NF-κB p65 in the nucleus by application of RLP. Our findings showing that cilostazol markedly prevented RLP-mediated IκBα degradation and activation of NF-κB in the nucleus indicated, as did BAY 11-7085, an IκB phosphorylation inhibitor (Pierce et al., 1997) provided strong evidence to support that cilostazol suppressed not only NF-κB activation but also translocation of the NF-κB p65 subunit into the nucleus, thereby inhibiting RLP-induced surface expression of adhesion molecules and MCP-1 in association with significant suppression of monocyte adhesion to the endothelial cells. We did not determine whether cilostazol suppresses RLP-activated NF-κB binding to its consensus binding elements of VCAM-1 and MCP-1 promoter genes. A change in inhibitor κ kinase-α/β expression, a major form of kinase that phosphorylates IκBα to degrade, remains undefined in this experiment.
Cilostazol has been documented to increase intracellular level of cyclic AMP by blocking its hydrolysis by type III phosphodiesterase (Kimura et al., 1985). Kim et al. (2002) have documented the effect of cilostazol to scavenge the hydroxyl and peroxyl radicals as determined by electron paramagnetic resonance and peroxyl radical absorbing technique. Most recently, Hong et al. (2003) have recently found that cilostazol increases the outward K+ current in SK-N-SH cells (human brain neuroblastoma cell line) by activating maxi-K channels (large conductance Ca2+-activated K+ channels) that is antagonized by iberiotoxin, a maxi-K channel blocker (Galvez et al., 1990).
Maxi-K channels are known to be activated by depolarization and increase in intracellular Ca2+. Increase in K+ currents through maxi-K channels hyperpolarizes the cell membrane (Latorre et al., 1989). Maxi-K channels when activated conduct outward K+ currents that accelerate the repolarization in the hippocampal pyramidal cells (Shao et al., 1999) and block the Ca2+ entry and minimize the neuronal depolarization in the ischemic cells (Gribkoff et al., 2001). Recently, we identified that TNF-α (50 ng/ml)-induced elevated cytosolic Ca2+ concentration was concentration dependently decreased by cilostazol (∼1-100 μM) in SK-N-SH cells, whereas TNF-α (50 ng/ml)-induced decreased mitochondrial Ca2+ and mitochondrial membrane potential were in turn elevated with increasing concentration of cilostazol, and all of these cilostazol effects were significantly antagonized by iberiotoxin (1 μM) (data not shown). Based on these facts with the present results, it is likely suggested that suppressions by cilostazol of the monocyte adhesion to HUVECs, expression of adhesion molecules and chemokine, activation of NF-κB, and degradation of IκB are mediated by the maxi-K channel opening. It remains to be clarified whether maxi-K channel opening by cilostazol is directly related with NF-κB activation.
Considering some reports that activation of NF-κB was inhibited by antioxidant compounds, such as N-acetyl-l-cysteine and pyrrolidine dithiocarbamate (Brand et al., 1997), it can be inferred that suppression by cilostazol of NF-κB activation is, at least in part, ascribed to its antioxidant action (Kim et al., 2002). On the other hand, it was also addressed that elevated cyclic AMP inhibited induction of NF-κB-dependent gene expression, but not nuclear translocation of p50/p65 heterodimers, in the human monocytic THP-1 cells and HUVECs (Ollivier et al., 1996). Currently, it remains undefined as to the direct relationships between increased cyclic AMP level and maxi-K channel opening induced by cilostazol in regulation of expression of adhesion molecules and chemokines through NF-κB-mediated transcription in the HUVECs.
Together, it is concluded that cilostazol inhibits RLP-stimulated expression of adhesion molecules and chemokines and suppresses monocyte adhesion to HUVECs by inhibition of LOX-1 receptor-coupled superoxide production and NF-κB-dependent nuclear transcription via mediation of the maxi-K channel opening (Fig. 9).
Footnotes
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doi:10.1124/jpet.104.077826.
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ABBREVIATIONS: Ox-LDL, oxidized low-density lipoprotein; RLP, remnant lipoprotein particle(s); VLDL, very low-density lipoprotein; LOX-1, lectin-like receptor for oxidized-low-density lipoprotein; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1; As-LOX-1, antisense-lectin-like receptor for oxidized-low-density lipoprotein; IκBα, inhibitory κBα; NF-κB, nuclear factor-κB; HUVEC, human umbilical vein endothelial cell; ELISA, enzyme-linked immunosorbent assay; LDL, low density lipoprotein; WT, wild-type; BAY 11-7085, (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile.
- Received September 13, 2004.
- Accepted November 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics