Abstract

Background: Early strut coverage after sirolimus-eluting stent (SES) implantation is associated with the activation of inflammation, but the underlying mechanisms are not completely understood. The present study aimed to identify the relationship between the anti-inflammatory cytokine interleukin (IL) 35 (IL-35) and early strut coverage in vivo and in vitro.

Methods: We utilized a retrospective study design to measure IL-35 levels in 68 stents from 68 patients with coronary artery disease and recorded serial optical coherence tomography (OCT) images (0 and 3 months) to assess stent endothelialization. The mechanism underlying the regulatory effects of IL-35 on macrophages and human umbilical vein endothelial cells (HUVECs) was also investigated. SESs were surgically implanted into the right common carotid arteries of 200 male New Zealand White rabbits receiving intravenous injections of IL-35 or a placebo.

Results: At the 3-month OCT evaluation, complete endothelium coverage was correlated with IL-35 levels. IL-35 induced the activation of an anti-inflammatory M2-like macrophage phenotype by targeting the signal transducer and activators of transcription (STAT)1/4 signalling pathway, and IL-35-treated macrophages induced endothelial proliferation and alleviated endothelial dysfunction. IL-35-treated New Zealand White rabbits with implanted SESs showed lower percentages of cross-sections with an uncovered strut, elevated mean neointimal hyperplasia (NIH) thickness, and inhibited inflammatory responses.

Conclusions: We investigated the effect of IL-35 expression on early stent endothelialization in vivo and in vitro and identified a crucial role for IL-35 in inducing the activation of an anti-inflammatory M2-like macrophage phenotype. The present study highlights a new therapeutic strategy for early stent endothelialization.

Introduction

Drug-eluting stents (DESs) are highly effective at reducing restenosis rates in patients with coronary artery disease (CAD) [1]. However, the use of DESs is limited by their associated delays in re-endothelialization and increased risks of late stent thrombosis [2,3]. Rapid endothelialization is crucial to guarantee the good performance of sirolimus-eluting stents (SESs) during in situ implantation. The primary mechanism of this phenomenon involves the recruitment of activated macrophages, as they are critical contributors to delayed early strut coverage and endothelial recovery [4,5]. Several classes of macrophages have been described based on their marker expression profiles, the production of specific factors, and their biological functions. Classically activated M1-like macrophages are typically induced by helper T cells 1 (Th1) cytokines or lipopolysaccharide (LPS) and are characterized by the expression of CD86 and CD274; they secrete the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin (IL) 6 (IL-6), and IL-1β and produce low levels of IL-10. The M2-like macrophages are induced by Th2 cytokines or glucocorticoids. M2-like macrophages are characterized by the expression CD163, low production of IL-12, and high production of both IL-10 and transforming growth factor-β (TGF-β) [6]. The direction of polarization of activated macrophages leads to vascular remodelling, superoxide production, and endothelial dysfunction. These changes are associated with delays in re-endothelialization and contribute to late neointimal instability and increased risks of late stent thrombosis.

IL-35, which consists of the IL-12 p35 subunit and Epstein–Barr virus-induced gene 3 (EBI3), is a member of the IL-12 cytokine family [7]. In contrast with other pro-inflammatory members of the IL-12 family, IL-35 exerts strong immunosuppressive effects that are comparable with IL-10 and TGF-β [7]. IL-35 plays important roles in atherosclerosis. Patients with CAD display significantly lower plasma IL-35 levels. The IL-35 level is associated with CAD risk factors, including the total cholesterol, low-density lipoprotein, and high-density lipoprotein cholesterol levels [8]. IL-35 functions to up-regulate the production of anti-inflammatory cytokines and down-regulate the expression of pro-inflammatory cytokines, leading to the inhibition of atherosclerotic lesions progression [9]. IL-35 also decreases the total number of macrophages and the M1/M2-like macrophage ratio [10], but the effect of IL-35 on the relationship between macrophage activation and early stent endothelialization remains unclear.

In the present study, we investigated the effect of IL-35 expression on stent endothelialization in vivo and in vitro. Moreover, we identified a crucial role for IL-35 in inducing an anti-inflammatory phenotype in activated macrophages by targeting the signal transducer and activators of transcription (STAT)1/4 signalling pathway and regulating endothelial dysfunction.

Experimental

Ethics statement

The present study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, China (KY 2017-077). All patients signed an informed consent form. The study was conducted in accordance with the Declaration of Helsinki. All rabbits were obtained from the Animal Centre of Harbin Medical University. All animals received humane care. The Hospital Scientific Affairs Committee on Animal Research and Ethics approved the study protocol (2013-Yan-144), and the methods were performed in accordance with ARRIVE guidelines and NC3Rs.

Patient population

The inclusion criteria for the present study were: (i) stable angina or unstable angina with only de novo lesions with a stenosis diameter >70% that was related to myocardial ischemia based on an objective analysis and (ii) a native vessel size of 2.5–3.5 mm based on a visual estimate that was able to be covered by a single stent (multiple lesions in the same patients were allowed if the lesions existed in different epicardial arteries) [11]. The exclusion criteria were [11]: (i) multiple lesions with a stenosis diameter >70% that were unable to be covered by a single stent in the same patient; (ii) any chronic illness, including cancer, liver cirrhosis, heart failure, and end-stage renal failure; (iii) concomitant inflammatory conditions, including connective tissue disease, inflammatory arthritis, and active infection; (iv) known intolerance to a study drug, metal alloy, or contrast media; and (v) lesions with an anatomy unsuitable for optical coherence tomography (OCT) imaging using the occlusion technique (proximal vessel size >3.5 mm or proximal lesions <15 mm from the ostium of each artery). Before the intervention, all patients were pretreated with a loading dose of 300 mg of aspirin and 600 mg of clopidogrel within 6 h of the percutaneous coronary intervention (PCI).

OCT definition

The intracoronary OCT imaging method was previously described [11,12]. Either a time-domain OCT system (M2/M3 System; LightLab Imaging, Westford, MA, U.S.A.) or a frequency-domain OCT system (C7-XR OCT Intravascular Imaging System; St. Jude Medical, St. Paul, MN, U.S.A.) was used in the present study. All OCT images were stored digitally and submitted to the Key Laboratories of Myocardial Ischemia at the Chinese Ministry of Education (Harbin, China) for off-line analyses [13]. The OCT image of each stent strut was classified into one of the following categories [14]: (i) well-opposed to the vessel wall with apparent neointimal coverage; (ii) well-opposed to the vessel wall without neointimal coverage; and (iii) malapposed to the vessel wall without neointimal coverage. A stent strut was classified as malapposed when the distance between its inner surface reflection and the vessel wall was >200 μm. This criterion was determined by adding the OCT axial resolution (20 μm) to the actual thickness of an SES strut (180 μm). The thickness of the area displaying neointimal hyperplasia (NIH) was measured as the distance between the endoluminal surface of the neointima and the luminal surface of the strut, and an uncovered strut was defined as having NIH thickness of 0 mm [15,16]. The percentage of uncovered struts was calculated as (number of uncovered struts/total number of struts) × 100. Complete endothelial coverage of the stent was defined as a stent with all analyzable struts covered by neointima. A cross-section was defined as having uncovered struts if one stent strut was uncovered on a cross-section, and a cross-section with an uncovered strut ratio >0.3 was defined when the ratio of uncovered struts to total stent struts per cross-section was >0.3 [17].

Cell culture

Monocytes were isolated from patients with stable angina or unstable angina 1 week after stenting and cultured with M-CSF (50 ng/ml, PeproTech, Rocky Hill, NJ, U.S.A.) for 7 days to induce macrophage differentiation. Cells were treated with normal medium for M0 macrophages supplemented with a polarizing mixture of LPS (50 ng/ml, Sigma–Aldrich, St. Louis, MO, U.S.A.) and IFN-γ (20 ng/ml, PeproTech, Rocky Hill, NJ, U.S.A.) for M1-like macrophages for 24 h. Some macrophages were treated with recombinant human IL-35 (rIL-35, 20 ng/ml), an anti-IL-12Rβ2 neutralizing antibody (2 μg/ml), anti-GP130 neutralizing antibody (100 ng/ml) (R&D Systems, Minneapolis, MN), or tofacitinib (5 nM, Cell Signaling Technology, Danvers, MA). Macrophages were generated and transfected with STAT1 and STAT4 short interfering (si)RNAs (60 nM; GenePharma) using Lipofectamine 2000 for 5 h before treatment with recombinant human cytokines. Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Gene Chemical Co., Ltd. (Shanghai, China). HUVECs were cultured in six-well culture plates and propagated in endothelial cell growth medium supplemented with or without TNF-α (20 ng/ml, R&D Systems) for 24 h.

Enzyme-linked immunosorbent assay

Plasma and supernatant cytokine levels were assessed using cytokine-specific enzyme-linked immunosorbent assay (ELISAs). The supernatant was obtained from each group of macrophages, and plasma was obtained from patients and rabbits after stent implantation. ELISAs were performed using rabbit cytokine-specific kits (IL-35 kit from CUSABIO, Wuhan, CN, for the detection of the rabbit protein and from BioLegend, San Diego, CA, U.S.A., for the detection of the human protein; IL-1β, IL-6, TNF-α, IL-10, IL-12, and TGF-β kits from Abcam, Cambridge, U.K., for the detection of the human proteins and from CUSABIO, Wuhan, CN, for the detection of the rabbit proteins). All assays were performed in triplicate.

Flow cytometry and apoptosis assays

The following PE- and FITC-conjugated antibodies were used to analyze macrophage phenotype-specific surface antigen expression: anti-human CD86, CD274, CD163 and isotype controls (eBioscience, San Diego, CA, U.S.A.). Macrophages were stained for 30 min at 4°C. For the apoptosis assay, HUVECs were stained with 5 μl of Annexin-FITC and 2 μl of propidium iodide (BD Biosciences, San Jose, CA, U.S.A.) and incubated for 15 min at room temperature, followed by the addition of 400 μl of binding buffer. A FACSCanto II flow cytometer was used to analyze positive cells. All assays were independently repeated at least three times.

BrdU assay

An HUVEC proliferation assay was performed according to the manufacturer’s instructions and previously described methods [18]. HUVECs (3 × 103 cells) in 100 μl of culture medium were added to 96-well round-bottomed plates and co-cultured with macrophages (1:25 ratio) for 24, 48, or 72 h. These co-cultures were incubated with 10 mM BrdU (Biotrak 2; Amersham, Little 7Chalfont, U.K.) for 24 h, and BrdU incorporation was quantified using a BrdU ELISA.

Quantificative RT-PCR

Total RNA was isolated from macrophages or the neointima using the TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the protocol recommended by the manufacturer. qRT-PCR was performed using a FAM-probe kit (GenePharma, Shanghai, CN) according to the manufacturer’s protocol in a 20-μl reaction containing 1 μg of purified RNA. Reverse-transcription reactions were incubated for 60 min at 37°C and 5 min at 85°C. The PCR protocol consisted of 40 cycles of 3 min at 95°C, 12 s at 95°C and 40 s at 62°C. The relative mRNA levels of the target genes were quantified using the 2−ΔΔCt method described by Livak and Schmittgen [19]. The primers used for real-time PCR are shown in Supplementary Table S1. Each sample was measured in triplicate.

Western blotting

Macrophages, HUVECs or neointimal samples were lyzed in RIPA buffer, and 30 µg of protein extracts were loaded and separated on Tris-glycine SDS/PAGE gels. The following targets were probed using the antibodies shown in Supplementary Table S2: IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, IL-12Rβ2, GP130, STAT1, p-STAT1, STAT4, p-STAT4 and β-actin (Abcam Cambridge, U.K.). ECL Prime Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ) and scanning densitometry (GS-710 imaging) were used to detect signals and acquire images. All data were obtained from three independent experiments.

Formation of tube-like structures

HUVECs were collected by treatment with trypsin for 1 min and then seeded in 24-well culture plates that had been precoated with Matrigel (BD Biosciences, San Jose, CA). After 6 h, cells were stained with 4 μg/ml Calcein AM dye (Thermo Fisher Scientific) and incubated at 37°C for 30 min. Tube formations were observed under a fluorescent microscope. Photographs were captured at 40× magnification, and tube-like formations were quantified using the ImageJ Angiogenesis Analyzer (http://rsb.info.nih.gov/ij/).

Rabbit model of carotid artery stenting, rIL-35 treatment, and OCT analysis

Male New Zealand White rabbits (3–4 kg, 3–4 months of age) were purchased from the Animal Centre of Harbin Medical University and surgical was performed in Second Affiliated Hospital of the Harbin Medical University Laboratory. Animals were provided free access to water and food. All animals received a subcutaneous injection of 5 mg/kg ketamine solution and 5% Inj Lidocaine for anaesthesia and analgesia [20]. After surgical exposure of the right carotid artery, the rabbits underwent implantation of a single SES (Partner®, Lepu Medical, Beijing, China) at 8–12 atm. The stents had nominal diameters of 2–2.75 mm, lengths of 8–15 mm, strut thicknesses of 0.18 mm, and an intended stent/artery ratio of 1.2:1. The animals received oral aspirin (40 mg) and clopidogrel (75 mg) 48 h before surgery [13]. One week after stent implantation, the animals received daily intravenous injections of rIL-35 (0.2 mg/kg, PeproTech) for 1 week, and the control group received saline injections. Then, the artery was imaged using OCT to study differences in strut coverage.

Histology

Histological assessments were performed using a previously validated method [1]. After follow-up stent imaging, rabbits were killed with an overdose of sodium pentobarbital. Carotid specimens were excised, fixed with formalin, and then embedded in methyl methacrylate. Four 2-mm-thick sections were obtained from each stent using a tungsten carbide knife. Sections (5-μm thickness) were cut using an automated microtome and stained with Haematoxylin and Eosin. Specimens were examined using a DMRAX2 photomicroscope (Leica Microsystems, Milan, Italy) and analyzed using Leica IM 500 image analysis software.

Statistical analysis

Quantificative variables are presented as mean values ± standard deviations, and qualitative variables are presented as total numbers and percentages. An independent two-sample t test or one-way analysis of variance (ANOVA) with the post hoc Student–Newman–Keuls test was used to assess differences between multiple sets of data. Categorical variables were also compared using the chi-square or Fisher’s exact test. Statistical significance was indicated for a two-sided P-value <0.05. All statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL, U.S.A.).

Results

IL-35 expression was up-regulated in vivo in patients in the stent strut coverage groups determined based on the OCT results

The baseline clinical characteristics of patients are summarized in Table 1. Here, 68 patients with 68 stents were evaluated using OCT immediately and 3 months after stent implantation. The patients were divided into two groups (covered vs uncovered) based on the strut coverage of stents, and representative OCT images are shown in Figure 1. OCT findings in the groups with different levels of strut coverage are shown in Table 2. At the 3-month OCT evaluation, the mean NIH area (P=0.004) was larger and the mean NIH thickness (P<0.001) was greater in the covered group. Significantly lower serum levels of the pro-inflammatory cytokine IL-1β (P=0.044) and higher levels of the anti-inflammatory cytokine IL-35 (P=0.024) were detected in the covered group than in the uncovered group (Table 2).

Representative OCT images obtained at the 3-month follow-up

Figure 1
Representative OCT images obtained at the 3-month follow-up

Completely covered struts (A,B) and markedly uncovered struts (C,D) were observed with SES at 3 months.

Figure 1
Representative OCT images obtained at the 3-month follow-up

Completely covered struts (A,B) and markedly uncovered struts (C,D) were observed with SES at 3 months.

Table 1
Baseline characteristics
Number variable68 (males: 48, females: 20)68 stents
Age (years) 61.06 ± 10.69 - 
Risk factor, n (%)   
Current smoker 40 (58.82) - 
Hypertension 34 (50.00) - 
Hyperlipidaemia 47 (69.12) - 
Diabetes mellitus 15 (22.06) - 
Disease, n (%)   
Angina pectoris 25 (36.76) 
Unstable angina 43 (63.23) 
Culprit lesion, n (%)   
LAD 38 (55.88) 
LCX 8 (11.76) 
RCA 22 (32.35) 
Mean reference vessel diameter (mm) 2.93 ± 0.35 - 
Stent length, mean (mm) - 25.74 ± 7.21 
Stent diameter, mean (mm) - 3.07 ± 0.39 
OCT findings   
Mean stent area (mm2- 6.40 ± 0.67 
Strut-level analysis   
Total number of stent struts - 8766 struts 
Malapposition, n (%) - 73 struts (0.83) 
IL-1β (ng/l) 39.40 ± 14.98  
IL-6 (ng/l) 148.37 ± 37.09  
IL-35 (ng/l) 32.22 ± 8.07  
TNF-α (μg/l) 9.39 ± 3.24  
TGF-β (ng/l) 169.88 ± 49.34  
Number variable68 (males: 48, females: 20)68 stents
Age (years) 61.06 ± 10.69 - 
Risk factor, n (%)   
Current smoker 40 (58.82) - 
Hypertension 34 (50.00) - 
Hyperlipidaemia 47 (69.12) - 
Diabetes mellitus 15 (22.06) - 
Disease, n (%)   
Angina pectoris 25 (36.76) 
Unstable angina 43 (63.23) 
Culprit lesion, n (%)   
LAD 38 (55.88) 
LCX 8 (11.76) 
RCA 22 (32.35) 
Mean reference vessel diameter (mm) 2.93 ± 0.35 - 
Stent length, mean (mm) - 25.74 ± 7.21 
Stent diameter, mean (mm) - 3.07 ± 0.39 
OCT findings   
Mean stent area (mm2- 6.40 ± 0.67 
Strut-level analysis   
Total number of stent struts - 8766 struts 
Malapposition, n (%) - 73 struts (0.83) 
IL-1β (ng/l) 39.40 ± 14.98  
IL-6 (ng/l) 148.37 ± 37.09  
IL-35 (ng/l) 32.22 ± 8.07  
TNF-α (μg/l) 9.39 ± 3.24  
TGF-β (ng/l) 169.88 ± 49.34  

Mean values (standard deviations) and % (n) are reported for continuous and categorical variables, respectively. Abbreviations: LAD, left anterior descending coronary artery; LCX, left circumflex coronary artery; MI, myocardial infarction; RCA, right coronary artery.

Table 2
OCT findings and serum cytokine profiles in patients at follow-up
Covered group (n=47)Uncovered group (n=21)P-value
OCT findings    
Mean stent area (mm26.44 ± 0.70 6.34 ± 0.60 0.470 
Mean luminal area (mm25.97 ± 0.68 6.03 ± 0.58 0.645 
Mean NIH area (mm20.47 ± 0.05 0.31 ± 0.08 <0.001 
Mean NIH thickness (mm) 0.054 ± 0.006 0.035 ± 0.007 <0.001 
Presence of thrombi, n (%) 3 (6.38) 2 (10.52) 0.641 
Serum cytokine levels    
IL-1β (ng/l) 205.14 ± 53.16 258.05 ± 74.26 0.006 
IL-6 (ng/l) 374.13 ± 33.10 381.16 ± 63.86 0.638 
IL-35 (ng/l) 26.49 ± 10.92 20.83 ± 6.28 0.009 
TNF-α (μg/l) 79.88 ± 20.67 83.22 ± 26.16 0.574 
TGF-β (ng/l) 12.94 ± 5.33 10.25 ± 5.65 0.063 
Covered group (n=47)Uncovered group (n=21)P-value
OCT findings    
Mean stent area (mm26.44 ± 0.70 6.34 ± 0.60 0.470 
Mean luminal area (mm25.97 ± 0.68 6.03 ± 0.58 0.645 
Mean NIH area (mm20.47 ± 0.05 0.31 ± 0.08 <0.001 
Mean NIH thickness (mm) 0.054 ± 0.006 0.035 ± 0.007 <0.001 
Presence of thrombi, n (%) 3 (6.38) 2 (10.52) 0.641 
Serum cytokine levels    
IL-1β (ng/l) 205.14 ± 53.16 258.05 ± 74.26 0.006 
IL-6 (ng/l) 374.13 ± 33.10 381.16 ± 63.86 0.638 
IL-35 (ng/l) 26.49 ± 10.92 20.83 ± 6.28 0.009 
TNF-α (μg/l) 79.88 ± 20.67 83.22 ± 26.16 0.574 
TGF-β (ng/l) 12.94 ± 5.33 10.25 ± 5.65 0.063 

Mean values (standard deviations) and % (n) are reported for continuous and categorical variables, respectively.

rIL-35 induces anti-inflammatory M2-like macrophage polarization in vitro

Inflammatory cells, such as macrophages or lymphocytes, play critical roles in neointimal formation and delay endothelialization after DES implantation [5]. We evaluated the expression levels of M1/M2 phenotypic markers and inflammatory cytokines in macrophages treated with rIL-35 compared with LPS and IFN-γ to investigate the effects of rIL-35 on macrophages. As expected, LPS and IFN-γ significantly induced the overexpression of M1-like macrophage markers (Figure 2A). The rIL-35-treated macrophages exhibited higher mRNA levels of M2-like macrophage markers, such as CD163, Ym1 and IL-10 (Figure 2B), while some M1-like macrophage markers, such as monocyte chemoattractant protein (MCP-1), IL-12p40, and inducible nitric oxide synthase (iNOS), were not obviously up-regulated (Figure 2A). rIL-35 increased the secretion of the anti-inflammatory cytokine TGF-β and inhibited pro-inflammatory cytokine secretion (Figure 2C–F). Based on the results of the FACS analysis, levels of the M1-like macrophage surface marker CD274 were increased and no effect on CD86 expression was observed in LPS+IFN-γ-treated macrophages, but the levels of these markers were decreased in rIL-35-treated macrophages (Figure 2G,H). The rIL-35-treated macrophages exhibited increased expression of CD163, a classic M2-like macrophage phenotypic marker. Macrophages were treated with rIL-35 after stimulation with LPS and IFN-γ. rIL-35 also partially induced the transformation of M1-like macrophages into M2-like macrophages. The polarization levels induced by LPS and IFN-γ were slightly inhibited after treatment with rIL-35 (Figure 2A,B). The rIL-35-treated macrophages stimulated with LPS and IFN-γ exhibited lower levels of M1-related markers than in cells treated with LPS and IFN-γ, but a slight increase in the levels of M2-like markers was observed. Analyses of the secretion of inflammatory cytokines (Figure 2C–F) detected using ELISAs and levels of macrophage surface markers (Figure 2G,H) using flow cytometry also produced the same results. Thus, rIL-35 induced M2-like macrophage polarization and inhibited pro-inflammatory cytokine secretion.

rIL-35 induces anti-inflammatory M2-like macrophage polarization

Figure 2
rIL-35 induces anti-inflammatory M2-like macrophage polarization

Human PBMCs were isolated and induced to differentiate into macrophages. Next, macrophages were treated with rIL-35 (20 ng/ml) and/or IFN-γ (20 ng/ml)+LPS (50 ng/ml) for 24 h. (A,B) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (CF) The cell supernatants of macrophages were analyzed using ELISAs. (G,H) Levels of CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs. The data are presented as means ± S.D.; n=6 samples pooled from three experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

Figure 2
rIL-35 induces anti-inflammatory M2-like macrophage polarization

Human PBMCs were isolated and induced to differentiate into macrophages. Next, macrophages were treated with rIL-35 (20 ng/ml) and/or IFN-γ (20 ng/ml)+LPS (50 ng/ml) for 24 h. (A,B) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (CF) The cell supernatants of macrophages were analyzed using ELISAs. (G,H) Levels of CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs. The data are presented as means ± S.D.; n=6 samples pooled from three experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

rIL-35 regulates M2-like macrophage polarization via the STAT1-STAT4 signalling pathway

IL-35 is unique compared with other cytokines in the IL-12 family because it induces a downstream signalling cascade through a GP130 (an IL-27 receptor component):IL-12Rβ2 (an IL-12 receptor component) heterodimer or through a GP130:GP130 and/or IL-12Rβ2:IL-12Rβ2 homodimer, which subsequently activate the STAT1 and STAT4 signalling pathways [21]. Macrophages treated with rIL-35 exhibited increased phosphorylation of STAT1 and STAT4 (Figure 3A–C), but the receptors GP130 and IL-12Rβ2 were not significantly altered (Supplementary Figure S1). The levels of phosphorylated STAT1/4 (Figure 3A–C) increased and the levels of phosphorylated NF-κB p65 (Figure 3A,D) and IκBα (Figure 3A,E) decreased 30 min and 2 h after rIL-35 treatment compared with the untreated group. A significant difference between the two groups was not observed at 30 min or 2 h, but the effect peaked at 24 h. A significant difference in the function of macrophages treated with different doses of rIL-35 was not observed.

IL-35 activates STAT1/4 signalling and inhibits NF-κB-mediated activation of inflammation

Figure 3
IL-35 activates STAT1/4 signalling and inhibits NF-κB-mediated activation of inflammation

(A) Macrophages were stimulated with rIL-35 (10, 20 or 40 ng/ml) for different times (30 min, 2 or 24 h), and cell lysates were subjected to Western blotting with anti-STAT1, p-STAT1, STAT4, p-STAT4, IκBα, p-IκBα, NF-κB p65, p-NF-κB p65 and β-actin antibodies. The phosphorylation level of STAT1 (B), STAT4 (C), NF-κB p65 (D) and IκBα (E) were analyzed by ratio to β-actin. The prefix ‘p’ indicates phosphorylation. Representative blots from three independent experiments are shown. *P<0.001.

Figure 3
IL-35 activates STAT1/4 signalling and inhibits NF-κB-mediated activation of inflammation

(A) Macrophages were stimulated with rIL-35 (10, 20 or 40 ng/ml) for different times (30 min, 2 or 24 h), and cell lysates were subjected to Western blotting with anti-STAT1, p-STAT1, STAT4, p-STAT4, IκBα, p-IκBα, NF-κB p65, p-NF-κB p65 and β-actin antibodies. The phosphorylation level of STAT1 (B), STAT4 (C), NF-κB p65 (D) and IκBα (E) were analyzed by ratio to β-actin. The prefix ‘p’ indicates phosphorylation. Representative blots from three independent experiments are shown. *P<0.001.

We then used the JAK-STAT activation inhibitor tofacitinib and STAT1/4 siRNAs to inhibit JAK-STAT phosphorylation or STAT1/4 mRNA expression. These treatments did not noticeably inhibit the effect of rIL-35 on the macrophage expression of M1-like macrophage-associated genes MCP-1, IL-12p40 and iNOS (Figure 4A–C). The increase in the expression of the M2-associated genes CD163, Ym1 and IL-10 was abrogated when the cells were pretreated with tofacitinib or STAT1/4 siRNAs (Figure 4D-F). Tofacitinib and STAT1/4 siRNAs reversed the increase in M2-like macrophage-associated anti-inflammatory mediator expression induced by rIL-35 (Figure 4G–J). Furthermore, the FACS analysis did not reveal significant changes in CD86 and CD274 levels after the rIL-35 treatment. Lower CD163 levels were observed after treatment with rIL-35 and tofacitinib or the STAT1/4 siRNAs than with rIL-35 alone (Figure 4K–N). Based on these data, rIL-35 induces M2-like phenotype polarization through the STAT1-STAT4 signalling pathway.

rIL-35 regulates M2-like macrophage polarization via STAT1-STAT4 signalling in vitro

Figure 4
rIL-35 regulates M2-like macrophage polarization via STAT1-STAT4 signalling in vitro

Macrophages were pretreated with tofacitinib (5 nM) for 1 h or transfected with si-STAT1/4 to inhibit the expression of STAT1/4 before stimulation with rIL-35 (20 ng/ml). (AF) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (GJ) Cell supernatants were analyzed using ELISAs to assess the production of IL-1β, TNF-α, IL-6 and TGF-β. (KN) Levels of CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs (the green scribing section). The results represent the means ± S.D. of three separate experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

Figure 4
rIL-35 regulates M2-like macrophage polarization via STAT1-STAT4 signalling in vitro

Macrophages were pretreated with tofacitinib (5 nM) for 1 h or transfected with si-STAT1/4 to inhibit the expression of STAT1/4 before stimulation with rIL-35 (20 ng/ml). (AF) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (GJ) Cell supernatants were analyzed using ELISAs to assess the production of IL-1β, TNF-α, IL-6 and TGF-β. (KN) Levels of CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs (the green scribing section). The results represent the means ± S.D. of three separate experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

The IL-35 receptor is critically involved in IL-35-induced M2-like macrophage polarization in vitro

We used specific antibodies to block GP130 and IL-12Rβ2 in macrophages before treatment with rIL-35 to investigate whether the IL-35 receptor is involved in the mechanism by which IL-35 regulates M2-like macrophage polarization. GP130 and IL-12Rβ2 blockade inhibited the phosphorylation of STAT1 and STAT4, and rIL-35-mediated STAT1 and STAT4 phosphorylation was completely inhibited (Figure 5A). The blockade of GP130 and IL-12Rβ2 attenuated the rIL-35-induced down-regulation of IL-1β, TNF-α and IL-6 expression and the loss of the elevated expression of the anti-inflammatory cytokine TGF-β (Figure 5B–E). Blockade of GP130 and IL-12Rβ2 in macrophages significantly inhibited the expression of M2 markers, such as CD163, Ym1 and IL-10, following rIL-35 stimulation (Figure 5G) and abrogated the inhibition of M1 markers, including MCP-1, IL-12p40 and iNOS (Figure 5F). Moreover, blockade of GP130 and IL-12Rβ2 attenuated the expression of CD163 in macrophages following rIL-35 stimulation (Figure 5H,I). Taken together, GP130 and IL-12Rβ2 are essential for rIL-35-induced M2-like macrophage polarization through activation of the STAT1-STAT4 pathway in macrophages.

The IL-35 receptor is critically involved in rIL-35-induced M2-like macrophage polarization in vitro

Figure 5
The IL-35 receptor is critically involved in rIL-35-induced M2-like macrophage polarization in vitro

Macrophages were pretreated with an anti-IL-12Rβ2 neutralizing antibody (2 μg/ml) and anti-GP130 neutralizing antibody (100 ng/ml) for 30 min, and then rIL-35 was added to the culture and incubated for another 24 h. (A) Cells were subjected to Western blotting assays with anti-STAT1, p-STAT1, STAT4, p-STAT4 and β-actin antibodies. (BE) Cell supernatants were analyzed using ELISAs. (F,G) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (H,I) Levels CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs (the green scribing section). The results represent the means ± S.D. of three separate experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5
The IL-35 receptor is critically involved in rIL-35-induced M2-like macrophage polarization in vitro

Macrophages were pretreated with an anti-IL-12Rβ2 neutralizing antibody (2 μg/ml) and anti-GP130 neutralizing antibody (100 ng/ml) for 30 min, and then rIL-35 was added to the culture and incubated for another 24 h. (A) Cells were subjected to Western blotting assays with anti-STAT1, p-STAT1, STAT4, p-STAT4 and β-actin antibodies. (BE) Cell supernatants were analyzed using ELISAs. (F,G) Levels of the MCP-1, IL-12p40, iNOS, CD163, Ym1 and IL-10 mRNAs were measured using qRT-PCR. (H,I) Levels CD86, CD274 and CD163 were assessed with FACS using specific mAbs or isotype-matched mAbs (the green scribing section). The results represent the means ± S.D. of three separate experiments. NS denotes not significant. *P<0.05, **P<0.01 and ***P<0.001.

rIL-35-induced M2-like macrophages promote endothelial proliferation in vitro

Because macrophages play essential roles in stent endothelialization, we next examined whether rIL-35-induced macrophages affected endothelial function. The rIL-35-induced macrophages promoted HUVEC proliferation to the greatest extent at 24, 48 and 72 h (Figure 6A) and significantly inhibited HUVEC apoptosis (Figure 6B,C). The rIL-35-induced macrophages significantly inhibited TNF-α-induced expression of the pro-inflammatory cytokine IL-12 and increased the expression of the anti-inflammatory cytokines IL-10 and TGF-β (Figure 6D–F). The NF-κB signalling pathway is a molecular cascade that governs the activation of inflammation and thus disrupts endothelial dysfunction [4]. We detected the inhibitory effect of rIL-35-induced macrophages on the phosphorylation of IκBα and NF-κB p65 in HUVECs stimulated with TNF-α, which simulates endothelial dysfunction. Based on our data, rIL-35 significantly inhibited IκBα and NF-κB p65 phosphorylation in macrophages (Figure 6G–I). Angiogenesis is essential for endothelialization after stent implantation. We examined whether rIL-35-induced macrophages regulate endothelial angiogenesis and found that rIL-35-induced macrophages drastically promoted the formation of tube-like structures by HUVECs in an in vitro assay system that mimics in vivo angiogenesis. The tube length of the group co-cultured with rIL-35-induced macrophages was increased by approximately 60% compared with the control HUVEC group (Figure 6J,K). These results conclusively revealed that rIL-35-induced macrophages are crucial regulators of endothelial dysfunction, including proliferation, apoptosis, inflammatory signalling pathway NF-κB activation, and angiogenesis.

The rIL-35-induced M2-like macrophages improve endothelial dysfunction in vitro

Figure 6
The rIL-35-induced M2-like macrophages improve endothelial dysfunction in vitro

HUVECs were treated with TNF-α (20 ng/ml) and then co-cultured with rIL-35-treated macrophages (1:25 ratio) for 24, 48, or 72 h. (A) Proliferation was analyzed using a BrdU assay. (B,C) HUVEC apoptosis was assessed using a combination of Annexin V and propidium iodide staining. (DF) Cell supernatants were collected and analyzed using ELISAs to determine IL-12, IL-10, and TGF-β levels. (GI) Cell lysates were subjected to Western blotting with anti-IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, and β-actin antibodies. (J,K) HUVECs were seeded on 24-well culture plates that had been precoated with Matrigel, incubated for 6 h, and the incubated with the Calcein AM dye for 30 min. Tube formations were observed under a fluorescent microscope. Photographs were captured at 40× magnification, and tube-like formations were quantified using the ImageJ Angiogenesis Analyzer. The data are representative of three independent experiments and are presented as means ± S.D. NS denotes not significant. ***P<0.001.

Figure 6
The rIL-35-induced M2-like macrophages improve endothelial dysfunction in vitro

HUVECs were treated with TNF-α (20 ng/ml) and then co-cultured with rIL-35-treated macrophages (1:25 ratio) for 24, 48, or 72 h. (A) Proliferation was analyzed using a BrdU assay. (B,C) HUVEC apoptosis was assessed using a combination of Annexin V and propidium iodide staining. (DF) Cell supernatants were collected and analyzed using ELISAs to determine IL-12, IL-10, and TGF-β levels. (GI) Cell lysates were subjected to Western blotting with anti-IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, and β-actin antibodies. (J,K) HUVECs were seeded on 24-well culture plates that had been precoated with Matrigel, incubated for 6 h, and the incubated with the Calcein AM dye for 30 min. Tube formations were observed under a fluorescent microscope. Photographs were captured at 40× magnification, and tube-like formations were quantified using the ImageJ Angiogenesis Analyzer. The data are representative of three independent experiments and are presented as means ± S.D. NS denotes not significant. ***P<0.001.

OCT reveals that rIL-35 promotes endothelialization in a rabbit stent implantation model

We subjected male New Zealand White rabbits to the implantation of a single SES and intravenous injections of rIL-35 to further define the roles of IL-35 in the activation of inflammation and early endothelialization in vivo. The experimental preparation of the animal model is depicted in Figure 7. Data obtained from serial OCT images showing the strut coverage in rabbits are summarized in Table 3. The rIL-35-treated animals showed lower percentages of cross-sections with any uncovered strut (57.38 ± 6.72 vs 62.24 ± 12.24%, P=0.001), cross-sections with ratios of uncovered to total strut >0.3 (10.17 ± 2.75 vs 11.83 ± 3.19%, P<0.001) and uncovered struts (7.89 ± 2.87 vs 9.19 ± 3.77%, P=0.009). An elevated mean NIH thickness (0.069 ± 0.006 vs 0.053 ± 0.021 mm, P<0.001) was also observed in animals receiving rIL-35. Thus, rIL-35 significantly promoted endothelial coverage of the struts.

Schematic showing the experimental design

Figure 7
Schematic showing the experimental design
Figure 7
Schematic showing the experimental design
Table 3
Procedural characteristics and OCT findings in rabbits
IL-35 groupControl groupP-value
Baseline    
Number of common carotid arteries treated 100 100 N/A 
Number of stents deployed 92 95 N/A 
Technical success rate (%) 92 95 0.568 
Number of deaths N/A 
Mean pre-stenting vessel diameter (mm) 2.53 ± 0.29 2.58 ± 0.35 0.496 
Follow-up    
Cross-sections with any uncovered strut (%) 57.38 ± 6.72 62.24 ± 12.24 0.001 
Cross-sections with ratios of uncovered to total strut >0.3 (%) 10.17 ± 2.75 11.83 ± 3.19 <0.001 
Presence of thrombi, n (%) 10 (10.87) 15 (15.79) 0.392 
Mean NIH thickness (μm) 69.33 ± 20.81 52.58 ± 22.43 <0.001 
Total number of stent struts 12119 12346 N/A 
Uncovered strut (%) 7.89 ± 2.87 9.19 ± 3.77 0.009 
IL-35 groupControl groupP-value
Baseline    
Number of common carotid arteries treated 100 100 N/A 
Number of stents deployed 92 95 N/A 
Technical success rate (%) 92 95 0.568 
Number of deaths N/A 
Mean pre-stenting vessel diameter (mm) 2.53 ± 0.29 2.58 ± 0.35 0.496 
Follow-up    
Cross-sections with any uncovered strut (%) 57.38 ± 6.72 62.24 ± 12.24 0.001 
Cross-sections with ratios of uncovered to total strut >0.3 (%) 10.17 ± 2.75 11.83 ± 3.19 <0.001 
Presence of thrombi, n (%) 10 (10.87) 15 (15.79) 0.392 
Mean NIH thickness (μm) 69.33 ± 20.81 52.58 ± 22.43 <0.001 
Total number of stent struts 12119 12346 N/A 
Uncovered strut (%) 7.89 ± 2.87 9.19 ± 3.77 0.009 

Mean values (standard deviations) and % (n) are reported for continuous and categorical variables, respectively. Abbreviation: N/A, not available.

rIL-35 inhibits inflammatory activation in vivo

Our previous studies confirmed a marked increase in the release of pro-inflammatory cytokines and a decrease in levels of the anti-inflammatory cytokine IL-10 in rabbits after stent implantation [13]. In this study, we further detected the activation of inflammation at different time points (7, 10, and 14 days) after injecting rIL-35 into animals that underwent stent implantation. Proteins isolated from the whole neointima revealed significantly decreased levels of IκBα and NF-κB p65 phosphorylation following the rIL-35 treatment (Figure 8A–C). According to the qRT-PCR results, rIL-35 increased the expression of the CD31 and VE-Cadherin mRNAs (Figure 8D,E). CD31 or VE-Cadherin expression in the normal blood vessel intima served as the positive control. Significantly lower levels of pro-inflammatory cytokines, including IL-1β, IL-6, and IL-12, were observed in the rIL-35-treated group, and the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly increased (Figure 8D). Based on our observations, the rIL-35 treatment strongly inhibits the activation of inflammation and regulates strut coverage in rabbits after stent implantation.

rIL-35 inhibits inflammatory activation and promotes endothelialization in vivo

Figure 8
rIL-35 inhibits inflammatory activation and promotes endothelialization in vivo

An SES was surgically implanted in the right common carotid arteries of 200 male New Zealand White rabbits, which received intravenous injections of rIL-35 or the placebo. (AC) The neointima around the stent was obtained and then subjected to Western blotting with anti-IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, and β-actin antibodies. (D,E) The neointima around the stent was obtained and used to measure the expression of the CD31 and VE-Cadherin mRNAs with qRT-PCR. CD31 or VE-Cadherin expression in the normal blood vessel intima served as the positive control. (F) Plasma was collected and analyzed using ELISAs to determine the IL-1β, IL-6, IL-12, IL-10, and TGF-β levels. The data are representative of three independent experiments and are presented as means ± S.D. ***P<0.001. (GJ) Histological features of stented lesions in rabbits from the control group and the corresponding OCT images. Haematoxylin and Eosin staining of right common carotid arteries: completely covered struts (G,I) and uncovered struts (H,J) were observed separately 1 week after stenting. Arrows indicate covered or uncovered metal struts. Original magnification, ×40.

Figure 8
rIL-35 inhibits inflammatory activation and promotes endothelialization in vivo

An SES was surgically implanted in the right common carotid arteries of 200 male New Zealand White rabbits, which received intravenous injections of rIL-35 or the placebo. (AC) The neointima around the stent was obtained and then subjected to Western blotting with anti-IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, and β-actin antibodies. (D,E) The neointima around the stent was obtained and used to measure the expression of the CD31 and VE-Cadherin mRNAs with qRT-PCR. CD31 or VE-Cadherin expression in the normal blood vessel intima served as the positive control. (F) Plasma was collected and analyzed using ELISAs to determine the IL-1β, IL-6, IL-12, IL-10, and TGF-β levels. The data are representative of three independent experiments and are presented as means ± S.D. ***P<0.001. (GJ) Histological features of stented lesions in rabbits from the control group and the corresponding OCT images. Haematoxylin and Eosin staining of right common carotid arteries: completely covered struts (G,I) and uncovered struts (H,J) were observed separately 1 week after stenting. Arrows indicate covered or uncovered metal struts. Original magnification, ×40.

Agreement between OCT and histopathology images

Thirteen representative OCT images and their corresponding histological cross-sections were randomly selected from 13 lesions to ascertain the agreement between OCT and histopathological findings. Compared with histopathology, OCT measurements of the NIH thicknesses and area were extremely accurate (Table 4 and Figure 8E–H).

Table 4
Agreement between OCT and histological findings
OCT (n=13)Histology (n=13)ICC (95% CI)P-value
Luminal area (mm23.57 ± 0.79 2.41 ± 0.38 0.644 (0.216–0.865) 0.004 
NIH area (mm20.44 ± 0.10 0.34 ± 0.08 0.938 (0.827–0.979) <0.001 
NIH thickness (mm) 0.049 ± 0.011 0.044 ± 0.011 0.961 (0.888–0.987) <0.001 
OCT (n=13)Histology (n=13)ICC (95% CI)P-value
Luminal area (mm23.57 ± 0.79 2.41 ± 0.38 0.644 (0.216–0.865) 0.004 
NIH area (mm20.44 ± 0.10 0.34 ± 0.08 0.938 (0.827–0.979) <0.001 
NIH thickness (mm) 0.049 ± 0.011 0.044 ± 0.011 0.961 (0.888–0.987) <0.001 

The intraclass correlation coefficient (ICC) was calculated to evaluate the agreement between the OCT and histological findings. Abbreviation: CI, confidence interval.

Discussion

Although the utilized DESs were effective at reducing the rates of in-stent restenosis, they were coated with antiproliferative drugs that exert undesirable effects, including contributing to poor endothelialization and inducing inflammatory responses [5]. DESs have been suggested to lead to an increased risk of delayed re-endothelialization associated with impaired vascular healing [22]. The lack of an endothelial monolayer and endothelial dysfunction disrupt the homeostatic regulation of thrombosis and leucocyte adhesion. Inflammatory cell infiltration and cytokine secretion facilitate endothelial cell growth and lead to the abnormal progression of NIH [4]. Delayed vascular healing has been a problem in the DES era. Accordingly, neointimal coverage at an earlier phase has attracted increasing attention [23,24]. Early neointimal coverage is expected to improve the safety of DESs; however, researchers have not clearly determined how quickly neointimal coverage begins after the implantation of DESs [25].

In patients with CAD, higher levels of IL-35 are produced as a specific immune response to counteract the local release of inflammatory cytokines and to decrease the activity of the ongoing immune processes within atherosclerotic plaques [8]. In addition, the plasma IL-35, IL-10, and TGF-β1 levels are dramatically decreased in patients with acute myocardial infarction [26], whereas the levels of pro-inflammatory cytokines are markedly increased, indicating that acute myocardial infarction is triggered by an imbalance of inflammatory cytokines. In the present study, significantly lower IL-35 levels were observed in the uncovered stent group than in the patients with complete endothelial coverage of the stent. Combined with the OCT evaluation of early strut coverage in the rabbit stenting model administered IL-35, our results suggest that IL-35 plays a crucial role in stent endothelialization.

Recently, tumour-derived IL-35 was reported to recruit monocytes and promote the polarization of monocytes into a pro-angiogenic phenotype [27]. Moreover, IL-35 not only decreases the number of macrophages but also the ratio of M1/M2 macrophages and influences the secretion of inflammatory cytokines [13]. The IL-35 treatment significantly reduced the number of M1-like macrophages, induced the polarization of M2-like macrophages and promoted anti-inflammatory cytokine secretion in the present study. Thus, rIL-35 exerted an immunosuppressive effect on macrophages. Therefore, IL-35 might be a potential therapeutic target for in-stent endothelialization by regulating macrophage function.

IL-35 binds to IL-35 receptors to initiate signal transduction and exert its biological functions. IL-35R activates STAT1 or STAT4 to mediate T-cell functions and activates STAT1 or STAT3 in B cells. Macrophages deficient in STAT3 inhibit angiogenesis and are unable to respond to IL-10 via STAT3 signalling [28]. Alternatively, in macrophages, STAT1 and NF-κB bind adjacent sites and work in concert to control the transcription of encoded genes [29]. According to our data, the IL-35 treatment enhanced the activation of STAT1 and STAT4 in the downstream signalling pathway in macrophages. We also confirmed the importance of IL-35R-mediated signalling in determining macrophage activation. IL-mediated signalling determines macrophage activation and affects the capacity of these cells to regulate vascular proliferation and endothelial recovery. IL-35-treated macrophages significantly increased endothelial proliferation and improved endothelial dysfunction. Collectively, IL-35 is important for endothelialization, and the potential mechanism of IL-35 is the polarization of macrophages to an anti-inflammatory phenotype by inducing the STAT1/4 signalling pathway to regulate endothelial dysfunction.

IL-35 may mediate the biological activities of different cell types via different receptors and STAT signalling pathways. Further studies are required to confirm those hypotheses. Many challenges also remain regarding the nature of the functional plasticity of IL-35-induced macrophages, as well as how IL-35-mediated regulation affects this process. Answers to these questions will help us identify methods that are potentially useful for controlling and guiding these regulatory responses and design effective therapies to ameliorate impairments in rapid endothelial recovery after the implantation of DESs.

Conclusion

In conclusion, IL-35 is associated with early strut coverage of SES, and the mechanisms underlying this process might regulate endothelialization by affecting macrophage activation, followed by the induction of endothelial proliferation and reduction in endothelial dysfunction in vivo and in vitro. The present study highlights a new therapeutic strategy for early stent endothelialization.

Clinical perspectives

  • Early strut coverage after SES implantation is associated with the activation of inflammation, but the underlying mechanisms are not completely understood. The present study aimed to identify the relationship between the anti-inflammatory cytokine IL-35 and early strut coverage in vivo and in vitro.

  • At the 3-month OCT evaluation, complete endothelium coverage was correlated with IL-35 levels. IL-35 induced an anti-inflammatory phenotype in activated macrophages by targeting the STAT1/4 signalling pathway, and IL-35-treated macrophages promoted endothelial proliferation and alleviated endothelial dysfunction. IL-35-treated New Zealand White rabbits with an implanted SES showed lower percentages of cross-sections with an uncovered strut, elevated mean NIH thickness, and inhibited inflammatory responses.

  • IL-35 is associated with early strut coverage of SES, and the mechanisms of this process might regulate endothelialization by affecting macrophage activation, followed by the induction of endothelial proliferation and alleviating endothelial dysfunction in vivo and in vitro. The present study highlights a new therapeutic strategy for inducing early endothelialization after stent implantation.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 81330033 (to B.Y.), 81771946 (to Y.S.)]; the National Key R&D Program of China [grant number 2016YFC1301100 (to B.Y.)]; the Key Laboratory of Myocardial Ischemia, Chinese Ministry of Education, Harbin, Heilongjiang Province, China [grant number KF201601 (to X.L.)]; the Graduate Student Innovation Foundation of Heilongjiang Province [grant number YJSCX2017-39HYD (to X.L.)]; the China Postdoctoral Science Foundation [grant number 2018M641870 (to R.Z.)]; and the Heilongjiang Postdoctoral Science Foundation [grant number LBH-Z18141 (to R.Z.)].

Author contribution

Conception and design: X.L., R.Z., Y.S., and B.Y. Development of the methodology: X.L., R.Z., J.H., and S.Y. Acquisition of data (provided animals, recruited and managed patients, provided facilities, and other tasks): J.W., M.Z., and S.F. Analysis and interpretation of data (for example, statistical analysis, biostatistics, and computational analysis): X.L., R.Z., X.W., X.H., H.L., and J.T. Writing, review, and/or revision of the manuscript: X.L., R.Z., and B.Y.

Abbreviations

     
  • ARRIVE

    animals in research: reportini in vivo experiments

  •  
  • BrdU

    bromodeoxyuridine

  •  
  • CAD

    coronary artery disease

  •  
  • DES

    drug-eluting stent

  •  
  • ELISA

    enzyme-linked immunosorbent assay

  •  
  • GP130

    glycoprotein

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • IFN-γ

    interferon-γ

  •  
  • IL

    interleukin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • JAK

    janus family tyrosine kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • MCP-1

    monocyte chemoattractant protein

  •  
  • M-CSF

    macrophage colony stimulating factor

  •  
  • NC3Rs

    national centre for the replacement, refinement and reduction of animals in research

  •  
  • NIH

    neointimal hyperplasia

  •  
  • OCT

    optical coherence tomography

  •  
  • RIPA

    radio immunoprecipitation assay

  •  
  • PE

    phycoerythroprotein

  •  
  • qRT-PCR

    quantitative real time transcription polymerase chain reaction

  •  
  • rIL-35

    recombinant human IL-35

  •  
  • SES

    sirolimus-eluting stent

  •  
  • STAT

    signal transducer and activators of transcription

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TNF-α

    tumour necrosis factor-α

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Author notes

*

These authors contributed equally to this work.