Acta Physiologica Sinica, April 25, 2003, 55(2): 128-134

Received 2002-08-12

Accepted 2002-11-27

This project was supported by grants from the Foundation of Guangzhou Science and Technology Committee, P.R.China (JB00000448165).

Corresponding author. Tel: +86-20-84113655 ; Fax:+86-20-84038377; E-mail: ls36@zsu.edu.cn

Research Paper

Recombinant human interleukin-10 inhibits proliferation of vascular smooth muscle cells stimulated by advanced glycation end products and neointima hyperplasia after carotid injury in the rat

OUYANG Ping, PENG Li-Sheng, YANG Hong, PENG Wen-Lie, WU Wen-Yan, XU An-Long*

Department of Biochemistry, School of Life Science, Sun Yat-sen University, Guangzhou 510275, China

Abstract: The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on proliferation of vascular smooth muscle cells (VSMCs) stimulated by advanced glycation end products (AGE) and neointima hyperplasia after rat carotid arterial injury. Rat aortic VSMCs were cultured and treated with rhIL-10 or AGE respectively, and then co-treated with rhIL-10 and AGE. Proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytomertry. Sprague-Dawley rats were treated with recombinant human IL-10 (rhIL-10) for 3 d after carotid arteries injury. The ratio of neointima to media area at the site of arterial injury was measured 28 d after balloon injury. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control, AGE stimulated VSMCs proliferation. rhIL-10 alone had no effect on VSMCs growth. With AGE stimulation, rhIL-10, at dose as low as 10 ng/ml, inhibited VSMCs growth (P<0.05). The cell number in G0/G1 phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone (P<0.01) by flow cytometry analysis. Compared with the control group of neointima hyperplasia in rats, the ratio of neointima to media area of recombinant human IL-10 group was reduced by 45% (P<0.01). The p44/42 MAPK activity was significantly enhanced by AGE. The AGE effects were opposed by rhIL-10. The anti-inflammatory cytokine rhIL-10 inhibits AGE-induced VSMCs proliferation. Recombinant human IL-10 also inhibited neointima hyperplasia after carotid artery injury in rats. The results suggest the possibility that recombinant human IL-10, as a potential therapeutic approach, prevents neointimal hyperplasia.

Key words: pathology; interleukin-10; muscle; smooth; vascular; advanced glycation end products; neointima hyperplasia

重组人白介素-10抑制晚期糖基化终产物诱导的大鼠

血管平滑肌细胞和血管新生内膜的增殖

欧阳平, 彭立胜, 杨红, 彭文烈, 吴文言, 徐安龙*

中山大学生命科学学院生物化学系, 广州 510275

摘要: 研究观察了重组人白介素10 (rhIL-10)对晚期糖基化终产物(AGE) 刺激下离体大鼠胸主动脉血管平滑肌细胞增殖及对SD大鼠血管损伤后新生内膜增殖的影响。体外培养大鼠主动脉血管平滑肌细胞, 采用 MTS/PES法确定血管平滑肌细胞的增殖状态; 应用流式细胞术测定细胞周期; 利用 p44/42 磷酸化抗 MAPK抗体的蛋白免疫印迹法测定p44/42 MAPK磷酸化蛋白表达。利用大鼠颈动脉血管损伤模型, 观察 rhIL-10对新生内膜增殖的影响。结果显示: (1) AGE处理组与对照组相比, AGE对血管平滑肌细胞增殖具有明显的刺激作用(P<0.05)。 rhIL-10单独应用对血管平滑肌细胞生长没有影响(P>0.05)。在AGE刺激下, 低至100 ng/ml的rhIL-10可抑制血管平滑肌细胞的生长(P<0.05)。 (2) 流式细胞术测定的结果显示, rhIL-10可以使AGE作用下的VSMC大部分处于G0/G1期, 与对照组相比有明显差异(P<0.01)。(3) AGE对p44/p42 MAPK磷酸化蛋白表达有显著的增强作用, 此作用可被rhIL-10抑制(P<0.001)。 (4) 大鼠颈动脉损伤后, rhIL-10治疗组的动脉血管新生内膜/中层面积比低于对照组约45％ (P<0.01)。表明抗炎细胞因子rhIL-10可抑制AGE诱导的大鼠血管平滑肌细胞增殖和血管新生内膜的增殖。

关键词: 病理学; 白介素10; 肌; 平滑; 血管; 晚期糖基化终产物; 新生内膜增殖

中图分类号: Q463

Accumulating evidence suggests that atherogenesis represents an exaggerated inflammatory response to vascular injury. After vessel injury, monocytes, platelets, and lymphocytes adhere to the vessel wall and release an array of cytokines and peptide growth factors. These growth-regulatory substances bind to their respective receptors and transduce signals that may influence the phenotype and growth of vascular smooth muscle cells (VSMCs), thus promoting development of advanced fibroproliferative lesions[1]. Smooth muscle cells in atheromatous lesions, but not in normal human arteries, express the human leukocyte antigen DR (HLA-DR)[2]. Encoded by the major histocompatibility complex (MHC), the HLA-DR glycoprotein participates in antigen presentation to T cells. As such, a local immune response, as suggested by T lymphcyte-VSMCs interactions, may contribute to atherogenesis. Manipulation of the anti-inflammatory products of T lymphocytes may provide a therapeutic strategy against pathologic vascular remodeling.

Intimal hyperplasia is a universal response of the arterial wall to balloon injury and the main mechanism of restenosis[3] after stent implantation[4], a technique used for the majority of percutaneous coronary interventions. Inflammatory cells play a key role in postinjury intimal hyperplasia. In the days after balloon angioplasty[5] or stenting[6], monocytes are recruited at the injury site, where they become activated macrophages. The magnitude of macrophage infiltration in stented lesions is correlated to subsequent intimal growth[7].

Modification of proteins by long-term incubation with glucose leads, through the formation of early stage products such as Schiff base and Amodori rearrangement products, to the formation of advanced glycation end products (AGE). AGE can alter protein function and disturb cellular metabolism. The occurrence of advanced glycation end products is considered today as the main pathogenic agent of atherosclerosis[8].

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a powerful inhibitory effect on monocytes. The principal routine function of IL-10 appears to limit and ultimately terminate inflammatory responses[9]. We studied the effect of recombinant human IL-10 (rhIL-10) on proliferation of rat VSMCs by AGE, and neointima hyperplasia after carotid arteries injury in rat.

1 MATERIALS AND METHODS

1.1 Materials. Hank′s balanced salt solution, Dulbecco′s modified Eagle′s medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), L-glutamine and antibiotic (penicillin G 10000 U/ml, streptomycin sulfate 10000 mg/ml) were obtained from GibcoBrl (Grand Island, NY, USA). Propidium iodide (PI), collagenase and elastase were obtained from Sigma Chemical (St Louis, Mo, USA). Recombinant human IL-10 protein was obtained from the expression and purification of rhIL-10 in Escherichia coli led to a development of stable and effective down stream techniques in our lab. The purity of recombinant human IL-10 was more than 98%. All cytokines were reconstituted in PBS with 0.5% FBS and diluted to desired concentrations with DMEM/5% FBS. Male Sprague-Dawley (SD) rats, weighing 0.30 to 0.4 kg, were obtained from the Animal Center of the First Military Medical University (Guangzhou, China). 2.0F Fogarty catheters were purchased from Baxter (Irvine, CA.USA).

1.2 Preparation of AGE-modified human serum albumin (AGE-HSA). AGE-modified human serum albumin was prepared in vitro as previously described. Briefly, 1.75 mg/ml of normal human serum albumin (Cortex Biochem, USA) was incubated at 37℃ for 8 weeks with 100 nmol/L D-glucose in 100 mmol/L phosphate buffer containing 200 U/ml penicillin, 70 μg/ml gentamicin,and 1.5 mmol/L PMSF. Samples incubated in an identical manner in the absence of glucose were used as controls. After incubation, all samples were dialyzed against phosphate buffer (pH 7.4).

1.3 Cell isolation and culture. SD rat VSMCs were isolated from the segments of thoracic aorta harvested. Vessels were opened longitudinally in DMEM supplemented with antibiotics. The vessels were digested with 0.5 mg/ml collagenase. Endothelial and adventitial layers were removed, and the remaining medial layer was placed in 1 mg/ml elastase solution for 1 h. After serial centrifugation and trypsinization the resulting cells were suspended in a tissue culture flask and placed in a 37℃, 5% CO2 incubator with a “complete media” containing DMEM, antibiotics, L-glutamine, and 10% FBS. Phase contrast microscopy revealed typical “hill and valley” morphology. Purity of isolation was demonstrated immunohistochemically with uniform phalloidin staining for F-actin and α-smooth muscle actin (Sigma).All studies were conducted using cells from passage 1 to 5．

1.4 Proliferation assay. Cells were trypsinized and plated with complete media in 1% gelatin coated 96-well microtiter plates at a density of 3000 cells/well. After 8 h the medium was changed to serum-free DMEM with glutamine and antibiotics. Serum-free conditions were maintained for 48 h to allow fro synchronized growth arrest. The medium was then changed into DMEM/5% FBS and the appropriate experimental agent. After 24 h rates of proliferation were determined with the CellTiter 96 assay (Promega, Madison, Wis. USA). A methoxyphenyl-tetrazolium salt (MTS)/phenazine ethosulfate (PBS) compound was bioreduced by cells into a colored formazan product that may be quantified colormetrically. The technique is equivalent to tritiated thymidine incorporation in determining viable cell numbers. 20 μl of MTS/PES was added to the appropriate wells and plates were incubated at 37℃ for 90 min. Absorbance was then recorded at 490 nm with a microtiter plate reader.Proliferation was subsequently expressed as absorbance (λ). Each experiment was done in quadruplicate.

1.5 Flow cytometric protocols for cell cycle analysis. VSMCs were harvested and single cell suspension in buffer (PBS+2% FBS) was prepared. Cells were washed and resuspended at 1×106 cells/ml. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube and 3 ml cold absolute ethanol was added. Cells were fixed for at least 1 h at 4℃. Cells were washed in PBS. One ml of prodium iodide staining solution was added to cell pellet and mixed well. 50 μg/ml of RNase A stock solution was added and incubated for 3 h at 4℃. VSMCs cycle analysis was performed by flow cytometry (Coulter Co. USA).

1.6 Western immunoblotting． Sprague-Dawley rat VSMCs culture were plated in 6-well culture plates at a density of 8×105 cells/well. Some of the cells were incubated with or without recombinant human IL-10 (100 ng/ml) in the presence of AGE (50 μg/ml) for 30 min after synchronized growth arrest. Immediately following culturing,VSMCs were washed in ice-cold PBS three times, scraped into ice-cold PBS, and lysed in an ice-cold lysis buffer containing 20 mmol/L Tris (pH 7.5), 150 mmolL NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L NaVO4 , 1 μg/ml and 1 mmol/L PMSF. After being incubated for 30 min on ice, samples were centrifuged at high speed for 15 min, and supernatants were collected. Total cellular protein was quantified with the Bio-Rad protein assay, and an aliquot of 100 μg of protein was separated by SDS-PAGE and transferred electrophoretically to Hybond polyvinylidene difluoride membranes. Determination of p44/42 MAPK activity was conducted with the p44/42 MAP Kinase Assay Kit (New England Biolabs, Beverly, MA, Catalog #9800) in strict accordance with the manufacturer′s instruction. The Immun-Star chemiluminescence substrate was used to develop the blots. Densitometric analysis was performed for all immunoblots using the Fluor-S Multimager with Quantity One software (Bio-Rad).

1.7 Effects of recombinant human IL-10 on the neointima proliferation after carotid artery injury in rat．

1.7.1 Construction of rat carotid injury model by balloon denudation． Male SD rats weighing 300-400 g were anesthetized with sodium pentobarbital (30 mg/kg IP), cut in the medium of cervix, sodium heparin 100 U/kg were injected via the external jugular vein, A 2F Forgaty catheter was inserted into the external carotid and pass down the left common carotid artery. The balloon was expanded with 30-second inflation at 1.5 atm. This procedure was repeated three times.

1.7.2 IL-10 Regimen． Five micrograms of rhIL-10 was injected intravenously 30 min before angioplasty, then 3 times daily for 72 h after angioplasty. Control animals received saline after the same injection protocol as mentioned above.

1.7.3 Fixation and removal of vessel segments. Three weeks after surgery, the rats were killed by an overdose of pentobarbital. For experiments involving histological examination of vessels, normal saline containing heparin was perfused for 15 min until the arterial tree was clear of blood. This is followed by perfusion fixation with 10% neutral buffered formalin. Cross sections from paraffin-embedded rat carotid arteries were stained with hematoxylin-eosin. Measurements of neointimal and medial corss-sectional area were determined by two independent reviewers in a double blinded fashion on four sections from each artery that spanned the 1.0 cm region of arterial injury using the Quantiment 520 image analysis system. Neointimal and medial boundaries were determined by internal elastic lamina and digital planimetry. Four measurements were averaged for each artery.

1.8 Statistical analysis． Data are presented as mean±SD. SPSS software was used in statistical analysis. Analysis of variance was used by ANVOA with Games-Howell test. Statistical significance was accepted within 95% confidence limits.

2 RESULTS

Treatment of VSMCs for 24 h with rhIL-10 alone had no effect on rat VSMCs proliferation (P>0.05 vs control at all doses, Fig.1). After growth arrest rat aortic VSMCs were stimulated with AGE for 24 h. AGE induced rat VSMCs growth. Compared to control, AGE induced VSMCs proliferation at a dose as low as 100 ng/ml (Table 1, P<0.05). AGE could not induce VSMCs proliferation at a dose of 10 ng/ml or 1 ng/ml. Dose-dependence of AGE could not be detected. When rhIL-10 incubated concomitantly with AGE, at a dose as low as 10 ng/ml, rhIL-10 inhibited AGE-induced VSMCs proliferation (0.989±0.217, P<0.05). More than 95% of the cells co-treated with rhIL-10 and AGE remained viable, which was demonstrated by trypan blue staining.

Cell cycle analysis showed that cell number in G0/G1 phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone group (Table 2, P<0.01) by flow cytometry analysis.

Table　１.Effects of advanced glycation end products on rat VSMCs proliferation (mean±SD, n=6)

Group	Ratio of cell proliferation (λ)
Control	0.664±0.196
AGE-HAS
100 μg/ml	1.094±0.079*
90 μg/ml	1.076±0.074*
80 μg/ml	1.107±0.254*
70 μg/ml	1.015±0.173*
60 μg/ml	0.995±0.367*
50 μg/ml	0.972±0.333*
40 μg/ml	1.037±0.154*
30 μg/ml	1.134±0.115*
20 μg/ml	0.963±0.116*
10 μg/ml	1.043±0.124*
100 ng/ml	0.954±0.322*
10 ng/ml	0.896±0.172
1 ng/ml	0.769±0.189

*P<0.05 vs control group.

Table　2.Effects of rhIL-10 and AGE co-treatment on cell cycle of rat VSMCs (mean±SD, n=6)

Group	G0/G1	S	G2/M
Control	81.7±2.6	9.5±0.6	8.8±1.5
AGE	75.2±2.9++	7.3±1.1	17.5±1.7
AGE+rhIL-10	82.4±1.5++	5.9±1.9	11.7±1.2

++P< 0.01 vs AGE group or AGE group vs control group.

In this study, to observe the effects of recombinant human IL-10 on neointima hyperplasia after carotid arteries injury in rat, we established a rat carotid injury model and the rats were treated with rhIL-10. Twenty-eight days after rat carotid injury, neointimal and medial areas were measured. A significant reduction in neointima/media area ratio was observed in arteries of the animals treated with rhIL-10 (45% reduction, P<0.01, Table 3 and Fig.2).

Table　3.Neointima/media ratio of rat carotid artery at d 28 after balloon injury (mean±SD, n=6)

Group	Neointima/media ratio
Arterial injury	1.264±0.114
rhIL-10+arterial injury	0.695±0.103**

**P<0.01 vs control group.

Data of Western immunoblotting showed that p44/42 MAPK phosphorylation was significantly induced within 30 min after AGE stimulation compared with control group (P<0.001, Fig.3). rhIL-10 moderately inhibited the activation of p44/p42 MAPK induced by AGE (n=3 for each condition; 311±54 vs 610±68; P<0.001; Fig.4).

Fig. 1. VSMCs proliferation in unstimulated cells (control) and in response to rhIL-10 did not affect unstimulated VSMCs’ growth.

Fig. 2.Effects of recombinant human interleukin-10 on proliferation of rat VSMCs stimualted by advanced glycation end products. n=6, *P<0.05 vs AGE-HSA group, separately.

Fig.3.Effects of recombinant human interleukin-10 on neointima hyperplasia after carotid artery was injured in the rat. A: Control group. B: rhIL-10 treatment group. Hematoxylin-eosin stain, 90×.

Fig.4.Inhibitory effects of recombinant human interleukin-10 on p44/42 mitogen-activated protein kinase phosphorylation by AGE in VSMCs. Line 1, control; line 2, AGE+rhIL-10; line 3, AGE.

3 DISCUSSION

The main findings of the present study are (1) AGE stimulated VSMCs proliferation. rhIL-10 alone had no effect on VSMCs growth. With AGE stimulation, rhIL-10, at dose as low as 100 ng/ml, inhibited VSMCs growth (P<0.05); (2) cell number in G0/G1 phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone group, assessed by flow cytometry analysis; (3) the p44/42 MAPK activity was significantly enhanced by AGE and the AGE effect was opposed by rhIL-10; (4) systemic administration of the anti-inflammatory cytokine rhIL-10 inhibited intimal hyperplasia after balloon injury in rats.

The machanisms of chronic vascular dysfunction have been much less studied than the acute response to proinflammatory cytokines. The transient induction of leukocyte adherence molecules, tissue factor and vascular hyperpermeability following exposure of endothelium to agents such as tumor necrosis factor-α and PDGF are a hallmark of the acute inflammatory response[10]. However, the situation is less defined in chronic vascular diseases such as diabetes. The inexorable accumulation of AGE in the vessel wall provides a means of distinguishing normal from diabetic vessels[11].The effects of advanced glycation end products can be summarized as follows: first, intracellular AGE can directly alter protein function in cells; second, extracellular AGE alters cell-matrix and cell-cell interactions; and finally, interaction of AGE with cellular receptors alters the level of gene expression. Immunolohistochemical analyses of human atherosclerotic lesions using a monoclonal anti-AGE antibody have demonstrated diffuse extracellular AGE-deposition as well as dense intracellular AGE-deposition in macrophage- and vascular smooth muscle cell-derived foam cells[12,13]. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily whose expression is upregulated at sites of diverse pathologies, from atherosclerosis to Alzheimer′s disease[14]. RAGE triggers a cascade of signaling mechanisms. Activation of JAK/STAT and phosphatidylinositol 3-kinase recruits pathways involving mitogen-activated protein kinases[15,16] In the case of mononuclear phagocytes, RAGE-dependent stimulation results in activated phenotype with increased chemotaxis and release of proinflammatory cytokines[17]. Our preliminary results show that AGE can stimulate VSMCs proliferation. The effect is opposed by rhIL-10.

IL-10 is an anti-inflammatory cytokine endogenously expressed in the human atherosclerotic plaque[18,19], with potent inhibitory effects on proinflammatory cytokine synthesis by activating monocytic cells[20].We observed that rhIL-10 inhibited AGE-stimulated VSMCs proliferation. Cell cycle analysis data show that cell number in G0/G1 phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone by flow cytometry analysis. IL-10 has been shown to inhibit both p44/42 and p38 MAPKs stimulated by LPS, CD40 ligation, or TNF-α in monocytes[21-23].To determine the effects of IL-10 and AGE on p44/42 MAPK activation in VSMCs, We pretreated VSMCs with either AGE or rhIL-10 alone or in combination, and examined phosphorylation status of p44/42 MAPK. The data show that rhIL-10 can significantly reduce p44/42 MAPK activation by AGE, which is statistically significant compared with pretreatment with AGE alone. These data suggest that the AGE stimulated VSMCs proliferation is attenuated by anti-inflammatory cytokine IL-10 via modulation of p44/42 MAPK.

After balloon angioplasty, additional inflammatory cells are recruited as part of the tissue phenomenon[24]. Experimental[6] and clinical[24] studies indicate that postangioplasty inflammation is remarkable. Therefore, inflammation plays a key role in the development of neointimal hyperplasia. In the present study, rhIL-10 reduced neointimal hyperplasia by 45% after balloon angioplasty in rats. In addition to being a monocyte deactivator, IL-10 may be protective against neointimal hyperplasia by several other functions, including inhibition of cell adhesion molecules[20], VSMCs proliferation, monocytes chemoattractant MCP-1[25], tissue factor[26], fibrinogen, metalloproteinase-9, T-lymphocyte granulocyte-macrophage colony-stimulation factor[27] and inducible nitric oxide synthase[28].

These results, however, should be interpreted with caution. The single injury model lacks the atherosclerotic substrate on which intima develops. Thus extrapolation of our data to human deserves further studies.

In conclusion, on the basis of recent finding that the inflammatory process in the atherosclerotic plaque may be regulated by a balance between proinflammatory and anti-inflammatory cytokines, our data indicate that the anti-inflammatory cytokine rhIL-10 inhibits AGE-induced VSMCs proliferation. Recombinant human IL-10 can also inhibit neointima hyperplasia after carotid artery injury in rat. The results suggest the possibility that recombinant human IL-10 prevents neointimal hyperplasia as a potential therapeutic approach.

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