Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits

Atherosclerosis xxx (2017) 1e9

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Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits Mitsumasa Sudo a, 1, Yuxin Li b, 1, Takafumi Hiro a, *, Tadateru Takayama a, Masako Mitsumata a, Masashi Shiomi c, Masahiko Sugitani d, Taro Matsumoto b, Hiroyuki Hao d, Atsushi Hirayama a a

Division of Cardiology, Department of Medicine, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan Division of Cell Regeneration and Transplantation, Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan c Institute for Experimental Animals, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan d Department of Pathology, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 December 2016 Received in revised form 28 May 2017 Accepted 22 June 2017 Available online xxx

Background and aims: Glucagon-like peptide-1 (GLP-1) is thought to inhibit development of aortic atherosclerosis and plaque formation. However, whether GLP-1 stabilizes fully developed atherosclerotic plaque or alters the complicated plaque composition remains unclarified. Methods: Ten Watanabe heritable hyperlipidemic (WHHL) rabbits were divided into GLP-1 receptor agonist treatment group and control group. After confirmation of atherosclerotic plaques in brachiocephalic arteries by iMap intravascular ultrasound (iMAP-IVUS), GLP-1 receptor agonist lixisenatide was administered to WHHL rabbits at 30 nmoL/kg/day for 12 weeks by osmotic pump. An equal volume of normal saline was administered in a control group. After evaluation by iMAP-IVUS at 12 weeks, brachiocephalic arteries were harvested for pathological histological analysis. Results: iMAP-IVUS analysis revealed larger fibrotic plaque components and smaller necrotic and calcified plaque components in the GLP-1 group than in the control group; %fibrotic area: 66.30 ± 2.06% vs. 75.14 ± 2.62%, p < 0.01, %necrotic area: 23.25 ± 1.87% vs. 16.17 ± 2.27%, p ¼ 0.02, %calcified area: 2.15 ± 0.24% vs. 1.00 ± 0.18%, p < 0.01), indicating that GLP-1 receptor agonist might modify plaque composition and increase plaque stability. Histological analysis confirmed that GLP-1 receptor agonist treatment improved smooth muscle cell (SMC)-rich plaque with increased fibrotic content. Furthermore, plaque macrophage infiltration and calcification were significantly reduced by GLP-1 receptor agonist treatment; %SMC area: 6.93 ± 0.31% vs. 8.14 ± 0.48%, p ¼ 0.02; %macrophage area: 9.11 ± 0.80% vs. 6.19 ± 0.85%, p < 0.01; %fibrotic area: 54.75 ± 1.63% vs. 69.60 ± 2.12%, p ¼ 0.02; %calcified area: 3.25 ± 0.67% vs. 0.75 ± 0.15%, p ¼ 0.02). Conclusions: GLP-1 receptor agonist inhibited plaque progression and promoted plaque stabilization by inhibiting plaque growth and modifying plaque composition. © 2017 Elsevier B.V. All rights reserved.

Keywords: Atherosclerosis Imaging Plaque Ultrasonics Diabetes mellitus

1. Introduction Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from the small intestine and stimulates the glucose-

* Corresponding author. E-mail address: [email protected] (T. Hiro). 1 These authors contributed equally to this work.

dependent insulin response through GLP-1 receptor [1]. GLP-1 receptor agonists, including lixisenatide, promote GLP-1 actions and are routinely used in drug therapy for type 2 diabetes [2,3]. Recent studies have elucidated the cardiovascular protective actions of GLP-1 and suggested novel use of this diabetes drug for various cardiovascular diseases [4e8]. Indeed, GLP-1 was shown to reduce vascular inflammation and prevent atherosclerosis in apolipoprotein E knockout (Apo E/) mice [9e11]. In these mouse studies,

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Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920

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M. Sudo et al. / Atherosclerosis xxx (2017) 1e9

GLP-1 was administered or overexpressed before the development of atherosclerosis, so what was observed was the inhibition of atherosclerotic plaque formation. It is unknown whether GLP-1 has a protective effect on fully developed atherosclerotic plaque, especially unstable plaque, which is a frequent complication of type 2 diabetes [12,13]. It also remains unclear whether GLP-1 modifies the components of fully developed plaque. An animal model of fully developed plaque and serial in vivo observations of plaque development are both needed to answer these questions. The Watanabe heritable hyperlipidemic (WHHL) rabbit is a mutant rabbit that shows spontaneous hyperlipidemia and later development of complicated atherosclerotic lesions [14e16]. iMAP intravascular ultrasound (iMAP-IVUS; Boston Scientific Corp. Natick, MA) is a color-mapping IVUS system that allows real-time cross-sectional identification of atherosclerotic plaque components [17]. We performed serial in vivo iMAP-IVUS imaging to investigate the effects of GLP-1 receptor agonist on fully developed atherosclerotic plaque in WHHL rabbits. 2. Materials and methods 2.1. Animals The experimental protocol was approved by the Animal Care and Use Committee of Nihon University. Ten male WHHL rabbit littermates (10e12 months of age) were obtained from Kobe University, Kobe, Japan. All rabbits were fed a normal chow (protein 17.5%, fat 4.0%, fiber 11.7%, ash 9.2%; CLEA Japan, Inc. Tokyo, Japan). The rabbits were housed in a room that was maintained at a constant temperature of 23 ± 2  C and humidity of 55± 5%. 2.2. Division of animals into a treatment group and a control group The WHHL rabbits were divided into two groups: one group (n ¼ 5) given GLP-1 receptor lixisenatide (Sanofi Co. Paris, France) at 30 nmoL/kg/day via ALZET osmotic pump (Model 2ML4; DURECT Corp. Cupertino, California, USA), and the other, a control group (n ¼ 5), given an equal volume of normal saline solution, also by osmotic pump. The pump was implanted subcutaneously on the back of each rabbit after inhalational induction of anesthesia (sevoflurane 2e5%, 4 L/min). The ALZET pump delivers solution continuously for only 4 weeks; therefore, we replaced the pump every 4 weeks. Before administration of the lixisenatide or saline solution and then at 12 weeks of treatment, blood samples were drawn from rabbits in both groups for measurement of blood sodium, blood triglycerides, blood GLP-1, blood glucose, and blood lixisenatide levels. Blood GLP-1 concentrations were determined by enzyme-linked immunosorbent assay (Cusabio Biotech Co. Ltd. Wuhan, China). 2.3. iMAP-IVUS analysis Before treatment and at 12 weeks, IVUS was performed in all rabbits for investigation of atherosclerotic plaques in the brachiocephalic arteries. After inhalation anesthesia (sevoflurane 2e5%, 4 L/min) was established, the right femoral artery was punctured with a 22G needle, and a 4F sheath was inserted. After administration of an intravenous bolus of 500 U unfractionated heparin, IVUS (Atlantis SR Pro 40-MHz Catheter; Boston Scientific Corp.) with iMAP (iLab; Boston Scientific Corp.) was performed over a coronary guide wire (ASAHI SION blue; Asahi Intecc Co. Ltd. Aichi, Japan). IVUS images were recorded continuously during an automatic pull-back maneuver at a constant speed of 0.5 mm/s. All images were analyzed offline with the use of a commercial software (QIvus 2.0; Medis Medical Imaging Systems bv, Leiden,

Netherlands). The external elastic membrane (EEM) area and lumen area were traced, and the plaque area was calculated as the EEM area minus the lumen area. The percentage of plaque area was then calculated as the plaque area divided by the EEM area. In iMAP-IVUS analysis, radiofrequency data are processed by autoregressive modeling and matched with a database of known radiofrequency signal profiles containing the characteristics of four tissue types [17e19]. The plaques are classified on the basis of four basic tissue components: fibrotic plaque (green), lipidic plaque (yellow), necrotic plaque (red), calcified plaque (blue). The percentage of each tissue component area was calculated as the particular component area divided by the plaque area. 2.4. Histologic analysis Immediately after the IVUS examination performed at 12 weeks, the rabbits were sacrificed, and whole-body systemic perfusion fixation was achieved by infusion of normal saline for 30 min followed by infusion of 10% buffered formalin for 30 min at 150 cmH2O. The brachiocephalic arteries were isolated and immersed in 10% buffered formalin for 24 h. Each specimen was embedded in paraffin, each blocks was cut at 2.5-mm intervals along the entire length of the artery, and sections of 4-mm thickness were then cut from the block. All sections were stained with hematoxylin and eosin, Elastica van Gieson, and Masson Trichrome. The lumen, internal elastic membrane (IEM), and EEM were traced. The vessel area was defined as the EEM area. The intima-media area was calculated as the EEM area minus the lumen area. Fibrotic tissue was characterized by bundles of collagen fibers with little or no lipid accumulation. Lipidic tissue was characterized by areas of loosely packed collagen bundles interspersed with extracellular lipid. A necrotic component was characterized by a hypocellular plaque cavity devoid of collagen and containing necrotic debris and cholesterol clefts or crystals. Calcified tissue was characterized by compact calcium crystals with intense staining [20,21]. The macrophage infiltration area, smooth muscle cell (SMC) area, and matrix metalloproteinase 9 (MMP-9) expression within the IEM area were analyzed immunohistochemically according to standard protocols, first with mouse monoclonal anti-rabbit macrophage antibody (Clone RAM-11; Dako UK Ltd. Ely, UK), mouse anti-human actin-smooth muscle monoclonal antibody (Clone 1A4; Dako UK Ltd.), and mouse monoclonal anti-MMP-9 antibody (Clone 56-2A4; Abcam PLC, Cambridge, UK), then with anti-mouse immunoglobulin-HRP (CSA II kit, Dako UK Ltd.). Specimens were viewed under a microscope (Olympus BX51, DP Controller Ver. 3.2.3.267; Olympus Corp. Tokyo, Japan) connected to a digital camera. Each tissue component was quantified as the ratio of tissue component area to intima-media area. Because thin-cap fibroatheroma (TCFA) is a type of unstable plaque, we also investigated the proportion of TCFA with a fibrous cap thickness of <65 mm [22]. All images were transferred into Photoshop CS6 (Ver. 13.0.1; Adobe Systems Incorporated, San Jose, California, USA) for pixel-based measurement. 2.5. Statistical analysis Discrete variables are presented as numbers and percentages. Continuous variables are presented as mean ± SE. Between-group differences in discrete variables were assessed by chi-square test or Fisher's exact test. Between-group differences in mean values were assessed by unpaired Student t-test or Wilcoxon rank sum test. Within-group differences in values obtained before treatment and those obtained at 12 weeks of treatment were assessed by paired Student t-test. Correlation between two parameters was assessed with Pearson's correlation coefficient. A two-sided p-value of <0.05 was accepted as statistically significant. JMP 9 version 9.0.0

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M. Sudo et al. / Atherosclerosis xxx (2017) 1e9

(SAS Institute Inc. Cary, North Carolina, USA) was used for all analyses. 3. Results 3.1. Blood chemistry values and body weight Laboratory values are listed on Table 1. The blood sodium level before treatment (baseline level) was higher in the GLP-1 group than in the Control group, and the blood triglyceride level at 12 weeks of treatment was lower in the GLP-1 group than in the Control group. Body weight and blood glucose levels before treatment and at 4, 8, and 12 weeks were comparable between the two groups (Supplementary Fig. 1A and B). The blood GLP-1 level before treatment and at 12 weeks was also comparable between the two groups (Control vs. GLP-1: before treatment, 16.78 ± 6.45 pg/mL vs. 13.96 ± 7.39 pg/mL, p ¼ 0.78; at 12 weeks, 24.46 ± 7.98 pg/mL vs. 11.71 ± 3.69 pg/mL, p ¼ 0.26) (Supplementary Fig. 1C). In the GLP-1 group, the blood lixisenatide concentration increased with treatment and reached a peak at 8 weeks (Supplementary Fig. 1D). 3.2. iMAP-IVUS After excluding images of poor quality and then matching images obtained before treatment and those obtained at 12 weeks of treatment, we analyzed 40 pairs of Control group iMAP-IVUS images and 35 pairs of GLP-1 group iMAP-IVUS images. The iMAPIVUS findings are summarized in Tables 2 and 3 and Figs. 1 and 2. Before treatment, average vessel, lumen, and plaque areas were larger in the GLP-1 group than in the Control group (Control vs. GLP-1: vessel area: 12.46 ± 0.36 mm2 vs. 14.24 ± 0.41 mm2, p < 0.01; lumen area: 7.17 ± 0.21 mm2 vs. 8.15 ± 0.29 mm2, p < 0.01; plaque area: 5.28 ± 0.23 mm2 vs. 6.09 ± 0.27 mm2, p ¼ 0.03) (Table 2). However, at 12 weeks of treatment, there was no difference in vessel, lumen, or plaque area between the two groups (vessel area: 12.22 ± 0.32 mm2 vs. 11.64 ± 0.32 mm2, p ¼ 0.20; lumen area: 5.72 ± 0.18 mm2 vs. 5.58 ± 0.26 mm2, p ¼ 0.26; plaque area: 6.49 ± 027 mm2 vs. 6.06 ± 0.27 mm2, p ¼ 0.26) (Table 2). In the Control group, plaque area was increased (Dplaque area ¼ þ1.22 ± 0.17 mm2, p < 0.01), lumen area was decreased (Dlumen area ¼ 1.45 ± 0.23 mm2, p < 0.01), and vessel area was unchanged (Dvessel area ¼ 0.23 ± 0.26 mm2, p ¼ 0.62) at 12 weeks, indicative of continuous plaque development without compensatory vessel enlargement (Table 3, Fig. 1). In the GLP-1

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group, plaque area was not increased (Dplaque area ¼ 0.03 ± 0.11 mm2, p ¼ 0.94), but both vessel area and lumen area were decreased (Dvessel area ¼ 2.61 ± 0.25 mm2, p < 0.01; Dlumen area ¼ 2.58 ± 0.21 mm2, p < 0.01) at 12 weeks of treatment, indicating that GLP-1 receptor agonist inhibited plaque growth and retarded positive vessel remodeling (Table 3 and Fig. 1). After 12 weeks, the fibrotic plaque component was decreased (D % Fibrotic area ¼ 7.63 ± 1.92%, p < 0.01), and the necrotic component was increased (D% Necrotic area ¼ þ7.40 ± 1.72%, p < 0.01) in the Control group (Table 3 and Fig. 2), indicative of plaque development and destabilization. With 12 weeks of GLP-1 receptor agonist treatment, the percentage of calcified component area decreased (D% Calcified area ¼ 0.91 ± 0.33%, p ¼ 0.04), and that of the fibrotic, lipidic, and necrotic component areas did not change (D% Fibrotic area ¼ 0.43 ± 2.00%, p ¼ 0.94; D% Lipidic area ¼ þ0.69 ± 0.52%, p ¼ 0.41; D% Necrotic area ¼ þ0.34 ± 1.74%, p ¼ 0.38), indicating that the GLP-1 receptor agonist treatment inhibited plaque destabilization (Table 3 and Fig. 2). Indeed, although the baseline percentages of the plaque component areas were similar between the two groups, the percentage of fibrotic component area was greater and the percentages of necrotic and calcified component areas were lower in the GLP-1 group than in the Control group at 12 weeks (Table 2). In comparing outcomes in the two groups, it appeared that the GLP-1 receptor agonist treatment significantly prevented the decrease of fibrotic components and the increase of necrotic components (Fig. 2).

3.3. Histology Forty-six Control group paraffin slices and 41 GLP-1 group paraffin slices were analyzed histologically. As with the iMAP-IVUS findings at 12 weeks, there were no differences in vessel and lumen areas between the two groups (Table 4; Control vs. GLP-1; vessel area: 8.35 ± 0.39 mm2 vs. 8.45 ± 0.31 mm2, p ¼ 0.84; lumen area: 2.52 ± 0.18 mm2 vs. 2.67 ± 0.14 mm2, p ¼ 0.32). Intima area, media area, intima area/media area, intima-media area, and percentage intima-media area were also comparable between the two groups (Table 4; Control vs. GLP-1; intima area: 4.44 ± 0.22 mm2 vs. 4.46 ± 0.27 mm2, p ¼ 0.97; media area 1.39 ± 0.05 mm2 vs. 1.33 ± 0.03 mm2, p ¼ 0.33; intima/media area 3.44 ± 0.23 vs. 3.43 ± 0.24, p ¼ 0.99; intima-media area: 5.83 ± 0.22 mm2 vs. 5.79 ± 0.27 mm2, p ¼ 0.91; % Intima-media area: 71.16 ± 0.97% vs. 68.01 ± 1.34%, p ¼ 0.06). The prevalence of TCFA was comparable between two groups (Table 4; Control vs. GLP-1; 15.2% vs. 4.9%,

Table 1 Laboratory values before treatment and at 12 weeks in the two study groups. Baseline

Total chol (mg/dl) LDL chol (mg/dl) HDL chol (mg/dl) Triglyceride (mg/dl) Total protein (g/dl) BUN (mg/dl) Creatinine (mg/dl) AST (U/l) ALT (U/l) LDH (U/l) Na (mmol/l) Cl (mmol/l) K (mmol/l)

At 12 weeks

Control (n ¼ 5)

GLP-1 (n ¼ 5)

p value

Control (n ¼ 5)

GLP-1 (n ¼ 5)

p value

1048.0 ± 70.0 847.0 ± 34.7 6.8 ± 0.4 232.2 ± 40.1 5.6 ± 0.2 19.2 ± 0.7 0.86 ± 0.09 28.6 ± 9.7 50.4 ± 13.1 271.4 ± 83.9 139.4 ± 0.7 100.0 ± 2.0 3.6 ± 0.2

1078.8 ± 92.9 823.2 ± 72.8 6.0 ± 0.0 245.2 ± 33.5 5.5 ± 0.1 24.6 ± 2.7 1.03 ± 0.10 21.4 ± 5.6 51.4 ± 12.3 121.0 ± 30.3 143.2 ± 1.3 103.2 ± 1.0 3.4 ± 0.2

0.80 0.78 0.10 0.81 0.64 0.06 0.25 0.54 0.96 0.13 0.04 0.18 0.58

904.8 ± 69.8 719.0 ± 39.3 7.0 ± 1.0 163.2 ± 25.3 5.7 ± 0.4 19.7 ± 0.3 0.93 ± 0.08 17.0 ± 3.3 92.6 ± 64.4 184.4 ± 37.9 140.8 ± 1.6 100.0 ± 1.1 3.2 ± 0.4

1118.0 ± 105.2 876.0 ± 63.0 8.0 ± 1.2 107.0 ± 4.6 6.0 ± 0.2 18.4 ± 2.1 0.83 ± 0.05 20.4 ± 1.5 36.4 ± 4.4 232.4 ± 41.1 143.4 ± 1.0 101.6 ± 1.6 3.6 ± 0.3

0.13 0.07 0.71 0.02 0.66 0.14 0.32 0.38 0.68 0.42 0.21 0.44 0.48

Values are shown as mean ± SE. Total chol, total cholesterol; LDL chol, low-density lipoprotein cholesterol; HDL chol, high-density lipoprotein cholesterol; BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT; alanine aminotransferase; LDH, lactic dehydrogenase.

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Table 2 iMAP-IVUS values before treatment and at 12 weeks in the two study groups. Baseline

2

Vessel area (mm ) Lumen area (mm2) Plaque area (mm2) % Plaque area % Fibrotic area % Lipidic area % Necrotic area % Calcified area

At 12 weeks

Control (n ¼ 40)

GLP-1 (n ¼ 35)

p value

Control (n ¼ 40)

GLP-1 (n ¼ 35)

p value

12.46 ± 0.36 7.17 ± 0.21 5.28 ± 0.23 42.04 ± 1.16 73.78 ± 1.64 6.60 ± 0.59 16.08 ± 1.25 1.55 ± 0.29

14.24 ± 0.41 8.15 ± 0.29 6.09 ± 0.27 42.70 ± 1.20 75.57 ± 1.90 4.97 ± 0.59 15.83 ± 1.45 1.91 ± 0.32

<0.01 <0.01 0.03 0.70 0.48 0.06 0.90 0.40

12.22 ± 0.32 5.72 ± 0.18 6.49 ± 0.27 52.09 ± 1.38 66.30 ± 2.06 6.20 ± 0.44 23.25 ± 1.87 2.15 ± 0.24

11.64 ± 0.32 5.58 ± 0.26 6.06 ± 0.27 52.14 ± 1.56 75.14 ± 2.62 5.67 ± 0.58 16.17 ± 2.27 1.00 ± 0.18

0.20 0.25 0.26 0.98 <0.01 0.45 0.02 <0.01

Values are shown as means ± SE. iMAP-IVUS, iMap™ intravascular ultrasound.

Table 3 Changes in the values of iMAP-IVUS from baseline to 12 weeks. Control (n ¼ 40)

Vessel area (mm2) Lumen area (mm2) Plaque area (mm2) % Plaque area % Fibrotic area % Lipidic area % Necrotic area % Calcified area

GLP-1 (n ¼ 35)

12 Weeks - Baseline

p value

12 Weeks - Baseline

p value

0.23 ± 0.26 1.45 ± 0.23 þ1.22 ± 0.17 þ10.16 ± 1.15 7.63 ± 1.92 0.53 ± 0.39 þ7.40 ± 1.72 þ0.60 ± 0.36

0.62 <0.01 <0.01 <0.01 <0.01 0.59 <0.01 0.12

2.61 2.58 0.03 þ9.44 0.43 þ0.69 þ0.34 0.91

<0.01 <0.01 0.94 <0.01 0.90 0.41 0.38 0.04

± ± ± ± ± ± ± ±

0.25 0.21 0.11 0.83 2.00 0.52 1.74 0.33

Data are presented as means ± SE. iMAP-IVUS, iMap™ intravascular ultrasound. p value: baseline vs. 12 weeks.

Fig. 1. Changes in vessel features and plaque components. Changes in vessel, lumen and plaque area after 12 weeks of treatment. **p < 0.05, baseline vs. 12 weeks; *p < 0.05, Control vs. GLP-1.

p ¼ 0.16). The percentages of macrophage-positive area and calcified deposits were lower in the GLP-1 group than in the Control group (Fig. 3A and B, % Macrophage area: 9.11 ± 0.80% vs. 6.19 ± 0.85%, p < 0.01, % Calcified area: 3.25 ± 0.67% vs. 0.75 ± 0.15%, p ¼ 0.02). The percentages of SMC area and fibrotic area were higher in the GLP-1 group than in the Control group (Fig. 3C and D; % SMC area: 6.93 ± 0.31% vs. 8.14 ± 0.48%, p ¼ 0.02; % Fibrotic area: 54.75 ± 1.63% vs. 60.60 ± 2.12%, p ¼ 0.02). The MMP-9 positive area (mm2/mm2) was comparable between the two groups (Fig. 3E;

Control vs. GLP-1; 17581 ± 3990 mm2/mm2 vs. 11675 ± 5485 mm2/ mm2, p ¼ 0.39). 3.4. iMAP-IVUS findings in comparison to histologic findings Representative iMAP-IVUS images (obtained before and at 12 weeks of treatment) and images of corresponding histologically stained specimens are shown in Fig. 4. Although all of the vessel, lumen, and intima-media areas in the histology images were

Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920

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Fig. 2. Changes in fibrotic, lipidic, necrotic, and calcified components after 12 week of treatment. **p < 0.05, baseline vs. 12 weeks; *p < 0.05, Control vs. GLP-1.

Table 4 Histological measurements at 12 weeks.

Vessel area (mm2) Lumen area (mm2) Intima area (mm2) Media area (mm2) Intima/media ratio Intima-media (mm2) % Intima-media area (%) Incidence of thin-cap fibroatheroma (%)

Control (n ¼ 46)

GLP-1 (n ¼ 41)

p value

8.35 ± 0.39 2.52 ± 0.18 4.44 ± 0.22 1.39 ± 0.05 3.44 ± 0.23 5.83 ± 0.22 71.16 ± 0.97 15.2

8.45 ± 0.31 2.67 ± 0.14 4.46 ± 0.27 1.33 ± 0.03 3.43 ± 0.24 5.79 ± 0.27 68.01 ± 1.34 4.9

0.84 0.32 0.97 0.33 0.99 0.91 0.06 0.16

% Intima-media area is defined as the ratio of intima-plaque area to vessel area. Data are presented as means ± SE.

smaller than those in the IVUS images (Supplementary Fig. 2), all areas measured by IVUS correlated clearly with the corresponding areas measured histologically (Supplementary Fig. 3AeF). Areas of positive macrophage staining in the histology images correlated with the necrotic, lipidic, and necrotic plus lipidic areas in the iMAP-IVUS images (Supplementary Fig. 3GeI), suggesting that intra-plaque inflammation promotes the formation of necrotic and lipidic plaques. 4. Discussion In this study, we investigated the effects of GLP-1 receptor agonist on fully developed atherosclerotic plaques in WHHL rabbits. The serial in vivo iMAP-IVUS observations revealed that GLP-1 receptor agonist treatment inhibited plaque growth, retarded positive vessel remodeling, and modified plaque components. Laboratory analysis clarified that GLP-1 receptor agonist reduced macrophage infiltration and calcium deposition. Thus, GLP-1 receptor agonist might inhibit plaque progression and promote plaque stabilization. Previous clinical studies have shown plaque inhibition and stabilization by intensive cholesterol lowering statin therapy [23e26]. However, it is not clear whether statin has similar plaque inhibition effects in patients without hyperlipidemia. Furthermore, the plaque stabilization effect of statin is delayed in patients with type 2 diabetes [27]. These issues highlight the need for new drugs to promote plaque inhibition in patients with diabetes, those with and those without hyperlipidemia. In the present study, GLP-1 receptor agonist was identified as a possible drug candidate that can be used instead of statin to achieve plaque inhibition and

stabilization, especially for patients with type 2 diabetes. Although recent studies have indicated that GLP-1 receptor agonist inhibits atherosclerosis development and plaque formation [8e11,28], as far as we know, ours is the first study to examine in an animal model whether GLP-1 receptor agonist promotes plaque stabilization. GLP-1 preserves endothelial function by inhibiting plasminogen activator inhibitor type-1 (PAI-1), vascular cell adhesion molecule (VCAM), and intracellular adhesion molecule-1 (ICAM-1) and by increasing endothelial nitric oxide synthase (eNOS) [7,29,30]. GLP-1 has been shown to reduce vascular inflammation and prevent atherosclerosis in Apo E/ mice [9e11]. As noted above, the GLP-1 in these studies was administered or overexpressed before the development of atherosclerosis and thus was shown to inhibit atherosclerotic plaque formation. The GLP-1 receptor agonist treatment-induced reduction of necrotic components might occur through inhibition of inflammation by GLP-1 [11,31,32]. Indeed, our analysis confirmed that administration of GLP-1 receptor agonist reduced macrophage infiltration and improved SMC-enriched plaques with increased fibrotic content. MMPs, such as MMP-9, have been reported to play key roles in atherosclerotic plaque formation and plaque instability [33,34]. We did not find that GLP-1 receptor agonist had an effect on MMP-9 expression in the atherosclerotic plaques. However, the blood triglyceride level at 12 weeks of treatment was lower in the GLP-1 group than in the Control group. These results are consistent with results of previously reported animal and clinical studies which GLP-1-based therapies inhibited triglyceride absorption [35] and reduced serum triglyceride levels [36]. The therapeutic action of GLP-1 in overcoming fully developed atherosclerosis might arise partially through the effect of GLP-1 on serum lipid profiles.

Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920

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Fig. 3. Histology. (A) Representative macrophage staining images and percentage of macrophage-positive area. (B) Representative EVG staining images of calcification and percentage of calcified components. (C) Representative SMC staining images and percentage of SMC-positive area. (D) Representative images of fibrotic area and percentage of fibrotic area. (E) Representative MMP-9 staining images and area of MMP-9-positive area. *p < 0.05, Control vs. GLP-1. EVG, Elastica van Gieson; SMC, smooth muscle cell.

Although pathological analysis is the gold standard for evaluation, serial in vivo intravascular imaging remains a necessary and important tool for observing plaque changes in vivo. In our study,

serial in vivo plaque observations were necessary. Although we randomly divided the littermate rabbits between the Control group and the GLP-1 group, the two groups were small, so there were

Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920

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Fig. 4. Representative iMAP-IVUS and histological staining images. (AeE) Control group images at the same cross-section. (A) Baseline iMAP-IVUS images, (B) iMAP-IVUS images at 12 weeks, (C) EVG staining, (D) SMC staining, and (E) macrophage staining. (FeJ) Images at the same cross-section in the GLP-1 group. (F) Baseline iMAP-IVUS images, (G) iMAP-IVUS images after 12 weeks of GLP-1 receptor agonist administration, (H) EVG staining, (I) SMC staining, and (J) macrophage staining. EVG, Elastica van Gieson; iMAP-IVUS, iMap™ intravascular ultrasound; SMC, smooth muscle cell.

differences in baseline vessel, lumen, and plaque areas between them. Without baseline iMAP-IVUS observation, these differences would not have been noticed but would have influenced the final measurements. Indeed, at 12 weeks, there were no differences in vessel, lumen, and plaque areas between the groups in either the iMAP-IVUS (Table 2) or pathology measurements. However, a comparison of the changes that occurred by 12 weeks of treatment revealed that GLP-1 receptor agonist inhibited plaque growth and retarded positive vessel remodeling. Our findings, taken together with those of many other studies, confirmed the protective effects of GLP-1 receptor against

atherosclerotic plaque [8e11,28e31,37,38]. However, the role of GLP-1 receptor agonist in atherosclerosis remains controversial. In a study conducted by Panjwani et al., GLP-1 did not attenuate atherosclerosis and macrophage accumulation in either the thoracic or abdominal aorta of diabetic Apo E/ mice, pointing to the importance of additional experimentation to identify the mechanisms and conditions linking GLP-1 receptor activation with atherosclerosis and macrophage migration [39]. Several clinical studies have suggested that circulating concentrations of GLP-1 are associated with coronary atherosclerosis [40,41]. However, we did not find a significant association between circulating

Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920

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M. Sudo et al. / Atherosclerosis xxx (2017) 1e9

concentrations of GLP-1 and coronary plaque progression in our animal model. Although the iMAP-IVUS images revealed a larger plaque area in the GLP-1 group than in the Control group before treatment, the blood GLP-1 concentration was comparable between the two groups. The discordance between the results of clinical studies and results of our animal study needs further investigation. iMAP is a newly developed IVUS tissue characterization system based on pattern recognition of radiofrequency signals, and ex vivo validation has shown accuracies at the highest levels of confidence of 97%, 98%, 95%, and 98% for necrotic, lipidic, fibrotic, and calcified regions, respectively [17]. The consistency between IVUS and histology findings in our study confirmed the reliability of in vivo iMAP-IVUS evaluation for both dimensional features and tissue characterization. All areas measured by iMAP-IVUS were smaller than the corresponding areas measured by histologic staining. The mismatch might have been the result of artifactual dimensional changes caused by fixation and histologic tissue processing factors such as dehydration, embedding, sectioning, and staining. These results are consistent with those of a previous study [42]. Animal models of atherosclerosis have been reported for several decades, and several newly described animal models, including mouse [43,44], rabbit [16], and pig [45] models, reflect human-like atherosclerosis with plaque rupture/instability. Animal models serve as tools of discovery aimed at uncovering mechanisms of and therapeutic approaches to plaque destabilization. Small animal models, such as mouse models, are suitable for investigating detailed mechanisms such as the signaling pathways. Large animal models, such as rabbit and pig models, are suitable for translational research, which bridges the gap between basic research and clinical application. The WHHL rabbit that we used, by virtue of the similarity of the size of its artery to that of human artery, is very appropriate for serial in vivo same-site intravascular imaging of the plaque development process. Our study results should be interpreted in light of our study limitations. Although our results and those of previous studies indicated that the effect of GLP-1 receptor agonist on plaque inhibition and stabilization might involve an anti-inflammatory effect [46e50], the detailed mechanisms by which this occurs were not investigated in this study, and they need to be clarified in future experiments. In addition, because of the fairly short time frame of our study, we used a fairly high dose of lixisenatide. Thus, the effect of long-term administration of lixisenatide at a clinical dose on fully developed atherosclerotic plaque needs to be investigated [51]. We also acknowledge that the dissimilarity in the evidence of atherosclerosis between our two groups of rabbits might have affected the subsequent changes in plaque area and vascular remodeling observed after 12 weeks of control or GLP-1 receptor agonist treatment. Furthermore, although complicated atherosclerotic lesions develop in WHHL rabbits, the disease in these animals does not perfectly mimic the coronary artery disease seen in patients with diabetes, in whom most atherosclerotic plaques develop slowly over many years. It is unknown whether our findings in WHHL rabbits predict outcomes in patients. Although the present animal study suggested GLP-1 receptor agonist as a possible drug candidate for plaque inhibition and stabilization, the clinical relevance of this observation needs further clinical investigation [40]. The results of our study suggest that GLP-1 receptor agonist has potential as a new candidate medicine for plaque inhibition and stabilization, especially for patients with type 2 diabetes. Conflict of interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

Financial support This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, including Grant-inAid for Young Scientists (B), No.23790883 (YL), Grant-in-Aid for Scientific Research (C), No. 25461093 (TH) and No. 25461094 (AH). Author contributions YL, TH, AH conceived the study and designed the experiments. MS, YL, TT, MM, MSu, MSh, TM, HH performed the experiments. MS, YL analyzed the data and drafted the manuscript. Acknowledgements We offer special thanks to Rie Takahashi and Yoshiki Taniguchi for their excellent technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2017.06.920. References [1] W. Kim, J.M. Egan, The role of incretins in glucose homeostasis and diabetes treatment, Pharmacol. Rev. 60 (2008) 470e512. [2] J. Rosenstock, M. Hanefeld, P. Shamanna, K.W. Min, G. Boka, P. Miossec, T. Zhou, I. Muehlen-Bartmer, R.E. Ratner, Beneficial effects of once-daily lixisenatide on overall and postprandial glycemic levels without significant excess of hypoglycemia in type 2 diabetes inadequately controlled on a sulfonylurea with or without metformin (GetGoal-S), J. Diabetes Complicat. 28 (2014) 386e392. [3] Y. Seino, A. Takami, G. Boka, E. Niemoeller, D. Raccah, PDY6797 investigators, Pharmacodynamics of the glucagon-like peptide-1 receptor agonist lixisenatide in Japanese and Caucasian patients with type 2 diabetes mellitus poorly controlled on sulphonylureas with/without metformin, Diabetes Obes. Metab. 16 (2014) 739e747. [4] L.A. Nikolaidis, D. Elahi, T. Hentosz, A. Doverspike, R. Huerbin, L. Zourelias, C. Stolarski, Y.T. Shen, R.P. Shannon, Recombinant glucagon-like peptide-1 increases myocardial glucose uptake and improves left ventricular performance in conscious dogs with pacing-induced dilated cardiomyopathy, Circulation 110 (2004) 955e961. [5] L.A. Nikolaidis, S. Mankad, G.G. Sokos, G. Miske, A. Shah, D. Elahi, R.P. Shannon, Effects of glucagon-like peptide-1 in patients with acute myocardial infarction and left ventricular dysfunction after successful reperfusion, Circulation 109 (2004) 962e965. [6] G.G. Sokos, L.A. Nikolaidis, S. Mankad, D. Elahi, R.P. Shannon, Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure, J. Card. Fail. 12 (2006) 694e699. [7] K. Ban, M.H. Noyan-Ashraf, J. Hoefer, S.S. Bolz, D.J. Drucker, M. Husain, Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways, Circulation 117 (2008) 2340e2350. [8] M. Arakawa, T. Mita, K. Azuma, C. Ebato, H. Goto, T. Nomiyama, Y. Fujitani, T. Hirose, R. Kawamori, H. Watada, Inhibition of monocyte adhesion to endothelial cells and attenuation of atherosclerotic lesion by a glucagon-like peptide-1 receptor agonist, exendin-4, Diabetes 59 (2010) 1030e1037. [9] H. Goto, T. Nomiyama, T. Mita, E. Yasunari, K. Azuma, K. Komiya, M. Arakawa, W.L. Jin, A. Kanazawa, R. Kawamori, Y. Fujitani, T. Hirose, H. Watada, Exendin4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury, Biochem. Biophys. Res. Commun. 405 (2011) 79e84. [10] M. Terasaki, M. Nagashima, T. Watanabe, K. Nohtomi, Y. Mori, A. Miyazaki, T. Hirano, Effects of PKF275-055, a dipeptidyl peptidase-4 inhibitor, on the development of atherosclerotic lesions in apolipoprotein E-null mice, Metabolism 61 (2012) 974e977. € llmann, F. Kahles, S. Reith, C. Lebherz, [11] M. Burgmaier, A. Liberman, J. Mo N. Marx, M. Lehrke, Glucagon-like peptide-1 (GLP-1) and its split products GLP-1(9-37) and GLP-1(28-37) stabilize atherosclerotic lesions in apoe(/) mice, Atherosclerosis 231 (2013) 27e435. [12] S.J. Nicholls, E.M. Tuzcu, S. Kalidindi, K. Wolski, W. Moon, I. Sipahi, P. Schoenhagen, S.E. Nissen, Effect of diabetes on progression of coronary atherosclerosis and arterial remodeling: a pooled analysis of 5 intravascular ultrasound trials, J. Am. Coll. Cardiol. 52 (2008) 255e262. € nnemaa, K. Pyo € r€ €, M. Laakso, Mortality from [13] S.M. Haffner, S. Lehto, T. Ro ala coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction, N. Engl. J. Med. 339

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Please cite this article in press as: M. Sudo, et al., Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits, Atherosclerosis (2017), http://dx.doi.org/ 10.1016/j.atherosclerosis.2017.06.920