1 Medical Clinic III, Department of Cardiology, Nephrology, Hypertension and Renal Failure, 2 Department of General Neurology, Hertie Institute for Clinical Brain Research and 3 Section of Radiobiology and Molecular Environmental Research, Department of Radiotherapy, Eberhard-Karls-University, D-72076 Tübingen, Germany
Correspondence and offprint requests to: Bernhard R. Brehm, MD, Hoppe-Seylerstr. 3, D-72076 Tübingen, Germany. Email: bernhard.brehm{at}onlinehome.de
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Abstract |
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Methods. Subtotally nephrectomized (SNX) rats were treated with an ETA receptor antagonist, an ETAB receptor antagonist, the angiotensin type 1 (AT1) receptor antagonist losartan (all 10 mg/kg body weight/day) or the angiotensin-converting enzyme (ACE) inhibitor trandolapril (0.1 mg/kg body weight/day) or received no medication (SNX) for 12 weeks. Then, aortal smooth muscle cells (SMCs) were isolated and cultivated. After incubation of SMCs with different growth factors (57 days), proliferation was measured using a bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA).
Results. Higher maximum levels of proliferation were found in SMCs from untreated SNX rats than in SMCs from control animals [platelet-derived growth factor-BB (PDGF-BB) 486.60±8.27 vs 346.74±4.60%, basic fibroblast growth factor (bFGF) 176.68±6.50 vs 123.71±1.49%, tumour necrosis factor- (TNF-
) 153.38±10.16 vs 122.27±1.41%]. Treatment with ET receptor antagonists or losartan attenuated growth factor-stimulated proliferation (PDGF-BB: ETA receptor antagonist, 135.71±1.08%; ETAB receptor antagonist, 122.72±0.58%; losartan: 103.69±1.83%, n = 8). SMCs from trandolapril-treated rats showed an increased response (PDGF-BB 663.48±7.00%, n = 8).
Conclusions. Treatment of SNX rats with ET receptor antagonists or losartan reduced growth factor-induced SMC proliferation in vitro. However, further investigations with uraemic patients have to clarify whether angiotensin or ET receptor antagonists inhibit the development of atherosclerosis.
Keywords: growth factors; proliferation; SMC; uraemia
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Introduction |
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During the past several years, experimental data have illuminated the role of inflammation in atherogenesis [3] with the recruitment of macrophages and T lymphocytes to the developing lesion, where they secrete numerous growth factors. Thereby, they are able to stimulate proliferation and migration of smooth muscle cells (SMCs).
In uraemia, pro-inflammatory cytokine and growth factor levels [e.g. tumour necrosis factor (TNF-), interleukin (IL)-1, IL-6, C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1)] are increased [4]. It is not known, however, whether SMCs in uraemia respond differently to growth factors. Moreover, the effect of ET-1 or angiotensin II antagonism on SMC proliferation rate has not been clarified to date.
In the present study, we investigated whether the proliferative response to several growth factors differs between aortal SMCs isolated from control and uraemic rats and how treatment of uraemic rats with different antihypertensive drugs influences the response to these growth factors.
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Methods |
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Cell culture
Primary cultures of SMCs were obtained by isolating the cells growing out from small pieces of the explanted aortas cultivated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 20% fetal calf serum (FCS; BioWhittaker) and 5% penicillin (100 U/ml)/streptomycin (100 µg/ml, Gibco) using cloning rings. A few days later, cell clones were isolated. Smooth muscle origin was confirmed immunocytochemically using a monoclonal antibody against smooth muscle -actin (Sigma-Aldrich).
Cell proliferation assay
From each animal, 27 subcultured SMC clones (passages 310) were grown to confluence, detached with trypsin, combined and seeded into 96-well microplates (10 000 cells/well) using 200 µl of DMEM + 5% FCS + 5% penicillin/streptomycin. After 24 h, the culture medium was replaced by medium containing different dilutions of platelet-derived growth factor-BB (PDGF-BB; 1013109 mol/l), basic fibroblast growth factor (bFGF; 10141010 mol/l), TNF- (1012109 mol/l), angiotensin II (1012107 mol/l) (all from Sigma-Aldrich), aldosterone (1012107 mol/l; Fluka) or ET-1 (1013107 mol/l; Calbiochem). After 5 (for PDGF-BB) or 7 (for all other substances) days, proliferation was determined by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in an 18 h period with a colorimetric cell proliferation enzyme-linked immunosorbent assay (ELISA; Roche) as described previously [5].
Real-time RTPCR
For the determination of angiotensin and ET receptor mRNA expression, total RNA was isolated from three confluent SMC cultures and from left ventricles of control and SNX animals using RNazol (Tel-Test). Purity and yield were assessed spectrophotometrically. Subsequently, aliquots of total RNA (1 µg) were reverse-transcribed with TaqMan® Reverse Transcription Reagents (Applied Biosystems) in a volume of 50 µl according to the manufacturer's instructions. Subsequently, 2 µl cDNA samples were amplified in a volume of 20 µl containing 1x SYBR-Green-PCR-Master Mix (Applied Biosystems) and the respective forward and reverse primers (300 nmol/l). The primers designed using Primer Express® Primer Design Software v2.0 and purchased from Invitrogen were: GAPDH forward, AACTCCCTCAAGATTGTCAGCAA; GAPDH reverse, GGCTAAGCAGTTGGTGGTGC; ETA receptor forward, ATTGCCCTCAGCGAACAC; ETA receptor reverse, CAACCAAGCAGAAAGACGGTC; ETB receptor forward, TGGAGCTGAGATGTGCAAGC; ETB receptor reverse, TGATCCCCACAGAAGCCTTC; AT1a receptor forward, GCCAGTTTGCCAGCTGTCAT; AT1a receptor reverse, CGCGCACACTGTGATATTGG; AT2 receptor forward, ATCCCTGGCAAGCATCTTATGT; AT2 receptor reverse, ATGTTGGCAATGAGGACAGACA. Polymerase chain reaction (PCR) amplification was performed in triplicate (2 min at 50°C, 10 min at 95°C, 15 s at 95°C and 1 min at 60°C for a total of 40 cycles) in an ABI PrismTM 7700 sequence detector (Applied Biosystems). The expression of treated (sample) cells relative to untreated (control) cells was determined using the delta-delta method presented by PE Applied Biosystems: ratio = 2(CTsample
CTcontrol) = 2
CT.
Statistical analysis
The effects of different growth factor concentrations on SMC proliferation were analysed using one-way ANOVA, followed by Dunnett's test. Bonferroni's multiple comparison test was used to determine the differences in proliferation between SMCs from different animals under the influence of one growth factor concentration. Statistical analyses of ET and AT receptor mRNA expression in control and SNX animals were conducted with unpaired t-test or Dunnett's test, respectively. P-values 0.05 were considered statistically significant. All data are presented as a percentage of the respective controls. The mean±SEM of at least three replicates was used for statistical comparison.
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Results |
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Decrease of angiotensin and endothelin receptor mRNA expression in cultured SMCs from SNX rats
In cultured SMCs from SNX rats, downregulation of AT1a, AT2, ETA and ETB receptor mRNA was determined by real-time RTPCR (AT1a receptor: SNX 74.5±5.45%, n = 9, P 0.05 vs control, 100± 3.24%, n = 12; trandolapril 42.4±3.77%, n = 6, P
0.05; losartan 110.7±12.84, n = 6, P
0.01; ETA antagonist 145±21.24%, n = 3, P
0.01; ETAB antagonist 39.2±2.47%, n = 3, P
0.05; AT2 receptor: SNX 28.1±3.14%, n = 6, P>0.05, vs control, 100± 10.21%, n = 9; trandolapril 183.4±44.55%, n = 6, P>0.05; losartan 424.0±105.0%, n = 4, P
0.01; ETA antagonist 166.2±50.6% n = 6, P>0.05; ETAB antagonist 806.6±139.6%, n = 3, P
0.01; ETA receptor: SNX 61.6±7.04%, n = 3, P
0.05 vs control, 100±7.91%, n = 9; ETB receptor: SNX 57.8±3.37%, n = 6, P
0.01 vs control, 100±3.24%, n = 9; data not shown).
Differences between control and SNX animals in terms of SMC proliferation
To study the effects of uraemia in vivo on the proliferation of isolated SMCs, the response of cultured SMCs from uraemic and control animals to PDGF-BB, bFGF, TNF-, angiotensin II, aldosterone and ET-1 was determined.
Low concentrations of PDGF-BB (1013 1012 mol/l), bFGF (10131012 mol/l) and TNF- (10121010 mol/l) increased growth in SMCs derived from the control animals (P
0.05 compared with cells treated with cytokine-free medium). SMCs derived from SNX animals showed no increase in proliferation when incubated with these concentrations. After incubation with high concentrations of the same growth factors, SMCs from SNX rats showed a higher increase in proliferation than control SMCs. TNF-
(109 mol/l) increased proliferation to 153.38± 10.16 vs 122.27±1.41% in control (n = 8, P
0.01, Figure 1a), 109 mol/l PDGF-BB resulted in 486.60± 8.27 vs 346.74±4.60% in control (n = 8, P
0.01, Figure 1b) and 1010 mol/l bFGF stimulated proliferation to 176.68±6.50% (n = 6) vs 123.71±1.49% (n = 4) in control (P
0.01, Figure 1c).
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Influence of treatment with ET receptor antagonists, losartan or trandolapril on SMC proliferation in comparison with untreated SNX rats
In SMCs from untreated SNX rats, PDGF-BB (109 mol/l) induced the maximum proliferation of 486.6±8.27% (as compared with unstimulated cells, n = 8, P 0.01). The increase in proliferation induced by PDGF-BB (109 mol/l) was significantly reduced in SMCs from SNX rats treated with the ETA receptor antagonist (135.71±1.08%, n = 8, P
0.01), the ETAB receptor antagonist (122.72±0.58%, n = 8, P
0.01) or the AT1 receptor antagonist losartan (103.69±1.83%, n = 8, P
0.01, Figure 2a).
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TNF- (109 mol/l) stimulated proliferation in SMCs from SNX animals to a maximum of 153.4± 10.16% (n = 8, P
0.01 compared with untreated cells). ETA receptor antagonism reduced the maximum response to 115.63±0.41% (n = 8, P
0.01), whereas ETAB receptor antagonism resulted in a higher SMC responsiveness to TNF-
(maximum 202±1.82%, n = 8, P
0.01). In contrast, after losartan treatment, TNF-
reduced SMC proliferation (minimum 77.82± 1.91%, n = 8, P
0.01, Figure 2c).
SMCs isolated after trandolapril treatment showed a greater increase in proliferation in response to PDGF-BB (109 mol/l), bFGF (1010 mol/l) and TNF- (109 mol/l) than cells originating from all other animals. The maximum response to PDGF-BB (109 mol/l) was 663.48±7.00% (n = 8, P
0.01 compared with SNX) (Figure 2d). bFGF-induced BrdU incorporation reached its peak at 568.81±17.94% (n = 4, P
0.01, Figure 2e). TNF-
stimulated SMC proliferation up to 356.34±10.43% (n = 8, P
0.01, Figure 2f).
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Discussion |
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In the present study, we found SMCs from SNX rats treated with the ETA receptor antagonist to show almost control levels of proliferation in response to PDGF-BB, bFGF and TNF-. This implies that via the ETA receptor, ET-1 might play a role in a certain sensitization of SMCs in vivo, making them more responsive to pro-atherogenic growth factors. SMCs from rats treated with the combined ETAB receptor antagonist also showed control-like reactions to PDGF-BB and bFGF. When stimulated with TNF-
, however, their proliferation was stronger than in SNX. This underlines the antiproliferative significance of the ETB receptor found by Murakoshi et al. [9] and shows the importance of a physiological balance between ETA and ETB receptors.
SMCs from rats treated with the ACE inhibitor trandolapril exhibited a stronger growth factor-stimulated proliferation than SMCs from any other animal. Treatment with trandolapril thus seemed to have sensitized the cells to PDGF-BB, bFGF and TNF-. This might arise from an increase in bradykinin synthesis under trandolapril leading to a sensitization of mitogen-activated protein kinases [10], which is essential for growth factor-mediated stimulation of DNA synthesis [11]. Accordingly, the augmented SMC proliferation in response to PDGF-BB, bFGF and TNF-
seen after trandolapril treatment might be due to a modified state of activation in these cells.
In contrast to our findings, several in vitro studies, and also in vivo models using balloon angioplasty, demonstrated a growth-inhibiting function of both ACE inhibitors and AT1 receptor antagonists [12]. However, several observations indicated that ACE inhibitors do not always reduce proliferation rates in smooth muscle cells. In a direct in vivo comparison of an AT1 receptor antagonist and the ACE inhibitor temocapril, Teng et al. [13] found that temocapril was not able to inhibit SMC proliferation in spontaneously hypertensive rats (SHRs) whereas it did in Wistar rats. Richter et al. showed in an in vivo model of transplant vasculopathy that the ACE inhibitor enalapril was able to reduce SMC proliferation in small intramyocardial arteries, but not in large epicardial vessels if given before intervention [14]. For an AT1 receptor antagonist, the same group could not demonstrate an antiproliferative function in large epicardial vessels. In our experiments, trandolapril even increased SMC proliferation under different growth factors. Furthermore, Wilson et al. [15] could demonstrate a comparable effect with an increased vessel stenosis using captopril in a model of neointima proliferation in a porcine coronary artery culture model. In these experiments, losartan inhibited neointima formation whereas captopril increased vessel stenosis by 200%. In an experiment with SHRs, Bravo et al. [16] found the proliferation of isolated carotid SMCs to be reduced after 16 weeks of losartan treatment but not after captopril medication. Additionally, cilazapril was demonstrated to inhibit neointimal formation in ballooned guinea pig carotid but not rabbit iliac arteries [17]. In porcine models of restenosis involving injury to the coronary arteries, neither cilazapril [18], enalapril [19], trandolapril nor captopril [20] were effective. Even worse were the results in patients with the ACE gene polymorphism DD, because when these patients were treated with an ACE inhibitor, an increased frequency of in-stent restenosis was found. This indicates that the growth-inhibitory action of ACE inhibitors depends on the genetic background of the individuals and also on the treatment regime [21]. In comparison with these in vivo analyses, our studies were done in vitro after isolation of aortal SMCs.
Limitations of the study
The evolution of atherosclerosis is a very complex mechanism involving SMCs, but also many other components. Rupture of atherosclerotic plaques has been identified as the proximate event in the majority of cases of acute ischaemic syndromes. Vulnerable plaques are characterized by a high lipid content, increased numbers of inflammatory cells and extensive adventitial and intimal neovascularity. The fibrous cap of an atherosclerotic plaque may become thin and rupture as a result of the depletion of matrix components through the activation of enzymes secreted by SMCs. This indicates that SMC proliferation represents only one component of atherosclerosis. Whether the inhibition of SMC proliferation in uraemia reduces the induction and progression of atherosclerosis cannot be fully answered by our investigations. However, in advanced coronary atherosclerotic plaques associated with unstable angina, an augmentation of inflammatory cell activity with significantly increased SMC areas has been shown. Therefore, the inhibition of SMC proliferation might represent one means of reducing susceptibility to plaque rupture.
In summary, we demonstrated that uraemia in the rat entails modifications in SMC responsiveness to various pro-atherogenic growth factors. These changes are still present when SMCs are isolated and subcultured. Consequently, certain factors present in the uraemic vasculature seem to induce permanent genetic changes in SMCs. Furthermore, we revealed that treatment of SNX rats with ET receptor antagonists or the AT1 receptor antagonist losartan more or less reduces accelerated proliferation of SMCs. Further investigations concerning the intracellular signalling mechanisms involved in the uraemia-related SMC modifications observed in this study should help to come to a deeper understanding of atherogenesis in uraemia.
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Acknowledgments |
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Conflict of interest statement. None declared.
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Notes |
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References |
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