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1U460 Institut National de la Santé et de la Recherche Médicale, 2Service d'Anatomo-Pathologie, and 3Département de Cardiologie, Centre Hospitalier Universitaire Bichat-Assistance Publique des Hopitaux de Paris, 75018 Paris, France
Submitted 25 September 2003 ; accepted in final form 25 March 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B/I-B system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-B
and I-B
degradation, NF-B activation, as well as the expression of the NF-B-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-B inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.
nuclear factor-B; intimal hyperplasia; cell migration and proliferation; interleukin-6
B/I-B system. Specifically, IL-10 inhibits the nuclear translocation of the transcription factor NF-B in activated monocytes (24), presumably via reduced proteolytic degradation of the NF-B specific inhibitor I-B (14). Several lines of evidence suggest that, in addition to its anti-inflammatory activity, IL-10 directly inactivates vascular cells (22). We explored the hypothesis that IL-10 inactivates vascular smooth muscle cells (SMCs), the principal cellular component of neointimal lesions induced by balloon injury or stenting in experimental models (26) and in humans (5), and that this effect involves the NF-B/I-B system.
The goals of the present study were to investigate whether 1) SMCs express the IL-10 receptor (IL-10R), 2) IL-10 modulates the NF-B/I-B system in SMCs in vitro and in vivo, and 3) IL-10 inhibits SMC activation and intimal hyperplasia after balloon injury of the rat aorta.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Wistar rats (Iffa Credo; Labresle, France), 350 g body wt, were used for in vitro preparation of SMC and in vivo studies. Animal protocols were in accordance with the European Community Standards on the care and use of laboratory animals (authorization no. 00577).
Cell Culture
Aortic SMCs were obtained from rat aortas, and their SMC identity was verified by immunohistochemistry with an anti -actin antibody (Dako; Glostrup, Denmark), as described (1).
RT-PCR
mRNA levels of IL-10R and IL-6, a prototypic NF-B-dependent gene (8), were evaluated by real-time RT-PCR assays using a Light Cycler (Roche Diagnostics; Meylan, France) with the FastStart DNA Master SYBR Green kit (Roche Diagnostics). For quantification, a standard curve was generated with various dilutions of the cDNA templates. mRNA levels were normalized to the mRNA levels of the -actin housekeeping gene. Oligonucleotide primers for IL-10R, IL-6, and -actin mRNA are shown in Table 1.
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The cells were stimulated with 1 µg/ml Escherichia coli LPS (Sigma; St. Louis, MO) for 45 min, with or without 24-h preincubation with 1 ng/ml murine IL-10 (mIL-10; kind gift of Dr. Alexandre Lebeaut, Schering-Plough Research Institute; Kenilworth, NJ). Cytoplasmic and nuclear protein extracts were prepared as described (4).
Harvested arteries were stripped off the adventitia, immediately snap frozen in liquid nitrogen, and kept at –80°C until use. The arteries were homogenized in cold lysis buffer with the use of a glass tissue grinder (Polylabo; Vineland, NJ).
Western Blot Analysis
Western blots were performed on cytoplasmic protein extracts from cultured SMCs and in total protein extracts from rat aortas using polyclonal antibodies against I-B and I-B (dilution 1/3,000, Tebu; Le Perray-en-Yvelines, France) or the IL-10R (dilution 1/2,000, Tebu), as described (7). Protein extracts obtained from rat spleens were used as positive controls for the detection of IL-10R.
EMSA
EMSA were performed on 20 µg of nuclear proteins with a commercial kit (Promega; Madison, WI), as described (7). The specificity of the reaction was assessed with 1) anti-p50 and anti-p65 monoclonal antibodies, and 2) 100-fold molar excess of cold oligonucleotide probes for NF-B or the irrelevant transcription factor SP-1.
IL-6 Secretion
The effect of mIL-10 on LPS-induced IL-6 release was measured in SMC-conditioned medium 6 h after LPS stimulation using a standard ELISA (BioSource; Camarillo, CA).
DNA Synthesis
DNA synthesis in SMCs was assessed by measurement of the incorporation of [3H]thymidine. Briefly, SMCs (20 x 104/cm2) were grown in 24-well plates to confluence in DMEM containing 10% fetal bovine serum. Cells were made quiescent by incubation with serum-free DMEM for 48 h. After being replenished with fresh DMEM, cells were incubated with 10 ng/ml basic FGF (bFGF) (PromoKine; Heidelberg, Germany) or both for 24 h. [3H]Thymidine (1 µCi) was added to each well and cells were incubated for 4 h. Incorporation of [3H]thymidine into the trichloroacetic acid-precipitable fraction was determined as described (21).
Cell Migration Assay
Smooth muscle cell migration was examined in 60 mm2 petri dishes containing 10% fetal bovine serum. Cells were grown to confluence, and the cells located in the center of the petri dish were removed with the transient application of a 6-mm2 disk of glass microfiber filters (Whatman International; Maidstone, UK). Cells were stimulated with 10 ng/ml bFGF, with or without 1 ng/ml mIL-10, for 24 h at 37°C in 5% CO2. Cells were washed with PBS, fixed in 4% paraformaldehyde, and stained with 10% Giemsa. The number of SMCs that had migrated from the edge to the center of the initially cell-free disk was determined microscopically.
Pharmacokinetics of mIL-10 In Vivo
Blood samples (2 ml) were collected on heparin from the tail vein of rats before, 30 min, 2, 6, 8, and 12 h after the first injection of saline (n = 3) or mIL-10 (1 µg/kg ip, n = 3; 5 µg/kg, n = 3; 20 µg/kg, n = 3). Blood samples were immediately centrifuged and plasma was kept at –80°C until analysis. Plasma IL-10 levels were measured in duplicate using a standard ELISA kit (Duoset, R&D systems; Abingdon, UK).
Experimental Model of Arterial Injury
Fifty-four rats underwent aortic balloon injury, as described (19). Treated rats (n = 26) received 20 µg/kg ip mIL-10 1 h before arterial injury, then daily until death. This mIL-10 dosage was based on the results of pharmacokinetic and NF-B activation studies (see RESULTS). Control rats (n = 25) received saline after the same injection protocol. Three rats were used as sham-operated controls. Animals were euthanized by pentobarbital overdose 6 h (mIL-10: n = 5, saline: n = 4, sham: n = 3), 4 days (mIL-10: n = 7, saline: n = 7), 7 days (mIL-10: n = 5, saline: n = 5), or 14 days (mIL-10: n = 9, saline: n = 9) after the procedure.
Morphometry and Immunohistochemistry
Seven and fourteeen days after arterial injury, arterial segments were pressure-perfusion fixed with 4% paraformaldehyde through a left ventricular puncture. Two serial 5-mm-long rings, spanning the entire infrarenal abdominal aorta, were cut and embedded in paraffin. Four 4-µm cross sections were cut from each ring, and stained with hematoxylin and eosin or orcein. Digital planimetry of orcein-stained arterial cross sections was performed as described (6).
Additional cross sections were immunostained with mouse monoclonal antibodies directed against the following: 1) ED-1, a marker of rat macrophage cytoplasm (dilution 1/100; Argene; Kidlington, UK); 2) PCNA, a nuclear proliferation marker (dilution 1/50, Castra; Newcastle, UK); or 3) smooth muscle -actin (dilution 1/100, Dako; Glostrup, Denmark), as described (6). For negative control experiments, primary antibodies were omitted.
Statistical Analysis
Data are expressed as means ± SD. EMSA and Western blot quantification were assessed by densitometric analyses of scanned films with the use of the NIH Image version 1.61 software. For RT-PCR analysis, the Light Cycler software was used and the results were expressed in arbitrary units and adjusted for -actin mRNA levels. A one-way ANOVA with post hoc Fisher's protected least-significant difference test was used to evaluate the effect of mIL-10 on I-B/NF-B levels, IL-6 mRNA and cytokine levels, and proliferation and migration of cultured SMCs. I-B levels in the aorta of mIL-10-treated versus control rats were compared with two-way ANOVA, which tested the effects of mIL-10, time, and the interaction of both. When the ANOVA indicated an overall difference between groups, specific intergroup differences at each time point, as well as intragroup variations over time, were tested using a one-way ANOVA and post hoc Fisher's protected least-significant difference test. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In vitro, the presence of IL-10R on SMCs was first demonstrated using RT-PCR (data not shown). Western blots were performed on SMC protein extracts with the use of an anti-IL-10R antibody displayed a band that migrated at 110 kDa (Fig. 1), consistent with the molecular weight of IL-10R (10). The same band was found in protein extracts obtained from rat spleens (data not shown). LPS stimulation of SMCs resulted in an increase of IL-10R expression (from 11.5 x 103 to 18 x 103 densitometric units; P < 0.05).
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Effects of mIL-10 on NF-B and I-B in SMCs
Stimulation of SMCs with LPS decreased cytosolic levels of I-B and I-B proteins (Western blots) and increased NF-B nuclear translocation (EMSA). Figure 2 shows that preincubation of SMCs with 1 ng/ml mIL-10 significantly reduced I-B and I-B degradation (–32% and –31%, respectively; P < 0.05 for both) and NF-B translocation (–75%, P < 0.005).
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B-Dependent IL-6 Gene Expression in SMCsThere was a 4.1-fold increase in IL-6 mRNA levels in LPS-stimulated SMCs (Fig. 3A), an effect that was reduced by 19% by mIL-10 (P = 0.01). Similarly, mIL-10 reduced IL-6 levels by 40% (P < 0.005) in the conditioned medium of LPS-stimulated SMCs (Fig. 3B).
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Incubation of SMCs with 10 ng/ml bFGF resulted in 3.3-fold increase in [3H]thymidine incorporation (data not shown). In presence of 1 ng/ml mIL-10, this increase was reduced by 60% (P < 0.0001). Similarly, mIL-10 reduced by 60% the bFGF-induced 6.1-fold increase (P < 0.0001) in SMC migration (Fig. 4).
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Pharmacokinetic of mIL-10 in rats.
Low basal levels of mIL-10 were detectable both in control and mIL-10-treated rats (
150 pg/ml). Thirty minutes after intraperitoneal injection of 20 µg/kg mIL-10, plasma mIL-10 concentrations increased >2,000-fold over baseline and then rapidly decreased but remained in the 1 ng/ml range (i.e., the mIL-10 concentration, which was shown to inhibit I-B degradation and NF-B activation in SMCs in vitro) for at least 12 h (Fig. 5). Lower mIL-10 dosages resulted in <1 ng/ml plasma mIL-10 concentrations as soon as 2 h after intraperitoneal injection (data not shown). Therefore, the 20 µg/kg mIL-10 dosage was subsequently used in the in vivo studies.
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B Activation and I-B Degradation After Balloon Injury
I-B and I-B levels decreased dramatically 6 h and 4 days after balloon injury of the rat aorta and then returned to baseline at 14 days (Fig. 6, A and C). In rats treated with mIL-10, I-B degradation was significantly reduced 6 h and 4 days after injury (two-way ANOVA: mIL-10 effect, P < 0.05; time effect, P < 0.005; interaction term, P = 0.7). A similar effect of mIL-10 on I-B levels was observed (two-way ANOVA: mIL-10 effect, P < 0.005; time effect, P < 0.005; interaction term, P = 0.6).
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B activation was detected by EMSA 6 h and 4 days after balloon injury (Fig. 6, B and D) but was no longer visible at 14 days. Treatment with mIL-10 reduced NF-B activation by 90% at 6 h and by 66% at 4 days (2-way ANOVA: mIL-10 effect, P < 0.005; time effect, P < 0.005; interaction term, P = 0.02). Effects of mIL-10 on Arterial Proliferative Activity after Balloon Injury
In this model, balloon injury of the infrarenal abdominal aorta results in full endothelial abrasion (data not shown). Seven days after balloon injury, reendothelialization of the injured aorta was partial at this time point without difference between the two groups (mIL-10: 54.3 ± 21.8 endothelial cells/section; saline: 54.3 ± 36.4 endothelial cells/section; P = 0.9). Proliferative activity in the intima and in the media, assessed by PCNA immunostaining, was reduced by 53% in mIL-10-treated rats (mIL-10: 109.6 ± 47.9 cells/cross section; saline: 206.6 ± 104.1 cells/cross section; P < 0.002).
Fourteen days after arterial injury, a mild intimal hyperplasia developed in saline-treated animals (Fig. 7). Aortas were almost reendothelialized without difference between the two groups (mIL-10: 121.7 ± 33.8 endothelial cells/section; saline: 113.9 ± 21.3 endothelial cells/section; P = 0.12). The intima was almost exclusively composed of SMCs and extracellular matrix, with virtually no macrophage (<1 cell/cross section). Intimal growth, expressed either as intimal area (mIL-10: 147 ± 11 µm2; saline: 441 ± 38 µm2; P < 0.005) or the intima-to-media ratio (mIL-10: 0.19 ± 0.02; saline: 0.75 ± 0.08; P < 0.005), was reduced by 75% in mIL-10-treated rats. In addition, treatment with mIL-10 resulted in a 70% reduction in the absolute number of SMCs in the intima (mIL-10: 112.3 ± 13 cells/cross section; saline: 380.0 ± 32 cells/cross section; P < 0.005), as well as 76% reduction in PCNA-positive cells in the intima and the media (mIL-10: 63.2 ± 7 cells/cross section; saline: 258.9 ± 21 cells/cross section; P < 0.005) 14 days after arterial injury.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B degradation and NF-B activation; and 3) mIL-10 inhibits SMC proliferation and migration, resulting in reduced intimal hyperplasia after balloon injury of the rat aorta. Overall, our results suggest a direct inhibitory effect of mIL-10 on vascular SMCs. Our data are in keeping with previous studies suggesting a direct inhibitory effect of IL-10 on SMC proliferation (22). In addition, we show that IL-10 inhibits SMC migration and we demonstrate for the first time that the IL-10R is present, and can be upregulated, in SMCs.
IL-10 is a known inhibitor of NF-B in macrophages (14). Our data extend this observation to vascular SMCs. NF-B activation in SMCs is a key feature of arterial response to balloon injury (16, 17), and hence, NF-B has been proposed as a potential intracellular target for novel therapeutic approaches against restenosis (15). A host of NF-B-dependent genes may be involved in postangioplasty intimal hyperplasia (15), including the proinflammatory gene IL-6 (12). Interestingly, recent studies suggest that IL-6 may facilitate SMC migration via cytoskeleton reorganization (25), providing a mechanism for the IL-10-induced inhibition of SMC migration observed in our study. Other NF-B-dependent genes that stimulate SMC proliferation (e.g., cyclin D1) or inflammation (e.g., intercellular adhesion molecule-1) may be the targets for IL-10 as well.
Whether the inhibitory effect of mIL-10 on intimal growth in the rat aorta stems from direct inactivation of SMC is unclear. The presence of the IL-10R in SMCs and the inhibitory effects of mIL-10 on NF-B activation, SMC proliferation and migration favor this hypothesis. Alternatively, the inhibitory effect of IL-10 on intimal hyperplasia may be an indirect consequence of IL-10-induced leucocyte inactivation. For example, IL-10 inhibits metalloproteinase-9 and stimulates tissue inhibitor of metalloproteinase-1 in macrophages (13), which in turn may reduce SMC migration in the intima. In addition, IL-10 is a potent inhibitor of IL-1 secretion by circulating monocytes (6), and thereby may inhibit IL-1-induced SMC proliferation. Finally, IL-10 has both negative (11) and positive (23) effects on interferon-
secretion on SMC proliferation and intimal growth in rats (9), the possibility that IL-10 influences intimal hyperplasia via an interferon--dependent pathway cannot be ruled out.
In the rat model, the inhibitory effect of IL-10 on intimal hyperplasia is mediated by SMC inactivation. Because, in the rat, intimal growth results almost exclusively from SMC migration and proliferation, with only marginal involvement of inflammatory cells, our data cannot be extrapolated to more relevant models of restenosis, in particular those incorporating the implantation of metallic stents. After a stenting procedure, inflammatory cells constitute up to 20% of intimal lesions (6), and, in this context, both the anti-inflammatory and anti-proliferative effects of IL-10 may operate.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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B and preservation of IB by interleukin-10 and interleukin-13. J Clin Invest 100: 2443–2448, 1997.[ISI][Medline]
B and IB system in injured arteries. Pathobiology 66: 311–320, 1998.[CrossRef][ISI][Medline]
B and IB- by aortic endothelium in an arterial injury model. Am J Pathol 148: 427–438, 1996.[Abstract]
-inducing factors but enhances NK cell production of IFN-. J Immunol 161: 4283–4288, 1998.B (NF B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem 270: 9558–9563, 1995.B is required for TNF--directed smooth muscle cell migration. FEBS Lett 508: 360–364, 2001.[CrossRef][ISI][Medline]
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P < 0.005 vs. LPS alone. D.U., densitometric units. B: EMSA (top) and bar graph (bottom) showing the inhibitory effect of 1 ng/ml mIL-10 on LPS-induced nuclear translocation of NF-
) or saline (
).
