Cardiovascular Research (2014) 102, 429–435 doi:10.1093/cvr/cvu028

Vessel wall, not platelet, P2Y12 potentiates early atherogenesis Laura E. West, Tanja Steiner, Heather M. Judge, Sheila E. Francis, and Robert F. Storey* Department of Cardiovascular Science, University of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX, UK Received 16 July 2013; revised 16 January 2014; accepted 24 January 2014; online publish-ahead-of-print 7 February 2014

Aims

Platelets have a fundamental role in atherothrombosis, but their role in early atherogenesis is unclear. The P2Y12 receptor is responsible for amplifying and sustaining platelet activation and P2Y12 inhibition is crucial in modulating the vessel wall response to injury. We therefore examined the role of platelet vs. vessel wall P2Y12 in early atherogenesis and considered the use of P2Y12 antagonists ticagrelor and clopidogrel in modulating this process. ..................................................................................................................................................................................... Methods ApoE2/2 and ApoE2/2 P2Y2/2 male mice underwent bone marrow transplantation and were fed a western diet for 4 12 weeks before assessing atherosclerotic burden. Compared with ApoE2/2 controls, platelet P2Y12 deficiency profoundly and results reduced platelet reactivity but had no effect on atheroma formation, whereas vessel wall P2Y12 deficiency significantly attenuated atheroma in the aortic sinus and brachiocephalic artery (both P , 0.001). ApoE2/2 and ApoE2/2 P2Y2/2 12 male mice fed western diet plus either twice-daily doses of ticagrelor (100 mg/kg) or daily clopidogrel (20 mg/kg) for 4 weeks exhibited no significant reduction in atheroma compared with control mice fed mannitol. Attenuated P-selectin expression confirmed platelet P2Y12 inhibition in drug-treated mice. ..................................................................................................................................................................................... Conclusions Despite its major contribution to platelet reactivity, platelet P2Y12 has no effect on early atheroma formation, whereas vessel wall P2Y12 is important in this process. Ticagrelor and clopidogrel effectively reduced platelet reactivity but were unable to inhibit early atherogenesis, demonstrating that these P2Y12 inhibitors may not be effective in preventing early disease.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Platelet † P2Y12 † Atherogenesis † Ticagrelor † Clopidogrel

1. Introduction Atherosclerosis is a chronic inflammatory disease, involving a complex network of cellular processes, and disease progression is well defined,1 – 4 yet novel cell roles continue to emerge. In addition to their fundamental role in atherothrombosis, platelets are known to interact with the endothelium and leucocytes and, upon activation, can elicit an inflammatory response in these cells via both direct adhesion and the release of pro-inflammatory cytokines from platelet a-granules.5 – 7 Furthermore, activated platelets can induce mitogenic effects on the vessel wall through secretion of growth factors.8,9 With their pro-atherogenic arsenal, platelets are increasingly regarded as discrete immune cells, yet the degree to which they contribute directly to plaque development in the early stages of disease remains to be fully established. Several studies have investigated various aspects of platelet involvement, including adhesion receptors and proteins,10 – 13 alluding to the notion that platelet activation potentiates lesion development.

Platelets possess receptors for many agonists, including thrombin, that initiate a platelet activation response and this is then amplified and sustained by the G protein-coupled receptor P2Y12. Consequently, inhibition of P2Y12 leads to a significant attenuation of both platelet aggregation and granule secretions.14 As a result, P2Y12 inhibitors, such as ticagrelor and clopidogrel, are essential in the treatment and prevention of thrombotic events in acute coronary syndromes.15 – 18 In addition, platelet P2Y12 inhibition has been shown to be crucial in modulating the vessel wall response to injury.19,20 Although predominantly expressed on platelets, P2Y12 is also expressed on vascular smooth muscle cells (VSMCs), where it mediates cell contraction21 and promotes pro-inflammatory and mitogenic responses via a thrombininduced pathway.22 Recent work has suggested that platelet P2Y12 and possibly vessel wall P2Y12 play a role in the development of mature atherosclerotic plaques in a mouse atherosclerosis model.23 Given this evidence, we hypothesized that P2Y12 activation may be important in promoting early atherogenesis, in contrast to later stages of disease

* Corresponding author. Tel: +44 114 2712154; fax: +44 114 271 1863. Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].

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Time for primary review: 27 days

430 when endothelial disruption may promote the role of platelets. In this study, we investigated the role of P2Y12 using a mouse model of atherogenesis and employed bone marrow transplantations to examine the contributions of platelet vs. vessel wall P2Y12 in early lesion formation. In addition, we considered the application of ticagrelor and clopidogrel treatment in the modulation of atherogenesis. Recent data show that ticagrelor also inhibits adenosine re-uptake by erythrocytes24 and blocks ADP-induced vasoconstriction in isolated arteries.25 Furthermore, ticagrelor offers a reduced mortality risk, compared with clopidogrel, in patients with acute coronary syndromes17 but the mechanisms for this mortality reduction remain to be established. We therefore also aimed to determine any non-P2Y12-mediated effects of ticagrelor or clopidogrel on atherogenesis.

Expanded methods including genotyping, bone marrow cell preparation, and flow cytometry can be found in Supplementary material online.

2.1 Animals Transgenic mice were obtained from in-house colonies derived from breeding pairs sourced externally. ApoE2/2 mice (JAX 2052) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and P2Y2/2 12 mice were derived from breeding pairs provided by Schering Plough Research Institute, NJ (courtesy of Dr M. Chintala). Both strains were developed on a C57BL/6 background and backcrossed 10 generations.26,27 ApoE2/2 P2Y2/2 (DK) 12 mice were generated from a cross of each parent colony and offspring were genotyped by polymerase chain reaction (PCR). Male mice were used for all experiments to avoid sex-related differences on atherogenesis. These were fed a standard chow diet (Harlan Laboratories, Indianapolis, IN, USA) until the commencement of each procedure aged 8 – 10 weeks, weighing 25 g. At the end of each procedure, mice were euthanized by an overdose of sodium pentobarbital via intraperitoneal injection. Experiments were performed in accordance with UK and European legislation under the Animals (Scientific Procedures) Act 1986 and European Directive 2010/63/EU.

2.2 A murine model of early atherogenesis Atherosclerosis was augmented by feeding male mice a high-fat (21%) western diet (829100; Special Diet Services, Braintree, UK) for 4 weeks, after which tissues were assessed for atherosclerotic burden. Bone marrow transplanted mice were allowed to recover for 5 weeks on a chow diet prior to switching to western diet. Animals were perfusion-fixed by intraventricular injection of 10% (v/v) buffered formalin and the thoracic aorta, brachiocephalic artery and aortic sinus were excised for histological and morphometric analysis.

2.3 Bone marrow transplantations Donor ApoE2/2 and DK mice were killed by cervical dislocation and the femurs and tibias removed under aseptic conditions. The bone marrow was washed and resuspended in Hank’s buffered salt solution (HBSS) to produce a single-cell suspension. ApoE2/2 and DK male mice were administered a lethal dose of whole-body irradiation totalling 11 Grays (1100 rads) split into two doses, 4 h apart. Irradiated mice then received 2 to 4 × 106 donor cells via tail vein injection. Two chimeric groups, in addition to control groups, were produced (see Supplementary material online, Table S2). This method has been successfully employed by our group in previous studies.19,28 The bone marrow P2Y12 phenotype was confirmed by P-selectin (CD62p) and activated GPIIbIIIa receptor (CD41/61) expression in response to thrombin receptor activating peptide (TRAP), which acts via the protease-activated receptor (PAR)4. Stimulation via the thrombin receptor induces a strong platelet activation response which is then amplified

further by P2Y12 and therefore provides a useful measure of P2Y12 inhibition or deficiency.19

2.4 Quantification of lesions in whole aortae Whole aortae were opened longitudinally, fixed in 4% (w/v) paraformaldehyde, and then stained with Oil Red O to identify areas of atheromatous lesion. NIS-elements software (Nikon Instruments, Kingston upon Thames, UK) was used to detect and quantify areas of positive staining. Lesion assessment was blinded and areas were expressed as a percentage of the total aortic surface area. Further measurements were performed to compare lesion area in the aortic arch vs. the descending aorta.

2.5 Quantification of lesions in the brachiocephalic artery and aortic sinus Tissue samples were processed and embedded into paraffin. The brachiocephalic artery was sectioned, typically collecting 10 × 5 mm sections at 100 mm intervals along the length of the artery. The aortic sinus was sectioned from the appearance of the valve leaflets until the beginning of the aorta, collecting 7 mm cross-sections and leaving no intervals. Sections were stained with Alcian Blue/Elastic van Gieson and analysed using NIS-elements software. One section per 150 mm interval (approximately) was assessed along the length of the brachiocephalic artery. For the aortic sinus, five sections were assessed, each with three valves present. In all cases, lesion assessment was blinded and areas were expressed as a ratio of total cross-sectional area (CSA) to account for differences in vessel/ sinus size and mean values for each mouse are presented in the results. In addition, sections were stained with Martius Scarlet Blue (MSB) to determine the collagen content of the lesions.

2.6 Immunohistochemistry Aortic sinus sections were stained for a-smooth muscle actin using standard immunohistochemical techniques. Briefly, sections were dewaxed, rehydrated, and blocked for endogenous peroxidases and non-specific antibody binding. Sections were incubated with a-smooth muscle actin primary antibody (M0851; Dako, Ely, UK; 1:150) for 1 h at room temperature followed by incubation with biotinylated anti-mouse IgG secondary antibody (Vector Labs, Peterborough, UK; 1:200) for 30 min, also at room temperature. Positive staining was detected by incubation with Vectastainw ABC-HRP (Vector Labs) and visualized by applying 3,3′ -diaminobenzidine tetrahydrochloride (DAB). Tissue was counterstained with Carazzi’s haematoxylin. Percentage positive staining was quantified using NIS-elements software.

2.7 Pharmacological inhibition of P2Y12 ApoE2/2 and DK male mice were fed western diet for 4 weeks in addition to either twice-daily doses of ticagrelor (BriliqueTM ; 100 mg/kg), daily clopidogrel (Plavixw; 20 mg/kg) or mannitol (control) in jelly. This method of voluntary drug delivery was adapted from a published method by Zhang.29 The doses of ticagrelor and clopidogrel were based on previous studies that established these as optimal for providing a high, sustained level of P2Y12 inhibition.19,20 Mannitol is a main constituent of ticagrelor and clopidogrel tablets and was therefore selected as a control. Mice were housed individually and trained for 1 week with twice-daily meals of jelly only before commencing drug treatment. Due to the short halflife of ticagrelor, mice were dosed twice-daily with ticagrelor and mannitol. Clopidogrel-fed mice were given one dose of clopidogrel and one dose of mannitol. All mice were observed daily to monitor jelly consumption and efficacy of drug delivery was determined by P-selectin expression in response to PAR-4 TRAP.

2.8 Platelet P-selectin and activated GPIIbIIIa receptor expression in diluted whole blood Blood was taken by cardiac puncture into hirudin (50 ml/mL, final concentration) before diluting in HEPES-Tyrode’s (HT) buffer with calcium chloride.

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2. Methods

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Aliquots of dilute blood were incubated with FITC anti-CD62p (BD Biosciences, Oxford, UK), PE anti-CD41/61 JON/A (Emfret Analytics, Eibelstadt, Germany), and increasing concentrations of PAR-4 TRAP (1 – 3 mmol/L; AYPGKF; Almac Sciences, East Lothian, UK) for 20 min at room temperature and analysed by flow cytometry. Expression was denoted as increase in median fluorescence of the entire platelet population over baseline values.

2.9 Coagulation tests

2.10 Statistical analysis Data are expressed as mean or individual values and presented with SEM. Independent samples’ t-test and one- or two-way ANOVA were performed with Tukey’s multiple comparison test using Prism (Version 6; GraphPad, San Diego, CA, USA) or SPSS software (Version 19; IBM, New York, NY, USA).

3. Results 3.1 Generation of an apoE2/2 P2Y2/2 12 colony To investigate the role of P2Y12 in early atherogenesis, a doubleknockout ApoE2/2 P2Y2/2 12 (DK) mouse model was created from crossing in-house ApoE2/2 and P2Y2/2 12 parent colonies. P2Y12 genotype was determined by PCR and further confirmed by a functional assay measuring platelet P-selectin and activated GPIIbIIIa receptor expression in response to PAR-4 TRAP. Platelets from DK mice demonstrated significantly attenuated P-selectin expression (P , 0.05 vs. ApoE; Figure 1A) and a reduction in the activated form of the GPIIbIIIa receptor (P , 0.001 vs. ApoE; Figure 1B) compared with ApoE2/2 mice in response to 1 mmol/L PAR-4 TRAP. A similar response was also seen at the higher concentration of TRAP. These results were consistent with our previous findings on P2Y2/2 mice.19 12

3.2 Vessel wall, not platelet, P2Y12 plays a role in early atherogenesis In order to assess the contribution of platelet and vessel wall P2Y12 in early atherogenesis, bone marrow transplantations were performed. In addition to ApoE2/2 and DK control groups, mice deficient in either platelet or vessel wall P2Y12 were generated. Thoracic aortae stained with Oil Red O demonstrated a non-significant trend towards reduced % atheroma in vessel wall P2Y12-deficient mice (Figure 2A and C ). However, when assessing the aortic arch alone, atheroma was significantly attenuated in these mice (Figure 2B): aortic arch lesion area was 6.7 + 0.83% in control mice, 4.33 + 0.4% in mice with deficient vessel wall and platelet P2Y12 (P , 0.05 vs. control), and 4.4 + 0.54% in mice with only deficient vessel wall P2Y12 (P , 0.05 vs. control). There was no significant effect of vessel wall P2Y12 in the descending aortae (Figure 2B). Mice with absence of only platelet P2Y12 exhibited no differences in lesion area compared with controls. A similar pattern was repeated in other areas of the arterial tree in mice whose vessels lacked P2Y12, an effect which was irrespective of platelet P2Y12 expression. In the brachiocephalic artery, lesion area,

Figure 1 P-selectin (A) and activated GPIIbIIIa receptor (B) expression (median fluorescence) in response to PAR-4 TRAP in platelets from ApoE2/2 and DK male mice (n ¼ 3 in each group). Data are mean + SEM; *P , 0.05, **P , 0.01, ***P , 0.001 (two-way ANOVA with Tukey’s multiple comparison).

represented as a ratio of the CSA, was 0.027 + 0.002 in control mice, 0.004 + 0.003 in mice with deficient vessel wall and platelet P2Y12 (P , 0.001 vs. control), and 0.006 + 0.003 in mice lacking only vessel wall P2Y12 (P , 0.001 vs. control) (Figure 2D). Absence of platelet P2Y12 alone appeared to have no effect on lesion size (0.021 + 0.004; P ¼ 0.56 vs. control). Analogous results were observed in the aortic sinus with notably less atheroma observed in mice deficient in vessel wall P2Y12 (Figure 2E and F ): aortic sinus lesion area was 0.071 + 0.008 in control mice, 0.024 + 0.003 in mice with deficient vessel wall and platelet P2Y12 (P , 0.001 vs. control), and 0.03 + 0.006 in mice lacking only vessel wall P2Y12 (P , 0.001 vs. control). MSB and a-smooth muscle actin staining for collagen and smooth muscle cells, respectively, within these aortic sinus lesions demonstrated little positive staining in those mice deficient in vessel wall P2Y12. In contrast, mice which did express vessel wall P2Y12 demonstrated much greater staining for both collagen and a-smooth muscle actin regardless of platelet P2Y12 expression (see Supplementary material online, Figures S1 and S2). The method employed in this study for bone marrow transplantation has successfully been used previously by our group.19,28 We further confirmed the bone marrow P2Y12 phenotype and therefore assessed the efficacy of transplantation by measuring P-selectin expression and the presence of activated GPIIbIIIa in response to PAR-4 TRAP in platelets from transplanted ApoE2/2 mice. Control mice transplanted with ApoE2/2 bone marrow expressed high levels of P-selectin and activated GPIIbIIIa over baseline values in response to PAR-4 TRAP (see Supplementary material online, Figure S3). This response, however, was considerably attenuated in ApoE2/2 mice transplanted with DK bone marrow

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Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured to assess the intrinsic (aPTT) and extrinsic (PT) coagulation pathways. Fibrinogen levels were a measure of the final common coagulation pathway. Blood was taken by cardiac puncture into 3.8% (final w/v) sodium citrate at a ratio of one part anticoagulant to nine parts blood, as used in previous murine studies.30 Samples were analysed by the Coagulation Department at the Royal Hallamshire Hospital (Sheffield Teaching Hospitals NHS Foundation Trust, UK).

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Figure 2 ApoE2/2 and DK male mice underwent bone marrow transplantation to generate chimeric groups: (1) ApoE into ApoE, (2) DK into ApoE, (3) DK into DK, and (4) ApoE into DK. Following 4 weeks on a western diet, aortae (n ¼ 12, 15, 12, and 12, respectively) were stained with Oil Red O (C) and lesion area was calculated as a percentage of surface area for the whole aorta (A) and the aortic arch and descending aorta (B). The brachiocephalic artery (D) (n ¼ 10, 13, 8, and 9) and aortic sinus (E) (n ¼ 13, 14, 9, and 10) were sectioned and stained with Alcian Blue/Elastic Van Gieson (F). Lesion area was quantified from at least five sections per mouse. Data are mean + SEM; *P , 0.05, **P , 0.01, ***P , 0.001 (A, D and E: one-way ANOVA with Tukey’s multiple comparison; B: two-way ANOVA with Tukey’s multiple comparison).

and thus confirmed the successful reconstitution of P2Y12-deficient donor bone marrow into recipient mice. In addition, we measured PT, aPTT, and fibrinogen levels to determine any variation in the potential for thrombin and fibrin generation between bone marrow transplanted mice and found no differences in these coagulation parameters (see Supplementary material online, Figure S4).

3.3 Ticagrelor and clopidogrel have no effect on early atherogenesis ApoE2/2 and DK male mice were fed either ticagrelor, clopidogrel, or mannitol (control) for 4 weeks, in addition to a western diet, to investigate the effects of pharmacological inhibition of P2Y12 on early

Vessel wall P2Y12 potentiates early atherogenesis

4. Discussion The role of platelets in atherogenesis and the potential therapeutic impact of P2Y12 antagonists on disease prevention are of great interest. In this study, we identify a previously unknown role of vessel wall P2Y12 in modulating early atherogenesis and therefore propose that it is vessel wall, rather than platelet, P2Y12 which plays a significant role in the

Figure 3 ApoE2/2 and DK male mice were fed western diet for 4 weeks in addition to either twice daily doses of ticagrelor (100 mg/ kg; n ¼ 10 and 9, ApoE and DK, respectively), daily clopidogrel (20 mg/kg; n ¼ 9 and 8) or mannitol (control; n ¼ 9 in both groups). Whole aortae (A) and aortic sinus (B) lesion area were quantified. Data are mean + SEM; ***P , 0.001 (Independent samples t-test); P ¼ NS (two-way ANOVA with Tukey’s multiple comparison).

Figure 4 P-selectin expression (median fluorescence) in response to 1 mmol/L PAR-4 TRAP in platelets from ApoE2/2 and DK male mice treated with either ticagrelor (n ¼ 7 and 6, ApoE, and DK, respectively), clopidogrel (n ¼ 5 and 6) or mannitol (control; n ¼ 7 and 6). Data are mean + SEM; ***P , 0.001 (two-way ANOVA with Tukey’s multiple comparison).

early stages of disease by promoting lesion development. Furthermore, we found that P2Y12 antagonists, ticagrelor and clopidogrel, were unable to inhibit these effects and so therefore may not offer a therapeutic strategy for the prevention of atherosclerosis in people without evidence of atherosclerotic disease. The pro-inflammatory capacity of platelets is evident and many studies have been conducted to examine their potential role in propagating lesion development, implicating roles for platelet adhesion proteins and receptors including P-selectin,11,13 GPIb,12 and VI.10 In addition, the deposition of cytokines, such as RANTES (CCL5) and platelet factor 4 (CXCL4), by activated platelets onto the endothelium has been attributed to increased leucocyte recruitment to atherosclerotic and injured vessels.6,13,31 With its central role in platelet reactivity, the P2Y12 receptor is an attractive candidate for investigation. In a recent study, Li et al. present evidence to suggest platelets promote lesion development via P2Y12-mediated granule release of P-selectin and platelet factor 4, thus amplifying leucocyte recruitment.23 Yet, despite this apparent role, we observed no such effect of platelet P2Y12 in our own model of early atherogenesis. To our knowledge, all platelet atherogenesis studies to date model established disease, choosing end points from 7 to more than 30 weeks on a chow or high-fat diet.10 – 12,32 Cholesterol clefts and fibrous caps are common features in these lesions and are typically associated with advanced plaques, including those which are susceptible to endothelial erosion and rupture.3 This is also true of the study by Li et al. which assessed atheroma in ApoE2/2 P2Y2/2 (DK) mice after 20 12 weeks on a high-fat diet, similar in composition to our own western diet.23 We chose to study a much earlier stage of atherogenesis, after only 4 weeks of feeding, where we could both accurately measure atheroma and also be confident of little risk of endothelial erosion. From our results, we see that it is vessel wall, rather than platelet, P2Y12 which is important in the development of early lesions, but perhaps this balance shifts as disease progresses. This hypothesis is supported by our previous work showing that platelet P2Y12, rather than vessel wall P2Y12, plays a profound role in the vessel wall response to injury.19 Consideration must also be made for the potential influence

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atherogenesis. Oil Red O staining of whole aortae showed no effect of ticagrelor or clopidogrel on lesion formation compared with controls for either mouse strains (Figure 3A). Likewise, further analysis of the aortic arch and descending aorta demonstrated no region-specific differences in lesion formation (data not shown). Histological analysis of the aortic sinus revealed similar results and showed no effect of the P2Y12 inhibitors on atherogenesis. An effect of P2Y12 on lesion size was still seen in the presence of the P2Y12 inhibitors when comparing DK mice with their ApoE2/2 counterparts (P , 0.001; Figure 3B). To assess platelet P2Y12 inhibition and therefore the efficacy of drug delivery, we measured P-selectin expression in response to 1 mmol/L PAR-4 TRAP. ApoE2/2 mice demonstrated significantly attenuated P-selectin expression when treated with either ticagrelor (P , 0.001 vs. control) or clopidogrel (P , 0.001 vs. control) (Figure 4), therefore verifying effective P2Y12 inhibition. As expected, reduced P-selectin expression was observed in all DK mouse treatment groups and there was no additional effect of ticagrelor or clopidogrel.

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Ticagrelor has been shown to block adenosine re-uptake by erythrocytes24 and so may have other, non P2Y12-mediated, vessel wall effects. Adenosine is known to induce nitric oxide production in both VSMC36 and endothelial cells,37 which suppresses adhesion molecule expression such as VCAM-138 and also inhibits VSMC proliferation.39 Furthermore, clinical studies show that ticagrelor offers a reduced mortality risk, compared with clopidogrel, in patients with acute coronary syndromes17 but the mechanisms for this mortality reduction remain to be established. Given ticagrelor’s potential athero-protective activity, we investigated non-P2Y12-mediated effects on atherogenesis using DK mice. Neither ticagrelor nor clopidogrel treatment had any effect on lesion development, indicating that they have no off-target effects that may influence early atherogenesis. However, consideration must also be made for the possible effects of P2Y12 deficiency on VSMC development and behaviour in this model, which may negate any effects on the vessel wall. In conclusion, these results demonstrate that platelet P2Y12 plays no role in early atherogenesis despite its dominant role in platelet reactivity and, similarly, the platelet P2Y12 inhibitors ticagrelor and clopidogrel have no effect at this stage of disease. This is in contrast to published data that document the involvement of platelet P2Y12 in later atherogenesis and its thrombotic complications.23 On the other hand, we have demonstrated an important role for vessel wall P2Y12 in early atherogenesis, particularly in those regions of the vasculature which are associated with turbulent blood flow. This work emphasizes the complexity of cellular roles within atherogenesis and how the nature and impact of these cell populations may change as disease evolves. Further investigation is necessary to clarify the extent to which vessel wall and platelet P2Y12 influence atherogenesis throughout disease progression to provide a clearer understanding of potential pharmacological targets in the prevention and management of atherosclerosis.

Supplementary material Supplementary material is available at Cardiovascular Research online. Conflict of interest: R.F.S. declares research grants, honoraria, and/or consultancy fees from Accumetrics, AstraZeneca, Bristol Myers Squibb, Daiichi Sankyo, Eisai, Eli Lilly, Iroko, Medscape, Novartis, Regeneron, Roche, and Sanofi Aventis. S.E.F. declares research grants from Novartis. The other authors have no disclosures.

Funding This work was supported by the University of Sheffield.

References 1. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–874. 2. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999;340:115 –126. 3. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol 1995;15:1512 – 1531. 4. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME et al. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol 1994;14:840 –856. 5. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998;391:591 – 594. 6. von Hundelshausen P, Weber KS, Huo Y, Proudfoot AE, Nelson PJ, Ley K et al. RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation 2001;103:1772 – 1777. 7. Gawaz M, Brand K, Dickfeld T, Pogatsa-Murray G, Page S, Bogner C et al. Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through

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of vessel wall P2Y12 on the results demonstrated by Li et al. 23 Only DK mice were chosen as recipients for their bone marrow transplantations and so further investigation looking at the role of vessel wall P2Y12 in later atherogenesis is warranted. Research into the role of the related platelet purine receptor P2Y1 in atherogenesis, which is more widely expressed than P2Y12, demonstrated a similar vascular wall effect on atherogenesis and so also supports the need for further exploration in this area.32 Although platelets are deemed to be the major source of P2Y12 in the body, the receptor has also been identified in VSMCs.21 In order to delineate the separate roles of platelet and vessel wall P2Y12 in early atherogenesis, we performed bone marrow transplantations, enabling the creation of chimeric mice expressing either vessel wall or platelet P2Y12. We found that, in the absence of vessel wall P2Y12, atheroma is attenuated throughout the arterial tree and appears to result in an earlier lesion phenotype with less collagen and smooth muscle cell content. Recent work by Rauch et al. links P2Y12 expression with VSMC mitogenesis via a thrombin-induced pathway and may provide a potential mechanism for the effect we observed.22 Interestingly, following exposure to thrombin, they found P2Y12 surface expression being significantly up-regulated and P2Y12-induced mitogenic effects enhanced. In an inflammatory setting such as atherogenesis, where thrombin generation is likely, it is probable that P2Y12-potentiated VSMC mitogenesis contributes to lesion progression. Indeed, P2Y12-positive VSMCs have been identified within atherosclerotic lesions.22 Furthermore, to eliminate the possible impact of differences in thrombin generation on the interpretation of our findings, we conducted PT and aPTT tests and found no difference in response between chimeric groups. The smaller lesions observed in these vessel wall P2Y12-deficient mice suggest that VSMC P2Y12 expression does influence lesion size, perhaps via VSMC mitogenesis, however this is yet to be explored. Absence of smooth muscle actin expression within these lesions, compared with positive staining seen in those mice which do express vessel wall P2Y12, lends some support to this hypothesis, although whether lesion progression is delayed or altered in the absence of VSMC P2Y12 remains to be seen. Indeed, little is known of the true effects of P2Y12 deficiency on VSMC development, phenotype or plasticity and so extensive investigation is required to find the exact mechanism behind these findings. Clopidogrel is widely used in the treatment of acute coronary syndromes; however, next-generation agents, such as ticagrelor, are increasingly employed due to their superior inhibitory effects and greater clinical efficacy. Although particularly effective at preventing thrombosis, evidence of their effects on atherogenesis is limited. Studies investigating clopidogrel treatment on atherosclerosis offer conflicting results33,34 and so we were interested to explore whether P2Y12 inhibitors were able to inhibit this vessel wall effect on atherogenesis. We found that, despite effective platelet P2Y12 inhibition, neither ticagrelor nor clopidogrel had any effect on early atherogenesis in ApoE2/2 mice. Studies have shown that ticagrelor35 and another reversibly binding P2Y12 inhibitor cangrelor21 are capable of inhibiting VSMC contraction in denuded arteries, but clopidogrel had no effect with the short half-life of the active metabolite being postulated as a potential contributory factor to this lack of response.21 Given our findings we deduce that, at doses which yield high levels of platelet P2Y12 inhibition, ticagrelor and clopidogrel are unable to inhibit VSMC P2Y12mediated effects on atherogenesis. Nonetheless, further investigation is needed to elucidate the action of these agents on VSMC P2Y12 receptors in vivo and deduce their ability to penetrate the endothelium.

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Vessel wall, not platelet, P2Y12 potentiates early atherogenesis.

Platelets have a fundamental role in atherothrombosis, but their role in early atherogenesis is unclear. The P2Y12 receptor is responsible for amplify...
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