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Predictive mouse model reflects distinct stages of human atheroma in a single carotid artery

  • JOYCE MS CHAN
    Correspondence
    Reprint request: Joyce M. S. Chan, Translational Cardiovascular Imaging Group, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), 11 Biopolis Way, #02-02 Helios, Singapore 138667.
    Affiliations
    Translational Cardiovascular Imaging Group, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore
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  • SUNG-JIN PARK
    Affiliations
    Translational Cardiovascular Imaging Group, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore
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  • MICHAEL NG
    Affiliations
    Translational Cardiovascular Imaging Group, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore
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  • WAY CHERNG CHEN
    Affiliations
    Bruker Singapore Pte. Ltd., Singapore
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  • JOANNE GARNELL
    Affiliations
    Translational Cardiovascular Imaging Group, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore
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  • KISHORE BHAKOO
    Affiliations
    Translational Imaging Laboratory, Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore
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Open AccessPublished:August 31, 2021DOI:https://doi.org/10.1016/j.trsl.2021.08.007
      Identification of patients with high-risk asymptomatic atherosclerotic plaques remains an elusive but essential step in preventing stroke. However, there is a lack of animal model that provides a reproducible method to predict where, when and what types of plaque formation, which fulfils the American Heart Association (AHA) histological classification of human plaques. We have developed a predictive mouse model that reflects different stages of human plaques in a single carotid artery by means of shear-stress modifying cuff. Validated with over 30000 histological sections, the model generates a specific pattern of plaques with different risk levels along the same artery depending on their position relative to the cuff. The further upstream of the cuff-implanted artery, the lower the magnitude of shear stress, the more unstable the plaques of higher grade according to AHA classification; with characteristics including greater degree of vascular remodeling, plaque size, plaque vulnerability and inflammation, resulting in higher risk plaques. By weeks 20 and 30, this model achieved 80% and near 100% accuracy respectively, in predicting precisely where, when and what stages/AHA types of plaques develop along the same carotid artery. This model can generate clinically-relevant plaques with varying phenotypes fulfilling AHA classification and risk levels, in specific locations of the single artery with near 100% accuracy of prediction. The model offers a promising tool for development of diagnostic tools to target high-risk plaques, increasing accuracy in predicting which individual patients may require surgical intervention to prevent stroke, paving the way for personalized management of carotid atherosclerotic disease.

      Abbreviations:

      ACAS (Asymptomatic Carotid Atherosclerosis Study), ACST (Asymptomatic Carotid Surgery Trial), AHA (American Heart Association), ApoE-/- (Apolipoprotein E knock-out mice), CD62P (P-selectin), CTA (Computed tomography angiography), CVD (Cardiovascular diseases), FLASH (Fast low-angle shot), HSS (High shear stress), IHC (Immunohistochemistry), IMR (Intima-media ratio), IPH (Intraplaque hemorrhage), JBA (Juxta-luminal black area), LCCA (Left common carotid artery), LRNC (Lipid rich necrotic core), LSS (Low shear stress), MAPK (Mitogen-activated protein kinase), MMP-9 (Matrix metalloproteinase-9), MOMA-2 (Anti-Macrophages/Monocytes Antibody), MRA (Magnetic resonance angiography), NF-kB (Nuclear factor- kappa-B), ORO (Oil red O), OSS (Oscillatory shear stress), PBS (Phosphate buffered saline), PCI (Percutaneous coronary intervention), PC MRA (Phase contrast magnetic resonance angiography), RCCA (Right common carotid artery), SD (Standard deviation), TIA (Transient ischemic attack), TOF MRA (Time-of-flight angiography), TRFC (Thinning/rupture of fibrous cap), VCAM-1 (Vascular cell adhesion molecule-1), VENC (Velocity encoding), WSS (Wall shear stress)
      At a Glance Commentary

       Background

      Identification of patients with high-risk asymptomatic plaques remains an elusive but essential step in preventing stroke. There is a lack of atherosclerosis animal model that can predict where, when and what types of plaque formation, which fulfils American Heart Association (AHA) histological classification of human plaques.

       Translational Significance

      We have developed a predictive mouse model that can generate clinically-relevant plaques of varying risk levels and AHA classification, along the same carotid artery with near 100% accuracy of prediction. The model offers a promising tool for development of diagnostic tools to target high-risk plaques, increasing accuracy in selecting high-risk patients for early stroke prevention.

      INTRODUCTION

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      A quest for advanced precision imaging tools to look beyond the lumen for in vivo identification of vulnerable atherosclerotic plaques is required imminently. Achieving this, however, will require establishing an animal model that produces both vulnerable plaques and stable plaques, that mirror clinically relevant pathologies within the same artery. Such a platform offers the potential for the development of novel imaging tools and therapeutic interventions.
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      Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.
      it was challenging to predict the exact locations, timepoints, and the types of plaques to be formed. Moreover, the models with additional manipulations such as Angiotensin II treatment
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      Endogenous angiotensin ii induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE−/− mice.
      and adenovirus-induced gene mutation
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      Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.
      increase the cost and time, limiting their use for high throughput screening. Previous mouse models
      • Cheng C
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      • Van Haperen R
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      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
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      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
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      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
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      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      have utilized the cuff to induce in vivo alterations of shear stress patterns in the common carotid artery in order to develop atherosclerotic lesions of different plaque phenotypes along the artery. It has been generally accepted that low shear stress (LSS) promotes formation of vulnerable, inflamed plaques, oscillatory shear stress (OSS) induces development of stable plaques, whilst high shear stress (HSS) protects against atherosclerosis.
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      The focus of these studies
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      , however, was on the high-risk vulnerable plaques. Information on low- or intermediate-risk plaques was minimally or not reported. Only limited regions, not the whole carotid artery, were evaluated in these models. Moreover, the plaque development in these models
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      were followed up longitudinally from days up to 12 weeks post-cuff implantation only and were not validated using the American Heart Association (AHA) histological classification of human plaques.
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      Furthermore, the accuracy of prediction in exact locations, timepoints, and the stages/types of plaques to be formed in these cuff-implantation models remained unexplored.
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      The large animal models include genetically modified rabbits and mini-pigs.
      • Shim J
      • Al-Mashhadi RH
      • Sørensen CB
      • Bentzon JF.
      Large animal models of atherosclerosis–new tools for persistent problems in cardiovascular medicine.
      The high cost of these models hinders their use for high throughput screening for drugs and diagnostics. To date, there is still a lack of an animal model that provides a reproducible and cost-effective method to predict exactly where, when and what types of plaques formation, that also fulfils the AHA histological classification of human plaques,
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      at a large scale.
      Here, to the best of our knowledge, we present the first large scale pre-clinical study that demonstrates an animal model that can generate clinically relevant plaques with varying degrees of (1) phenotypes, (2) risk levels, and (3) stages/types (reflecting AHA classification of human plaques Type II–VI
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      ) at specific anatomical locations along the same artery, with high reproducibility and accuracy of prediction.

      MATERIAL AND METHODS

       Overview of study design, animal and cuff implanted animal model

      All animal experimental procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee in Biological Resource Center at A*STAR, Singapore. The overview of study design is shown in Supplementary Fig 1 A. ApoE−/−(apolipoprotein E knock-out mice [Taconic Biosciences]) were used to develop the cuff implanted atherosclerosis model. The shear-stress modifying cuff (Promolding B.V., Netherlands) and surgical implantation procedure were described in previous studies.
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      The cone-shaped inner lumen of the cuff generated the defined regions of LSS upstream, HSS within the cuff, and OSS downstream within the common carotid artery (Supplementary Fig 1B). At 8 weeks of age, ApoE−/− mice (n = 50) were started on atherogenic diet (Altromin C1061, 1% cholesterol, 1% Na-cholate) and continued till the end of the study. At 9 weeks old, in vivo MRI of carotid arteries using time-of-flight angiography sequence and velocity mapping were performed 1 week before cuff placement. At 10 weeks of age, surgical implantation of the shear-stress modifying cuff was performed on the right common carotid artery (RCCA), where the left common carotid artery (LCCA) was left untreated as internal control. At 11 weeks old, in vivo MRI of carotid arteries using the same time-of-flight angiography and velocity mapping sequence were repeated 1 week post-cuff placement to measure the velocity changes induced by the cuff. The cuffs were implanted on the RCCA of the animals for different durations (ie, 10 weeks (n = 10), 15 weeks (n = 10), 20 weeks (n = 10), 30 weeks (n = 10), 50 weeks (n = 10)] before histological evaluation. In vivo MRI of carotid arteries screening using the same time-of-flight angiography sequence was performed in 3 mice randomly selected in each group to ensure the cuff was in place before histological evaluation.

       Animal imaging and analysis

       In vivo magnetic resonance imaging of carotid arteries and velocity map

      ApoE−/− mice were imaged with a 11.7 T MRI system (Bruker, BioSpec) before cuff placement, under anesthetization by inhalation of 1.5%–2% isoflurane. Scans were carried out using a 40 mm inner diameter transmit-receive volume coil (Bruker). A 3-dimensional fast low-angle shot (FLASH) gradient-echo based time-of-flight angiography sequence was used with the following parameters: TR: 12.0 ms; TE: 2 ms; FOV: 24 × 24 × 10 mm; acquisition matrix: 256 × 256 × 64; averages: 4; slab thickness: 10 mm; flip angle: 20°; acquisition time: 10 minutes 54 seconds. A field-of-view saturation band was placed on the rostral end of the imaging volume to negate signal from veins. Adopting the same scan geometry and orientation, a 3-dimensional FLASH based phase contrast magnetic resonance angiography sequence was used to quantify the velocity of blood flow in the carotid arteries with the following parameters: TR: 20.0 ms; TE: 3.5 ms; FOV: 24 × 24 × 10 mm; acquisition matrix: 256 × 256 × 64; averages: 2; slab thickness: 10 mm; flip angle: 20°; velocity encoding: 60 cm/s; 3 orthogonal directions, acquisition time: 10 minutes 55 seconds.

       Shear stress calculations

      The wall shear stress (WSS) at specific points along the artery were calculated using equation (1), with assumptions that blood is a Newtonian fluid, there is laminar flow, and the flow is in a non-compressible cylindrical tube.
      WSS=τ=4ηνd
      (1)


      Where mouse blood viscosity η = 10 mPa*s, ν is blood velocity and d is the lumen diameter.
      • Reneman RS
      • Hoeks APG.
      Wall shear stress as measured in vivo: consequences for the design of the arterial system.
      ,
      • Windberger U
      • Bartholovitsch A
      • Plasenzotti R
      • Korak KJ
      • Heinze G.
      Whole blood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian species: reference values and comparison of data.
      The MRI velocity map images were analyzed for peak velocity and lumen diameter using the ImageJ software.
      Detailed shear stress measurements, velocity map processing, measurements of peak velocity and diameter, histology, immunohistochemical staining, measurements of the intima-media ratio (IMR), plaque size, vascular remodeling ratio and vulnerability index as well as statistical analysis are available in Supplementary Methods.

      RESULTS

       Both low shear stress and oscillatory shear stress induce atherosclerotic plaque formation, whereas high shear stress protects against atherosclerosis

      Firstly, the shear stress levels were calculated in both untreated and cuff-implanted carotid arteries in mice (Fig 1, Supplementary Fig 2 and 3). The shear stress modifying cuff induced 5 different shear stress fields along the RCCA (compared to the baseline shear stress in the untreated RCCA), with a decrease in shear stress in R1, R2, and R3 of 36.27%, 25.56% and 11.19%, respectively. In contrast, there was increased shear stress of 125.06% inside the cuff (R4), and vortices with OSS (24.26% increase) downstream from the cuff (R5). To study the effect of the 5 different shear stress fields on plaque vulnerability and progression, serial-sectioned samples of the entire carotid arteries showed that atherosclerotic plaques had already developed by week 10 at the bifurcation sites (Supplementary Fig 4). These are natural sites of shear stress related plaque initiation
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      as confirmed in LCCA without cuff implantation (Supplementary Fig 4). By week 15, plaque lesions had developed in the LSS (R1 and R3) and the vortices/OSS (R5) regions in the cuff-implanted carotid artery. By weeks 20, 30, and 50, the lesion areas had markedly expanded in the LSS regions (R1, R2, R3; Figs 2 and 3). In the vortices/OSS region (R5), a gradual increase in lesion area was also observed from week 15 onward, but these were considerably smaller than the lesions in LSS regions (R1, R2, R3; Figs 2 and 3). No atherosclerotic lesions were present in the HSS region (R4) in all animals (n = 50) at any point up to 50 weeks after cuff implantation, whereas all animals (n = 40) developed lesions under LSS and OSS regions from week 15 onwards. No plaque lesion was observed in the LCCA (except at bifurcation sites), any point up to 50 weeks.
      Fig 1
      Fig 1In vivo MRA, velocity mapping and shear stress calculations of carotid arteries of ApoE-/- mouse 1 week post cuff implantation. (A) In vivo MRA of carotid arteries of ApoE-/- mouse. (B, C) MR velocity mapping of right and left common carotid arteries of ApoE-/- mouse. Scale bar 1 mm, colour bar: Velocity (cm/s) (D) 5 regions of different flow dynamics and shear stress. The local values for shear stress in 5 different regions (R1-5) were calculated based on MR velocity mapping measurements. (E) Table of mean values of wall shear stress in different regions.
      Fig 2
      Fig 2Pattern of progressive atherosclerosis model. Representative histological images and in vivo MRA of RCCA of ApoE-/- mouse at different stages of disease. (A–D) The closer to the bottom of the cuff-implanted artery (towards R1), the lower the magnitude of shear stress, the more unstable and higher risk the plaques become. (C, D) From week 30 onwards, a definitive, specific pattern was observed: low-risk, stable plaques were found in R5 (c,d); no plaques in R4 (g,h); intermediate-risk plaques in R3 (k,l); high-risk plaques in R1 and R2 (o,p,s,t).
      Fig 3
      Fig 3Correlation between plaque size, vascular remodeling and magnitude of shear stress. (A) The plaque sizes increase in all respective regions (R1, R2, R3, R5) as the cuff implanted in the artery longer. From week 20 onwards, magnitude of shear stress is significantly inversely correlated with plaque size and the correlation becomes stronger as atherosclerosis progressed. (B) Similarly, the vascular remodeling increase in all respective regions (R1, R2, R3, R5) as the cuff implanted in the artery longer. From week 20 onwards, magnitude of shear stress is significantly inversely correlated with vascular remodeling with stronger correlations as atherosclerosis advanced.

       Low shear stress induces larger plaques and greater vascular remodeling than oscillatory shear stress

      The atherosclerotic plaque size in the LSS region (R1, R2, R3) were remarkably larger than those in the OSS region (R5) from week 30 post-cuff placement (Figs 2 and 3, A). Plaque areas in both LSS (R1,R2,R3) and OSS (R5) regions were noticeably more extensive than those in the HSS (R4) region and the equivalent region in LCCA (Figs 2 and 3, A, Supplementary Fig 3). No significant difference in IMR was detected between the HSS (R4) region and the equivalent region in LCCA (Fig 2, Supplementary Fig 3).
      At weeks 15 and 20, similar IMR and vascular remodeling were found in R1 and R3 regions, whereas R2 was relatively spared of plaque lesions. As atherosclerosis progressed to weeks 30 and 50, a more definitive vascular remodeling pattern was observed in the LSS region; the plaques that were formed furthest away from the cuff (ie, R1: most proximal, most upstream field) were larger in size and demonstrated greater vascular remodeling compared to those formed closer to the cuff (ie, R3). (Week 30: the vascular remodeling at R1, R2 and R3: 13.1-fold, 10.0-fold, and 9.2-fold. Week 50: R1, R2 and R3: 17.5-fold, 15.6-fold, and 13.7-fold; Fig 3, B).

       Low shear stress induces development of vulnerable and inflamed plaques, whereas oscillatory shear stress induces stable lesions

      Serial histological analyses including immunohistochemistry of the entire carotid arteries was performed to evaluate the effect of the 5 different shear stress fields on plaque vulnerability and inflammation at different timepoints. From week 15 onwards, higher lipid content was observed in the plaques located in the LSS regions (R1, R2 and R3) than in those in the OSS region (R5) (Fig 4, A and Fig 6, A). From week 30 onwards, a more definitive pattern was observed in the LSS regions; the plaques that were formed furthest away from the cuff (in R1) bore the highest lipid content compared with those in R2, which in turn contained more lipid content than plaques closest to the cuff (R3; Figs 4, A and 6, A). Moreover, the other high-risk plaque features seen in patients (Stary stage V and VI set by the AHA Committee on Vascular Lesions
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      ), including lipid rich necrotic core (LRNC) and thin fibrous cap, were also consistently observed in the plaques formed in R1 and R2, occasionally in R3 plaques and absent in R5 plaques (Figs 2, 4, A and 10).
      Fig 4
      Fig 4Lipid, collagen and smooth muscle cell content in the plaques in all regions (R1-5) of RCCA. (A) At week 15 and week 20, higher lipid content (oil red O stain) was observed in the plaques located in the LSS regions (R1, R2 and R3) than in those in the OSS region (R5). From week 30 onwards, a more definitive pattern was observed in the LSS regions: the plaques formed in R1 bore the highest lipid content compared with those in R2, which in turn contained more lipid content than plaques in R3. (B) The tunica adventitia of all samples were stained well by Masson's trichrome stain for collagen. From week 15 onwards, a lower content of collagen was observed in the plaques in LSS regions (R1, R2, R3) as compared to those in the OSS region (R5). From week 30 onwards, in the LSS regions, plaques in R1 and R2 has a minimal collagen content compared with plaques in R3 and R5, which has the highest collagen content. (C) From week 15 onwards, the plaques in LSS regions (R1, R2, R3) contained minimal thin layers of vascular smooth muscle cells (SMA) in the cap of the plaques. By contrast, in the plaques located at the OSS region (R5), vascular smooth muscle cells were significantly more abundant and uniformly distributed in the intima. From week 30 onwards, the plaques formed in R1 contained the least vascular smooth muscle cells compared with those in R2, the plaques at R3 contained the highest smooth muscle cell content.
      The distribution of vascular smooth muscle cells and collagen in the plaques demonstrated an opposite pattern to that of lipid in the lesions. From week 15 onwards, the plaques in LSS regions (R1, R2, R3) contained minimal thin layers of vascular smooth muscle cells and collagen in the cap of the plaques. By contrast, in the plaques located at the OSS region (R5), vascular smooth muscle cells and collagen were observed to be abundant and uniformly distributed in the intima (Figs 4, B, C, and 6, B, C). From week 30 onwards, a more definitive pattern was also observed in the LSS regions; the plaques formed in R1 contained the least vascular smooth muscle cells and collagen. Interestingly, the area closer to the cuff, the plaques expressed more smooth muscle cells and collagen, with plaques at R3 expressing the highest smooth muscle cell and collagen content (Figs 4, B, C and 6, B, C).
      To further differentiate the different types of plaques, antibodies against a targeted cohort of inflammatory biomarkers were used to study the inflammatory activities within the plaques. At weeks 15 and 20, there were no significant differences in the expression of the MOMA-2, P-selectin (CD62P) and MMP-9 in the plaques at LSS regions (R1, R3) compared with those in the OSS region (R5; Figs 5 and 7, A–D). At week 20, highest expression of VCAM-1 was found in the plaques formed furthest away from the cuff in R1 compared with those in R2, which in turn were more inflamed than plaques closest to the cuff in R3. VCAM-1 was found to be the earliest inflammatory biomarkers to demonstrate such pattern of differential expression by the plaques along the same carotid artery. From week 30 onwards, plaques with different degree of inflammation were formed along the same carotid artery depending on their position relative to the cuff. Similar to the pattern of plaque vulnerability, highest expression of all inflammatory biomarkers (ie, MOMA-2, CD62P, VCAM-1 and MMP-9) was found in the plaques formed furthest away from the cuff in R1 compared with those in R2, which in turn were more inflamed than plaques closest to the cuff in R3 (Figs 5 and 7, A–D).
      Fig 5
      Fig 5Expression of inflammatory biomarkers (ie, P-selectin [CD62P], VCAM-1, MOMA-2 and MMP-9) in the plaques in all regions (R1-5) of RCCA. At weeks 15 and 20, no significant differences in the expression of MOMA-2, CD62P and MMP-9 in the plaques at LSS regions (R1, R3) was observed, compared with those in the OSS region (R5). At week 20, highest expression of VCAM-1 was found in the plaques formed furthest away from the cuff in R1 compared with those in R2, which in turn were more inflamed than plaques closest to the cuff in R3. From week 30 onwards, highest expression of all inflammatory biomarkers (ie, MOMA-2, CD62P, VCAM-1 and MMP-9) was found in the plaques formed furthest away from the cuff in R1 compared with those in R2, which in turn were more inflamed than plaques closest to the cuff in R3.

       Magnitude of shear stress has significant inverse correlation with vascular remodeling, plaque size, vulnerability and inflammation

      The correlations between magnitude of shear stress and plaque size, vulnerability and inflammation were evaluated. From week 20 onwards, magnitude of shear stress was significantly inversely correlated with plaque size and the correlation became stronger as atherosclerosis progressed (Fig 3, A, Week 20: R2 = 0.62, P < 0.01; Week 30: R2 = 0.79, P < 0.01; Week 50: R2 = 0.84, P < 0.01). A similar inverse correlation between magnitude of shear stress and vascular remodeling with stronger correlations as atherosclerosis advanced was demonstrated (Fig 3, B, Week 20: R2 = 0.70, P < 0.01; Week 30: R2 = 0.75, P < 0.01; Week 50: R2 = 0.79, P < 0.01). Magnitude of shear stress was significantly inversely correlated with lipid content, one of the “destabilizing” components in the plaque (Fig 6, A, Week 30: R2 = 0.61, P < 0.05; Week 50: R2 = 0.64, P < 0.01), but significantly correlated with collagen and smooth muscle cell content, the “stabilizing” components in the plaque (Fig 6, B: collagen content: Week 30: R2 = 0.60, P < 0.05; Week 50: R2 = 0.64, P < 0.01; Fig 6, C: smooth muscle cell content: Week 30: R2 = 0.79, P < 0.01; Week 50: R2 = 0.74, P < 0.01). Further, magnitude of shear stress was also significantly inversely correlated with the expression of all inflammatory biomarkers (Fig 7, A–D, CD62P: Week 30: R2 = 0.69, P < 0.01; Week 50: R2 = 0.89, P < 0.01; VCAM-1: Week 20: R2 = 0.85, P < 0.01; Week 30: R2 = 0.83, P < 0.01; Week 50: R2 = 0.88, P < 0.01; MOMA-2: Week 30: R2 = 0.89, P < 0.01; Week 50: R2 = 0.82, P < 0.01; MMP-9: Week 30: R2 = 0.82, P < 0.01; Week 50: R2 = 0.83, P < 0.01).
      Fig 6
      Fig 6Correlation between plaque vulnerability and magnitude of shear stress. (A) From week 30 onwards, magnitude of shear stress is significantly inversely correlated to the lipid content (oil red O stain), the “vulnerable” component in the plaque. (B, C) From week 30 onwards, magnitude of shear stress is significantly correlated with collagen (Masson's trichrome stain) and smooth muscle cell contents, both the “stabilizing” components in the plaque.
      Fig 7
      Fig 7Correlation between plaque inflammation and magnitude of shear stress. (A-D) From week 30 onwards, magnitude of shear stress is significantly inversely correlated with the expression of all inflammatory biomarkers (ie, MOMA-2, CD62P, VCAM-1 and MMP-9). Amongst all these biomarkers, VCAM-1 is the earliest one that demonstrates a significant inverse correlation with magnitude of shear stress as early as week 20.
      In summary, from week 15 onwards, the plaques formed in LSS regions (R1, R2, R3) were found to have significantly greater plaque size, vascular remodeling, plaque vulnerability and inflammation than those in OSS region (R5). From week 30 onwards, a specific pattern of plaques with different phenotypes were formed within the same carotid artery depending on their position relative to the cuff. The further away from the cuff proximally (more upstream), the lower the magnitude of shear stress, the more unstable the plaques, with greater degree of vascular remodeling, plaque size, plaque vulnerability and inflammation.

       Pattern of progressive translational atherosclerosis model

      The plaques in all regions (R1 to R5) at all timepoints (10 to 50 weeks post-cuff implantation) were examined and quantitatively analyzed based on the features described in the AHA Classification on Vascular Lesions (ie, Type II: foam cells; Type III: lipid pools; Type IV: lipid core; Type V: necrotic core; Type VI: surface defect, hemorrhage).
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      ,
      • Gibbons RJ
      • Balady GJ
      • Bricker JT
      • et al.
      ACC/AHA 2002 guideline update for exercise testing: summary article. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1997 Exercise Testing Guidelines).
      A pattern of this progressive atherosclerosis animal model are summarized in Figs 2, 8, 9, 10 and Supplementary Fig 4. At week 10, high-risk, vulnerable plaques with necrotic cores (Type V) and surface defects (Type VI) were found in all histological sections in R1. 40% of histological sections showed Type VI plaques with surface defects, 10% of which also bear necrotic cores. The remaining 60% sections in R1 revealed Type V plaques with necrotic cores. No plaques were observed in R2 to R5 at week 10. Similarly, at week 15, high-risk, vulnerable plaques bearing necrotic cores (Type V) and surface defects (Type VI) were also found in all histological sections in R1. 50% of histological sections showed Type VI plaques with surface defects, 30% of which also bear necrotic cores. The remaining 50% sections revealed Type V plaques with necrotic cores. In R2, high-risk plaques bearing necrotic cores (Type V) were found in only 10% of the histological sections and no plaques were found in the remaining 90%. In R3, 60% of the sections showed a mixture of 60% of advanced plaques with lipid cores (Type IV) and necrotic cores (Type V) and 40% of stable plaques with lipid pools (Type III). No plaques were observed in the remaining 40% of the sections. In R4, no plaques were observed. In R5, high-risk plaques with lipid cores (Type IV) and necrotic cores (Type V) were found in half of all sections. Majority of these high-risk plaques also bear lipid pools and foam cells. The remaining half of all sections revealed low-risk plaques with foam cells (Type II) and lipid pools (Type III).
      Fig 8
      Fig 8Summary of progressive translational atherosclerosis model. The plaques in all regions (R1-5) were examined and classified based on the features described in the AHA Classification on Vascular Lesions.
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      ,
      • Gibbons RJ
      • Balady GJ
      • Bricker JT
      • et al.
      ACC/AHA 2002 guideline update for exercise testing: summary article. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1997 Exercise Testing Guidelines).
      Fig 9
      Fig 9(A) Quantitative analysis of plaque features described in the AHA Classification on Vascular Lesions. Individual plaque features described in the AHA Classification on Vascular Lesions
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      ,
      • Gibbons RJ
      • Balady GJ
      • Bricker JT
      • et al.
      ACC/AHA 2002 guideline update for exercise testing: summary article. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1997 Exercise Testing Guidelines).
      , that is foam cell (FC), lipid pool (LP), lipid core (LC), necrotic core (NC), surface defect (SurDef) and hemorrhage (HE), were examined and quantitatively analysed in all regions (R1 to R5) at all timepoints (10 to 50 weeks post-cuff implantation). In R1 and R2, greater proportion of plaques displaying SurDef and HE towards week 50. In R3, at least 50% of the samples showed stable plaques from week 20 to 50. In R5, LC and NC components in plaques were reduced at week 20, and stable plaques were formed at weeks 30 and 50. (B) Plaque vulnerability index. Vulnerability index was used to quantify the degree of vulnerability in the plaques in R1 to R5 at all timepoints.
      • Di Gregoli K
      • Somerville M
      • Bianco R
      • et al.
      Galectin-3 identifies a subset of macrophages with a potential beneficial role in atherosclerosis.
      The higher the vulnerability index, the more vulnerable and unstable the plaques. In R1 and R2, the plaques become increasingly vulnerable and unstable as atherosclerosis progresses towards week 50. By contrast, the plaques in R3 and R5 become more stable at weeks 30 and 50.
      Fig 10
      Fig 10Histological images of RCCA of ApoE−/− mouse at 30 weeks after implantation of shear stress modifying cuff. (a, b, c) In R5, oscillatory shear stress induced development of low-risk plaques with foam cells (FM) (a), reflecting features of human plaque AHA classification type II. These plaques with stable phenotype were also characterized by low macrophage content (b) and high smooth muscle cell content (c). (d, e, f) In R4, high shear stress (within the cuff) protected the carotid artery against atherosclerosis. No atherosclerotic lesion (d), or MOMA stain for macrophage (e) was observed in the vessel wall. Smooth muscle cell content was observed only in the tunica media (f). (g, h, i) In R3, low shear stress (LSS) induced development of intermediate-risk plaques with multiple lipid pools (LP) and FM (g), reflecting features of human plaque AHA classification type III. The plaques also revealed moderate macrophage content (h) and smooth muscle cell content at the cap of the plaque (i). (j, k, l) In R3, LSS induced development of high-risk plaques with multiple lipid cores (LC) (j), reflecting features of human plaque AHA classification type IV. These plaques with unstable, vulnerable phenotype were also characterized by high macrophage content (k) and low levels of smooth muscle cell content (l). (m, n, o) In R1, R2, LSS induced development of high-risk plaques with multilayered necrotic cores (NC) and LC (m), reflecting features of human plaque AHA classification type V. These plaques with unstable, vulnerable phenotype were also characterized by significantly high macrophage content (n) and low levels of smooth muscle cell content in the fibrous cap of plaque (o). (p,q,r) In R1, R2, LSS induced development of high-risk plaques with lipid rich necrotic core (NC) and intraplaque hemorrhage (He) (p), reflecting features of human plaque AHA classification type VI. These plaques with unstable, vulnerable phenotype were also characterized by significantly high macrophage content (q) and minimal levels of smooth muscle cell content (r).
      As atherosclerosis progressed to week 20, a more definitive pattern was observed. All plaques in R1 were high-risk plaques with surface defect (Type VI) and/or necrotic core (Type V) features. 40% of the sections showed Type VI plaques with surface defects, 12% of which also bear necrotic cores. The remaining 60% sections revealed Type V plaques with necrotic cores. Majority of these high-risk plaques also bear lipid cores, lipid pools and foam cells. In R2, high-risk plaques displaying necrotic cores (Type V) were found in 80% of the histological sections and no plaques were found in the remaining 20%. In R3, 50% of the sections showed high-risk plaques with lipid cores (Type IV) and necrotic cores (Type V), whereas the remaining 50% revealed low-risk plaques with lipid pools (Type III). In R5, low-risk plaques with foam cells (Type II) and lipid pools (Type III) were observed in 80% of the sections. The remaining 20% revealed high-risk plaques with lipid cores (Type IV) and necrotic cores (Type V).
      From week 30 onwards, a definitive, specific pattern was observed: all sections in R1 and R2 were high-risk plaques (AHA Type V and VI). At week 30 and 50, high-risk, vulnerable plaques with necrotic core (Type V), and/or surface defect and hemorrhage (Type VI) features were found in all histological sections in R1 and R2. At week 30, 2% of the sections showed plaques with hemorrhage, 38% revealed plaques with surface defect in R1. Majority of these complex plaques also bear necrotic core. At week 50, the proportion of the complex plaques increased with 12% of the sections showed intraplaque hemorrhage, 48% revealed plaques with surface defect in R1. Similarly, in R2, the proportion of the complex plaques is higher in week 50 than week 30, with 10% of the sections showed intraplaque hemorrhage, 38% revealed plaques with surface defect. In R3, all sections were a mixture of high-risk plaques with necrotic cores and/or lipid cores (∼44% AHA Type IV and V) or stable plaques with lipid pools (∼56% AHA Type III) at week 30 and 50. No plaques were found in R4. All sections in R5 were low-risk, stable plaques (AHA Type II and III) at week 30 and 50. Furthermore, vulnerability index was used to quantify the degree of vulnerability in the plaques in R1 to R5 at all timepoints,
      • Di Gregoli K
      • Somerville M
      • Bianco R
      • et al.
      Galectin-3 identifies a subset of macrophages with a potential beneficial role in atherosclerosis.
      highlighting the differential plaque development in this model (Fig 9, B).
      In summary, in R1 and R2, the advanced plaques progressed to become complex plaques with greater proportion of plaques displaying surface defect and hemorrhage towards week 50. In R3, at least 50% of the sections revealed stable plaques from week 20 to 50. In R5, it showed plaque regression with 50% of sections showing advanced plaques with necrotic cores and/or lipid cores at week 15, reduced to 20% at week 20. Stable plaques were formed at week 30 and 50.

      DISCUSSION

      In this study, we describe the development of an animal model that provides a reliable and reproducible method to predict exactly where, when and what types of plaques will form. The model generates a specific pattern of clinically-relevant plaques with different risk levels along the same artery depending on their position relative to the cuff. The closer to the bottom of the cuff-implanted artery, the lower the magnitude of shear stress, the more unstable the plaques with higher grade of AHA classification, and greater degree of vascular remodeling, plaque size, plaque vulnerability and inflammation, resulting in higher risk plaques. Although the relation between shear stress and atherogenesis has been well documented, to the best of our knowledge, it is the first large scale pre-clinical study to demonstrate that by actively modifying the magnitude of shear stress, plaques with varying degree of phenotypes, types of AHA classification and risk levels can be developed in specific locations along the single artery in a reproducible manner with near 100% accuracy of prediction.
      Previous studies
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      have utilized the shear-stress modifying cuff to generate vulnerable plaques in LSS region, stable plaques in OSS region, and atherosclerotic disease-free area in HSS region within the common carotid artery. Building on the earlier studies,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      we have further investigated the following 5 areas in this cuff-implantation animal model, and to the best of our knowledge, this is the first study to:
      (1) Investigate the variations in the magnitude of shear stress in 5 different regions of the whole carotid artery. Previous studies
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      have investigated the shear stress in 3 regions of the carotid artery in this cuff-model, that is, LSS region proximal to the cuff, HSS within the cuff, and OSS distal to cuff, while other studies
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      ,
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      only investigated the shear stress in 1 region of the carotid artery. In this study, in addition to the OSS region in R5 and HSS region in R4, the LSS region was subdivided into 3 different LSS regions (R1, R2, R3). The shear stress magnitude in R1 was lowest, followed by that in R2, which was in turn lower than that in R3 (shear stress magnitude in R1<R2<R3). This would allow further investigation in the relationship between the magnitude of LSS and the subsequent plaque development. (2) Elucidate the relationship between the magnitude of LSS and the subsequent development of plaques examined by clinically-relevant parameters, including types of AHA classification and risk levels of plaques, to approximate this animal model for clinical translation. Previous studies have only investigated the effect of shear stress on plaque inflammation,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      vulnerability
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      and phenotypes,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      but not on the types of AHA Classification on Vascular Lesions.
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      (3) Follow up the plaque development longitudinally from early stage to very advanced disease stage; (4) Develop the progressive translational atherosclerosis model in which the plaques in all regions (R1-5) were examined and classified based on the features described in the AHA Classification on Vascular Lesions.
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      Our study has longitudinally followed up and examined the plaque development using clinically-relevant parameters (inflammation, vulnerability, phenotypes, types of AHA classification, and risk levels) from early, intermediate, advanced to very advanced disease stage (10, 15, 20, 30, 50 weeks post-cuff implantation) in all 5 regions of the whole carotid artery in this cuff-implantation animal model. Specifically, the plaques in all 5 regions in all disease stages were examined using AHA classification of human plaques
      • Stary HC
      • Chandler AB
      • Dinsmore RE
      • 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.
      as benchmark to approximate this animal model for clinical translation. Previous studies have only followed up the plaque development longitudinally from days
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      ,
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      to weeks,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      with longest follow-up period up to 12 weeks post-cuff implantation
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      in 1
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      ,
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      or 3
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      regions of the carotid artery in this cuff-model. Plaques in these studies
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      • Sasaki T
      • Kuzuya M
      • Nakamura K
      • et al.
      A simple method of plaque rupture induction in apolipoprotein E-deficient mice.
      • von der Thüsen JH
      • van Vlijmen BJM
      • Hoeben RC
      • et al.
      Induction of atherosclerotic plaque rupture in apolipoprotein E−/− mice after adenovirus-mediated transfer of p53.
      were not systematically examined and classified using AHA classification of human plaques. (5) Examine the accuracy of prediction in precisely where (regions R1-5), when (timepoint after cuff placement) and what stages/types (AHA Type II–VI) and risk levels (high/intermediate/low-risk) of plaques develop in the atherosclerosis animal model.
      Firstly, we provide evidence that variations in the 5 different shear stress fields profoundly affect the development of atherosclerotic plaques with different phenotypes and risk levels along the same artery. We conclude that both LSS and vortices with OSS are highly proatherogenic; whilst HSS protects against atherosclerosis. This is based on observations that, (1) plaque lesions were developed from week 15 onwards in all of the cuff-implanted animals, under both hemodynamic conditions (LSS: R1,2,3; OSS: R5); (2) plaques were absent in all the straight vessel segments of contralateral, untreated LCCA, except at the sites of bifurcations; and (3) plaques were absent in all the HSS field (R4) in cuff-implanted carotid arteries. Moreover, we conclude that LSS (R1,2,3) induces development of larger, more vulnerable and inflamed plaques with greater vascular remodeling, whilst OSS (R5) induces formation of stable plaques. These findings concur with the generally accepted notion that LSS induces development of vulnerable, inflamed plaques, OSS promotes formation of stable plaques, whilst HSS protects against atherosclerosis.
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      The shear stress values in this study (LSS [R1: 4.6 N/m2, R2: 5.5 N/m2, R3: 7.0 N/m2], HSS: [R4: 18.9 N/m2], OSS: [R5: 10.0 N/m2]) are also similar to those in the previous cuff-implantation models (LSS: 10 N/m2, HSS: 10–25 N/m2, OSS: 14 N/m2]).
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      ,
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      Recent studies showed that activation of endoplasmic reticulum stress in mechano-regulation has been associated with promoting endothelial inflammation at arterial regions of athero-susceptible shear stress.
      • Bailey KA
      • Haj FG
      • Simon SI
      • Passerini AG.
      Atherosusceptible shear stress activates endoplasmic reticulum stress to promote endothelial inflammation.
      The 2 key proinflammatory signalling pathways, the mitogen-activated protein kinase pathway and nuclear factor- kappa-B pathway have also been identified as playing a significant role in endothelial activation at athero-susceptible sites with low/OSS. By contrast, HSS was shown to suppress activation of these pathways and protect against lesion development.
      • Warboys CM
      • Amini N
      • de Luca A
      • Evans PC.
      The role of blood flow in determining the sites of atherosclerotic plaques.
      ,
      • Passerini AG
      • Polacek DC
      • Shi C
      • et al.
      Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta.
      Here we provide further evidence that the magnitude of LSS has statistically significant inverse correlations with (1) plaque size, (2) vascular remodeling, (3) plaque vulnerability, and (4) plaque inflammation, which in turn indicate the risk levels of the individual plaques. These correlations also become stronger for cuffs implanted in the carotid arteries for longer periods, especially after 30 weeks. The prominent role of local shear stress in the arterial remodeling and atherogenic processes in the plaques observed in this study is remarkably similar to the observations in the previous cuff-implantation mouse models,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      • von der Thüsen JH
      • van Berkel TJ
      • Biessen EA.
      Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.
      atherosclerotic pig model
      • Chatzizisis YS
      • Jonas M
      • Coskun AU
      • et al.
      Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress: an intravascular ultrasound and histopathology natural history study.
      ,
      • Chatzizisis YS
      • Baker AB
      • Sukhova GK
      • et al.
      Augmented expression and activity of extracellular matrix-degrading enzymes in regions of low endothelial shear stress colocalize with coronary atheromata with thin fibrous caps in pigs.
      and patients in the PREDICTION study.
      • Stone PH
      • Saito S
      • Takahashi S
      • et al.
      Prediction of progression of coronary artery disease and clinical outcomes using vascular profiling of endothelial shear stress and arterial plaque characteristics: the PREDICTION Study.
      In previous cuff-implantation mouse models, LSS was associated with (1) larger plaque size
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • Kuhlmann MT
      • Cuhlmann S
      • Hoppe I
      • et al.
      Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.
      ; (2) greater outward vascular remodeling
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ; (3) increased plaque inflammation (ie, higher expression of inflammatory markers for macrophages,
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      VCAM-1,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      CD62P
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      and MMP
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ); (4) increased plaque vulnerability (ie, higher “destabilizing” lipid content
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ,
      • Pedrigi RM
      • Mehta V V
      • Bovens SM
      • et al.
      Influence of shear stress magnitude and direction on atherosclerotic plaque composition.
      but lower “stabilizing” smooth muscle cell
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      and collagen content
      • Cheng C
      • Tempel D
      • Van Haperen R
      • et al.
      Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
      ,
      • Chan JMS
      • Monaco C
      • Wylezinska-Arridge M
      • et al.
      Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.
      ). In the pig model, LSS was an independent predictor responsible for the formation of rupture-prone thin cap fibroatheromas, and the vulnerability of the atherogenic phenotype was inversely correlated with the magnitude of local shear stress.
      • Chatzizisis YS
      • Jonas M
      • Coskun AU
      • et al.
      Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress: an intravascular ultrasound and histopathology natural history study.
      ,
      • Chatzizisis YS
      • Baker AB
      • Sukhova GK
      • et al.
      Augmented expression and activity of extracellular matrix-degrading enzymes in regions of low endothelial shear stress colocalize with coronary atheromata with thin fibrous caps in pigs.
      Furthermore, the crucial proatherogenic role of LSS in patients was highlighted by the observation in the PREDICTION Study where LSS provides a substantial independent prognostication of plaque progression and clinical worsening of luminal obstruction treated with a percutaneous coronary intervention. By contrast HSS was not associated with plaque progression or progressive luminal obstruction.
      • Stone PH
      • Saito S
      • Takahashi S
      • et al.
      Prediction of progression of coronary artery disease and clinical outcomes using vascular profiling of endothelial shear stress and arterial plaque characteristics: the PREDICTION Study.
      It is noteworthy that amongst the 4 inflammatory biomarkers, VCAM-1 was shown to be the best early biomarker to track inflammation in high-risk vulnerable plaques as it showed a strong and significant correlation as early as 20 weeks post-cuff placement whilst the other inflammatory biomarkers (ie, MOMA-2, CD62P and MMP-9) revealed this correlation only after 30 weeks. This corroborates previous studies that VCAM-1 expression is one of the earliest inflammatory events in atherogenesis.
      • Galkina E
      • Ley K.
      Vascular adhesion molecules in atherosclerosis.
      VCAM-1 mediates firm adhesion of monocytes, preceding their transmigration to the nascent lesion.
      • Galkina E
      • Ley K.
      Vascular adhesion molecules in atherosclerosis.
      VCAM-1 expression is present at atherosclerosis-susceptible sites, even before macroscopic disease becomes apparent, with perpetual expression in advanced plaques.
      • Galkina E
      • Ley K.
      Vascular adhesion molecules in atherosclerosis.
      Therefore VCAM-1 has been exploited as a valuable target to develop molecular imaging tool for atherosclerosis.
      • McAteer MA
      • Mankia K
      • Ruparelia N
      • et al.
      A leukocyte-mimetic magnetic resonance imaging contrast agent homes rapidly to activated endothelium and tracks with atherosclerotic lesion macrophage content.
      The histological sections were validated using AHA classification of human plaques as benchmark to approximate this animal model for clinical translation. By week 20 and 30, this model achieved 80% and near 100% accuracy of prediction respectively, in precisely where (regions R1-5), when (timepoint after cuff placement) and what stages/types (AHA Type II–VI) and risk levels (high/intermediate/low-risk) of plaques develop along the same carotid artery of the same animal. The near 100% accuracy of prediction has significant importance because it means that 30 weeks after cuff placement, there is near 100% chance to find high-risk plaques (AHA Type V and VI) in R1 and R2, a mixture of high-risk plaques (AHA Type IV and V) or stable plaques (AHA Type II and III) in R3, no plaques in R4, and low-risk, stable plaques (AHA Type II and III) in R5. Moreover, pivotal features of human vulnerable plaque development, such as LRNC and thinning/rupture of fibrous cap (TRFC) were also consistently observed in the plaques formed in R1 and R2 from 30 weeks onwards. Intraplaque hemorrhage (IPH), however, was infrequent. In a meta-analysis of clinical studies,
      • Gupta A
      • Baradaran H
      • Schweitzer AD
      • et al.
      Carotid plaque MRI and stroke risk: a systematic review and meta-analysis.
      patients bearing these high-risk carotid plaque features (ie, LRNC, IPH and TRFC) detected by MRI were reported to have 3-times, 4.5-times and 6-times higher risk for stroke or transient ischemic attack respectively. Another meta-analysis showed that patients bearing carotid plaques with high lipid or intraplaque hemorrhage content, detected as echolucent plaques by ultrasound had a 2.3 times higher risk of stroke than those with echogenic plaques, across all stenosis severities.
      • Gupta A
      • Kesavabhotla K
      • Baradaran H
      • et al.
      Plaque echolucency and stroke risk in asymptomatic carotid stenosis: a systematic review and meta-analysis.
      Furthermore, a large clinical study reported that the size of a juxta-luminal black area (corresponds to the area of lipid core close to plaque lumen) in ultrasound images of asymptomatic carotid plaques is linearly related to the risk of stroke and can be used in risk stratification models.
      • Kakkos SK
      • Griffin MB
      • Nicolaides AN
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      Therefore, the animal model developed herein also demonstrated a specific pattern of plaques with different types of AHA classification and risk levels that closely resemble in patients along the single carotid artery, depending on their position relative to the cuff.
      With the high accuracy of prediction and direct clinical relevance to patients with carotid artery disease, this model not only saves cost from repeated imaging and histology to confirm the presence of plaques, but also well serves the purpose of (1) high throughput screening for diagnostic probes and drugs, (2) development of endovascular devices, (3) discovery of novel theranostic targets in the high-risk plaques, offering a clear advantage over other conventional and large animal models.
      • Gargiulo S
      • Gramanzini M
      • Mancini M.
      Molecular imaging of vulnerable atherosclerotic plaques in animal models.
      ,
      • Shim J
      • Al-Mashhadi RH
      • Sørensen CB
      • Bentzon JF.
      Large animal models of atherosclerosis–new tools for persistent problems in cardiovascular medicine.
      The model provides a valuable platform for diagnostic tools to test their targeting capabilities to (1) differentiate the high-risk plaques from the stable ones in the single artery, eliminating the local hemodynamic variation and animal-to-animal variation; (2) track the plaque progression from early to advanced stage, and (3) monitoring the response to new therapies. In the development of endovascular therapy, for example, drug-eluting balloon/stent, the model can be used to test novel drug release formulation, evaluation of entrapment efficiency and sustained bioavailability of the drugs in both high-risk and low-risk plaques before moving to large animal models. This model can be extended to atherosclerotic disease in all vascular beds, including intracranial, coronary and peripheral arteries, not solely the carotid artery.
      In conclusion, we have developed and characterized a predictive animal model of progressive atherosclerosis that was histologically validated at a large scale. This model can generate clinically-relevant plaques with varying degree of (1) phenotypes, (2) types of AHA classification, and (3) risk levels in specific locations along the same carotid artery in a reproducible manner with near 100% accuracy of prediction. This unique animal model offers a promising tool for development of therapeutics and diagnostic tools to target the high-risk vulnerable plaques (but not the stable ones), allowing accurate risk stratification of individual patients, increasing accuracy in predicting which patients need or do not need surgical intervention to prevent stroke, affording the opportunity for early treatment for stroke prevention. This animal model could pave the way for personalized management of carotid artery disease as it facilitates development of diagnostic tools for therapy selection, response monitoring and follow-up therapy planning of individual patients.

      ACKNOWLEDGMENTS

      Conflicts of Interest: All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare.
      This study was supported by the core fund from Institute of Bioengineering and Bioimaging, Agency for Science, Technology and Research (A*STAR), Singapore .
      Author contributions are as follows: J. M. S. Chan designed experiments, analyzed the data and wrote the manuscript; S. J. Park and M. Ng performed experiments, collected and analyzed the data; W. C. Chen designed MR experiments and analyzed the MR data; J. Garnell performed experiments and collected the data; K. Bhakoo was involved in experimental design, data analysis and revision of the manuscript.
      No editorial support was required in the preparation of this manuscript. All authors have read the journal authorship agreement and the manuscript has been reviewed and approved by all authors. The authors thank Nikon Imaging Centre, Singapore for their guidance and support in this study.

      DATA AVAILABILITY

      The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

      Appendix. Supplementary materials

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