Endogenous NO Blockade Enhances Tissue Factor Expression via Increased Ca2+ Influx Through MCP-1 in Endothelial Cells by Monocyte Adhesion
Objective— Ca2+ plays an important role in tissue factor (TF) gene expression. We investigated the role of endogenous nitric oxide (NO) in the induction of TF expression in endothelial cells (ECs) by monocyte adhesion and the mechanisms of NO action.
Methods and Results— Inhibition of endogenous NO by Nω-nitro-l-arginine methyl ester (l-NAME) enhanced TF promoter activity and protein expression induced in human coronary ECs by monocyte adhesion, as well as EC surface TF activity. l-NAME also induced monocyte chemoattractant protein-1 (MCP-1) expression, which was blocked by an NO donor, NOC18. Exogenous MCP-1 enhanced TF expression induced by monocyte adhesion, whereas adenovirus-mediated expression of the mutant MCP-1, 7ND, abolished the l-NAME enhancement of TF expression induced by monocyte adhesion. Monocyte attachment to l-NAME–treated ECs increased Ca2+ influx, which was prevented by NOC18, anti–MCP-1 antibody or 7ND. These results indicate that the binding of increased MCP-1 induced by endogenous NO blockade to CCR2 mediated the enhancement of Ca2+ influx only when monocytes adhered to ECs, which upregulated TF expression in ECs triggered by monocyte adhesion.
Conclusion— MCP-1/CCR2 may play a role in Ca2+ influx-dependent TF regulation in the monocyte–EC interaction in the impairment of NO synthesis.
In the early stage of atherosclerosis, monocyte chemoattractant protein-1 (MCP-1) and adherent molecules are induced in endothelial cells (ECs) under conditions of endothelial dysfunction. These result in monocyte adhesion to the ECs, which plays a crucial role in the progression of atherosclerosis.1,2 MCP-1 also triggers firm adhesion of monocytes to vascular ECs under low flow conditions.3 Namiki et al reported that local overexpression of MCP-1 at the vascular wall induces macrophage accumulation in rabbits.4 In addition, Boring et al showed that the deficiency of CCR2, a receptor for MCP-1, attenuates monocyte/macrophage recruitment at vascular inflammatory sites.5 These observations indicate that MCP-1/CCR2 plays a central role in the monocyte–EC interaction of atherogenesis.
Nitric oxide (NO) synthesized in ECs contributes to normal vascular function, including vascular relaxation, antiinflammation, and antithrombogenicity. Yang et al reported that enhanced production of NO reduces endotoxin- and cytokine-induced tissue factor (TF) expression.6 Endothelial NO synthesis is inhibited by Nω-nitro-l-arginine methyl ester (l-NAME) which increases MCP-1 in ECs in vitro.7 In vivo studies with a rat model showed that inhibition of NO synthesis by l-NAME increased MCP-1 expression and induced increased arterial thrombogenicity including upregulated expression of TF and the resultant thrombin generation.2,8
Intracellular Ca2+ concentration ([Ca2+]i) consists of intracellular mobilization from Ca2+ stores and influx via the plasma membrane. The upregulated expression of TF is inhibited by 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,Nc,Nc-tetraacetic acid tetraacetoxymethylester (BAPTA-AM) chelation of Ca2+ in cultured smooth muscle cells, indicating that Ca2+ signaling is involved in the induction of TF gene expression.9 Ziegelstein et al showed that endothelial Ca2+ signaling, including intracellular Ca2+ mobilization and Ca2+ influx, is induced by monocyte attachment.10 We recently showed that monocyte adhesion upregulates TF expression mediated via the RhoA/NF-κB pathway in ECs.11 However, little is known about the relationships between MCP-1/CCR2 and Ca2+ signaling in TF expression of the monocyte–EC interaction.
In the present study, we examined this using human coronary ECs and peripheral blood monocytes, and found that MCP-1 induced by l-NAME increased Ca2+ signaling via Ca2+ influx in ECs when monocytes adhered to ECs and that the increased Ca2+ entry enhanced the TF expression in ECs compared with non–l-NAME treatment in response to monocyte adhesion.
The sources of most of the conventional reagents for the present study have been described previously.11,12 l-NAME and BAPTA-AM were purchased from Sigma Chemical Co, recombinant human MCP-1 and anti–MCP-1 neutralizing antibody from Genzyme/Techne, and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), an NO donor, from Dojindo Laboratories. Human factor VIIa and factor X were provided by Dr Tomohiro Nakagaki (The Chemo-Sera Therapeutic Research Institute, Kumamoto, Japan). The fluorogenic substrate for factor Xa, Boc-Ile-Glu-Gly-Arg-methylcoumarylamide (MCA) was obtained from the Protein Research Foundation. Nifedipine and nicardipine, l-type Ca2+ channel blockers, were generously provided by Bayer Ltd (Leverkusen, Germany) and Yamanouchi Pharmaceutical Co Ltd (Tokyo, Japan), respectively.
Isolation of Human Peripheral Blood Monocytes
Human peripheral blood monocytes were isolated as described previously.11 Informed consent for experimental examination of human peripheral blood was obtained from all the volunteers, and ethical approval was obtained for the study at Fukushima Medical University.
Human coronary arterial ECs were cultured according to the supplier’s instructions (Clonetics Incand Sanko Junyaku Co Ltd) as described previously.11,12 ECs were pretreated with l-NAME or l-arginine overnight, and 1×104 or 5×105 monocytes were added to confluent ECs in 24-well or 60-mm dishes for immunoassay, Western blotting and TF activity. Supernatants and ECs were used for immunoassays and Western blotting of TF and MCP-1, respectively. The conditioned medium from the coculture of ECs and monocytes or from MCP-1–treated monocytes for 18 hours was added to ECs to determine TF expression. ECs were cultured in 35-mm glass bottom dishes (MatTek Co), and [Ca2+]i was measured in ECs when monocytes attached to them. Culture conditions for promoter analysis are described below.
Enzyme-linked immunosorbent assay (ELISA) for TF and MCP-1 in the medium was performed using ELISA kits (IMUBIND Tissue Factor ELISA Kit, American Diagnostic Inc and AN′ALYZA Human MCP-1, Genzyme/Techne) as described previously.13
Western blotting was performed as described previously.11–13 ECs were washed with PBS three times to minimize the contamination of monocytes. We used mouse monoclonal antibody to human TF (Enzyme Research Laboratories) diluted 1:500, goat polyclonal antibody to human MCP-1 (Genzyme/Techne) diluted 1:500, mouse monoclonal antibody to human CD14 (Santa Cruz Biotechnology) diluted 1:500, and goat polyclonal antibody to human CCR2 (ab1668, Abcam Ltd) diluted 1:1000.14 NF-κB activation was also determined by Western blotting as described previously.11
Determination of EC Surface TF Activity
TF activity was determined as factor X activation by factor VIIa/TF complex on ECs after monocyte adhesion as described previously.15 After 6 hours of incubation, the cells were washed three times with HEPES-buffered saline (20 mmol/L HEPES, 150 mmol/L NaCl, pH 7.5) containing 5 mmol/L CaCl2. Aliquots of a mixture of 1 μL of 10 nmol/L factor VIIa, 5 μL of 400 nmol/L factor X, and 194 μL of HEPES-buffered saline containing 5 mmol/L CaCl2 were added to the washed cells and incubated for 1 hour at 37°C. The generated factor Xa was determined using 100 μL of 200 μmol/L Boc-Ile-Glu-Gly-Arg-MCA. The reaction was determined by measuring the absorbance at 390 nm (excitation) and 444 nm (emission) with SPECTRA microplate reader (Molecular Devices Corporation).
DNA Transfection and Luciferase Assay
The luciferase reporter construct TF/Luc contains the human TF promoter region between −383 and +121 as described previously.11
Measurement of [Ca2+]i
Endothelial [Ca2+]i was measured in response to monocyte attachment or thrombin in the absence or presence of external Ca2+ as described previously.16 ECs were incubated with 10−4 mol/L l-NAME in the absence or presence of anti–MCP-1 antibody (250 μg/mL) overnight. Fura-2–loaded ECs were perfused with prewarmed (37°C) Tyrode solution with or without external Ca2+ in the absence or presence of 20 ng/mL MCP-1, and isolated monocytes were added. [Ca2+]i was also measured in the ECs transfected with LacZ or the dominant negative human MCP-1, 7ND, as described below, in response to monocyte attachment. Ni2+ was used as a nonspecific Ca2+ channel inhibitor.
Adenovirus Gene Transfer
ECs were infected with adenoviruses encoding 7ND (N-terminal MCP-1 deletion variant), which binds to the receptor for MCP-1 (CCR2) and subsequently blocks MCP-1–mediated biological activities, or LacZ at a multiplicity of infection of ≈50, as described previously.2,11
The optical densities of individual immunoblots were analyzed using the National Institutes of Health (Bethesda, MD) IMAGE program as described previously.11,12
Statistical analyses were performed using ANOVA with Scheffé post hoc test if appropriate. A value of P<0.05 was considered significant.
TF Expression in ECs Triggered by Monocyte Adhesion
Western blotting showed that l-NAME alone had no effect on TF expression in ECs (Figure 1A, lane 2), and that monocyte adhesion to ECs induced an increase in the expression of TF compared with ECs cultured alone (Figure 1A, lane 3). Pretreatment of ECs with l-NAME overnight enhanced the increased expression of TF caused by monocyte adhesion (Figure 1A, lane 4). Similar results were obtained by ELISA of the culture medium in 4 independent experiments (ECs; control, ECs+l-NAME; 102±14%, ECs+monocytes (Mo); 210±20%, ECs+l-NAME+Mo; 351±34%, P<0.01, control versus ECs+Mo and ECs+l-NAME+Mo, P<0.05, ECs+Mo versus ECs+l-NAME+Mo, n=4, control values; 48±10 pg/mL).
Promoter analysis revealed that l-NAME treatment had no effect on the promoter activity of TF in ECs cultured alone (Figure 1B, lane 2). Addition of monocytes increased the activity of the TF promoter in ECs after 6 hours of incubation, and pretreatment of ECs with l-NAME overnight significantly augmented the increased promoter activity of TF in ECs induced by monocyte adhesion (Figure 1B, lanes 3 and 4), indicating that the upregulation of the TF gene expression in ECs in response to monocytes was at the transcriptional level.
We also measured EC surface TF activity in response to monocyte adhesion (Figure 1C). A similar result by TF activity was obtained to those of TF expression and promoter activity. Monocyte adhesion induced 45±13% and 81±20% increases in EC surface TF activity in control and l-NAME–treated ECs, respectively (Figure 1C, lanes 3 and 4).
We examined the effects on TF expression of adding monocytes and the conditioned medium from the coculture for 18 hours. At least as determined by Western blotting, adding monocytes alone showed a negligible band (Figure 1D, lane 1), and the addition of the conditioned medium to ECs did not significantly increase the expression of TF (Figure 1D, lanes 2 and 3). In addition, we further examined the expression of relatively monocyte-specific antigen CD14 to clarify that the TF being measured is coming from ECs rather than residual monocytes/macrophages. Western blotting revealed the negligible effect of monocyte adhesion on CD14 expression in ECs after the cultures were washed extensively to remove the monocytes (data not shown).
Effect of l-NAME on MCP-1 Production in ECs
Western blotting demonstrated that 10−4 mol/L l-NAME significantly increased the expression of MCP-1 in ECs cultured alone (Figure 2A, lane 2). A similar result was obtained by ELISA of the culture medium (Figure 2B, lane 2). Coincubation with 500 μmol/L NOC18, an NO donor, prevented the increased production of MCP-1 in ECs induced by 10−4 mol/L l-NAME (Figure 2A and 2B, lane 3).
Effect of MCP-1 on TF Expression
To clarify the relationship between l-NAME–induced MCP-1 production and enhancement of monocyte adhesion-triggered TF expression in ECs, we examined the effect of pretreatment with MCP-1 overnight on TF expression in ECs cultured alone and in those cocultured with monocytes. The concentrations of MCP-1 in the medium of ECs cultured alone and after adding monocytes were 2.5±0.4 ng/mL and 4.3±0.6 ng/mL of 4 separate experiments, respectively. Western blotting revealed that various concentrations of MCP-1 (2.5, 5, 20, and 100 ng/mL) alone did not change the expression of TF in ECs cultured alone (20 ng/mL MCP-1; Figure 3, lane 2). Monocyte adhesion increased the TF expression in ECs as described above (Figure 1), and MCP-1 augmented the increased expression of TF caused by monocyte adhesion (Figure 3, lanes 3 and 4). These results were consistent with those of the l-NAME experiments (Figure 1), suggesting that MCP-1 mediates l-NAME–induced enhancement of TF expression triggered by monocyte adhesion.
We further determined whether conditioned medium from MCP-1–treated monocytes for 18 hours induced an increase of TF expression without the necessity for monocyte adhesion, indicating no significant increase in TF expression in ECs alone stimulated with the MCP-1–treated monocyte-conditioned medium (Figure 3B).
Effect of Blockade of CCR2 on Monocyte Adhesion–Triggered TF Expression
To clarify the role of MCP-1 in TF expression in l-NAME–treated ECs caused by monocytes, we examined the expression of CCR2 in the ECs used in this study and transfected 7ND into ECs to block CCR2, followed by l-NAME treatment and adding monocytes. Figure 4A shows the expression of CCR2 in the ECs in addition to the control expression by monocytes as determined by Western blotting. Importantly, 7ND prevented the augmentation of the increased TF expression caused by monocyte adhesion in l-NAME–treated ECs (Figure 4B, lane 3).
Correlation Between Ca2+ Signaling and TF Expression
We examined the relationship between Ca2+ signaling and TF expression. Without decreasing cell viability (94±3% versus 96±2% of control, not significant), BAPTA-AM (10 μmol/L), an intracellular Ca2+ chelator, prevented the upregulation of TF expression in l-NAME–treated ECs induced by monocyte adhesion as determined by Western blotting (Figure 5A, lane 3). Representative immunoblots are shown at the top of Figure 5A. Similar results were obtained from promoter analysis (Figure 5B). Monocyte adhesion to l-NAME–treated ECs increased the phosphorylation of NF-κB p65, whereas Ca2+ chelation prevented the activation of NF-κB p65 caused by monocyte adhesion in l-NAME–treated ECs (Figure 5C, lane 3). These results indicate that Ca2+ plays a crucial role in TF expression at a site upstream of NF-κB.
Correlation Between Ca2+ Influx and MCP-1
To investigate the mechanism by which l-NAME augments the increased TF expression caused by monocyte adhesion, we focused on the correlation between Ca2+ influx and MCP-1/CCR2. First, we measured endothelial [Ca2+]i in response to monocyte attachment in the presence or absence of external Ca2+. Monocyte attachment induced a rapid and transient increase in [Ca2+]i in ECs. Mean peak [Ca2+]i values increased by 26±5% in the presence of external Ca2+ compared with those in its absence (n=40, each group, P<0.0001, Figure 6A), indicating that the increase in the presence of external Ca2+ was attributable to Ca2+ influx through the plasma membrane in response to monocyte attachment. In the absence of external Ca2+, incubation of ECs with 10−4 mol/L l-NAME or exogenous MCP-1 did not alter the [Ca2+]i peak values in ECs when monocytes attached to them (Figure 6B, lanes 2 and 3). In contrast, in the presence of external Ca2+, there was an increase in [Ca2+]i in response to monocyte attachment in l-NAME–treated cells compared with the control (Figure 6C, lane 2). Coincubation with NOC18 prevented the [Ca2+]i increase caused by l-NAME in ECs triggered by monocytes (Figure 6C, lane 3). The l-NAME–induced [Ca2+]i increase was also abolished by incubating ECs with anti–MCP-1 overnight (Figure 6C, lane 4).
To further examine the role of MCP-1/CCR2 in the [Ca2+]i increase in the presence of external Ca2+ in response to monocyte attachment, we transfected the 7ND gene into ECs. Transfection of the LacZ gene into ECs did not significantly affect the [Ca2+]i increase caused by l-NAME in response to monocyte attachment (Figure 6D, lane 2). 7ND reversed the l-NAME–induced enhancement of [Ca2+]i increase in response to monocyte attachment to ECs (Figure 6D, lane 3).
Ni2+ (5 mmol/L) blocked the Ca2+ influx in response to monocyte attachment in the presence of external Ca2+ (Figure 6E, lane 4). The [Ca2+]i increase induced by l-NAME or MCP-1 in ECs in response to monocyte attachment was also completely blocked by Ni2+ (Figure 6E, lanes 5 and 6).
To mimic the effect of monocyte attachment on Ca2+ influx in ECs, we used thrombin as another agonist of Ca2+ influx. Thrombin (1 U/mL) induced a rapid increase in [Ca2+]i in the presence or absence of external Ca2+ in ECs. Exogenous MCP-1 (20 ng/mL) augmented the Ca2+ influx caused by thrombin in the presence of external Ca2+, but not the intracellular Ca2+ mobilization in the absence of external Ca2+ (Figure 6F), although MCP-1 alone did not induce Ca2+ influx or intracellular Ca2+ mobilization in ECs.
Moreover, nifedipine or nicardipine did not change the monocyte attachment–induced increase in [Ca2+]i in l-NAME–treated ECs, suggesting that it is unlikely that l-type voltage-dependent Ca2+ channels are involved (data not shown).
In this study we showed that treatment of ECs with l-NAME not only enhanced the upregulation of TF expression triggered by monocyte adhesion but also the Ca2+ influx in response to monocyte attachment. Our data also showed that monocyte attachment-induced increase in Ca2+ influx was mediated via MCP-1/CCR2 in l-NAME–treated cells.
In the present study, blockade of endogenous NO by l-NAME induced modest but clear increases in endothelial TF promoter activity, TF expression, and surface TF activity in response to monocyte adhesion by 71±9%, 119±18%, and 81±20%, respectively (Figure 1), although the degree of increase was modest compared with those induced by other agonists such as lipopolysaccharide (LPS), interleukin (IL)-1β, and Ca2+ ionophore in ECs.6,17 Especially, EC surface TF activity as determined by factor X activation increased in response to monocyte adhesion in l-NAME–treated ECs, indicating that the modest but definite upregulation of coagulation cascade. These results, to our knowledge, are the first in the literature to demonstrate the significance of endogenous NO blockade in TF gene expression induced in ECs by monocyte adhesion at least in vitro using the various assay systems as described above. This would suggest that monocyte adhesion to NO-impaired ECs promotes the procoagulability of the vascular wall in the initial steps of atherosclerosis. The issue that monocyte adhesion induces significant amounts of TF in NO-impaired ECs in vivo remains to be elucidated. Because l-NAME produced MCP-1 and CCR2 was expressed in ECs used in this study which were consistent with previous reports.7,18–20 Thus, we determined whether or not MCP-1 directly induces TF gene expression in ECs. There was no significant difference in TF promoter activity and TF expression in response to the various concentrations of MCP-1 in ECs cultured alone, suggesting that MCP-1 does not induce TF directly in ECs. The medium from MCP-1–treated monocytes did not augment TF expression in ECs cultured alone, indicating no involvement of MCP-1–derived other factor(s) from monocytes. However, in response to monocyte adhesion, the elevated or exogenous MCP-1 augmented the increased expression of TF in l-NAME–treated ECs, which was prevented by blockade of CCR2 by 7ND. This suggests that MCP-1 only works when monocytes adhere to ECs.
The induction of TF is Ca2+-dependent in various cells.9,17 In this study, TF expression was not altered by l-NAME in ECs cultured alone with no significant change in [Ca2+]i. When monocytes were attached, there were increases in endothelial [Ca2+]i and NF-κB activation in l-NAME–treated ECs compared with untreated ECs (Figures 5C and 6⇑C). BAPTA-AM prevented the upregulation of TF expression caused by monocyte adhesion as well as the increased activation of NF-κB in l-NAME–treated ECs (Figure 5). These results indicate that Ca2+ signaling is upstream of NF-κB in monocyte adhesion to ECs. Ca2+ experiments performed in the present study demonstrated that l-NAME–treated ECs had increased Ca2+ influx unrelated to voltage-dependent l-type Ca2+ channel when monocytes were attached, and that an NO donor reversed the effect of l-NAME on the increased Ca2+ influx in this monocyte–EC interaction.
Thus, we clarified the relationship between MCP-1/CCR2 and Ca2+ influx in the monocyte–EC interaction. l-NAME treatment and exogenous MCP-1 augmented the Ca2+ influx caused by monocyte attachment in ECs, but not the intracellular Ca2+ mobilization. Blockade of CCR2 by 7ND or an antibody to MCP-1 prevented the increase in monocyte attachment–induced Ca2+ influx in ECs caused by l-NAME. Our results show, again for the first time, that the increased endothelial MCP-1 caused by NO blockade augmented Ca2+ influx in response to monocyte attachment, which may serve to enhance TF gene expression at the transcriptional level. For a better understanding of the mechanism involved, we used thrombin as another agonist of Ca2+ influx. Exogenous MCP-1 also enhanced the Ca2+ influx induced by thrombin in ECs (Figure 6F). These findings suggest that the binding of increased MCP-1 to G-protein–coupled receptor CCR2 may directly enhance the signaling to the Ca2+ channel which is responsible for the Ca2+, only when ECs are stimulated with agonists such as monocyte attachment and thrombin. Mirzadegan et al showed that CCR2 antagonists inhibit MCP-1–stimulated Ca2+ influx in CCR2-transfected cells.21 Taken together, our results suggest that MCP-1 may play a role in Ca2+ influx involving CCR2-activated channel. The detailed mechanism by which MCP-1/CCR2 modulates agonist-mediated Ca2+ influx induced in ECs by monocytes attachment must be elucidated.
In a rat model, increased MCP-1 expression is responsible for macrophage accumulation in the vascular wall induced by chronic blockade of NO synthesis by l-NAME.2 Using the same model of long-term inhibition of NO increased arterial thrombogenicity along with upregulated expression of TF.8 Our study may thus provide a possible mechanism involving MCP-1/CCR2-mediated Ca2+ influx for the increased TF expression in the monocyte–EC interaction in their animal model in vivo. The present study suggests the role of MCP-1/CCR2 in procoagulant activity in the vascular wall, which is critical for the development of early atherosclerosis and the formation of vulnerable plaques.1,5In conclusion, our study presents evidence that endogenous NO blockade increases endothelial Ca2+ influx in response to monocyte attachment, and that this is mediated via MCP-1/CCR2, which causes upregulation of TF in ECs. This indicates that impairment of NO synthesis enhances TF expression via inflammation-dependent Ca2+ influx in the monocyte–EC interaction.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14370232) and a Research Grant for Cardiovascular Diseases from the Ministry of Health, Labor, and Welfare of Japan.
- Received November 22, 2004.
- Accepted July 1, 2005.
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