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Abstract

Background:

The purpose of the study was the comparative assessment of ticagrelor and clopidogrel effects on carotid post-balloon injury (PBI) and on post carotid artery stenting (CAS) rate of in-stent restenosis (ISR) and in-stent thrombosis in atherosclerotic rabbits.

Methods:

Forty-eight New Zealand white rabbits on high-fat diet were randomized into 4 groups: A1: PBI and clopidogrel (30 mg/kg/d), A2: PBI and ticagrelor (21 mg/kg twice daily), B1: PBI, CAS, and clopidogrel (30 mg/kg/d), B2: PBI, CAS, and ticagrelor (21 mg/kg twice daily). All rabbits received orally aspirin (10 mg/kg/d) and interventions were performed in their right carotid arteries (RCAs). Optical coherence tomography (OCT) and carotid angiography were performed at end point, while platelet aggregation and lipid profile were measured. After euthanasia both carotids were obtained for histological examination.

Results:

In B1 group, 3 rabbits presented thrombotic total occlusion of the stents, while none such episode was observed in B2 group. The neointimal areas in RCAs, calculated by OCT, did not differ between A1 and A2 groups, and between B1 and B2 groups (P > .05). From the histological findings, the intima/(media + intima) percentage (%) in RCAs of balloon-injured rabbits did not present any difference between groups (P = .812). Similarly, the immunohistochemically determined accumulation of endothelial cells and macrophages on vascular walls was equivalent between groups (P > .05).

Conclusion:

Following carotid balloon injury and stenting, clopidogrel and ticagrelor did not show any differential effects on the extent of neointimal formation and ISR in atherosclerotic rabbits receiving aspirin. Three thrombotic stent occlusions were noted in the clopidogrel treatment group, but this finding was not statistically significant.

Keywords

ticagrelor clopidogrel neointimal hyperplasia in-stent restenosis in-stent thrombosis

Introduction

Percutaneous transluminal carotid angioplasty followed by stent implantation (carotid artery stenting [CAS]) is considered an expanding method in the carotid artery disease worldwide.¹ The infrequent, yet important, early complications of post-balloon injury (PBI), neoatherosclerosis,^2,3 in-stent restenosis (ISR), and in-stent thrombosis (IST) may limit clinical efficacy, leading to recurrent cerebrovascular events.^4,5 The platelets’ role in the pathogenesis of restenosis and/or thrombosis early after endovascular interventions has been studied extensively.^6

-9 Biochemical interactions of blood platelets with injured arterial wall or stent material consist the main underlying mechanism.¹⁰ Combination of aspirin and clopidogrel, as dual antiplatelet therapy, administered in patients undergoing either percutaneous coronary intervention (PCI)¹¹ or CAS^12,13 is known for alleviating platelet aggregation and adhesion.

Clopidogrel is a P2Y12 receptor inhibitor and its active metabolite selectively targets the adenosine diphosphate (ADP)–induced stimulation of platelet function, thereby inhibiting platelet aggregation. Its administration has been associated with lower cardiovascular events rate^14,15 and reduced post-vascular injury neointimal hyperplasia.^16,17 Alternatively, ticagrelor, a novel, reversibly binding P2Y12 receptor inhibitor provides significant and more consistent platelet aggregation inhibition function than typical regimen of clopidogrel in patients with either stable coronary artery diseases or acute coronary syndrome.^18,19 Compared to clopidogrel, ticagrelor appears more efficient to reduce ischemic events in patients undergoing PCI.¹³ In case of peripheral arterial disease and revascularization (eg, carotid, femoral arteries), the potential protective role of ticagrelor is under investigation.

In the study, we comparatively assess the impact of ticagrelor versus clopidogrel, as part of dual antiplatelet therapy, on PBI, ISR, and IST in an atherosclerotic rabbit model undergoing carotid balloon-injury accompanied or not by CAS.

Materials and Methods

Animals and Housing Conditions

Forty-eight male New Zealand rabbits, of a body weight between 2.5 and 3 kg, were used. The study was conducted in the Biomedical Research Foundation of the Academy of Athens and was approved by the Animal Care Committee according to the European Union Act and Greek Law (permit no 1153/12-05-2015). The rabbits were housed individually in cages on stainless steel racks at a room temperature of 21 ± 2 °C and relative humidity of 50 ± 5%. A 12-hour light/dark cycle was maintained. All animals had unlimited access to water and were fed ad libitum (rabbit chow 2930, Harlan Laboratories, Inc) during the acclimatization period prior to the onset of the protocol.

All animals were fed Western-type, high-fat (1%, wt/wt: Mucedola) diet for 8 weeks, while on the fourth week they were randomly assigned, while maintaining the high-fat diet (HFD), into the following 4 equal groups (n = 12):

Group A1: It underwent carotid artery balloon injury and received clopidogrel for 4 weeks.

Group A2: It underwent carotid artery balloon injury and received ticagrelor for 4 weeks .

Group B1: It underwent carotid artery balloon injury and stenting and received clopidogrel for 4 weeks.

Group B2: It underwent carotid artery balloon injury and stenting and received ticagrelor for 4 weeks.

Dosage was 30 mg/kg daily for clopidogrel (Pharmathen) and 21 mg/kg twice a day for ticagrelor (AstraZeneca). The dose of ticagrelor was previously determined by a pharmacokinetic study, under the guidance of scientific colleagues in Astra Zeneca company. Aspirin (Bayer Hellas) was administered orally (10 mg/kg/d) to all animals, starting 7 days prior to the procedure up until the sacrifice at the end of the experimentation period. Loading doses of dual antiplatelet agents (aspirin 30 mg/kg plus clopidogrel 120 mg/kg or ticagrelor 42 mg/kg) were given 2 hours before the procedure. All drugs were administered via esophageal gavage.

Surgical Procedure

Induction of anesthesia for each animal was performed with intramuscular injection of ketamine (25 mg/kg, Imalgene 1000; Merial) and midazolam (0.5 mg/kg, Dormicum; Roche Hellas), 30 minutes before surgery. A dose of cefuroxime (25 mg/kg, Zinacef; Covis Pharmaceuticals, Inc) was also injected intramuscularly to each rabbit, preprocedural. Maintenance of anesthesia was supported during the procedure with continuous isoflurane administration (1.5%-2%, IsoFlo; Zoetis) via face mask with simultaneous oxygen supply . Blood sampling was performed via an intravenous (IV) catheter placed in the middle ear artery. Monitoring of electrocardiography, oxygen saturation, and vital signs was conducted during surgery (DASH 2000 Pro, GE Healthcare). Immediately before endothelial denudation, each animal received 100 IU/kg of heparin (IV, LEO Pharmaceuticals Products).

After sedation, rabbits were placed at a supine position on the operating table and connected to the veterinary ventilator (MDS Matrx Model 2000; Hallowell EMC). The surgical field was sterilized using topical 10% povidone–iodine (Betadine, Lavipharm). For local anesthesia, a 1% lidocaine (Xylocaine, AstraZeneca) solution was injected subcutaneously. A lateral skin incision, approximately 4 cm in length, was performed 0.5 and 1 cm from the midline to expose the right carotid artery (RCA), and a 3-0 black silk was tied at the most distal (cephalic) part of the carotid artery for its dilation, thereby making it easy to puncture. A deflated 5F introducer sheath (2.25 × 8 mm; Cook Medical) was inserted via the proximal part and was advanced to the level of the carotid bifurcation through a coronary guide wire (Medtronic Inc). A deflated balloon catheter was then advanced into the RCA under the guidance of angiography. The balloon was inflated at 10 atm for 30 seconds. For the carotid stent groups, the stent delivery system was then advanced up to the injury site and a bare metal stent (BMS; Medtronic Inc, Cobalt Alloy, 2.25 × 8 mm) was deployed. After removing the catheter the incision was sutured-closed.

At the end of the procedure, angiography was conducted in order to document vessel patency. After recovery, each animal was given an intramuscular injection of the anti-inflammatory drug Rimadyl (Carprofen) (approximately 4 mg/kg; Laboratories Pfizer Ltda).

Patency of Arteries

On the 28th day after initial injury, we evaluated the patency of either the balloon-injured or the stent-implanted arteries in all groups using carotid angiography (C-arm; Philips BV Libra, Philips Medical Systems) and optical coherence tomography (OCT; St Jude Medical).

An intravascular OCT imaging catheter (DragonFly Duo, St Jude Medical) was inserted through the sheath in the RCA and over the guidewire. Optical coherence tomography serial images of the carotid artery were acquired in an automated pullback format at a rate of 1 mm/s and 15 frames/s during intermittent contrast medium flush through the guiding catheter. They were then analyzed by computerized morphometry (DICOM Image Software), to measure lumen area, perimeter, and mean diameter of each carotid artery section. For the stent groups, those recordings were calculated for pre, distal, mid, and post stent areas. The evaluation of the OCT and angiography results was conducted by 2 independent investigators, blinded to the protocol groups.

Histology

At end point, all rabbits were humanely euthanized by administrating an overdose of intravenous sodium pentobarbital (Dolethal; Vetoquinol Ireland Limited). The injured and stented arterial segments as well as the corresponding left carotid artery, serving as control, were excised and fixed in 10% buffered formaldehyde and each specimen was embedded in a paraffin block. Histological sections (5-µm thickness) were cut from the proximal and distal ends and midpoint of the carotid arteries with a Fully Automated Rotary Microtome (Leica Biosystems GmbH). For the stented segments, we used hard tissue blades (C.L. Sturkey, Inc).

For each staining per rabbit, 3 nonconsecutive sections (at equal intervals of 50 μm) were obtained following a standardized protocol to allow the colocalization of the measured variables. Quantitative analysis of luminal, neointimal, and medial areas was performed on sections stained with hematoxylin and eosin. Neointimal formation was calculated as follows: [intima/(media + intima)] × 100 (%) and results were reported as percentage. Macrophages and endothelial cells (ECs) detection were also performed on nonconsecutive tissue sections. This was achieved by applying immunohistochemical methods using monoclonal antibodies against RAM-11 (Dako, 1:50) and PECAM (CD-31, 1:50), antigens, respectively. All images of the cross sections of regions of interest were assessed using Image Pro Plus software version 4.1 (Media Cybernetics). All observations and grading were performed by one investigator blinded to the research protocol.

Platelet Aggregation

Platelet function in whole blood, collected from the marginal ear vein, was assessed using the Multiplate Analyzer (Roche Diagnostics) according to the manufacturer’s instructions. Adenosine diphosphate (20 mL, ADPtest) was added to induce platelet aggregation measured as electrical impedance. The following parameters were recorded: Aggregation, measured in AU (aggregation units), area under the curve, measured in U = AU × min ([1U = 10AU × min], and velocity, measured in AU/min. All blood samples were analyzed at 30 minutes after sampling, while assays were performed before euthanasia at 1 hour and 4 hours after the last dose of ADP inhibitors.

Lipid Profile Analysis

The route of blood collection was the marginal ear vein and was performed at baseline and at end point of experimentation, just before euthanasia. Plasma samples were initially stored in deep freezer (−78 °C) and were later used in order to assess total cholesterol, triglycerides, and high-density lipoprotein cholesterol concentrations using an enzymatic method (Chemwell 2910, Awareness Technology Inc).

Statistical Analysis

In order to compare the differences between groups at the end of the study, a paired t test or a 1-way analysis of variance with post hoc tests data analyses were performed. When multiple pairwise comparisons were needed, we used the Tukey test, while when there was violation of data normal distribution, nonparametric Kruskal-Wallis, and Mann-Whitney tests were applied. All tests were 2 sided and a value of P < .05 was considered statistically significant. Results are expressed as the mean ± standard deviation and analyzed by SPSS (version 22.0; IBM Corp).

Results

Patency of Arteries

Inability to obtain adequate arterial access for the OCT catheter was encountered in 3 rabbits in the B1 group, due to IST. In PBI rabbits, the OCT calculated neointimal areas in RCAs did not differ between A1 and A2, while the mid-stent neointimal areas was similar in B1 and B2 groups, as well. Perimeter, mean diameter, and lumen area of each carotid artery were found with no statistically significant differences between all groups (P > .05; Table 1 and Figure 1).

Table 1.

Results Derived From Optical Coherence Tomography (OCT), Histological Analysis of the Right Carotid Arteries, and Blood Samples Measurements.

	Groups
	A1	A2	P value (A1 vs A2)	B1	B2	P value (B1 vs B2)
OCT parameters
Mean neointimal area (mm²)	4.20 ± 0.96	3.78 ± 1.31	.781	1.74 ± 0.51	1.45 ± 0.62	.654
Perimeter (mm)	5.46 ± 1.21	4.36 ± 2.04	.670	7.93 ± 1.05	7.92 ± 0.69	.991
Histological parameters
Intima/intima + media thickness (%)	68.48 ± 12.31	64.22 ± 13.22	.712	23.51 ± 6.20	19.85 ± 5.78	.356
Macrophages stained-area (%)	11.40 ± 1.85	13.50 ± 4.21	.389	17.54 ± 4.68	17.28 ± 6.53	.948
Endothelial cells–stained area (%)	16.42 ± 4.53	23.72 ± 9.45	.213	23.16 ± 8.25	21.57 ± 7.16	.780
Blood parameters
Total cholesterol (mg/dL)	776 ± 238	833 ± 378	.791	888 ± 415	791 ± 334	.541
Triglycerides (mg/dL)	185 ± 67	145 ± 52	.289	166 ± 67	179 ± 82	.496
HDL (mg/dL)	48.75 ± 13.39	49,67 ± 26.972	.876	63.75 ± 18.167	44.42 ± 15.86	.528
LDL (mg/dL)	762.65 ± 223.61	782.42 ± 326.48	.761	844 ± 308.73	674.42 ± 385.76	.604
Platelet aggregation (AUC)	20.83 ± 8.53	15.38 ± 5.22	.002	8.95 ± 2.78	6.55 ± 1.47	.005

Abbreviations: AUC, area under the curve; CAS, carotid artery stenting.

Groups: A1) Carotid balloon injury and clopidogrel (30 mg/kg/d), A2) Carotid balloon injury and ticagrelor (21mg/kg twice daily), B3) Carotid balloon injury and CAS and clopidogrel (30 mg/kg/d), B4) Carotid balloon injury and CAS and ticagrelor (21 mg/kg twice daily). The stained area for macrophages and endothelial parameters is expressed as the percentage of the whole neointimal area.

Figure 1.

Optical coherence analysis of carotid arteries after balloon-injury or stent implantation: (A) right carotid artery (RCA) clopidogrel-treated group, (B) RCA ticagrelor-treated group, and (C-D) In-stent thrombosis in clopidogrel-treated rabbit. 97 × 96 mm (300 × 300 DPI).

Histology

The effect of clopidogrel and ticagrelor treatment on neointimal formation was evaluated in all animal groups. Left carotid arteries lesions showed negligible differences (P > .05). As expected, injured RCAs showed more extended neointimal formation than intact left carotid arteries. However, we did not detect any significant differences (P > .05) between treatments (ticagrelor vs clopidogrel) on neointimal formation (Figure 2).

Figure 2.

Balloon injury–induced stenosis in right carotid arteries (RCAs). Left carotid arteries (LCAs) served as controls (hematoxylin and eosin staining): (A) RCA clopidogrel-treated group, (B) RCA ticagrelor-treated group, (C) LCA clopidogrel-treated group, and (D) LCA ticagrelor-treated group.

Based on immunohistochemistry analysis, macrophages numbers did not differ between drug therapy (clopidogrel vs ticagrelor, P > .05) and independent of type of intervention (balloon-injury with or without stenting). Similarly, ECs accumulation was equivalent along all groups (P > .05; Table 1).

Platelet Aggregation

Platelet aggregation was increased in all groups at the 4-hour sampling. However, post hoc comparison revealed a statistically significant lower platelet activation in ticagrelor-treated compared to clopidogrel-treated counterparts (P < .01). Velocity of platelet aggregation did not differ significantly among therapy groups (Table 1).

Lipid Profile Analysis

Biochemical evaluation of the parameters measured at baseline, before surgery, and at the end. Total cholesterol and triglycerides concentrations were elevated due to HFD, with no statistical differences between groups (P > .05) in the end of experimentation. On the other hand, HDL levels remained ultimately unchanged along time (P > .05). Finally, as long as LDL levels are concerned, all groups were found with higher values at end point when compared with baseline but there were no statistically significant differences between groups (P > .05).

Discussion

In the present experimental study, the choice of either clopidogrel or ticagrelor did not affect the amount of neointimal formation and ISR in atherosclerotic rabbits undergoing, respectively, balloon injury or balloon injury plus CAS. Similarly, the ECs and inflammatory cells infiltration of both injured and stented carotid arteries were not affected by the selected type of P2Y12 inhibitor for treatment. However, a significant portion of clopidogrel-treated rabbits, undergoing CAS, showed IST unlikely to ticagrelor therapy. The latter could be explained by the higher platelet aggregation observed in clopidogrel rather than ticagrelor group.

It is well established that BMS have been highly associated with ISR and IST than new-generation drug-eluting stents.²⁰ Regarding the exclusive usage of either balloon angioplasty or BMS implantation in carotid artery stenosis, numerous experimental studies have been conducted to assess the preventive impact of a wide variety of pharmaceutical agents on neointimal hyperplasia in the context of post-balloon angioplasty or ISR or IST in carotid arteries.^21
-23 However, no firm conclusions have been drawn. On the other hand, a plethora of evidence have indicated dual antiplatelet therapy as an effective measure to reduce ISR or IST in patients undergoing coronary stenting, even in the long term.²⁴ Taken into consideration the above data, we tested whether the potential protective action of P2Y12 inhibitors in coronary arteries may be extrapolated to carotid artery atherothrombosis using a valid rabbit model.^25,26 Indeed, the intima/media ratio of the RCA and the neointimal area of the injured artery was thicker in comparison with the same area in the left carotid artery in our balloon-injured groups. It is well known that neointimal hyperplasia formation begins within 5 days following balloon injury and maintains for at least 4 weeks until a maximum intima/media ratio has been attained.²⁷ After 4 weeks of balloon injury, ticagrelor-treated and clopidogrel-treated groups showed equal intima/media ratio and neointimal areas. Similarly, in stented groups, the degree of ISR did not differ between therapeutic modalities. A previous porcine study demonstrated the superiority of ticagrelor over either clopidogrel or prasugrel in reducing the neointimal hyperplasia after drug-eluting stents implantation in pig’s coronary arteries.²⁸ The authors ascribed this difference to the pleiotropic effects of ticagrelor other than platelet P2Y12 inhibition. In a carotid anastomosis animal model, Sürer et al documented less neointimal hyperplasia with ticagrelor treatment compared to control animal group, indicating a potential antiproliferative action.²⁹

To our knowledge, this is the first study evaluating the differential effects of clopidogrel and ticagrelor as preventive drugs of neointimal hyperplasia, ISR, and IST after carotid angioplasty or CAS. Previously, statins have shown counterregulatory impact on carotid neointimal hyperplasia.³⁰ In our study, ticagrelor therapy failed to reduce neointimal hyperplasia and ISR compared to clopidogrel, despite a more effective reduction in platelet aggregation levels. Perhaps, platelet aggregation is not involved in neointimal hyperplasia process in large carotid/peripheral arteries as much as it is in smaller coronary arteries or anastomoses.^31
-33 Hence, no differential effects on carotid neointimal hyperplasia are expected between different P2Y12 inhibitors regimens. Apart from platelet aggregation, factors such as, inflammation, blood flow disturbances, and various growth factors, may contribute to neointimal hyperplasia.³⁴ The damaged area is coated with platelets and macrophages releasing thrombotic (fibrinogen and von Willebrand factor) and growth factors (platelet-derived growth factor and transforming growth factor) or impairing the release of anticoagulants from ECs,³⁵ narrowing the arterial lumen. In our study, we did not detect any differential effects of ticagrelor and clopidogrel on ECs and macrophages contents in PBI or stented carotids. Hence, our study demonstrated that the potential “pleiotropic,” antiproliferative action of ticagrelor did not influence ECs triggering or inflammation to a greater extent than clopidogrel. Thus, the choice of ticagrelor or clopidogrel may have not any impact on the amount of neointimal hyperplasia and ISR prevention in carotid arteries. Unambiguously, further investigation is required to explain how P212Y inhibitors affect homeostasis of large peripheral arteries, like carotids.

Total occlusion of the ipsilateral carotid artery was noted in 3 of 12 clopidogrel-treated rabbits undergoing CAS. No thrombotic occlusions were observed in stented rabbits receiving ticagrelor. Thrombotic complications were identified using real-time, in vivo, OCT images in each stent. Both groups had received before intervention the related loading doses of either ticagrelor or clopidogrel, while aspirin administration was uninterrupted. In-stent thrombosis represents an uncommon, but detrimental complication of CAS. Several factors are involved, like antiplatelet therapy resistance or discontinuation and inherent or acquired thrombotic predisposition.³⁶ In our study, platelet aggregation was further reduced in ticagrelor than clopidogrel group. Although 25% of clopidogrel-treated rabbits developed IST, the absence of such complication in the ticagrelor group did not drive to a statistically significant difference between those groups. These are preliminary data and future studies should investigate whether there is a drug effect of P2Y12 inhibitors on IST, in the clinical setting of peripheral arteries angioplasty and BMS implantation.

There are several limitations in our study. First, the present study did not have the statistical power to identify a significant direct, inverse relationship between ticagrelor and IST. Second, we did not use additional biomarkers, for example, CRP, hsCRP, HbA_1c, fasting glucose, and so on, to assess the underlying inflammatory or other mechanisms associated with neointimal hyperplasia or IST/ISR. Third, this is an animal study and clinical extrapolation should be done with caution.

Conclusion

Clopidogrel and ticagrelor seem to have equal effects concerning neointimal formation and ISR prevention in atherosclerotic rabbits after carotid artery balloon injury with or without stent implantation. Our preliminary data implicate a higher preventive efficacy of ticagrelor rather than clopidogrel against IST in rabbits undergoing CAS. The clinical significance of our findings requires further investigation.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was funded by AstraZeneca Company, Sweden, which also supplied the pure substance of ticagrelor. Clopidogrel substance and aspirin were kindly granted by Pharmathen Pharmaceuticals Company, Greece and Bayer Hellas Company, Greece, respectively.

ORCID iD

Marianna Stasinopoulou

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Carotid Stent Restenosis and Thrombosis in Rabbits: The Effect of Antiplatelet Agents

Abstract

Background:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and Methods

Animals and Housing Conditions

Surgical Procedure

Patency of Arteries

Histology

Platelet Aggregation

Lipid Profile Analysis

Statistical Analysis

Results

Patency of Arteries

Histology

Platelet Aggregation

Lipid Profile Analysis

Discussion

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

References