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Prostaglandins and Other Lipid Mediators

Original research article

The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE−/− mice Daniel F.J. Ketelhuth a , Andreas Hermansson a , Hanna Hlawaty b , Didier Letourneur b , Zhong-qun Yan a , Magnus Bäck a,c,∗ a

Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, 17176 Stockholm, Sweden INSERM U1148 (ex U698), Bichat Hospital, 75018 Paris, France c Department of Cardiology, Karolinska University Hospital, 17176 Stockholm, Sweden b

a r t i c l e Article history: Available online xxx Keywords: Atherosclerosis Inflammation Lipoxygenase Leukotriene receptor

i n f o

a b s t r a c t Leukotriene B4 (LTB4 ) induces proinflammatory signaling through BLT receptors expressed in atherosclerotic lesions. Either genetic or pharmacological targeting of the high affinity LTB4 receptor, BLT1 , reduces atherosclerosis in different mouse models. The low affinity BLT2 receptor for LTB4 may transduce additional pro-atherogenic signaling, but combined BLT1 and BLT2 receptor antagonism has not previously been explored in atherosclerosis. The aim of the present study was to unravel the effects of the BLT receptor antagonist BIIL284 in apolipoprotein E deficient mice in terms of atherosclerotic lesion size and composition, as well as on arterial matrixmetalloproteinase (MMP) activity and plasma cytokines. Oral administration of BIIL284 (0.3–3 mg/kg) dose-dependently decreased atherosclerotic lesion size after 12 weeks. In addition, significantly smaller aortic lesions were observed in mice treated with BIIL284 (3 mg/kg) for 24 weeks. The reduced atherosclerosis was associated with less lesion smooth muscle cells, less arterial MMP activities and lower plasma levels of TNF-␣ and IL-6. Taken together, these results suggest a therapeutic value of BLT receptor antagonism in atherosclerosis. © 2015 Published by Elsevier Inc.

Introduction Leukotriene B4 (LTB4 ) induces proinflammatory signaling through cell membrane receptors, denoted BLT1 and BLT2 , which represent the high- and low affinity LTB4 receptors, respectively [1]. In human atherosclerotic lesions, three major cell types express BLT receptors, namely monocytes/macrophages, endothelial cells and vascular smooth muscle cells [2]. Mechanistic studies have supported that LTB4 -induced effects on these target cells may be involved in the development and progression of atherosclerosis [3,4]. Furthermore, increased LTB4 production has been correlated to early signs of atherosclerosis [5]. Targeted deletion of the BLT1 receptor in hypercholesterolemic mice reduces atherosclerotic lesion size [6] and smooth muscle cell content compared with their BLT1 +/+ counterparts [7]. Also pharmacological BLT1 receptor antagonism by means of CP105,696 reduces atherosclerosis in mice deficient in either apolipoprotein E (ApoE) or the low density lipoprotein receptor (LDLR) [8].

∗ Corresponding author at: Center for Molecular Medicine, Karolinska University Hospital, L8:03, 17176 Stockholm, Sweden. Tel.: +46 8 51770000; fax: +46 8313147. E-mail address: [email protected] (M. Bäck).

Special attention has been devoted to LTB4 signaling in the accelerated atherosclerosis observed in subjects with obstructive sleep apnea (OSA) [4,9,10]. In line with this, the increased atherosclerotic lesion size observed in ApoE−/− mice subjected to intermittent hypoxia (a murine model of OSA) is absent in BLT1 and ApoE double knock-out mice [11]. Taken together, these studies convincingly demonstrated a major role of LTB4 signaling in atherosclerosis by means of its high affinity BLT1 receptor. In contrast, the BLT2 receptor antagonist LY255283 did not alter atherosclerotic lesion size after 8 weeks treatment in ApoE−/− mice [12]. Nevertheless, LY255283-treated mice exhibited reduced vascular oxidative stress and improved endothelial function compared with untreated mice [12], suggesting that targeting also the BLT2 receptor could have additional beneficial effects in atherosclerosis. The latter suggestion is supported by results obtained in BLT1 and BLT2 double knock-out mice, which have revealed non-redundant roles of these receptors in other disease models [13]. However, no previous study has evaluated the effects of combined BLT1 and BLT2 receptor antagonism in atherosclerosis. BIIL284 is a prodrug, and its active metabolite BIIL315 has been characterized as potent non-selective BLT receptor antagonists in vitro [14]. Oral administration of BIIL284 inhibits LTB4 -induced responses in vivo [14] as well as Mac-1 expression in patients with

http://dx.doi.org/10.1016/j.prostaglandins.2015.05.007 1098-8823/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Ketelhuth DFJ, et al. The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE−/− mice. Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.05.007

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rheumatoid arthritis [15]. BIIL284 almost abolishes intimal hyperplasia in rats 14 days subsequent to balloon injury of the carotid artery [2] and reduces in-stent stenosis in hypercholeterolemic rabbits [16]. Given the above-mentioned emerging evidence of a major role of LTB4 signaling in atherosclerosis [17], the present study was designed to evaluate the effects of BIIL284 in ApoE-deficient mice. Materials and methods Animals All animal experiments were approved by the Regional Ethics Committee for Animal Research in Stockholm. Male ApoE−/− mice from Taconic (Ry, Denmark) were fed standard mouse chow until 8 weeks of age, followed by standard chow (D12102, Research Diets Inc, New Brunswick, NJ) supplemented with or without BIIL284 for either 12 or 24 weeks. The dosing of BIIL was either 0.3 or 3 mg/kg per day. Mice were sacrificed under gas anesthesia at either 20 or 32 weeks of age. After heart punction and vascular perfusion with sterile RNase-free PBS, the heart, aorta and carotid arteries were removed. The hearts were frozen in OCT medium for histological analysis using a standardized protocol [18]. The thoracic aortas were formalin-fixed, opened longitudinally, pinned out and stained with Sudan IV (Merck AG, Darmstadt, Germany). En face aortic lesion and total aortic area were quantified using Histolab software. The abdominal aortas were snap-frozen on dry ice and stored at −80 ◦ C until RNA extraction.

PCR Total RNA was isolated using RNeasy (Qiagen) and concentrations were measured spectrophotometrically (Nanodrop 1000, Thermo Fisher Scientific). RNA quality was analyzed on a 2100 Bioanalyzer (Agilent). Reverse-transcription was performed with Superscript-II (Invitrogen) and random hexamers. Real time PCR was performed on a 7900HT Fast Real-Time PCR system (PerkinElmer Applied Biosystems) using TaqMan Assay-on-Demand from Applied Biosystems. Results were calculated as 2−CT obtained by comparing the threshold cycle (CT) for the gene of interest with that obtained using TATA-binding protein (TBP) as housekeeping gene. Statistical analysis All results are expressed as mean ± SEM. Statistically significant differences were determined using a Student’s t-test for pairwise comparisons, and a one- or two way analysis of variances (ANOVA) followed by Tukey’s test for multiple comparisons. Analyses were performed using SigmaPlot version 12 (Systat Software Inc). Results Pharmacokinetics The plasma concentrations of the active metabolite BIIL315 are shown in Table 1. At 20 weeks of age, the concentrations of BIIL315 were significantly higher in the high dose (3 mg/kg BIIL284) compared with the low dose (0.3 mg/kg BIIL284) treatment group.

Plasma analysis

Plasma lipids

A blood sample was obtained from each mouse by heart puncture at the time of sacrifice. Plasma concentrations of the active BIIL284-metabolite, BIIL315 were determined as previously described [15,16]. Plasma cholesterol and triglycerides were measured using enzymatic colorimetric kits (Randox Lab. Ltd. Crumin, UK). Plasma concentrations of selected inflammatory (IL-6, MCP-1, and TNF-␣) and anti-inflammatory (IL-10) cytokines were measured using flow cytometry using BDTM Cytometric Bead Array assay (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions.

The results of the lipid measures are shown if Table 2. BIIL284 did not significantly alter the levels of either cholesterol or triglycerides.

Immunohistochemistry Cryosections of the aortic root were used for immunohistochemical stainings with antibodies against vascular smooth muscle cells (␣-SM-actin), macrophages (CD68) and T-lymphocytes (CD3). Stained areas were quantified using Histolab image analysis software. Gelatin zymography Carotid arteries were harvested from 20 weeks old ApoE−/− mice treated for 12 weeks with either chow or chow containing BIIL284 (0.3 and 3 mg/kg). Arterial samples (n = 6) were flushed with saline, cleaned from adipose tissue and opened longitudinally before incubation in serum free-medium at 37 ◦ C. The conditioned media was collected at 24, 48, 72 and 96 h of incubation and stored at −20 ◦ C until analysis. Gelatinolytic activities of 20 ␮L conditioned media normalized by mg of arterial tissue were analyzed by gelatin zymography as previously described [16]. Results are expressed in OD units obtained from densitometric analyses of scanned gelatine gels using Scion Image Software.

Atherosclerosis The results of the en face quantifications of Sudan IV staining in the thoracic aorta are shown in Fig. 1. There were significantly larger lesions in 32-compared with 20 weeks old mice. In 20-week old mice, there was a dose-dependent decrease in the proportion of Table 1 Plasma concentrations of the active BIIL284-metabolite, BIIL315, at the time of sacrifice. Treatment

Dose

n

Treatment time (week)

BIIL315 (ng/mL)

Control BIIL284 BIIL284 Control BIIL284

– 0.3 mg/kg 3 mg/kg – 3 mg/kg

10 10 10 10 9

8–20 8–20 8–20 8–32 8–32

0±0 1.3 ± 0.2 70.4 ± 49 0±0 23.5 ± 3.4

Table 2 Plasma lipid levels.

Mice (20 weeks old) Control BIIL284 (0.3 mg/kg; 12 weeks) BIIL284 (3 mg/kg); 12 weeks) Mice (32 weeks old) Control BIIL284 (3 mg/kg; 32 weeks)

Cholesterol

Triglycerides

(mmol/L)

(mmol/L)

n

12 ± 0.8 11 ± 0.4 12 ± 1.9

1.2 ± 0.09 1.4 ± 0.14 1.1 ± 0.13

(9) (9) (8)

7.6 ± 0.5 7.0 ± 0.4

1.5 ± 0.13 1.7 ± 0.18

(10) (10)

Please cite this article in press as: Ketelhuth DFJ, et al. The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE−/− mice. Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.05.007

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significant decreases in proMMP-2, MMP-2 and proMMP-9 activities in arteries derived from BIIL284-treated compared with non-treated mice (Fig. 3). This effect was dose-dependent with the most pronounced effects observed in mice treated with high dose BIIL284 (3 mg/kg). MMP and TIMP mRNA levels Changes in mRNA levels of matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitor of MMP (TIMP)-1 and -2 assessed in the abdominal aorta by real time TaqMan PCR are shown in Fig. 3. There was a trend toward decreased MMP-9, and increased TIMP-1 expression, which did not reach statistical significance (Fig. 3). Likewise, there were no significant differences between the different treatment groups in terms of MMP-2 and TIMP-2 (Fig. 3). Plasma cytokines

Fig. 1. En face analysis of thoracic aortic atherosclerosis in 20 and 32 week old ApoE−/− mice fed standard chow without (Control) or with BIIL284 (0.3 and 3 mg/kg) for either 12 or 24 weeks as indicated. A, untreated; B, BIIL284 0.3 mg/kg; C, BIIL 3 mg/kg (A–C 12 weeks treatment); D untreated; E, BIIL 3 mg/kg (D–E 24 weeks treatment). * P < 0.05 vs. 20-week control † P < 0.05 vs. 32-week control.

the thoracic aorta covered by lesions in mice treated with BIIL284 (either 0.3 or 3 mg/kg) compared with controls (Fig. 1). In addition, the higher dose of BIIL284 (3 mg/kg) was evaluated after 24 weeks of treatment after which treated mice exhibited less lesions compared with untreated mice (Fig. 1). Cellular composition BIIL284 significantly and dose-dependently reduced the area stained by ␣SM -actin in the aortic root (Fig. 2) as compared with control. In contrast, there were no significant difference in the proportion of macrophages in the aortic root lesions, as determined by CD68 staining (Fig. 2). The areas stained by the antibody against the T-lymphocyte marker CD3 were small and not significantly different between treatment groups (data not shown). Gelatinase activity Gelatinase activity of proMMP-2 (72 kDa), MMP-2 (68 kDa) and proMMP-9 (92 kDa) were detected in carotid artery supernatants (n = 6; Fig. 3), whereas MMP-9 was undetectable. There were

Plasma levels of the pro-inflammatory cytokines TNF-␣ and IL-6 were significantly lowered upon long-term treatment with BIIL284 (3 mg/kg; Fig. 4). However, no difference between groups was observed for the levels of either MCP-1 or the anti-inflammatory cytokine IL-10 (Fig. 4). Discussion In the present study, the BLT receptor antagonist BIIL284 inhibited atherosclerosis progression in ApoE-deficient mice. The observed reduction in smooth muscle cells, carotid artery MMP activity and plasma cytokine concentrations may contribute to the anti-atherosclerotic effects of this BLT receptor antagonist. In one published study of BIIL284 pharmacokinetics in humans, plasma levels of BIIL315 in the range of

- mice.

Leukotriene B4 (LTB4) induces proinflammatory signaling through BLT receptors expressed in atherosclerotic lesions. Either genetic or pharmacological ...
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