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ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-κB activity and AT1 receptor expression
BBRC Biochemical and Biophysical Research Communications 325 (2004) 532–540 www.elsevier.com/locate/ybbrc
ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-jB activity and AT1 receptor expression Alexander Schmeissera,*,1, Oliver Soehnleinb,1, Thomas Illmerd, Hanns-Martin Lorenzc, Saeed Eskafib, Olaf Roerickb, Christoph Gablerc, Ruth Strasserd, Werner G. Danielb, Christoph D. Garlichsb b c
a Medical Clinic II, University of Technology, Dresden, Germany Medical Clinic II, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany Medical Clinic III, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany d Medical Clinic I,University of Technology, Dresden, Germany
Received 12 October 2004
Abstract Objective. Angiotensin converting enzyme (ACE) inhibitors significantly improve survival in patients with atherosclerosis. Although ACE inhibitors reduce local angiotensin II (AngII) formation, serine proteases form AngII to an enormous amount independently from ACE. Therefore, our study concentrates on the effect of the ACE-inhibitor ramiprilat on chemokine release, AngII receptor (ATR) expression, and NF-jB activity in monocytes stimulated with AngII. Methods and results. AngII-induced upregulation of IL-8 and MCP-1 protein and RNA in monocytes was inhibited by the AT1R-blocker losartan, but not by the AT2R-blocker PD 123.319. Ramiprilat dose-dependently suppressed AngII-induced upregulation of IL-8 and MCP-1. The suppressive effect of ramiprilat on AngII-induced chemokine production and release was in part caused by downregulation of NF-jB, but more by a selective and highly significant reduced expression of AT1 receptors as shown in monocytes and endothelial cells. Conclusion. In our study we demonstrated for the first time that ramiprilat reduced expression of AT1R in monocytes and endothelial cells. In addition, ramiprilat downregulated NF-jB activity and thereby reduced the AngII-induced release of IL-8 and MCP-1 in monocytes. This antiinflammatory effect, at least in part, may contribute to the clinical benefit of the ACE inhibitor in the treatment of coronary artery disease. 2004 Elsevier Inc. All rights reserved. Keywords: Coronary artery disease; Inflammation; Angiotensin II; Ramiprilat; Angiotensin receptor
In the development and progression of atherosclerosis and its complications such as myocardial infarction and stroke, local and systemic inflammatory processes play a key role [1,2]. Recent work has shown that angiotensin II (AngII), the most important component of the renin–angiotensin system (RAS), has significant *
Corresponding author. E-mail address: [email protected] (A. Schmeisser). 1 These authors have contributed equally to the study.
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.059
proinflammatory functions in the vascular wall, including the production of reactive oxygen species, inflammatory cytokines, and adhesion molecules (reviewed in [3]). An important link between AngII and inflammation is the nuclear factor-jB (NF-jB), a transcription factor stimulated by AngII mainly via AT1 receptor subtype (AT1R). AT1R is a 7-transmembrane G-protein-coupled receptor expressed on endothelial cells, monocytes, and vascular smooth muscle cells (VSMCs) [4]. The AngII-mediated stimulation of NF-jB via AT1R in
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monocytes/macrophages and VSMCs results in the upregulation of the proinflammatory and angiogenic cytokines such as MCP-1, IL-6, and several growth factors [5]. Besides the circulating RAS, there are numerous local tissue AngII-generating systems. Indeed, high levels of angiotensin converting enzyme (ACE) expression and AngII have been shown in experimental and human lesions [6–8]. ACE, AngII, and its receptors are colocalized especially in areas of inflammation in human atherosclerotic lesions [8]. In addition, recent data indicate that inflammatory cells can release enzymes that generate AngII, including ACE from monocytes/macrophages [8,9], cathepsin G from neutrophils [10], and chymase from mast cells [11]. The accumulation of high local levels of AngII, of which 90% in atherosclerotic aortas are chymase-dependent [4]. activates AngII-receptors on different cell types, leading to progressive lesion formation via proliferation of smooth muscle cells, formation of foam cells, and facilitation of thrombosis. Evidence from experimental [12] and clinical [13,14] trials supports the role of direct vascular effects of tissue ACE-inhibition. Despite the theoretical limit of a pharmacological ACE-inhibition resulting from alternative AngII-producing pathways, recent clinical trials have consistently documented the salutary effects of this class of agents in treating and preventing cardiovascular disease, with impressive reductions in coronary and cerebral vascular events. Particularly the heart outcomes prevention evaluation (HOPE) study has provided additional compelling data regarding the beneficial effects of ACE inhibition (ramipril) in vascular diseases and its complications [6]. Considering the possibility of alternatively produced AngII within atherosclerotic vessels, different trials studied the effects of selective inhibition of the AT1R signalling in comparison with conventional ACE inhibition. The OPTIMAAL trial, as the clinical landmark study in this context, compared the effects of the ACE inhibitor captopril with the selective AT1R blocker losartan in high risk patients after myocardial infarction. Surprisingly, the results demonstrated a non-significant difference between ACE inhibition and AT1R blockade in total mortality in favour of the ACE inhibitor [15]. The exact antiatherosclerotic mechanism of ACE-inhibition, however, is still unknown. Although ACE is an enzyme critical for the formation of AngII, it is also responsible for the destruction of the vasodilator bradykinin. But currently there is no strong evidence that ACE inhibitors may exert their antiatherogenic effect primarily through indirect effects on kinin metabolism with subsequent NO production [3]. Therefore, our study hypothesized that ACE inhibitors, independently from the suggested incomplete inhibition of the tissue AngII formation, can modulate
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the local vascular AngII-mediated inflammatory action by influencing the signal cascade in AT1R bearing cells of the atherosclerotic plaque. To prove this concept in vitro, ACE-inhibitor-treated (ramiprilat) human monocytic cells were examined for the production of different chemokines after addition of exogenous AngII.
Materials and methods Cell culture. THP-1 cells, a monocytic cell line, were cultured in RPMI 1640 (Biochrom) containing 10% FCS (Biochrom) at a density of up to 1 · 106 cells/mL. Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood drawn from healthy volunteers using density gradient centrifugation. After centrifugation the PBMC layer was removed, washed twice with PBS, and resuspended in RPMI 1640 containing 10% autologous serum. After a period of adherence (45 min) lymphocytes were washed out. The monocytesÕ purity assayed by FACS analysis was always more than 80%. Cell culture of human umbilical vein endothelial cells (HUVEC) was performed as described previously. The umbilical vein was incubated with 0.1% dispase, HUVEC were washed out and cultured in endothelial basal medium with endothelial cell growth supplement (PromoCell). Ramiprilat was a gift from Aventis. Losartan, PD 123.319, AngII, and LPS were purchased from Sigma. RT-PCR analysis. RNA Isolation including DNase digestion was performed by using Qiagen RNeasy Mini kit (Qiagen) following the manufacturerÕs protocol. For RT-PCR the ThermoScript One-Step System from Invitrogen was established. The Master Mix was prepared according to the manufacturerÕs instructions. cDNA synthesis and predenaturation was performed with one cycle of 60 C for 30 min and 95 C for 5 min, PCR amplification was performed with 29 cycles of 15 s at 95 C, 30 s at 57 C, and 30 s at 70 C. The gene specific primers were as follows: IL-8 sense: 5 0 -GGACAAGAGCCAGGAAGAAACC-3 0 ; IL-8 antisense: 5 0 -CTTCAAAAACTTCTCCACAAC-3 0 ; MCP-1 sense: 5 0 -GATGCAATCAATGCCCCAGTC-3 0 ; MCP-1 antisense: 5 0 -TTG CTTGTCCAGGTGGTCCAT-3 0 ; and GAPDH sense: 5 0 -GAAGGT GAAGGTCGGAGT-3 0 ; GAPDH antisense: 5 0 -GAAGATGGTGA TG GGATTTC-3 0 . PCR products were run on 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Gels were documented using the MultiImage Light cabinet (Alpha Innotech). Assays for MCP-1 and IL-8. Supernatants from human monocytes and THP-1 cells (treated as indicated) were collected and assessed for MCP-1 content by commercially available enzyme immunoassays (R&D Systems) according to the manufacturerÕs instructions. For determination of IL-8, supernatants were analysed by using the Random Access Immunoassay Analyser (DPC Biermann) and a commercially available Immulite Kit for IL-8 (DPC Biermann) following the manufacturerÕs protocol. Flow cytometry .After incubation of THP-1 cells and HUVEC with ramiprilat, cells were washed twice with PBS and incubated with 0.5% BSA in PBS for 30 min at 4 C. One microgram per milliliters of either anti human AT1R (306, SantaCruz), anti human AT2R (H-143, SantaCruz) or rabbit IgG (Sigma) was added for 30 min at 4 C. After washing, cells were incubated with mouse anti-rabbit-FITC conjugated antibody (Sigma). Cells were immediately assayed in Becton–Dickinson FACS Calibur (Becton–Dickinson). Results are expressed as mean fluorescence intensity (MFI). Western blot. After treatment of THP-1 cells and HUVEC with ramiprilat, cells were washed with PBS and lysed with 0.1 mL/106 cells of lysis buffer (50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1.9 mg/mL aprotinin, and 0.5 mg/ mL leupeptin, pH 7.4). After having assayed the protein concentration
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the proteins (30 lg per lane) were separated by 10% SDS–PAGE under reducing conditions and transferred to a PVDF membrane. Non-specific binding was blocked by 10% dried milk in TBS-Tween (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) overnight at 4 C. Incubation with anti-AT1R (1:500) or anti-AT2R (1:500) was followed by incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (1:5000). Staining was visualized by enhanced chemiluminescence system (Amersham). To confirm equal protein loading membranes were stained with Ponceau S. Electrophoretic mobility shift assay. Double-stranded DNA oligonucleotide probes (Promega) were labelled according to manufacturerÕs protocol using T4 polynucleotide kinase (Promega) in the presence of [c-P32]ATP (Amersham-Buchler) and 1· kinase buffer (Promega). Labelled oligos were purified on Sephadex G25 columns (PharmaciaBiotech) as recommended by the distributor. Labelled probes were incubated with 10 lg of nuclear protein for 30 min in binding buffer (Promega) in the presence of 2 lg poly(dI/dC) (Pharmacia). The samples were run on 5% non-denaturing polyacrylamide gels. Gels were dried and exposed to a Fuji imaging plate. Fuji FLA-2000 system and AIDA 2.0 software were used for semiquantification. Protein probes for supershift assays were preincubated with a supershifting antibody for 45 min on ice prior to the incubation period with the labelled probe. For binding inhibition of NF-jB the following antibodies were used: p65(A), p50(MLS), c-rel(N466)X, Rel-B (C19)X, and p52(447)X—all rabbit polyclonal antibodies (SantaCruz). Probes were subsequently treated as described above for conventional electrophoretic mobility shift assay (EMSA) analysis. Statistics. Data are presented as means ± SD. Statistical significance was calculated using StudentÕs t test for paired samples. A value of p < 0.05 was considered significant.
Results Angiotensin II induces MCP-1 and IL-8 expression in monocytes and THP-1 cells via AT1R Experiments that aimed at changes on protein level were carried out on both, human monocytes and THP-1 cells, whereas changes on RNA level were examined with THP-1 cells only. We stimulated human monocytes and THP-1 cells with 500 nM AngII, what we found to be the optimal dose in our experimental setting. Stimulation of human monocytes with 500 nM AngII for 24 h caused a significantly increased release of IL-8 and MCP-1 as compared to untreated cells: IL-8 concentration in poststimulatory supernatants increased more than twofold compared to control (p < 0.01, Fig. 1A). MCP-1 concentration in poststimulatory supernatants was several fold enhanced by AngII (p < 0.01, Fig. 1C). Since AngII mediates its function via two receptors, AT1R and AT2R, we coincubated monocytes with AngII and the AT1R antagonist losartan or the AT2R antagonist PD 123.319. Losartan completely abolished AngII-mediated stimulation of IL-8 and MCP-1 release (p < 0.01, Figs. 1A–C). PD 123.319, on the other hand,
Fig. 1. AngII enhances chemokine release and production in monocytes and THP-1 cells. (A,C) Influence of AngII (500 nM) and cotreatment with losartan (1 lM, 24 h) or PD 123.319 (1 lM) on concentration of IL-8 (A) and MCP-1 (C) in poststimulatory supernatants of human monocytes. n = 3. *p < 0.05 between the specific experiments. (B,D) RT-PCR analysis for IL-8 (B) and MCP-1 (D) shows the results of three independent experiments. THP-1 cells were stimulated with AngII (500 nM) and cotreated with losartan (1 lM) or PD 123.319 (1 lM) where indicated. GAPDH PCR shows equal amount of RNA.
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did not significantly influence IL-8 secretion (p = 0.06; Fig. 1A), but even enhanced MCP-1 release (p < 0.05; Fig. 1C). Similar results were obtained from experiments in THP-1 cells (data not shown). To distinguish enhanced de novo chemokine production from increased release of chemokines we performed PCR analysis (Figs. 1B and D). Stimulation of THP-1 cells with 500 nM AngII for 4 h showed a significant increase of RNA for IL-8 and MCP-1. Again, AngII-induced upregulation of IL-8 and MCP-1 production were blocked by losartan but not by PD 123.319. Ramiprilat reduces AngII-induced production and release of IL-8 and MCP-1 in monocytes and THP-1 cells In monocytes not stimulated with AngII ramiprilat did not significantly alter the release of IL-8 and MCP-1 (IL-8: p = 0.1, 1 lM ramiprilat vs. control; p = 0.05, 10 lM ramiprilat vs. control; MCP-1: p = 0.1, 1 lM ramiprilat vs. control; p = 0.07, 10 lM ramiprilat vs. control, Figs. 2A and B) as did losartan (IL-8: p = 0.07 vs. control; MCP-1: p = 0.5 vs. control, Figs. 2A and B), whereas PD 123.319 significantly enhanced IL-8 and MCP-1 levels (IL-8: p < 0.01 vs. control; MCP-1: p < 0.01 vs. control, Figs. 2A and B). In contrast, in AngII-stimulated monocytes, cotreatment with ramiprilat caused a dose-dependent reduction of chemokine production (Figs. 3B and D) and release (Figs. 3A and C) as compared with cells treated with AngII only (Fig. 3A; IL-8: 63.3 ± 5.8 to 38.4 ± 3.1 pg/ mL with 1 lM ramiprilat (p < 0.01), to 33 ± 3.7 pg/mL with 10 lM ramiprilat (p < 0.01); MCP-1: 82.2 ± 4.6 to 74.8 ± 7 pg/mL with 1 lM ramiprilat (p = 0.2), to 27.8 ± 4.5 pg/mL with 10 lM ramiprilat (p < 0.01)). This corresponds to the production of IL-8 and MCP1 RNA after cotreatment with ramiprilat. Here, 10 lM
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ramiprilat triggered a significant reduction of IL-8 and MCP-1 RNA, whereas pretreatment with 1 lM ramiprilat did not show an effect on MCP-1 RNA but on IL-8 RNA production (Figs. 3B and D). Ramiprilat partially suppresses AngII-mediated NF-jB activation AngII-dependent increase in IL-8 and MCP-1 can be mediated intracellularly by increased production or activity of NF-jB [3]. In our EMSAs, AngII significantly increased NF-jB activation in THP-1 cells (Fig. 4, p < 0.05). However, pretreatment of THP-1 cells with ramiprilat demonstrated a non-significant trend for reduced NF-jB activity (p = 0.06). As HMG-CoA reductase inhibitors (statins) are known to suppress AngII-mediated NF-jB activity, simvastatin was used as a control in our experiment. However, even simvastatin did not significantly lower NF-jB activity in AngII-treated THP-1 cells. Ramiprilat downregulates AT1-receptor expression on THP-1 cells and HUVEC Next, we tested the effect of ramiprilat on the expression of AT1R and AT2R. FACS analysis of surfacebound receptor expression revealed that incubation of THP-1 cells with ramiprilat significantly reduced the expression of AT1R in a dose-dependent manner (10 lM ramiprilat vs. control, 24 h treatment, p < 0.01, Fig. 5A), whereas the expression of AT2R was hardly affected. Lower, but still significant, effects on the AT1R were observable using a shorter treatment period (10 h, data not shown) or lower concentrations of ramiprilat (1 lM, p < 0.01, Fig. 5A). Corresponding results were obtained from Western blot analysis of whole cell lysates of THP-1 cells treated with different concentrations of ramiprilat (Fig. 5B). Again, ramiprilat signifi-
Fig. 2. Effect of losartan, PD 123.319, and ramiprilat on basal release of IL-8 (A) and MCP-1 (B) in non-stimulated monocytes. Native monocytes were treated with losartan (1 lM), PD 123.319 (1 lM), and ramiprilat (1 and 10 lM) each for 24 h. Histograms represent IL-8 (A) and MCP-1 (B) concentration thereafter. *p < 0.05 between the specific experiments.
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Fig. 3. Effect of ramiprilat treatment on chemokine production and release in AngII-stimulated monocytes. (A,C) Treatment of monocytes with AngII (500 nM) and where indicated cotreatment with ramiprilat (1 and 10 lM) for 24 h. Histograms show IL-8 (A) and MCP-1 (C) concentration. n = 3. *p < 0.05. (B,D) RT-PCR for IL-8 (B) and MCP-1 (D) in THP-1 cells treated with AngII (500 nM) for 4 h and additionally treated with ramiprilat (1 and 10 lM) for 24 h where indicated. Densitometric analysis of band intensities is representative for three independent experiments. LPS was used as positive control.
cantly lowered AT1R, while AT2R expression remained unchanged. As it was shown that statins downregulate AT1R we used simvastatin as a control for our experiments. In the same experimental setting as above, simvastatin downregulated AT1R expression on the surface of THP-1 cells (Fig. 5C). AT2R expression, however, was not affected (data not shown). To investigate whether the effect of ramiprilat on AngII receptors is specific for monocytes we performed similar experiments in endothelial cells: the expression of AT1R was significantly lowered by a 24 h treatment of HUVEC with 1 and 10 lM ramiprilat as shown by FACS analysis (Fig. 6A). AT2R expression did not significantly change upon ramiprilat treatment (data not shown). Similar results were obtained in Western blot analysis of whole cell lysate (Fig. 6B).
Discussion The main finding of our study is that the AngII– AT1R-mediated production of the proinflammatory and angiogenic chemokines IL-8 and MCP-1 was significantly and dose-dependently suppressed by pretreatment of monocytes with the ACE inhibitor ramiprilat, the active metabolite of ramipril. The effects of ramiprilat were independent from the suppression of AngII production in the monocytes themselves, because the spontaneous and unstimulated monocytic production of IL-8 and MCP-1 was influenced neither by the ramiprilat treatment nor by the application of the AT1R blocker losartan (Fig. 2). Furthermore, the commonly used stimulation doses of AngII (100–500 nM) are manifold higher than the spontaneous production of AngII by monocytes themselves [8]. Moreover, we used AngII
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Fig. 4. Effects of AngII and ramiprilat on NF-jB activity in human monocytes analysed by EMSA. Monocytes were incubated with 500 nM AngII for 4 h. In parallel experiments, cells were pretreated with ramiprilat 1 lM for 24 h and then stimulated with AngII. Competition assays with a 100fold excess of unlabeled or mutant NF-jB show specific NF-jB complexes. Left panel shows a representative autoradiogram from four different EMSA experiments with similar results. Positions of specific NF-jB complexes and free oligonucleotide are indicated (arrows). Right panel shows values of mean ± SD obtained by densitometric analysis. *p < 0.05 vs. control.
Fig. 5. Effect of ramiprilat and simvastatin on expression of AT1R and AT2R in THP-1 cells. (A) FACS analysis of AT1R/AT2R expression. Histogram overlays are representative of four independent histograms. Bars in the chart show the relative reduction of receptor expression as compared with the basal MFI, which was set to 100%. n = 4, *p < 0.05. (B) Western blot analysis of AT1R/AT2R expression. Bands are representative of three independent experiments. Ponceau S staining below shows equal amount of protein loaded on each lane. (C) The effect of different concentrations of simvastatin on AT1R expression in THP-1 cells. The expression was analysed by FACS. The chart presents the relative AT1R expression as compared with the basal MFI, which was set to 100%. n = 3, *p < 0.05.
and not AngI, which is the substrate of ACE, as stimulating agent. That indicates that the AngII–AT1R-induced monocytic chemokine production was inhibited by ramiprilat by a mechanism other than the simple suppression of the AngII-production. For the observed phenomenon, we assume at least two underlying
mechanisms. To the best of our knowledge we could demonstrate for the first time that the basal cellular AT1R-expression of the monocytes was dose- and time-dependently downregulated by ramiprilat, whereas the AT2R expression remained unaffected. These results were reproduced to a comparable extent in HUVEC,
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Fig. 6. Effect of ramiprilat treatment on AT1R expression in HUVEC. (A) Histogram overlay shows AT1R expression on resting HUVEC and in HUVEC after treatment with 10 lM ramiprilat as well as staining with an isotype control antibody. Bars show AT1R expression analysed by FACS compared to basal MFI, which was set to 100%. n = 3, *p < 0.05. (B) Western blot illustrates AT1R/AT2R expression in whole cell lysates of ramiprilat treated HUVEC. Bands are representative of three independent experiments.
meaning that the AT1R downregulating effect of ramiprilat is not specific for monocytes. Moreover, the ramiprilat-mediated downregulation of AT1R-expression in the monocytic cell line THP-1 was in a similar range as the downregulation by simvastatin. We used a statin as positive control, because it directly downregulates AT1R by reducing the half-life of the AT1R mRNA in VSMCs [9]. Because in our study AngII stimulation of monocytes was performed after their pretreatment
with ramiprilat over 24 h, the suppression of the AngII-induced MCP-1 and IL-8 production is at least partly explained by the downregulation of AT1R-expression. In addition, since monocytic AT2R-expression remained unchanged after ramiprilat pretreatment, AngII binding may be shifted relatively more in direction of the AT2R. This finding is of importance, because many studies have demonstrated that AT2R inhibits various functions mediated via AT1R [10,11]. Our study emphasizes this phenomenon by several observations. First, the AngII–AT1R-stimulated MCP-1 and IL-8 production was completely abolished by AT1R-blockade. In contrast, chemokine production after stimulation with AngII was further and markedly increased by antagonizing AT2R with PD 123.319. Second, basal monocytic MCP-1 and IL-8 production was significantly altered by blockade of AT2R, but not the AT1R subtype. These data support the hypothesis that AT2R inhibits the function of the AT1R subtype also with regard to the production of specific chemokines in human monocytes. The underlying mechanisms for the ramiprilat-induced selective AT1R-downregulation in monocytes and in HUVEC remain unclear. Previous studies demonstrated the modulation of the AT1R-expression by various agonists, most of which induce profound alterations in AT1R mRNA turnover. One suggestion for our observation refers to increased bradykinin levels under ACE-inhibition. Monocytes/macrophages are known to express the bradykinin receptor 2 (B2R). Bradykinin stimulation of peritoneal macrophages induced the secretion of superoxide radicals, arachidonic acid, and prostaglandin E2 (PGE2) via the B2R-subtype. In addition, PGE2 increased the concentration of cAMP in mononuclear phagocytes. Enhanced levels of superoxide radicals and cAMP finally lead to destabilization of the AT1R mRNA with subsequent downregulation of the receptor protein in rat VSMCs. Most studies analysing the transcriptional regulation of the AT1R were done in mouse and rat models. However, sequence alignment of the rat and human 5 0 regulatory regions revealed only 35% overall nucleotide identity. Care must therefore be taken in applying data from rat models to humans. With regard to the supposed effects of ACE inhibitors on the activation of NF-jB and AP-1, it is of interest that the human AT1R-promoter bears an AP-1 binding element but not an NF-jB binding element. The latter was found only in rats. In fact, it was shown that the phorbol ester PMA activates the human AT1R promoter in VSMCs via an AP-1 element. In addition, a prominent role for PKC/MAPK and Ets proteins in AT1R regulation has been proposed [12]. In conclusion, these data suggest that the ACE-inhibitor-mediated inhibition of NF-jB, marginally shown also in our experiments, is not directly responsible for the observed AT1R downregulation.
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This background leads to a second potential mechanism that could be responsible for the immunomodulatory effect of ramiprilat after monocytic stimulation with AngII. Some experimental studies used numerous ACE-inhibitors (captopril, enalapril, and quinapril, respectively) in vitro and in vivo, and demonstrated the inhibition of the redox-sensitive transcription factors NF-jB and AP-1 with consecutive downregulation of MCP-1, tissue factor, IL-8, and other growth factors and cytokines [13,16]. Both marker chemokines (IL-8, MCP-1) used in our experiments are chemokines which are regulated by these two redox-sensitive transcription factors after stimulation with AngII [5,17]. Ramiprilat significantly inhibited the production of these chemokines and showed a trend for inhibition of the activity of NF-jB (p = 0.06). The explanation for it could be related to the merely modest activation of monocytic NF-jB by AngII, which therefore can be inhibited by ramiprilat only marginally. Recently published data about the activation of NF-jB also through the AT2R subtypes does not seem to be relevant in our cell system and for the measured chemokines, because the entire effect of AngII was abolished by the selective AT1R blocker losartan in these studies. AT1R and ACE expression is dramatically increased in atherosclerotic plaques, particularly in macrophages at the vulnerable fibrous cap [18,19]. This correlates with signs of plaque instability such as inflammation, apoptosis, intraplaque-neovascularization, and production of cytokines and growth factors. Many studies strongly indicated the antiatherogenic potential of agents that interfere with the renin–angiotensin system, such as ACE inhibitors or AngII receptor blocker. In our experiments we demonstrated that the ACE-inhibitor ramiprilat suppresses the AngII–AT1R proinflammatory signalling mainly by downregulation of the AT1R expression in human monocytes and endothelial cells and, to a lower extent, by inhibition of the AngIIinduced NF-jB activation. In conclusion, these data could provide a new antiinflammatory and therefore plaque-stabilizing mechanism to explain the particularly favourable outcome of clinical studies with ACE-inhibitors in patients with coronary and cerebrovascular atherosclerosis. The main limitation of our studies is that the data are mainly based on in vitro experiments. To prove the supposed importance of our data, future studies will have to confirm them in an in vivo setting.
Acknowledgments This work was supported by Aventis and the Interdisciplinary Center for Clinical Research (IZKF, Project No. B29) of the University Erlangen-Nuremberg. The authors thank Sabine Kesting, Doris Flick (both Medi-
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cal Clinic II, Erlangen, Germany), and Silke Winkler (Medical Clinic III, Erlangen, Germany) for excellent technical assistance.
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ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-jB activity and AT1 receptor expression Alexander Schmeissera,*,1, Oliver Soehnleinb,1, Thomas Illmerd, Hanns-Martin Lorenzc, Saeed Eskafib, Olaf Roerickb, Christoph Gablerc, Ruth Strasserd, Werner G. Danielb, Christoph D. Garlichsb b c
a Medical Clinic II, University of Technology, Dresden, Germany Medical Clinic II, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany Medical Clinic III, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany d Medical Clinic I,University of Technology, Dresden, Germany
Received 12 October 2004
Abstract Objective. Angiotensin converting enzyme (ACE) inhibitors significantly improve survival in patients with atherosclerosis. Although ACE inhibitors reduce local angiotensin II (AngII) formation, serine proteases form AngII to an enormous amount independently from ACE. Therefore, our study concentrates on the effect of the ACE-inhibitor ramiprilat on chemokine release, AngII receptor (ATR) expression, and NF-jB activity in monocytes stimulated with AngII. Methods and results. AngII-induced upregulation of IL-8 and MCP-1 protein and RNA in monocytes was inhibited by the AT1R-blocker losartan, but not by the AT2R-blocker PD 123.319. Ramiprilat dose-dependently suppressed AngII-induced upregulation of IL-8 and MCP-1. The suppressive effect of ramiprilat on AngII-induced chemokine production and release was in part caused by downregulation of NF-jB, but more by a selective and highly significant reduced expression of AT1 receptors as shown in monocytes and endothelial cells. Conclusion. In our study we demonstrated for the first time that ramiprilat reduced expression of AT1R in monocytes and endothelial cells. In addition, ramiprilat downregulated NF-jB activity and thereby reduced the AngII-induced release of IL-8 and MCP-1 in monocytes. This antiinflammatory effect, at least in part, may contribute to the clinical benefit of the ACE inhibitor in the treatment of coronary artery disease. 2004 Elsevier Inc. All rights reserved. Keywords: Coronary artery disease; Inflammation; Angiotensin II; Ramiprilat; Angiotensin receptor
In the development and progression of atherosclerosis and its complications such as myocardial infarction and stroke, local and systemic inflammatory processes play a key role [1,2]. Recent work has shown that angiotensin II (AngII), the most important component of the renin–angiotensin system (RAS), has significant *
Corresponding author. E-mail address: [email protected] (A. Schmeisser). 1 These authors have contributed equally to the study.
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.059
proinflammatory functions in the vascular wall, including the production of reactive oxygen species, inflammatory cytokines, and adhesion molecules (reviewed in [3]). An important link between AngII and inflammation is the nuclear factor-jB (NF-jB), a transcription factor stimulated by AngII mainly via AT1 receptor subtype (AT1R). AT1R is a 7-transmembrane G-protein-coupled receptor expressed on endothelial cells, monocytes, and vascular smooth muscle cells (VSMCs) [4]. The AngII-mediated stimulation of NF-jB via AT1R in
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monocytes/macrophages and VSMCs results in the upregulation of the proinflammatory and angiogenic cytokines such as MCP-1, IL-6, and several growth factors [5]. Besides the circulating RAS, there are numerous local tissue AngII-generating systems. Indeed, high levels of angiotensin converting enzyme (ACE) expression and AngII have been shown in experimental and human lesions [6–8]. ACE, AngII, and its receptors are colocalized especially in areas of inflammation in human atherosclerotic lesions [8]. In addition, recent data indicate that inflammatory cells can release enzymes that generate AngII, including ACE from monocytes/macrophages [8,9], cathepsin G from neutrophils [10], and chymase from mast cells [11]. The accumulation of high local levels of AngII, of which 90% in atherosclerotic aortas are chymase-dependent [4]. activates AngII-receptors on different cell types, leading to progressive lesion formation via proliferation of smooth muscle cells, formation of foam cells, and facilitation of thrombosis. Evidence from experimental [12] and clinical [13,14] trials supports the role of direct vascular effects of tissue ACE-inhibition. Despite the theoretical limit of a pharmacological ACE-inhibition resulting from alternative AngII-producing pathways, recent clinical trials have consistently documented the salutary effects of this class of agents in treating and preventing cardiovascular disease, with impressive reductions in coronary and cerebral vascular events. Particularly the heart outcomes prevention evaluation (HOPE) study has provided additional compelling data regarding the beneficial effects of ACE inhibition (ramipril) in vascular diseases and its complications [6]. Considering the possibility of alternatively produced AngII within atherosclerotic vessels, different trials studied the effects of selective inhibition of the AT1R signalling in comparison with conventional ACE inhibition. The OPTIMAAL trial, as the clinical landmark study in this context, compared the effects of the ACE inhibitor captopril with the selective AT1R blocker losartan in high risk patients after myocardial infarction. Surprisingly, the results demonstrated a non-significant difference between ACE inhibition and AT1R blockade in total mortality in favour of the ACE inhibitor [15]. The exact antiatherosclerotic mechanism of ACE-inhibition, however, is still unknown. Although ACE is an enzyme critical for the formation of AngII, it is also responsible for the destruction of the vasodilator bradykinin. But currently there is no strong evidence that ACE inhibitors may exert their antiatherogenic effect primarily through indirect effects on kinin metabolism with subsequent NO production [3]. Therefore, our study hypothesized that ACE inhibitors, independently from the suggested incomplete inhibition of the tissue AngII formation, can modulate
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the local vascular AngII-mediated inflammatory action by influencing the signal cascade in AT1R bearing cells of the atherosclerotic plaque. To prove this concept in vitro, ACE-inhibitor-treated (ramiprilat) human monocytic cells were examined for the production of different chemokines after addition of exogenous AngII.
Materials and methods Cell culture. THP-1 cells, a monocytic cell line, were cultured in RPMI 1640 (Biochrom) containing 10% FCS (Biochrom) at a density of up to 1 · 106 cells/mL. Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood drawn from healthy volunteers using density gradient centrifugation. After centrifugation the PBMC layer was removed, washed twice with PBS, and resuspended in RPMI 1640 containing 10% autologous serum. After a period of adherence (45 min) lymphocytes were washed out. The monocytesÕ purity assayed by FACS analysis was always more than 80%. Cell culture of human umbilical vein endothelial cells (HUVEC) was performed as described previously. The umbilical vein was incubated with 0.1% dispase, HUVEC were washed out and cultured in endothelial basal medium with endothelial cell growth supplement (PromoCell). Ramiprilat was a gift from Aventis. Losartan, PD 123.319, AngII, and LPS were purchased from Sigma. RT-PCR analysis. RNA Isolation including DNase digestion was performed by using Qiagen RNeasy Mini kit (Qiagen) following the manufacturerÕs protocol. For RT-PCR the ThermoScript One-Step System from Invitrogen was established. The Master Mix was prepared according to the manufacturerÕs instructions. cDNA synthesis and predenaturation was performed with one cycle of 60 C for 30 min and 95 C for 5 min, PCR amplification was performed with 29 cycles of 15 s at 95 C, 30 s at 57 C, and 30 s at 70 C. The gene specific primers were as follows: IL-8 sense: 5 0 -GGACAAGAGCCAGGAAGAAACC-3 0 ; IL-8 antisense: 5 0 -CTTCAAAAACTTCTCCACAAC-3 0 ; MCP-1 sense: 5 0 -GATGCAATCAATGCCCCAGTC-3 0 ; MCP-1 antisense: 5 0 -TTG CTTGTCCAGGTGGTCCAT-3 0 ; and GAPDH sense: 5 0 -GAAGGT GAAGGTCGGAGT-3 0 ; GAPDH antisense: 5 0 -GAAGATGGTGA TG GGATTTC-3 0 . PCR products were run on 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Gels were documented using the MultiImage Light cabinet (Alpha Innotech). Assays for MCP-1 and IL-8. Supernatants from human monocytes and THP-1 cells (treated as indicated) were collected and assessed for MCP-1 content by commercially available enzyme immunoassays (R&D Systems) according to the manufacturerÕs instructions. For determination of IL-8, supernatants were analysed by using the Random Access Immunoassay Analyser (DPC Biermann) and a commercially available Immulite Kit for IL-8 (DPC Biermann) following the manufacturerÕs protocol. Flow cytometry .After incubation of THP-1 cells and HUVEC with ramiprilat, cells were washed twice with PBS and incubated with 0.5% BSA in PBS for 30 min at 4 C. One microgram per milliliters of either anti human AT1R (306, SantaCruz), anti human AT2R (H-143, SantaCruz) or rabbit IgG (Sigma) was added for 30 min at 4 C. After washing, cells were incubated with mouse anti-rabbit-FITC conjugated antibody (Sigma). Cells were immediately assayed in Becton–Dickinson FACS Calibur (Becton–Dickinson). Results are expressed as mean fluorescence intensity (MFI). Western blot. After treatment of THP-1 cells and HUVEC with ramiprilat, cells were washed with PBS and lysed with 0.1 mL/106 cells of lysis buffer (50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1.9 mg/mL aprotinin, and 0.5 mg/ mL leupeptin, pH 7.4). After having assayed the protein concentration
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the proteins (30 lg per lane) were separated by 10% SDS–PAGE under reducing conditions and transferred to a PVDF membrane. Non-specific binding was blocked by 10% dried milk in TBS-Tween (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) overnight at 4 C. Incubation with anti-AT1R (1:500) or anti-AT2R (1:500) was followed by incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (1:5000). Staining was visualized by enhanced chemiluminescence system (Amersham). To confirm equal protein loading membranes were stained with Ponceau S. Electrophoretic mobility shift assay. Double-stranded DNA oligonucleotide probes (Promega) were labelled according to manufacturerÕs protocol using T4 polynucleotide kinase (Promega) in the presence of [c-P32]ATP (Amersham-Buchler) and 1· kinase buffer (Promega). Labelled oligos were purified on Sephadex G25 columns (PharmaciaBiotech) as recommended by the distributor. Labelled probes were incubated with 10 lg of nuclear protein for 30 min in binding buffer (Promega) in the presence of 2 lg poly(dI/dC) (Pharmacia). The samples were run on 5% non-denaturing polyacrylamide gels. Gels were dried and exposed to a Fuji imaging plate. Fuji FLA-2000 system and AIDA 2.0 software were used for semiquantification. Protein probes for supershift assays were preincubated with a supershifting antibody for 45 min on ice prior to the incubation period with the labelled probe. For binding inhibition of NF-jB the following antibodies were used: p65(A), p50(MLS), c-rel(N466)X, Rel-B (C19)X, and p52(447)X—all rabbit polyclonal antibodies (SantaCruz). Probes were subsequently treated as described above for conventional electrophoretic mobility shift assay (EMSA) analysis. Statistics. Data are presented as means ± SD. Statistical significance was calculated using StudentÕs t test for paired samples. A value of p < 0.05 was considered significant.
Results Angiotensin II induces MCP-1 and IL-8 expression in monocytes and THP-1 cells via AT1R Experiments that aimed at changes on protein level were carried out on both, human monocytes and THP-1 cells, whereas changes on RNA level were examined with THP-1 cells only. We stimulated human monocytes and THP-1 cells with 500 nM AngII, what we found to be the optimal dose in our experimental setting. Stimulation of human monocytes with 500 nM AngII for 24 h caused a significantly increased release of IL-8 and MCP-1 as compared to untreated cells: IL-8 concentration in poststimulatory supernatants increased more than twofold compared to control (p < 0.01, Fig. 1A). MCP-1 concentration in poststimulatory supernatants was several fold enhanced by AngII (p < 0.01, Fig. 1C). Since AngII mediates its function via two receptors, AT1R and AT2R, we coincubated monocytes with AngII and the AT1R antagonist losartan or the AT2R antagonist PD 123.319. Losartan completely abolished AngII-mediated stimulation of IL-8 and MCP-1 release (p < 0.01, Figs. 1A–C). PD 123.319, on the other hand,
Fig. 1. AngII enhances chemokine release and production in monocytes and THP-1 cells. (A,C) Influence of AngII (500 nM) and cotreatment with losartan (1 lM, 24 h) or PD 123.319 (1 lM) on concentration of IL-8 (A) and MCP-1 (C) in poststimulatory supernatants of human monocytes. n = 3. *p < 0.05 between the specific experiments. (B,D) RT-PCR analysis for IL-8 (B) and MCP-1 (D) shows the results of three independent experiments. THP-1 cells were stimulated with AngII (500 nM) and cotreated with losartan (1 lM) or PD 123.319 (1 lM) where indicated. GAPDH PCR shows equal amount of RNA.
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did not significantly influence IL-8 secretion (p = 0.06; Fig. 1A), but even enhanced MCP-1 release (p < 0.05; Fig. 1C). Similar results were obtained from experiments in THP-1 cells (data not shown). To distinguish enhanced de novo chemokine production from increased release of chemokines we performed PCR analysis (Figs. 1B and D). Stimulation of THP-1 cells with 500 nM AngII for 4 h showed a significant increase of RNA for IL-8 and MCP-1. Again, AngII-induced upregulation of IL-8 and MCP-1 production were blocked by losartan but not by PD 123.319. Ramiprilat reduces AngII-induced production and release of IL-8 and MCP-1 in monocytes and THP-1 cells In monocytes not stimulated with AngII ramiprilat did not significantly alter the release of IL-8 and MCP-1 (IL-8: p = 0.1, 1 lM ramiprilat vs. control; p = 0.05, 10 lM ramiprilat vs. control; MCP-1: p = 0.1, 1 lM ramiprilat vs. control; p = 0.07, 10 lM ramiprilat vs. control, Figs. 2A and B) as did losartan (IL-8: p = 0.07 vs. control; MCP-1: p = 0.5 vs. control, Figs. 2A and B), whereas PD 123.319 significantly enhanced IL-8 and MCP-1 levels (IL-8: p < 0.01 vs. control; MCP-1: p < 0.01 vs. control, Figs. 2A and B). In contrast, in AngII-stimulated monocytes, cotreatment with ramiprilat caused a dose-dependent reduction of chemokine production (Figs. 3B and D) and release (Figs. 3A and C) as compared with cells treated with AngII only (Fig. 3A; IL-8: 63.3 ± 5.8 to 38.4 ± 3.1 pg/ mL with 1 lM ramiprilat (p < 0.01), to 33 ± 3.7 pg/mL with 10 lM ramiprilat (p < 0.01); MCP-1: 82.2 ± 4.6 to 74.8 ± 7 pg/mL with 1 lM ramiprilat (p = 0.2), to 27.8 ± 4.5 pg/mL with 10 lM ramiprilat (p < 0.01)). This corresponds to the production of IL-8 and MCP1 RNA after cotreatment with ramiprilat. Here, 10 lM
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ramiprilat triggered a significant reduction of IL-8 and MCP-1 RNA, whereas pretreatment with 1 lM ramiprilat did not show an effect on MCP-1 RNA but on IL-8 RNA production (Figs. 3B and D). Ramiprilat partially suppresses AngII-mediated NF-jB activation AngII-dependent increase in IL-8 and MCP-1 can be mediated intracellularly by increased production or activity of NF-jB [3]. In our EMSAs, AngII significantly increased NF-jB activation in THP-1 cells (Fig. 4, p < 0.05). However, pretreatment of THP-1 cells with ramiprilat demonstrated a non-significant trend for reduced NF-jB activity (p = 0.06). As HMG-CoA reductase inhibitors (statins) are known to suppress AngII-mediated NF-jB activity, simvastatin was used as a control in our experiment. However, even simvastatin did not significantly lower NF-jB activity in AngII-treated THP-1 cells. Ramiprilat downregulates AT1-receptor expression on THP-1 cells and HUVEC Next, we tested the effect of ramiprilat on the expression of AT1R and AT2R. FACS analysis of surfacebound receptor expression revealed that incubation of THP-1 cells with ramiprilat significantly reduced the expression of AT1R in a dose-dependent manner (10 lM ramiprilat vs. control, 24 h treatment, p < 0.01, Fig. 5A), whereas the expression of AT2R was hardly affected. Lower, but still significant, effects on the AT1R were observable using a shorter treatment period (10 h, data not shown) or lower concentrations of ramiprilat (1 lM, p < 0.01, Fig. 5A). Corresponding results were obtained from Western blot analysis of whole cell lysates of THP-1 cells treated with different concentrations of ramiprilat (Fig. 5B). Again, ramiprilat signifi-
Fig. 2. Effect of losartan, PD 123.319, and ramiprilat on basal release of IL-8 (A) and MCP-1 (B) in non-stimulated monocytes. Native monocytes were treated with losartan (1 lM), PD 123.319 (1 lM), and ramiprilat (1 and 10 lM) each for 24 h. Histograms represent IL-8 (A) and MCP-1 (B) concentration thereafter. *p < 0.05 between the specific experiments.
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Fig. 3. Effect of ramiprilat treatment on chemokine production and release in AngII-stimulated monocytes. (A,C) Treatment of monocytes with AngII (500 nM) and where indicated cotreatment with ramiprilat (1 and 10 lM) for 24 h. Histograms show IL-8 (A) and MCP-1 (C) concentration. n = 3. *p < 0.05. (B,D) RT-PCR for IL-8 (B) and MCP-1 (D) in THP-1 cells treated with AngII (500 nM) for 4 h and additionally treated with ramiprilat (1 and 10 lM) for 24 h where indicated. Densitometric analysis of band intensities is representative for three independent experiments. LPS was used as positive control.
cantly lowered AT1R, while AT2R expression remained unchanged. As it was shown that statins downregulate AT1R we used simvastatin as a control for our experiments. In the same experimental setting as above, simvastatin downregulated AT1R expression on the surface of THP-1 cells (Fig. 5C). AT2R expression, however, was not affected (data not shown). To investigate whether the effect of ramiprilat on AngII receptors is specific for monocytes we performed similar experiments in endothelial cells: the expression of AT1R was significantly lowered by a 24 h treatment of HUVEC with 1 and 10 lM ramiprilat as shown by FACS analysis (Fig. 6A). AT2R expression did not significantly change upon ramiprilat treatment (data not shown). Similar results were obtained in Western blot analysis of whole cell lysate (Fig. 6B).
Discussion The main finding of our study is that the AngII– AT1R-mediated production of the proinflammatory and angiogenic chemokines IL-8 and MCP-1 was significantly and dose-dependently suppressed by pretreatment of monocytes with the ACE inhibitor ramiprilat, the active metabolite of ramipril. The effects of ramiprilat were independent from the suppression of AngII production in the monocytes themselves, because the spontaneous and unstimulated monocytic production of IL-8 and MCP-1 was influenced neither by the ramiprilat treatment nor by the application of the AT1R blocker losartan (Fig. 2). Furthermore, the commonly used stimulation doses of AngII (100–500 nM) are manifold higher than the spontaneous production of AngII by monocytes themselves [8]. Moreover, we used AngII
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Fig. 4. Effects of AngII and ramiprilat on NF-jB activity in human monocytes analysed by EMSA. Monocytes were incubated with 500 nM AngII for 4 h. In parallel experiments, cells were pretreated with ramiprilat 1 lM for 24 h and then stimulated with AngII. Competition assays with a 100fold excess of unlabeled or mutant NF-jB show specific NF-jB complexes. Left panel shows a representative autoradiogram from four different EMSA experiments with similar results. Positions of specific NF-jB complexes and free oligonucleotide are indicated (arrows). Right panel shows values of mean ± SD obtained by densitometric analysis. *p < 0.05 vs. control.
Fig. 5. Effect of ramiprilat and simvastatin on expression of AT1R and AT2R in THP-1 cells. (A) FACS analysis of AT1R/AT2R expression. Histogram overlays are representative of four independent histograms. Bars in the chart show the relative reduction of receptor expression as compared with the basal MFI, which was set to 100%. n = 4, *p < 0.05. (B) Western blot analysis of AT1R/AT2R expression. Bands are representative of three independent experiments. Ponceau S staining below shows equal amount of protein loaded on each lane. (C) The effect of different concentrations of simvastatin on AT1R expression in THP-1 cells. The expression was analysed by FACS. The chart presents the relative AT1R expression as compared with the basal MFI, which was set to 100%. n = 3, *p < 0.05.
and not AngI, which is the substrate of ACE, as stimulating agent. That indicates that the AngII–AT1R-induced monocytic chemokine production was inhibited by ramiprilat by a mechanism other than the simple suppression of the AngII-production. For the observed phenomenon, we assume at least two underlying
mechanisms. To the best of our knowledge we could demonstrate for the first time that the basal cellular AT1R-expression of the monocytes was dose- and time-dependently downregulated by ramiprilat, whereas the AT2R expression remained unaffected. These results were reproduced to a comparable extent in HUVEC,
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Fig. 6. Effect of ramiprilat treatment on AT1R expression in HUVEC. (A) Histogram overlay shows AT1R expression on resting HUVEC and in HUVEC after treatment with 10 lM ramiprilat as well as staining with an isotype control antibody. Bars show AT1R expression analysed by FACS compared to basal MFI, which was set to 100%. n = 3, *p < 0.05. (B) Western blot illustrates AT1R/AT2R expression in whole cell lysates of ramiprilat treated HUVEC. Bands are representative of three independent experiments.
meaning that the AT1R downregulating effect of ramiprilat is not specific for monocytes. Moreover, the ramiprilat-mediated downregulation of AT1R-expression in the monocytic cell line THP-1 was in a similar range as the downregulation by simvastatin. We used a statin as positive control, because it directly downregulates AT1R by reducing the half-life of the AT1R mRNA in VSMCs [9]. Because in our study AngII stimulation of monocytes was performed after their pretreatment
with ramiprilat over 24 h, the suppression of the AngII-induced MCP-1 and IL-8 production is at least partly explained by the downregulation of AT1R-expression. In addition, since monocytic AT2R-expression remained unchanged after ramiprilat pretreatment, AngII binding may be shifted relatively more in direction of the AT2R. This finding is of importance, because many studies have demonstrated that AT2R inhibits various functions mediated via AT1R [10,11]. Our study emphasizes this phenomenon by several observations. First, the AngII–AT1R-stimulated MCP-1 and IL-8 production was completely abolished by AT1R-blockade. In contrast, chemokine production after stimulation with AngII was further and markedly increased by antagonizing AT2R with PD 123.319. Second, basal monocytic MCP-1 and IL-8 production was significantly altered by blockade of AT2R, but not the AT1R subtype. These data support the hypothesis that AT2R inhibits the function of the AT1R subtype also with regard to the production of specific chemokines in human monocytes. The underlying mechanisms for the ramiprilat-induced selective AT1R-downregulation in monocytes and in HUVEC remain unclear. Previous studies demonstrated the modulation of the AT1R-expression by various agonists, most of which induce profound alterations in AT1R mRNA turnover. One suggestion for our observation refers to increased bradykinin levels under ACE-inhibition. Monocytes/macrophages are known to express the bradykinin receptor 2 (B2R). Bradykinin stimulation of peritoneal macrophages induced the secretion of superoxide radicals, arachidonic acid, and prostaglandin E2 (PGE2) via the B2R-subtype. In addition, PGE2 increased the concentration of cAMP in mononuclear phagocytes. Enhanced levels of superoxide radicals and cAMP finally lead to destabilization of the AT1R mRNA with subsequent downregulation of the receptor protein in rat VSMCs. Most studies analysing the transcriptional regulation of the AT1R were done in mouse and rat models. However, sequence alignment of the rat and human 5 0 regulatory regions revealed only 35% overall nucleotide identity. Care must therefore be taken in applying data from rat models to humans. With regard to the supposed effects of ACE inhibitors on the activation of NF-jB and AP-1, it is of interest that the human AT1R-promoter bears an AP-1 binding element but not an NF-jB binding element. The latter was found only in rats. In fact, it was shown that the phorbol ester PMA activates the human AT1R promoter in VSMCs via an AP-1 element. In addition, a prominent role for PKC/MAPK and Ets proteins in AT1R regulation has been proposed [12]. In conclusion, these data suggest that the ACE-inhibitor-mediated inhibition of NF-jB, marginally shown also in our experiments, is not directly responsible for the observed AT1R downregulation.
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This background leads to a second potential mechanism that could be responsible for the immunomodulatory effect of ramiprilat after monocytic stimulation with AngII. Some experimental studies used numerous ACE-inhibitors (captopril, enalapril, and quinapril, respectively) in vitro and in vivo, and demonstrated the inhibition of the redox-sensitive transcription factors NF-jB and AP-1 with consecutive downregulation of MCP-1, tissue factor, IL-8, and other growth factors and cytokines [13,16]. Both marker chemokines (IL-8, MCP-1) used in our experiments are chemokines which are regulated by these two redox-sensitive transcription factors after stimulation with AngII [5,17]. Ramiprilat significantly inhibited the production of these chemokines and showed a trend for inhibition of the activity of NF-jB (p = 0.06). The explanation for it could be related to the merely modest activation of monocytic NF-jB by AngII, which therefore can be inhibited by ramiprilat only marginally. Recently published data about the activation of NF-jB also through the AT2R subtypes does not seem to be relevant in our cell system and for the measured chemokines, because the entire effect of AngII was abolished by the selective AT1R blocker losartan in these studies. AT1R and ACE expression is dramatically increased in atherosclerotic plaques, particularly in macrophages at the vulnerable fibrous cap [18,19]. This correlates with signs of plaque instability such as inflammation, apoptosis, intraplaque-neovascularization, and production of cytokines and growth factors. Many studies strongly indicated the antiatherogenic potential of agents that interfere with the renin–angiotensin system, such as ACE inhibitors or AngII receptor blocker. In our experiments we demonstrated that the ACE-inhibitor ramiprilat suppresses the AngII–AT1R proinflammatory signalling mainly by downregulation of the AT1R expression in human monocytes and endothelial cells and, to a lower extent, by inhibition of the AngIIinduced NF-jB activation. In conclusion, these data could provide a new antiinflammatory and therefore plaque-stabilizing mechanism to explain the particularly favourable outcome of clinical studies with ACE-inhibitors in patients with coronary and cerebrovascular atherosclerosis. The main limitation of our studies is that the data are mainly based on in vitro experiments. To prove the supposed importance of our data, future studies will have to confirm them in an in vivo setting.
Acknowledgments This work was supported by Aventis and the Interdisciplinary Center for Clinical Research (IZKF, Project No. B29) of the University Erlangen-Nuremberg. The authors thank Sabine Kesting, Doris Flick (both Medi-
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cal Clinic II, Erlangen, Germany), and Silke Winkler (Medical Clinic III, Erlangen, Germany) for excellent technical assistance.
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