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CARDIOVASCULAR

Angiotensin II Type 1 Receptor Antagonists Inhibit Basal As Well As Low-Density Lipoprotein and Platelet-Activating Factor-Stimulated Human Monocyte Chemoattractant Protein-1

Department of Medicine, University of Western Australia, and the West Australian Institute for Medical Research, Perth, Western Australia

Received December 19, 2002; accepted February 24, 2003.

	Abstract

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Monocyte chemoattractant protein-1 (MCP-1) is a potent chemotactic agent for monocytes and other cells and is thought to be involved in atherosclerosis, recruiting monocytes to the subendothelial space or to the site of inflammation. Angiotensin II has been demonstrated, at least in animal models, to stimulate MCP-1 expression. We investigated the effect of the angiotensin II type 1 (AT1) receptor antagonists irbesartan and losartan on MCP-1 production by freshly isolated human monocytes. Irbesartan and losartan inhibited basal MCP-1 production in a dose-dependent manner. Low-density lipoprotein (LDL) stimulated MCP-1 in a concentration-dependent manner, with 200 µg/ml LDL protein giving a 2-fold increase in MCP-1. Irbesartan and losartan dose dependently blocked LDL-stimulated MCP-1. An angiotensin II type 2 receptor antagonist, S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid (PD123319), had no significant effect on basal MCP-1 levels or LDL-stimulated MCP-1. After noting homology between the AT1 receptor and the platelet-activating factor (PAF) receptor, we showed that irbesartan inhibited both [³H]PAF binding to human monocytes and carbamyl-PAF stimulation of MCP-1. However, irbesartan affinity for the PAF receptor was 700 times less than PAF, suggesting that there may be another mechanism for irbesartan inhibition of PAF-stimulated MCP-1. This is the first report showing that AT1 receptor antagonists inhibit basal as well as LDL- and PAF-stimulated MCP-1 production in freshly isolated human monocytes.

Monocyte/macrophages make up the bulk of infiltrated leukocytes in atherosclerotic plaque and are considered to be the main inflammatory mediators in atherosclerosis. Numerous studies suggest that MCP-1 has an important role in the infiltration of monocytes into lesions (Nelken et al., 1991; Yla-Herttuala et al., 1991; Yu et al., 1992). Mice lacking MCP-1 exhibited attenuated atherosclerosis and monocyte accumulation in the artery (Gu et al., 1998). Deletion of the receptor for MCP-1 resulted in decreased lesion formation (Boring et al., 1998).

Ang II has been implicated in the pathogenesis of atherosclerosis. Ang II can stimulate production of reactive oxygen species and increase expression of proinflammatory gene products, and both oxidative stress and inflammation are thought to have a role in atherogenesis (Griendling and Alexander, 1997; Ross, 1999). Studies in experimental models of atherosclerosis have shown that inhibition of angiotensin-converting enzyme or blockade of AT1 receptors decreases atherosclerosis (Schuh et al., 1993; Keidar et al., 1997; Makaritsis et al., 1998).

Ang II has been shown to increase MCP-1 mRNA expression in cultured monocytic U937 cells and in rat thoracic aortic vascular smooth muscle cells (Hernandez-Presa et al., 1997). Ang II can also activate MCP-1 gene transcription and stimulates MCP-1 mRNA in rat aortic smooth muscle cells (Chen et al., 1998). The increase in MCP-1 mRNA can be prevented by the AT1 receptor antagonist losartan (Chen et al., 1998).

We investigated the effect of Ang II and the AT1 receptor antagonists irbesartan and losartan on the production of MCP-1 by human monocytes. Ang II had no effect on MCP-1 levels in our study, perhaps due to its metabolism during incubation with monocytes. Irbesartan and losartan inhibited both basal and stimulated release of MCP-1 possibly through a non-AT1 receptor-related mechanism.

Platelet-activating factor (PAF) is a phospholipid with proinflammatory and thrombogenic properties, and PAF has been shown to stimulate MCP-1 production (Sugano et al., 2001). We studied the effect of the AT1 receptor antagonists irbesartan and losartan on [³H]PAF binding and carbamyl-PAF (c-PAF) stimulation of MCP-1 in human monocytes.

Our results suggest a mechanism for inhibition of atherosclerosis by AT1 receptor antagonists involving inhibition of basal MCP-1 levels and blockade of the effects of PAF and perhaps other structurally related molecules, resulting in decreased MCP-1 production and subsequent cell migration.

	Materials and Methods

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Materials. RPMI 1640 medium, penicillin/streptomycin, and fetal calf serum were purchased from Invitrogen (Carlsbad, CA). Media and other reagents were made up in sterile water (Baxter Health-care, Old Toongabbie, NSW, Australia). The fetal calf serum was heat-inactivated before use. Ficoll-Paque and PD 10 Sephadex G-25 columns were purchased from Amersham Biosciences AB (Uppsala, Sweden). Dynabeads [M-450 CD2 (pan T)] were purchased from Dynal (Victoria, Australia). Carbamyl PAF (PAF agonist) was purchased from BIOMOL Research Laboratories (Sapphire Bioscience, St. Crows Nest, NSW, Australia). Radioactively labeled 1-O-[³H]octadecyl)[1-O-[³H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine] (PAF) (6.03 TBq/mmol) was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). The AT2 receptor antagonist PD123319 was purchased from Sigma/RBI (Natick, MA). WEB 2086 (PAF antagonist) was from Boehringer Ingelheim GmbH (Ingelheim, Germany). Irbesartan was a gift from Bristol-Myers Squibb Co. (Stamford, CT). Losartan was a gift from Merck Sharp and Dohme (South Granville, NSW, Australia). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cells. Human peripheral blood mononuclear cells were prepared by centrifuging blood (containing 1 mg/ml EDTA) from healthy volunteers, on Ficoll-Paque at 500g for 30 min at 20°C. Cells in the resultant interface layer were further purified by washing with centrifuged plasma and then with Hanks' balanced salt solution (HBSS) without calcium or magnesium, centrifuging at 100g for 10 min at 4°C to remove platelets. The cells were counted and CD2 Dynabeads added to remove T cells, according to the manufacturer's instructions. This resulted in a cell population enriched in monocytes (60% CD14+) and limited possible pre-experimental activation of cells if adherence had been used for purification. The cells were counted and viability assessed with trypan blue. Cells were resuspended in HBSS or in RPMI 1640 medium containing 2% penicillin/streptomycin and 1% heat-inactivated fetal calf serum (HIFCS), before plating in Falcon 24-well culture plates (BD Biosciences, Franklin Lakes, NJ) at 1 to 2 x 10⁶/well (0.8–1.0 ml/well). Reagents were added and the cells were incubated overnight at 37°C in 5% CO₂. The following day, the medium was removed, centrifuged to pellet any cells, the supernatant stored at –80°C, and cell viability assessed using trypan blue.

LDL Preparation. Blood from healthy volunteers was collected into EDTA (1 mg/ml). LDL was isolated immediately from fresh plasma by density gradient ultracentrifugation as described previously (Croft et al., 1991). Briefly, plasma density was increased to 1.07 by addition of NaCl and then a four-step gradient was constructed over the plasma using the following densities: 1.8 ml of 1.063 kg/l NaCl, 1.8 ml of 1.04 kg/l NaCl, 1.8 ml of 1.02 kg/l NaCl, and 2.1 ml of water. Samples were ultracentrifuged at 205,000g (average) for 20 h using a Centrikon T-1190 ultracentrifuge (Kontron Instruments, Milan, Italy). The LDL band was collected by aspiration and passed through a PD10 Sephadex column (Pharmacia AB, Uppsala, Sweden) to remove the excess salt and the majority of the EDTA. The LDL was stored in the dark at 4°C. LDL protein was determined by a modification of the Lowry method (Markwell et al., 1978) using BSA protein standard (Sigma-Aldrich). Just before use, isolated LDL was passed through a second PD10 column to remove the remaining EDTA.

Assay for MCP-1. Human MCP-1 OptEIA (BD PharMingen, San Diego, CA) and Costar enzyme immunoassay/radioimmunoassay one-half-area flat bottom 96-well plates (Corning Glassworks, Corning, NY) were used to measure MCP-1 levels in cell supernatants. The sensitivity of the assay was 30 pg/ml, the intra-assay coefficient of variation was 10%, and the interassay coefficient of variation was 12%.

PAF Binding Assay. Cells were washed twice with cold HEPES-Tyrode's buffer (Ishii et al., 1997) containing 0.25% (w/v) fatty acid-free human serum albumin (HSA) and kept on ice. Cells (1–2 x 10⁶/200-µl/tube) were preincubated for 5 min with (nonspecific binding) or without (total binding) unlabeled PAF (10 µM) or test reagents before addition of [³H]PAF (4 nM final concentration) and incubation on ice for 10 min. Cells were then washed three times with the cold buffer and solubilized with 1% Triton X-100. Radioactivity associated with the cells was measured with liquid scintillation counting. Separate experiments were carried out using varying concentrations of [³H]PAF, and saturation binding data were analyzed with nonlinear regression using GraphPad Prism 3 (GraphPad Software Inc., San Diego, CA). Competitive binding experiments were carried out measuring the binding of 4 nM [³H]PAF in the presence of varying concentrations of irbesartan and unlabeled PAF, and IC₅₀ (inhibitory concentration 50%) concentrations were determined for each ligand using GraphPad Prism 3.

Searching GenBank. GenBank was searched using human AT1 receptor GenBank accession no. AAB34644 [GenBank] .

Statistics. Results are expressed as mean ± S.E.M., and data were analyzed using the paired samples t test. Data for Fig. 8 was expressed as percentage of control (100%) values before the t test because of large variability in MCP-1 levels between monocyte populations.

View larger version (21K): [in this window] [in a new window]   Fig. 8. AT1 receptor antagonists inhibit carbamyl-PAF-stimulated MCP-1. Cells were isolated as described under Materials and Methods and resuspended in HBSS (A). Cells were preincubated for 20 min with varying concentrations of irbesartan or WEB 2086 or medium (control), and then c-PAF (0.5 µM) was added and the cells incubated overnight. Results are expressed as percentage of control levels and are the mean ± S.E. from three to five experiments. B, cells were resuspended in RPMI 1640 medium + 1% HIFCS and preincubated for 20 min with irbesartan (50 µM), losartan (100 µM), WEB 2086 (50 µM), or medium (control). Varying concentrations of c-PAF were added and the cells incubated overnight. Results are expressed as percentage of control levels (mean = 4015 ± 2276 pg/ml) and are the mean ± S.E. from two to five experiments.

	Results

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Time Course of MCP-1 Production. Isolated human monocytes were incubated at 37°C, and cell supernatants were collected at varying time points. MCP-1 production increased over time (Fig. 1), so future incubations were carried out overnight.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Time course of MCP-1 production in human monocyte supernatants. Human peripheral blood mononuclear cells were isolated as described under Materials and Methods and resuspended in HBSS. The cells were incubated at 37°C, and cell supernatants were collected after 2-, 4-, 8-, and 20-h incubation. Results are the mean of two experiments.

Effect of Ang II and Irbesartan on MCP-1 Production by Human Monocytes. Human monocytes in HBSS were preincubated for 20 min with or without irbesartan (50 µM) and then incubated overnight with or without Ang II (10^–7 M) (Fig. 2). Levels of MCP-1 produced by cells incubated with DMSO (vehicle for irbesartan) were not different from control cells incubated with medium alone. There was no significant effect of Ang II on basal MCP-1 production. Irbesartan reduced basal MCP-1 by more than 90% under these conditions.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Inhibition of MCP-1 in human monocytes by the AT1 receptor antagonist irbesartan (IRB). IRB inhibits and Ang II has no effect on basal MCP-1. Cells were isolated as described under Materials and Methods and resuspended in HBSS. After preincubation for 20 min with IRB (50 µM, AT1 antagonist), DMSO (D) (0.05%, vehicle for IRB), or medium (control, C), Ang II (AII, 10–7 M) was added, and the cells incubated overnight. Results are the mean ± S.E. from six experiments. **, p = 0.001 versus control.

AT1 Receptor Antagonists Dose Dependently Inhibit Basal and LDL-Stimulated MCP-1 Production. Fig. 3A shows that increasing concentrations of irbesartan resulted in increasing inhibition of basal MCP-1 production by human monocytes. At 15 µM irbesartan, MCP-1 was reduced by more than 60% (p < 0.02), whereas at 50 µM irbesartan, MCP-1 inhibition was greater than 95% (p = 0.001). A similar effect was seen with losartan, another AT1 antagonist, at concentrations 2 times higher than irbesartan (Fig. 3B).

View larger version (22K): [in this window] [in a new window]   Fig. 3. AT1 receptor antagonists inhibit basal and LDL-stimulated MCP-1. A, monocytes were isolated as described under Materials and Methods, resuspended in HBSS, and preincubated for 20 min with 0, 5, 15, or 50 µM irbesartan (IRB). Varying concentrations of LDL were added, and the cells were incubated overnight. Results are the mean ± S.E. from two to four experiments. B, isolated cells were preincubated for 20 min with 0, 25, 50, or 100 µM losartan. Medium or LDL (200 µg/ml final concentration) was added, and the cells were incubated overnight. Results are the mean of two experiments.

Addition of increasing concentrations of LDL before the overnight incubation resulted in dose-dependent increases in MCP-1, with 200 µg/ml LDL protein resulting in a 2-fold increase in MCP-1 levels compared with control cells (p < 0.04) (Fig. 3A). LDL-stimulated MCP-1 levels were reduced by irbesartan in a dose-dependent manner (200 µg/ml LDL plus 50 µM irbesartan, p < 0.02; 100 µg/ml LDL plus 50 µM irbesartan, p < 0.02; 50 µg/ml LDL plus 50 µM irbesartan, p < 0.03) (Fig. 3A). Losartan dose dependently reduced MCP-1 levels stimulated with 200 µg/ml LDL (Fig. 3B).

Specificity of AT1 Antagonist. To determine whether this result was specific for AT1 receptor antagonists, the effect of an AT2 receptor antagonist, PD123319, was examined. Figure 4 shows that the AT2 antagonist had no significant effect on basal MCP-1 levels, whereas the same concentration of AT1 antagonist almost completely blocks MCP-1 production. The LDL-stimulated increase in MCP-1 was blocked by the AT1 antagonist, but the same concentration of AT2 antagonist had no significant effect. These data suggest that the effect on both basal and LDL-stimulated MCP-1 is due mainly to the AT1 type receptor antagonist.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Specificity of AT1 receptor antagonists. Cells were isolated as described under Materials and Methods, resuspended in HBSS, and preincubated for 20 min with irbesartan (IRB) (50 µM, AT1 antagonist), PD123319 (PD) (50 µM, AT2 antagonist), DMSO (D) (0.05%, vehicle), or medium (control, C). Medium or LDL (200 µg/ml protein final concentration) was added, and the cells were incubated overnight. Results are the mean ± S.E. from seven experiments. **, p < 0.005 versus control; ##, p = 0.001 versus C + LDL.

Possible Binding of Irbesartan to Other Receptors. The inhibition of basal MCP-1 production by irbesartan and losartan suggested the possibility that these reagents are able to bind to cell receptors other than AT1. A previous study (Raiden et al., 1997) reported that losartan blocked the binding of [³H]fMLP to the fMLP receptor, which was found to have 25 to 30% structural homology with the AT1 receptor. The PAF receptor is in the same family of chemoattractant receptors as the fMLP receptor. A search conducted using GenBank found 22% sequence similarities between the angiotensin receptor and the PAF receptor. This raised the possibility that irbesartan and losartan may bind to the PAF receptor.

Irbesartan Inhibits PAF Binding to Human Monocytes. To determine whether irbesartan binds to the PAF receptor, human monocytes were preincubated with irbesartan, losartan, WEB 2086 (PAF receptor antagonist), or unlabeled PAF (nonspecific binding) before addition of [³H]PAF (Fig. 5). Irbesartan and losartan dose dependently inhibited [³H]PAF binding, indicating that they bind to the PAF receptor. Their efficacy was similar to the PAF antagonist WEB 2086 in our study. PAF is very hydrophobic and exhibits high nonspecific binding to the lipid bilayer of plasma membranes (Chao and Olson, 1993), which may account for some of the uninhibitable [³H]PAF binding.

View larger version (15K): [in this window] [in a new window]   Fig. 5. AT1 receptor antagonists inhibit [3H]PAF binding to human monocytes. Cells were isolated as described under Materials and Methods, resuspended in HEPES-Tyrode's + 0.25% HSA, and preincubated with unlabeled PAF (10 µM, nonspecific binding); varying concentrations of irbesartan (IRB), losartan (LOS), or WEB 2086 (WEB); or medium before addition of [3H]PAF as described under Materials and Methods. Bound [3H]PAF cpm was calculated by subtracting nonspecific counts (bound in the presence of unlabeled PAF) from total [3H]PAF counts bound. Results are the mean ± S.E. from three or four experiments. *, p < 0.05 versus control.

Competitive Binding. Varying concentrations of [³H]PAF were added to human monocytes to determine binding parameters (Fig. 6). Data were analyzed using nonlinear regression in GraphPad Prism 3 to obtain B_max (36,280) and K_D (41 nM) values for [³H]PAF.

View larger version (14K): [in this window] [in a new window]   Fig. 6. [3H]PAF monocyte binding parameters. Cells were isolated as described under Materials and Methods, resuspended in HEPES-Tyrode's + 0.25% HSA, and varying concentrations of [3H]PAF were added as described under Materials and Methods. Bound [3H]PAF cpm was calculated as described in Fig. 5. Results are the mean ± S.E. from five experiments. Saturation binding data were analyzed with nonlinear regression using GraphPad Prism 3, and the data were displayed as a Scatchard plot (inset).

Competitive binding experiments were carried out using irbesartan and unlabeled PAF (Fig. 7), and the IC₅₀ value (inhibitory concentration 50%) determined. K_i (affinity of PAF receptor) was calculated from K_D and IC₅₀ values using Prism. Using five different monocyte preparations, unlabeled PAF gave a mean IC₅₀ value of 6.4 ± 1.6 x 10^–8 M and a K_i value of 5.8 ± 1.5 x 10^–8 M. Irbesartan gave a mean IC₅₀ value of 49.0 ± 4.3 x 10^–6 M and a K_i value of 43.1 ± 3.9 x 10^–6 M. These results indicated that irbesartan had 700 to 800 times lower affinity for the PAF receptor than unlabeled PAF.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Competitive binding curves for irbesartan and PAF. Cells were isolated as described under Materials and Methods, resuspended in HEPES-Tyrode's + 0.25% HSA, and preincubated with unlabeled PAF (10 µM, nonspecific binding), varying concentrations of irbesartan or unlabeled PAF, or medium before addition of [3H]PAF as described under Materials and Methods. Bound [3H]PAF cpm was measured, and IC50 values were determined for each ligand using GraphPad Prism 3. Shown are two examples of competition binding curves.

Irbesartan Inhibits PAF-Stimulated MCP-1 Production. Human monocytes were preincubated with irbesartan, losartan, or the PAF antagonist WEB 2086, and then incubated overnight with the stable PAF agonist carbamyl-PAF, and supernatant MCP-1 levels were measured. The stable metabolite of PAF was required for stimulation of MCP-1, probably because native PAF is degraded during incubation with cells. This experiment was carried out in HBSS (Fig. 8A) and RPMI 1640 medium containing 1% HIFCS, a more physiological medium (Fig. 8B). In serum-free HBSS (Fig. 8A), irbesartan dose dependently inhibited basal (control plus irbesartan, p < 0.002 for all irbesartan concentrations) and carbamyl-PAF (c-PAF)-stimulated (control plus c-PAF, p < 0.03; c-PAF plus irbesartan, p < 0.002 for all irbesartan concentrations) MCP-1 production. WEB 2086 also dose dependently inhibited basal (control plus WEB 2086, p < 0.05 for all WEB 2086 concentrations) and c-PAF-stimulated (c-PAF plus 20 or 50 µM WEB 2086, p < 0.009) MCP-1 release, but was less effective than irbesartan.

Carbamyl-PAF stimulated monocyte MCP-1 production in a dose-dependent manner (Fig. 8B) (control plus c-PAF 1 µM, p < 0.04). Figure 8B shows that in RPMI 1640 medium containing 1% HIFCS, irbesartan, and losartan inhibited c-PAF MCP-1 stimulation, similar to the PAF antagonist WEB 2086 (c-PAF 1 µM plus irbesartan, p < 0.04; plus losartan, p < 0.09; plus WEB 2086, p < 0.04) (c-PAF 0.6 µM plus irbesartan, p < 0.05; plus losartan, p < 0.04; plus WEB 2086, p < 0.07). Basal MCP-1 levels were significantly reduced in the presence of irbesartan, losartan, and WEB 2086 (control plus irbesartan, p < 0.001; plus losartan, p < 0.001; plus WEB 2086, p < 0.002). Thus, the receptor or mechanism involved in the inhibition of human monocyte MCP-1 is sensitive to LDL, PAF, AT1 antagonists, and PAF antagonists.

PAF Receptor Antagonist Inhibits Basal and LDL-Stimulated MCP-1 Production. Fig. 9 shows that increasing concentrations of WEB 2086 resulted in increasing inhibition of basal MCP-1 production. At 10 µM WEB 2086, there was a 20% reduction (p < 0.05), whereas 50 µM WEB 2086 resulted in a 60% decrease (p < 0.009) in basal MCP-1. Irbesartan was more effective, with 10 µM irbesartan reducing basal MCP-1 levels by 40% (p < 0.05), 20 µM giving 75% reduction (p < 0.02), and 50 µM reducing levels by greater than 95% (p < 0.008). LDL (200 µg/ml) significantly increased MCP-1 production 2-fold (p < 0.01). WEB 2086 inhibited LDL stimulated MCP-1 levels by 30% (p < 0.02) at 20 µM and by 50% (p < 0.02) at 50 µM. Irbesartan was again more effective, reducing LDL-stimulated MCP-1 by 50% (p < 0.02) at 10 µM, 70% (p < 0.001) at 20 µM, and greater than 95% (p < 0.002) at a concentration of 50 µM.

View larger version (20K): [in this window] [in a new window]   Fig. 9. PAF receptor antagonist inhibits basal and LDL-stimulated MCP-1. Monocytes were isolated as described under Materials and Methods, resuspended in HBSS, and preincubated for 20 min with 0, 10, 20, or 50 µM irbesartan or WEB 2086. Medium or LDL (200 µg/ml final concentration) was added, and the cells were incubated overnight. Results are mean ± S.E. from three experiments.

	Discussion

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Recent studies suggest that AT1 receptor antagonists used in the treatment of hypertension may also be beneficial in the treatment of atherosclerosis. Lesion development in Apo E-deficient mice was significantly reduced by treatment with the AT1 antagonists irbesartan (Dol et al., 2001) and losartan (Keidar et al., 1997). Irbesartan-treated mice had less macrophages in the lesion area, suggesting irbesartan inhibits monocyte/macrophage influx into the vessel wall. Irbesartan treatment also decreased MCP-1 mRNA levels and MCP-1 immunostaining in the lesion area (Dol et al., 2001). Treatment of patients with coronary artery disease with irbesartan reduced levels of the inflammatory markers vascular cell adhesion molecule-1, tumor necrosis factor-, and superoxide (Navalkar et al., 2001). Lipid peroxidation, superoxide levels, and monocyte-binding capacity were reduced in subjects with coronary artery disease receiving irbesartan (Khan et al., 2001).

Our in vitro studies with freshly isolated human monocytes suggest that AT1 receptor antagonists may inhibit the inflammatory component of atherosclerosis. We found that irbesartan and losartan inhibited basal production of the inflammatory marker MCP-1 by human monocytes in the absence of any stimulant. Yanagitani et al. (1999) showed that the AT1 antagonist CV11974 decreased basal levels of peroxide production in macrophages. This may be related to our findings, because reactive oxygen species, which include peroxide and superoxide, are involved in MCP-1 production (De Keulenaer et al., 2000).

Ang II stimulated MCP-1 mRNA in the cultured monocytic cell line U937 and in cultured rat thoracic aortic vascular smooth muscle cells (Hernandez-Presa et al., 1997). In rat aortic smooth muscle cells, Ang II stimulated MCP-1 mRNA and the increase was prevented by losartan (Chen et al., 1998). We found that in freshly isolated human monocytes, Ang II had no effect on MCP-1 protein levels, whereas the AT1 receptor antagonists irbesartan and losartan inhibited basal MCP-1. It is possible that the absence of an effect of Ang II was caused by its degradation during the 20-h incubation with cells. It may be necessary to use a stable derivative of Ang II to see an effect under these conditions. It is also possible that there was basal release of Ang II by the cells and that MCP-1 stimulation by basal Ang II was inhibited by irbesartan and losartan. These hypotheses were not examined in our study. Our results suggested the possibility that inhibition of basal MCP-1 was independent of the AT1 receptor. Raiden et al. (1997) reported that the AT1 receptor antagonist losartan inhibited neutrophil recruitment and activation by fMLP, by inhibiting neutrophil binding of fMLP through a mechanism independent of losartan binding to AT1 receptors. The AT1 receptor and the high-affinity receptor for fMLP share 25 to 30% sequence identity.

The receptors for Ang II, fMLP, PAF, complement protein fragment 5a, and the chemokines belong to the family of seven-transmembrane-domain rhodopsin-like G protein-coupled receptors (Murphy, 1994). On searching GenBank, we found that the AT1 receptor has 22% homology with the PAF receptor. We showed that irbesartan and losartan inhibited both binding of [³H]PAF to monocytes and PAF stimulation of MCP-1 in monocytes. When irbesartan binding to the human monocyte PAF receptor was examined more closely, it was found that the affinity of the PAF receptor for irbesartan (43 µM) was about 700 times less than affinity for PAF (0.06 µM). This suggests that the inhibition of PAF-stimulated MCP-1 by irbesartan may be partially independent of irbesartan binding to the PAF receptor.

PAF stimulates thromboxane A₂ (TXA₂) production (Ishizuka et al., 1994), a TXA₂ analog stimulates MCP-1 production and TXA₂ receptor antagonists inhibit PAF-induced MCP-1 in human umbilical vein endothelial cells (HUVECs) (Ishizuka et al., 2000). These studies suggest that the TXA₂ receptor may be involved in PAF-stimulated MCP-1 in HUVECs. Irbesartan inhibited TXA₂-induced vasoconstriction in canine coronary arteries and human platelet aggregation, and high concentrations of irbesartan significantly inhibited TXA₂ receptor antagonist binding (Li et al., 2000). However, the affinity of the TXA₂ receptor for irbesartan (10 µM) was 1400 times less than the affinity for the TXA₂ analog (7 nM) (Li et al., 2000). These findings suggest that inhibition of PAF-stimulated MCP-1 by irbesartan may be partially independent of irbesartan binding to the TXA₂ receptor. Irbesartan was 2-fold more potent than losartan in the inhibition of TXA₂ analog-induced vasoconstriction (Li et al., 2000) as it was in the inhibition of MCP-1 production in monocytes in the current study. Other AT1 antagonists may behave differently to irbesartan and losartan, depending on their binding properties.

A 300-mg dose of irbesartan results in human plasma concentrations of around 10 µM irbesartan (Pool et al., 1998). Our results suggest that this concentration of irbesartan inhibits basal and LDL-stimulated MCP-1 production from monocytes (Figs. 3A and 9). Sugano et al. (2001) showed that 10 nM carbamyl-PAF stimulated MCP-1 in cultured human uterine cervical fibroblasts, and this stimulation of MCP-1 was abolished by coincubation with 10 µM (1000-fold excess) WEB 2170, a PAF receptor antagonist, in medium containing 0.1% BSA. We showed that 1 µM carbamyl-PAF stimulated MCP-1 production in human monocytes. This stimulation was inhibited by 50 µM (50-fold excess) WEB 2086, a PAF receptor antagonist, and by 50 µM of the AT1 receptor antagonist irbesartan (Fig. 8B), in medium containing 1% serum, a more physiological environment with more potentially complicating factors than 0.1% BSA.

The involvement of PAF in atherosclerosis was suggested in a study where the PAF receptor antagonist WEB 2086 inhibited fatty streak development in LDL receptor null mice fed a western diet (Subbanagounder et al., 1999). They found that the in vitro inhibitory effects of WEB 2086 on monocyte binding to endothelial cells did not seem to be due to blocking the PAF receptor. Similarly, in our experiments, the inhibitory effects of irbesartan, losartan, and WEB 2086 on carbamyl-PAF stimulation of MCP-1 may not be due entirely to blocking the PAF receptor.

Ether- and ester-containing PAF-like lipids are generated during oxidation of LDL (Tokumura et al., 1996), and PAF and PAF-like oxidized phospholipids have been shown to activate monocytes via the PAF receptor (Heery et al., 1995; Tokumura et al., 1996; Lehr et al., 1997). Hayek et al. (2000) have shown that the AT1 antagonist losartan significantly reduced human monocyte-derived macrophage uptake of oxidized LDL and also decreased CD36 (an ox LDL receptor) mRNA expression. These reports raise the possibility that LDL incubated overnight with monocytes, as in our study, becomes oxidized and PAF-like lipids are generated and stimulate MCP-1 production. LDL may not be working exclusively through the PAF receptor because the PAF receptor antagonist did not completely inhibit LDL stimulated MCP-1 (Fig. 9), although it blocked PAF-stimulated MCP-1 (Fig. 8A). It is possible that other PAF receptor antagonists with different binding characteristics may show greater inhibition.

Our study shows that the AT1 receptor antagonists irbesartan and losartan decreased basal MCP-1 levels in human monocytes possibly through a mechanism independent of binding to the AT1 receptor. LDL-stimulated and carbamyl-PAF-stimulated MCP-1 levels in human monocytes were reduced by these AT1 receptor antagonists (Figs. 3, 8, and 9) through a mechanism partially independent of binding to the PAF receptor. Another possible mechanism of action is reduction of MCP-1 levels by nitric oxide. It has been shown (Kalinowski et al., 2002) that AT1 receptor antagonists stimulate nitric oxide release in rat platelets and HUVECs. Increased nitric oxide release in the presence of AT1 receptor antagonists may decrease MCP-1 levels because nitric oxide has been shown to inhibit MCP-1 production (Zeiher et al., 1995; Tsao et al., 1997). Li et al. (2000) found that a nitric-oxide synthase inhibitor had no effect on inhibition of TXA₂-induced vasoconstriction by irbesartan. However, the effect of a nitric-oxide synthase inhibitor on the reduction of monocyte MCP-1 production by irbesartan has not been studied.

Because PAF and Ang II may both be involved in atherosclerosis, AT1 receptor antagonists may inhibit atherosclerosis through more than one pathway; blocking AT1 receptors and the effects of Ang II, as well as inhibiting the effects of PAF or PAF-like lipids through some as yet unidentified mechanism. We have shown that the AT1 receptor antagonists irbesartan and losartan inhibit basal as well as LDL- and PAF-stimulated MCP-1 production by human monocytes, important inflammatory mediators in atherosclerosis. At atherogenic sites, LDL and/or LDL oxidation products as well as PAF are likely to be present. AT1 receptor antagonists may reduce the levels of MCP-1 at these sites by inhibiting basal release of MCP-1 and by blocking the stimulation of MCP-1 production by these molecules. Lower levels of MCP-1 may result in less circulating monocytes entering the vessel wall. In this way, AT1 receptor antagonists may inhibit the inflammatory component of atherosclerosis. Further work is required to determine the mechanism of the anti-inflammatory effect of AT1 receptor antagonists.

	Acknowledgements

 
We thank Kitya Dufall for excellent technical assistance.

	Footnotes

 
This work was supported in part by a grant from Bristol-Myers Squibb (to L.J.B.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.102.047795.

ABBREVIATIONS: MCP-1, monocyte chemoattractant protein-1; LDL, low-density lipoprotein; Ang II, angiotensin II; AT1, angiotensin II type 1; PAF, platelet-activating factor; AT2, angiotensin II type 2; c-PAF, carbamyl-PAF; HBSS, Hanks' balanced salt solution; HIFCS, heat-inactivated fetal calf serum; BSA, bovine serum albumin; HSA, human serum albumin; DMSO, dimethyl sulfoxide; fMLP, N-formylmethionyl-leucyl-phenylalanine; TXA₂, thromboxane A₂; HUVEC, human umbilical vein endothelial cell; PD123319, S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid; WEB 2086, 3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f][1,2,4]-triazolo-[4,3-a][1,4]-diazepine-2-yl]-1-(4-morpholynil)-1-propionate; CV11974, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxic acid.

Address correspondence to: Julie Proudfoot, Department of Medicine, Box X2213 GPO, University of Western Australia, Perth 6847, Western Australia. E-mail: jproudft{at}cyllene.uwa.edu.au

	References

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