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Atherosclerosis 236 (2014) 277e285

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Atherosclerosis

journal homepage: www.elsevier .com/locate/atherosclerosis

The interleukin-1b modulator gevokizumab reduces neointimalproliferation and improves reendothelialization in a rat carotiddenudation model

François Roubille a, David Busseuil a, Yanfen Shi a, Walid Nachar a,T�eodora Mihalache-Avram a, M�elanie Mecteau a, Marc-Antoine Gillis a, Genevi�eve Brand a,Gabriel Th�eberge-Julien a, Mathieu R. Brodeur a, Anne-Elen Kernaleguen a,M�elania Gombos a, Eric Rh�eaume a, b, Jean-Claude Tardif a, b, *

a Research Centre, Montreal Heart Institute, Montreal, Quebec, Canadab Department of Medicine, Universit�e de Montr�eal, Montreal, Quebec, Canada

a r t i c l e i n f o

Article history:Received 5 July 2013Received in revised form20 June 2014Accepted 11 July 2014Available online 21 July 2014

Keywords:GevokizumabInterleukin-1bNeointima formationEndotheliumCarotid artery denudation

* Corresponding author. Montreal Heart Institute,treal, Quebec H1T 1C8, Canada. Tel.: þ1 514 376 3330

E-mail address: jean-claude.tardif@icm-mhi.org (J

http://dx.doi.org/10.1016/j.atherosclerosis.2014.07.0120021-9150/© 2014 Elsevier Ireland Ltd. All rights rese

a b s t r a c t

Objective: Excessive neointima formation often occurs after arterial injury. Interleukin-1b (IL-1b) is apotent pleiotropic cytokine that has been shown to regulate neointimal proliferation. We investigatedthe effects of the IL-1b modulator gevokizumab in a rat carotid denudation model.Methods: SpragueeDawley rats were subjected to balloon denudation of the right carotid artery andwere then randomized to receive a single subcutaneous infusion immediately after balloon injury ofsaline (control group, n ¼ 13) or gevokizumab (gevokizumab groups, n ¼ 15 in each group: 1, 10 and50 mg/kg). We evaluated the treatment effects on carotid intima-media thickness (IMT) using ultraso-nography, on endothelial regrowth using Evans Blue staining and on inflammatory response using his-tology. We also assessed the effects of IL-1b and gevokizumab on human umbilical vein endothelial cells(HUVEC) and rat smooth muscle cells.Results: We found that carotid IMT, in the proximal part of the denuded artery at day 28, was decreasedby gevokizumab 1 mg/kg compared with controls. Neointima area and the intima/media area ratio wereboth reduced in the gevokizumab 1 mg/kg-treated group. Gevokizumab at the 1 mg/kg dose alsoimproved endothelial regrowth. No effect was observed with gevokizumab 10 or 50 mg/kg. Gevokizumabalso decreased the inflammatory effect of IL-1b in in vitro cell experiments and protected HUVECs fromIL-1b's deleterious effects on cell migration, apoptosis and proliferation.Conclusion: A single administration of gevokizumab 1 mg/kg improves endothelial regrowth and reducesneointima formation in rats following carotid denudation, at least in part through its beneficial effects onendothelial cells.

© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Arterial endothelial denudation following angioplasty leads torestenosis, corresponding to an accumulation of inflammatory cellsresulting in the proliferation of smooth muscle cells (SMC) whichform neointimal tissue at the injury site [1]. Inhibition of proin-flammatory pathways has been shown to decrease neointimal

5000 Belanger Street, Mon-x3612; fax: þ1 514 593 2500..-C. Tardif).

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formation, highlighting the role of post-denudation inflammationin vascular responses [2,3].

Interleukin-1b (IL-1b) is a potent proinflammatory cytokinesecreted by different cell types including macrophages and endo-thelial cells [4], playing an important role in the pathophysiology ofneointima formation [5,6]. In the absence of the natural antagonistof the IL-1 receptor, post-injury intimal proliferation is increased asdemonstrated in IL-1Ra-deficient mice [7]. IL-1b mediates the up-regulation of adhesion molecule expression on endothelial cellsand smooth muscle cells [8,9], thereby increasing recruitment andextravasation of inflammatory cells, particularly monocytes, andactivation of macrophages via the activation of the inflammasome

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285278

[10]. Importantly, the inflammasome can in turn induce IL-1bproduction [11]. Beyond neointima formation, atherosclerotic pla-que formation is decreased in animal models by genetic disruptionof the IL-1b gene [12] or IL-1 receptor (IL-1R) [13] or pharmaco-logical inhibition [14]. Therefore, our main hypothesis is thatmodulating IL-1b bioactivity using a biological drug specificallytargeting IL-1b (an anti-IL-1b antibody) could favorably affectreendothelialization and neointimal proliferation after arterialdenudation, possibly by reducing systemic and local inflammation(hs-CRP, IL-6, TNF-a) and also by regulating expression of adhesionmolecules such as VCAM-1 and ICAM-1.

Many therapies targeting IL-1 signaling have been developed totarget auto-inflammatory diseases and more recently cardiovas-cular diseases [15]. Gevokizumab, a potent anti-IL-1b neutralizingantibody, binds to a unique IL-1b epitope that is proximal to, butdoes not overlap with the receptor/ligand interface. Gevokizumabbinding to IL-1b may reduce the rate of assembly of the activesignaling complex rather than prohibit its assembly entirely [16].Gevokizumab is therefore considered a modulator and not ablocker of IL-1b signaling, which may result in the reduction ofpathologically high activity while allowing homeostatic signalingin biologically important pathways [17]. Gevokizumabwas recentlyshown to reduce atherosclerotic plaque formation in ApoE-deficient mice [18].

In the current study, we investigated the effects of threedifferent dosages (1, 10 and 50 mg/kg) of a single infusion of thisbiological drug gevokizumab on reendothelialization and neo-intimal formation in a rat carotid denudation model.

2. Materials and methods

2.1. Animals and experiments

Animal care and procedures complied with the CanadianCouncil on Animal Care guidelines and were approved by theinstitutional ethics committee for animal research. Fifty-eight(58) male SpragueeDawley rats (3 months of age, weighing330e360 g) were fed a 0.5% cholesterol-enriched diet starting 2days before the injury, throughout the experimental protocol.Animals were anesthetized with continuous isoflurane (Abbott,Montreal, Canada) administration (induction 2.5%e3.0%, 1.5 L/min oxygen, followed by 1.5e2.0%, 2 L/min oxygen) and heparin(100 IU in each animal) was injected into the right jugular vein.To maintain analgesia, buprenorphine (100 mg/kg, Schering-Plough, Hertfordshire, UK) was administered i.v. before the sur-gery and given once daily for 3 days after surgery. Electrocar-diogram was monitored and thermoregulation was achievedduring the procedure.

The right common, external and internal carotid arteries wereexposed by a neck incision under a stereo-microscope (LeicaM80, Leica Microsystems, Wetzlar, Germany). Meanwhile, bloodflow was temporarily interrupted by ligation of the common andinternal carotid arteries using surgical thread. The external ca-rotid artery was partially cut at about 2 mm distally from thearterial bifurcation with micro-scissors. A balloon angioplastycatheter (balloon diameter 1.5 mm, length 15 mm, Maverick,Medtronic, Minneapolis, MN, USA) was introduced through thecut of the external carotid into the common carotid artery andwas advanced to the proximal edge of the omohyoid muscle. Theballoon was then inflated with saline and rotated three times toensure uniformity of the extent of balloon injury. The balloonwas then deflated and removed. The external carotid artery wasligated and blood flow was restored through the common andinternal carotid arteries. Rats were then randomly assigned toreceive a single subcutaneous injection of either isotype IgG2

control preparation at the dose of 10 mg/kg (control group,n ¼ 13) or gevokizumab at the dose of 1 mg/kg (gevokizumab1 mg/kg group, n ¼ 15) or 10 mg/kg (gevokizumab 10 mg/kggroup, n ¼ 15) or 50 mg/kg (gevokizumab 50 mg/kg group,n ¼ 15) immediately after balloon injury. Ultrasonographic ex-aminations of both left and right carotid arteries were performedat baseline, at day 14 and day 28 post-denudation. Animals wereweighed regularly throughout the experimental protocol. Afterthe final ultrasonogram, rats were sacrificed by exsanguinationunder anesthesia 28 days after vascular injury. Right carotid ar-teries were harvested for histological analyses. Blood sampleswere obtained through the abdominal aorta duringexsanguination.

2.2. Gevokizumab

Gevokizumab aliquots were provided by Institut de RecherchesInternationales Servier (Suresnes, France) and were kept at 4 �C(see Supplementary methods for details).

2.3. Ultrasonographic determination of carotid intima-mediathickness

B-mode ultrasound imaging of carotid arteries was performed atbaseline before carotid injury, 14 and 28 days after the injury.Special care was taken to acquire similar imaging during follow-upstudies by the same experienced operator. Six cardiac cycles wereused to obtainmean values for all measurements, with the operatorbeing blinded to treatment assignment (see Supplementarymethods for details).

2.4. Evans blue staining and determination of endothelial regrowth

Endothelial regrowth was evaluated by Evans blue staining.Twenty-eight days after carotid denudation and gevokizumab orcontrol treatment, 5 rats/group received an intravenous injection of5% Evans blue dye solution in the caudal vein (50 mg/ml/kg; Sigma,Saint Louis, MO, USA) 5 min before sacrifice (see Supplementarymethods for details).

2.5. Histomorphometry

The intima and media cross-sectional areas of the carotid ar-teries were measured and the intima/media area ratios werecalculated. All measurements were performed by two experiencedinvestigators blinded to randomized treatment assignment (seeSupplementary methods for details).

2.6. Immunohistochemistry

We assessed the levels of leucocytes (CD45), monocytechemotactic protein-1 (MCP-1) and vascular cell adhesionmolecule-1 (VCAM-1) on carotid paraffin-embedded sections (seeSupplementary methods for details).

2.7. Blood analyses

Serum soluble intercellular adhesion molecule-1 (sICAM-1)levels were measured at baseline and day 28 after carotid injury byenzyme-linked immunosorbent assay (ELISA) (see Supplementarymethods for details). Total cholesterol and triglyceride levels weremeasured with an automated filter photometer system (DimensionRxL Max; Dade Behring, Deerfield, IL, USA).

Table 1Serum levels of total cholesterol and glycemia.

Control Gevokizumab 1 mg/kg Gevokizumab 10 mg/kg Gevokizumab 50 mg/kg

Baseline 28 days Baseline 28 days Baseline 28 days Baseline 28 days

Total cholesterol (mM) 2.62 ± 0.09 1.96 ± 0.11 2.37 ± 0.09 1.93 ± 0.14 2.53 ± 0.08 1.84 ± 0.08 2.56 ± 0.08 1.86 ± 0.09Triglycerides (mM) 0.88 ± 0.12 1.00 ± 0.28 1.02 ± 0.13 1.31 ± 0.30 1.10 ± 0.11 0.73 ± 0.10 1.06 ± 0.09 1.01 ± 0.15Glycemia (mM) 10.6 ± 0.5 11.5 ± 0.8 10.6 ± 0.4 10.7 ± 0.7 10.5 ± 0.4 11.2 ± 0.5 10.2 ± 0.4 11.1 ± 0.9

Total cholesterol was significantly reduced in all groups at day 28 compared with baseline (P < 0.05), but there were no significant differences between treatment groups.Triglyceride levels significantly decreased from baseline to 28 days only in the 10 mg/kg-treated group. Glycemia significantly increased from baseline to 28 days only in thecontrol group, but the differences between treatment groups were not significant.

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285 279

2.8. mRNA quantification

An additional group of normal animals (no surgery nor gevo-kizumab treatment, n ¼ 6) was added for RNA analyses (seeSupplementary methods for details).

2.9. Cell culture experiments

Primary rat vascular smooth muscle cells (SMC) were isolatedfrom aortas of male SpragueeDawley rats. Human umbilical veinendothelial cells (HUVECs) and SMCs were treated with IL-1b inpresence or absence of gevokizumab or the IgG2 control antibody.

Fig. 1. Gevokizumab 1 mg/kg treatment reduces carotid intima-media thickness (IMT), at tmeasured using ultrasonography at days 14 and 28 in the (a) proximal segment, (b) mid-segmproximal, mid and distal carotid artery segments. Control group, n ¼ 10; gevokizumab 1 mg**P < 0.01.

Endothelial cell migration was evaluated using an in vitro scratchwound assay. Proliferation was assessed with an MTT assay. Flowcytometry was used to quantitate the apoptosis rate and theexpression of adhesion molecules using anti-human E-selectin andVCAM-1 antibodies (BD Biosciences). mRNA expression wasassessed by qPCR (see Supplementary methods for details).

2.10. Statistical analyses

All analyses were performedwith SAS version 9.3 or higher (SASInstitute Inc., Cary, NC, USA) and conducted at the 0.05 significancelevel. Continuous variables are presented as adjusted

he proximal level at day 28. Intima-media thickness ratio of right/left carotid arteriesent and (c) distal segment; (d) average of right/left intima-media thickness ratio of the/kg, n ¼ 12; gevokizumab 10 mg/kg, n ¼ 13; gevokizumab 50 mg/kg, n ¼ 12. *P < 0.05,

Fig. 2. Gevokizumab 1 mg/kg improves endothelial regrowth at day 28. (a)Longitudinally-opened common right carotid arteries stained with Evans blue at day28 after balloon denudation from a control animal and a gevokizumab 1 mg/kg-treatedanimal. Blue staining (non-endothelialized surface) was quantified in the denudedcarotid artery segment and presented as (b) endothelial regrowth at day 28 aftertreatment with control or gevokizumab. **P ¼ 0.009, n ¼ 5 in each group. (For inter-pretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285280

mean ± standard error of the mean (SEM) (see Supplementarymethods for details).

3. Results

Animals with a denuded right carotid artery were randomlyassigned to one of the 4 treatment groups: control or gevokizumabat dosages of 1, 10 or 50 mg/kg. In vivo, gevokizumab at 1 mg/kgyielded significant beneficial effects on every endpoint, in contrastto the higher dosages.

3.1. Body weight and blood analyses

Body weight increased consistently with age, without any sig-nificant differences between groups (Supplementary Fig. 1). Totalcholesterol levels significantly decreased between baseline and 28days in each group, with similar values among groups (Table 1).Triglyceride levels significantly decreased from baseline to 28 daysin the 10 mg/kg-treated group only (Table 1).

3.2. Low-dose gevokizumab reduces carotid intima-mediathickness

We first assessed by ultrasonography the right/left carotid ar-tery IMT ratio obtained on day 14 and day 28 at the proximal(Fig. 1a), mid (Fig. 1b), and distal levels (Fig. 1c), of the injured ca-rotid segment. At day 14, we observed a decreased right/left IMTratio in the gevokizumab 1 mg/kg-treated group compared withcontrols, at the distal level (2.25 ± 0.26 vs 3.17 ± 0.29, P ¼ 0.009)(Fig. 1c), and after averaging all 3 levels studied (Fig. 1d, P ¼ 0.047).We did not observe any difference at the proximal and mid-levelswhen compared with controls (respectively, P ¼ 0.255 andP ¼ 0.159).

At day 28, we found IMT to be significantly decreased in thegevokizumab 1 mg/kg-treated group compared with controls, atthe proximal level (1.87 ± 0.20 vs 2.58 ± 0.19, P ¼ 0.026) (Fig. 1a).We did not observe significant differences at mid and distal levels(Fig. 1b and c), or when combining the data from all 3 levels studied(P ¼ 0.095) (Fig. 1d). Carotid artery elasticity measured by

ultrasonography was similar for all four treatment groups (data notshown).

3.3. Low-dose gevokizumab improves endothelial regrowth

We evaluated the re-endothelialization of the denuded portionof the carotid artery by Evans blue staining (Fig. 2a). Endothelialregrowth was significantly improved by 3-fold in the gevokizumab1 mg/kg-treated group compared with controls (31.3 ± 2.8% vs10.8 ± 2.8%, P ¼ 0.009) (Fig. 2b). Given the improved endothelialregrowth in the gevokizumab 1 mg/kg-treated group, we assessedneointimal tissue formation using morphometric analysis.

3.4. Low-dose gevokizumab reduces neointimal tissue formation

We assessed neointimal tissue formation on HPS-stained sec-tions at 5 different levels in the proximal half of the injured carotidartery (Fig. 3a). Neointimal area was reduced by 25% in the gevo-kizumab 1 mg/kg-treated group compared with controls(162,375 ± 14,195 vs 217,601 ± 14,303 mm2, P ¼ 0.016) (Fig. 3b). Insupport of these findings, gevokizumab 1 mg/kg treatment led to asignificant decrease of the intima/media area ratio assessed bymorphometry as compared with controls (1.19 ± 0.11 vs 1.63 ± 0.14,P ¼ 0.015) (Fig. 3c), corresponding to a reduction of 27%.

3.5. Impact of gevokizumab on inflammation

Inflammation is one of the main determinants of intimal pro-liferation [19]. We therefore evaluated systemic as well as in situinflammation. In situ, the total number of cells was quantified in theneointimal tissue of HPS-stained sections at 5 different levels in theproximal half of the injured carotid artery. Compared with controls,the total number of cells per section was significantly reduced inthe gevokizumab 1 mg/kg-treated group (P ¼ 0.006) as well as inthe 50 mg/kg-treated group (P ¼ 0.049) (Fig. 4a).

There was a 56% reduction in neointimal CD45-positive cellsurface in the gevokizumab 1 mg/kg-treated group compared withcontrols (Fig. 4b), which did not reach statistical significance(P ¼ 0.23), probably due to the small sample size (n ¼ 10 and 8,respectively). Gevokizumab treatment did not have an effect on thelevels of proteins involved in the IL-1b inflammatory pathways,including VCAM-1 and MCP-1 (data not shown). We also evaluatedsICAM-1 and did not find any significant difference betweentreated groups (data not shown).

Finally, we evaluated changes in the expression of inflammatoryand adhesion genes using qRT-PCR. We assessed the expression ofthe following genes in a carotid segment from normal animals andin the distal half of the injured carotid artery of control andgevokizumab-treated animals: Ccl2, Icam1, Vcam1, Tnf, Il6, Il-1b, Il-1Ra, and I11r1. Although the expression of most of the inflamma-tory genes tested (except ICAM-1 and VCAM-1) was significantlyincreased compared with normal animals, there were no differ-ences observed following treatment (Supplementary Fig. 2). We didnot find any differences in the circulating levels of inflammatorycytokines (IL-1b, TNF-〈, IL-6, data not shown).

3.6. In vitro assessment of the anti-inflammatory effects ofgevokizumab

To further assess the efficacy of gevokizumab and to appraisewhether the effects observed in vivomay have been due to its anti-inflammatory effects on endothelial or smooth muscle cells, wetreated HUVECs and rat SMCs with IL-1b in presence or absence ofgevokizumab and measured the expression of adhesion moleculesresponsible for inflammatory cell infiltration.

Fig. 3. Gevokizumab 1 mg/kg reduces neointima proliferation at day 28. Morphometric analyses of carotid arteries at day 28 after denudation and treatment with gevokizumab orcontrol. (a) HPS-stained cross-sections of carotid arteries from a control animal, gevokizumab 1 mg/kg, gevokizumab 10 mg/kg and gevokizumab 50 mg/kg-treated animals(magnification �10); (b) neointimal tissue surface quantification, *P ¼ 0.016; (c) intima/media area ratio of right/left carotid artery, *P ¼ 0.015. Control group, n ¼ 8; gevokizumab1 mg/kg, n ¼ 10; gevokizumab 10 mg/kg, n ¼ 10; gevokizumab 50 mg/kg, n ¼ 10.

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285 281

In the experiments using HUVECs, cell exposure to gevokizumabalone or an IgG2 antibody alone did not exert any effect on the cellsurface expression of E-selectin or VCAM-1 as assessed by flowcytometry (data not shown). As expected, exposure of HUVECs toIL-1b induced an increase in expression of E-selectin and VCAM-1(P < 0.01 and P < 0.05, respectively, Fig. 5). The IL-1b-induced in-crease in HUVEC expression of E-selectinwas inhibited by both lowand higher doses of gevokizumab (P < 0.01, Fig. 5a), whereas forVCAM-1, this overexpression was numerically decreased by a lowdose of gevokizumab (0.5 ng/mL, P ¼ 0.063) and significantlydecreased with higher doses (5 ng/mL and 50 ng/mL, P < 0.01).

In rat SMC experiments, gevokizumab alone or the IgG2 controlantibody alone did not exert any significant effect on the mRNAexpression for ICAM-1 and VCAM-1 (data not shown). IL-1b

induced a significant overexpression of the mRNAs for ICAM-1 andVCAM-1 (Supplementary Fig. 3), which was significantly decreasedby gevokizumab (5 ng/mL or 50 ng/mL, P < 0.001, SupplementaryFig. 3).

3.7. In vitro assessment of deleterious effects of IL-1b on endothelialcell migration, apoptosis and proliferation and their blockade bygevokizumab

To assess the effect of IL-1b on endothelial cell migration, weperformed a scratch wound assay where HUVECs were exposed toIL-1b. As presented in Fig. 6, wound closure was inhibited by 25.1%by IL-1b alone (p < 0.0001) and by 16.4% for IL-1b in presence ofcontrol IgG2 antibody (p ¼ 0.0013); in contrast, there was no

Fig. 4. Gevokizumab 1 mg/kg decreases total cell number and CD45-positive area inthe neointimal tissue at day 28. (a) Total cell number quantification in the neointimaltissue on HPS-stained cross-sections of denuded carotid arteries (total number persection). *P ¼ 0.049, **P ¼ 0.006. Control group, n ¼ 8; gevokizumab 1 mg/kg, n ¼ 10;gevokizumab 10 mg/kg, n ¼ 10; gevokizumab 50 mg/kg, n ¼ 10); (b) CD45-labeledcross-sections of denuded carotid arteries from a control animal and a gevokizumab1 mg/kg-treated animal (magnification �10); (c) CD45-positive cell area quantificationin the neointimal tissue of denuded carotid arteries (gevokizumab 1 mg/kg group(n ¼ 10) versus controls (n ¼ 8), P ¼ 0.23).

Fig. 5. Gevokizumab decreases the IL-1b-induced overexpression of endothelial adhesion mflow cytometry; (b) VCAM-1 expression in HUVECs assessed by flow cytometry. The percentcell surface proteins under the different conditions relative to that of IL-1b stimulated cells, N

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285282

significant difference between control cells (not exposed to IL-1b orgevokizumab) and cells exposed to both IL-1b and gevokizumab.Gevokizumab improved IL-1b-inhibited endothelial cell migration(P ¼ 0.0021) whereas the IgG2 control antibody did not exert asignificant effect. Direct comparison of the percent difference tocontrol cells between cells exposed to both IL-1b and gevokizumaband those exposed to IL-1b and IgG2 control antibody almostreached statistical significance (p ¼ 0.0573, not represented inFig. 6a).

Exposure of HUVEC cells to IL-1b resulted in other deleteriouseffects by increasing the percentage of cells undergoing apoptosis.Fig. 6b shows that IL-1b caused a 2.2-fold increase in late apoptosisof HUVECs whereas gevokizumab blocked 53% of this increase(p ¼ 0.0027). When HUVECs were treated with the IgG2 controlantibody, there was no effect on IL-1b-induced apoptosis. The ef-fects of treatments with gevokizumab and IgG2 control antibody onIL-1b-induced apoptosis were significantly different (P ¼ 0.0006).

Conversely, IL-1b decreased significantly the proliferation ofHUVECs when compared to unexposed control cells (P ¼ 0.0167,Fig. 6c). The addition of the IgG2 control antibody did not exert anysignificant effect on Il-1b-induced inhibition of proliferation. Incontrast, gevokizumab improved significantly the proliferation ofHUVECs exposed to Il-1b (P ¼ 0.0021). There was also a significantdifference between the cells exposed to IL-1b in presence ofgevokizumab versus those in presence of the control IgG2 antibody(P ¼ 0.0038).

4. Discussion

We have shown that a single infusion of the biological drugspecifically targeting IL-1b gevokizumab at 1 mg/kg consistentlyimproves endothelial regrowth, decreases neointimal formationand reduces intima-media thickness in a rat carotid denudationmodel. In vitro experiments show that gevokizumab may decreasethe IL-1b-induced expression of several inflammatory mediatorsand adhesion molecules by both endothelial and smooth musclecells. Additionally, the in vitro experiments also demonstrateddeleterious effects of IL-1b on endothelial cell migration, apoptosisand proliferation which could be significantly attenuated by gevo-kizumab. Our findings underline the critical role of IL-1b signalingin re-endothelialization and neointima proliferation and highlight anew strategy to treat injured arteries.

The anti-IL-1b treatment in this rat model of carotid denudationhad amajor impact on re-endothelialization, whichwas found to be

olecules in vitro in cultured HUVECs. (a) E-selectin expression in HUVECs assessed byages presented here report the ratios of the percent of cells significantly expressing the¼ 5 experiments, triplicates for each experiment. Means and SEM are presented. IL-1b

increased significantly the expression of both E-selectin and VCAM-1, when compared to controls (P < 0.01 and P < 0.05, respectively). The IgG2 control antibody alone did not exertany significant effect. Gevokizumab at a dose of 0.5 ng/mL decreased the IL-1b-stimulated expression of E-selectin (P < 0.01) but not VCAM-1 (P]NS), whereas higher doses (5 and50 ng/mL) significantly decreased the IL-1b-induced expression of both E-selectin and VCAM-1 (P < 0.01). IL-1b: IL-1b at the dose of 50 U/mL; Gev 0.5: gevokizumab at the dose of0.5 ng/mL; Gev 5: gevokizumab at the dose of 5 ng/mL; Gev 50: gevokizumab at the dose of 50 ng/mL; IgG 0.5: IgG2 at the dose of 0.5 ng/mL; IgG2 5: IgG at the dose of 5 ng/mL; IgG50: IgG2 at the dose of 50 ng/mL *P < 0.05, **P < 0.01.

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Fig. 6. Gevokizumab improves in vitro characteristics associated with better re-endothelialization in cultured HUVECs. (a) The effect of gevokizumab on migration of humanumbilical vein endothelial cells (HUVECs) exposed to IL-1b was assessed using a scratch wound assay. Briefly, HUVECs monolayers were scratched and then incubated for 16 h withor without IL-1b, gevokizumab or IgG2. Representative images are shown and the lower panel shows quantification of wound closure for n ¼ 6 experiments. (b) Apoptosis assayusing an Annexin V/PI staining and flow cytometry quantification. HUVECs were treated for 24 h in presence of IL-1b and gevokizumab or IgG2 control. Late apoptosis represent cellspositive for both Annexin V and PI. n ¼ 6 experiments. (c) An MTT assay was used to measure the proliferation and viability of HUVECs after 48 h of IL-1b treatments with or withoutgevokizumab or IgG2. n ¼ 5 experiments. IL-1b: IL-1b 50 U/mL; Gev: gevokizumab 50 ng/mL; IgG: IgG2 50 ng/mL *P < 0.05, **P < 0.01, ***P < 0.001. Means and SEM are presented.

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285 283

F. Roubille et al. / Atherosclerosis 236 (2014) 277e285284

2.9 times more complete in the gevokizumab 1 mg/kg-treatedgroup compared with controls. As supported by our in vitro ex-periments, the local production of IL-1b after carotid denudationhas deleterious effects on endothelial cell migration and prolifer-ation/apoptosis and these effects can be alleviated by gevokizumab.The benefit of gevokizumab on re-endothelialization comparesfavorably with that of other interventions with a positive impact onendothelialization in rat carotid injury models such as local VEGFdelivery [20] or treatment with the phosphodiesterase inhibitorcilostazol [21]. In vitro experiments demonstrated that gevokizu-mab can exert powerful anti-inflammatory effects on both endo-thelial and smooth muscle cells. Reduced expression of VCAM-1and inflammatory mediators could result in a more physiologicreendothelialization associated with a healthier endothelium: forinstance, in an in vitro study onmigration of HUVEC and coverage ofstents, VCAM-1 decreasewas correlated to better cellular migration[22]. Treatment with gevokizumab 1 mg/kg also reduced neo-intimal formation after carotid denudation, as measured by histo-morphometry. The chemotherapy agent paclitaxel decreasedneointima formation by more than 50% in a rat model of carotiddenudation [23], but in contrast could delay reendothelialization,as corroborated by in vitro studies [24,25].

In the current study, we did not find any differences in thecirculating levels of inflammatory cytokines (IL-1b, TNF-a, IL-6,data not shown). However, given that the current model involvesa localized vascular lesion; its effect on circulating cytokines isexpected to be modest. Therefore, the local tissue production ofproinflammatory cytokines should be of greater interest than sys-temic release. With regards to RNA expression in carotid arterytissue, proinflammatory genes were increased in both the controland treated animals when compared with normal rats. These re-sults support the validity of our model, as it corroborates theincreased local inflammation occurring in denuded carotid arteries.We did not find any difference in the carotid tissue mRNA expres-sion of proinflammatory genes between the treated and controlanimal groups; whether this could be attributed to subtle localeffects remains to be determined. Of note, only the distal half of thedenuded carotid arteries could be analyzed for gene expression.The timing of such analyses might also be crucial. We cannotexclude that only mild inflammatory processes were still active at28 days. An earlier window for RNA and histological analyses couldhave also been of interest. Interestingly, in our in vitro experiments,the IL-1b-induced increase in HUVEC and rat SMC expression ofadhesion molecules was inhibited by gevokizumab.

In our in vivo animal study, themost effective gevokizumab dosewas 1 mg/kg, whereas non-significant effects were observed with10 and 50mg/kg doses. The effect of a high dose of gevokizumab onIL-1b signaling remains to be further investigated. Our findingswith the 1 mg/kg dose are however in line with previous datashowing better efficacy at 1 mg/kg than with 10 mg/kg for somemarkers of atherosclerosis in apoE knockout mice [18]. Further-more, in our in vitro experiments, gevokizumab decreased the IL-1b-induced overexpression of endothelial adhesion molecules inHUVECs without an obvious doseeresponse relationship. Thiscould reflect the complex impact of a cytokine modulating agentwith differing effects at different doses. Whether dosages lowerthan 1 mg/kg could be of value in our model also remains to bedetermined.

4.1. Study limitations

Our study has limitations. First, potential early changes of in-flammatory markers induced by gevokizumab may have beenmissed as blood samples were only taken at the time of sacrifice.Second, although the carotid denudation model is widely used in

rats, the differences in severity of injury between animals should beconsidered. To address this issue, only one experienced operatorperformed all the experiments and was blinded to the randomizedtreatment assignment. Given that the 15 mm-long balloon intro-duced in the common carotid artery was bearing against theomohyoidmuscle, the proximal region of the denuded vessel mighthave been the most injured one; this further highlights theobserved benefit of gevokizumab on neointimal formation in thisregion.

4.2. Conclusion

A single infusion of the IL-1b modulator gevokizumab at 1 mg/kg leads to significant improvement of reendothelialization ofdenuded carotid arteries and reduction of neointimal formation, atleast in part through its beneficial effects on endothelial cells.

Funding sources

The Institut de Recherches Internationales Servier provided thedrug and funding for the mentioned research activities. This workwas supported by grants from the F�ed�eration Française de Car-diologie (FR). Dr. Tardif holds the Canada Research Chair (tier 1) intranslational and personalized medicine and the Universit�e deMontr�eal research chair in atherosclerosis.

Conflict of interest

The Institut de Recherches Internationales Servier provided thedrug and specific grants for the mentioned research activities. BothDrs. Roubille and Tardif have received honoraria for lectures fromServier.

Acknowledgments

The authors gratefully acknowledge the statistical analysis byClifford Ekwempe, MSc, and Marie-Claude Guertin, PhD, at theMontreal Heart Institute Coordinating Center (MHICC). Specialthanks to Ekaterini Kritikou, PhD, for scientific review and editingof the current manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.atherosclerosis.2014.07.012.

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