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Innovation in medical imaging to improve disease staging, therapeutic intervention, and clinical outcomes

  • Marwa Daghem
    Correspondence
    Corresponding author. Centre for Cardiovascular Science, Chancellor's Building, 49 Little France Crescent, Edinburgh, EH16 4SA, Scotland.
    Affiliations
    British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
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  • David E. Newby
    Affiliations
    British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
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      Highlights

      • Calcification plays an important role in the pathogenesis of atherosclerosis and begins early on in the disease.
      • Variable calcification patterns are associated with different histopathological and clinical features.
      • Modern imaging strategies allow assessment of morphological coronary calcification as well as the underlying early biological changes and activity of calcification.

      Abstract

      Calcification plays an important role in the pathogenesis of atherosclerosis and begins early on in the disease process. The presence of calcium has long been seen as a surrogate marker of atherosclerosis and is a well-established predictor of cardiac risk. Evidence suggests that different calcification patterns are associated with different histopathological and clinical features. At the patient level, the presence of macrocalcification, as assessed by the coronary calcium score, confers worst outcomes. At the plaque level, microcalcification rather than macrocalcification denotes plaque vulnerability. Improved non-invasive imaging modalities may allow for a more comprehensive assessment of atherosclerotic calcification and help identify patients at increased risk of clinical sequelae.

      Keywords

      Abbreviations:

      VSMC (vascular smooth muscle cell), RANK (receptor activator of nuclear factor kappa-Β), BMP (bone morphogenetic protein), OPG (osteoprotegerin), OCT (optical coherence tomography), IVUS (intravascular ultrasound), CAC (coronary artery calcium score), CT (computed tomography), PET (positron emission tomography), 18F–NaF (18F-sodium fluoride)

      1. Introduction

      Calcification plays an important role in the pathogenesis of atherosclerosis and begins early on in the disease process. The presence of calcium has long been seen as a surrogate marker or atherosclerosis and is a well-established predictor of cardiac risk [
      • Budoff M.J.
      • Shaw L.J.
      • Liu S.T.
      • Weinstein S.R.
      • Mosler T.P.
      • Tseng P.H.
      • et al.
      Long-term prognosis associated with coronary calcification: observations from a registry of 25,253 patients.
      ]. In recent years, coronary calcification has come under renewed attention, with growing evidence suggesting that different calcification patterns are associated with different histopathological and clinical features.
      Traditional computed tomography calcium scoring measures visible calcium deposition in the coronary arteries – otherwise known as macrocalcification. The current thinking is that macrocalcification identifies a high-risk vulnerable patient rather than a vulnerable plaque or vulnerable vessel. On the other hand, microcalcification consists of micro-deposits of calcium (smaller than 50 μm), which cannot be detected by conventional CT and is thought to represent the early stages of the process and may in fact indicate plaque vulnerability. In the transition from microcalcification to macrocalcification, small discrete nodules of calcium (up to 3 mm) appear termed “spotty calcification”. Recent data suggest that plaque rupture events are more common in less calcified lesions with higher degree of local inflammation rather than in densely calcified, healed atherosclerotic plaque. The so called “calcium paradox” is still a topic of considerable debate. Improved non-invasive imaging modalities have shed light on the mechanisms regulating the evolution of atherosclerosic calcification and helped identify patients at increased risk of clinical sequelae.
      This review will focus on coronary calcification: the underlying pathogenesis and molecular mechanism, implications with regards to plaque progression and the relationship of the extent and patterns of calcification to plaque morphology. We will explore the established and emerging imaging modalities and the potential implications on diagnosis, risk stratification, and patient care.

      2. Pathogenesis

      Atherosclerosis is an inflammatory process defined by intimal or subintimal lipid deposits forming fatty streaks. Progressive lesions develop a necrotic core with abundant macrophages, foam cells, cellular debris and extravasation of erythrocytes from newly formed fragile capillaries [
      • Libby P.
      • Theroux P.
      Pathophysiology of coronary artery disease.
      ]. Throughout this process of plaque development, the composition of the fibrous cap (crucial in determining the plaque's structural integrity) is also in a state of flux. A thick cap is associated with relative plaque stability [
      • Stary H.C.
      • Chandler A.B.
      • Glagov S.
      • Guyton J.R.
      • Insull W.
      • Rosenfeld M.E.
      • 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.
      ]. However, inflammation via the action of matrix metalloproteinases drives decreased synthesis and increased breakdown of collagen, resulting in thinning and weakening of the cap and increasing the risk of plaque rupture and exposure of its thrombogenic constituents to the blood [
      • Libby P.
      Current concepts of the pathogenesis of the acute coronary syndromes.
      ].
      The body has several healing mechanisms that seek to stabilise atherosclerotic plaques including calcification. Calcification starts early on in the atherosclerotic process and is observed in lesions with pathological intimal thickening [
      • Stary H.C.
      The sequence of cell and matrix changes in atherosclerotic lesions of coronary arteries in the first forty years of life.
      ]. It is believed to occur as a healing response to intense necrotic inflammation and it is useful to consider the calcification in two stages. It is postulated that microcalcification represents the early stages of the process and is triggered by intense inflammation within the lipid core of the atheromatous plaque [
      • New S.E.P.
      • Aikawa E.
      Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification.
      ]. Microcalcifications are associated with markers of plaque vulnerability, such as intraplaque haemorrhage [
      • Lin R.
      • Chen S.
      • Liu G.
      • Xue Y.
      • Zhao X.
      Association between carotid atherosclerotic plaque calcification and intraplaque hemorrhage: a magnetic resonance imaging study.
      ], and its presence in the fibrous cap might promote cavitation-induced plaque rupture [
      • Kelly-Arnold A.
      • Maldonado N.
      • Laudier D.
      • Aikawa E.
      • Cardoso L.
      • Weinbaum S.
      Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries.
      ]. In contrast, macrocalcification represents the end stages of disease with the formation of homogeneous or sheet-like calcification which effectively walls off the inflamed necrotic core and stabilises the plaque by serving as a barrier to inflammation and rupture [
      • Burke A.P.
      • Weber D.K.
      • Kolodgie F.D.
      • Farb A.
      • Taylor A.J.
      • Virmani R.
      Pathophysiology of calcium deposition in coronary arteries.
      ].
      Pathomorphologically, atherosclerotic calcification typically affects the arterial intimal layers in association with macrophages, lipids and vascular smooth muscle cells (VSMC) [
      • Demer L.L.
      • Tintut Y.
      Vascular calcification: pathobiology of a multifaceted disease.
      ] which should be distinguished from calcification in arterial medial layers causing elastin mineralisation and subsequent loss of elasticity and is often associated with renal failure, diabetes mellitus, hypercalcaemia, and hyperphosphataemia [
      • Johnson R.C.
      • Leopold J.A.
      • Loscalzo J.
      Vascular calcification: pathobiological mechanisms and clinical implications.
      ]. The same conditions associated with medial calcification – namely diabetes mellitus and chronic kidney disease - are also associated with accelerated atherosclerosis.
      Whilst the exact underlying molecular mechanisms of atherosclerotic calcification are largely unknown, histological studies have highlighted the complexity of the cellular interactions involved in vascular calcification; a process that involves positive and negative regulators that orchestrate cellular recruitment, differentiation, and function [
      • Demer L.L.
      • Watson K.E.
      • Boström K.
      Mechanism of calcification in atherosclerosis.
      ]. A number of resident and circulating cells are subjected to such processes including mesenchymal stem cells, macrophages and vascular smooth muscle progenitor cells. They have all been shown to undergo osteoclastic differentiation [
      • Demer L.L.
      • Watson K.E.
      • Boström K.
      Mechanism of calcification in atherosclerosis.
      ]. This process is triggered by two main cytokines: monocyte colony-stimulating factor and the ligand for receptor activator of nuclear factor (NF)-κB (RANKL).
      Initial calcification is thought to result from apoptosis of smooth muscle cells: a process triggered by pro-inflammatory cytokines released from local activated macrophages. Calcifying extracellular vesicles are released with formation of micro-deposits of calcium (smaller than 50 μm), of which hydroxyapatite is the main component [
      • Kelly-Arnold A.
      • Maldonado N.
      • Laudier D.
      • Aikawa E.
      • Cardoso L.
      • Weinbaum S.
      Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries.
      ,
      • Durham A.L.
      • Speer M.Y.
      • Scatena M.
      • Giachelli C.M.
      • Shanahan C.M.
      Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness.
      ]. Macrophage-derived matrix vesicles also play a role in the process of microcalcification [
      • New S.E.P.
      • Goettsch C.
      • Aikawa M.
      • Marchini J.F.
      • Shibasaki M.
      • Yabusaki K.
      • et al.
      Macrophage-derived matrix vesicles: an alternative novel mechanism for microcalcification in atherosclerotic plaques.
      ] resulting in larger punctate appearance. Microcalcifications coalesce into a larger mass and become spotty calcification (<3 mm). These can then go on to coalesce into larger masses forming macrocalcific deposits. This homogeneous or sheet-like calcification effectively walls off the inflamed necrotic core. However, calcified sheets may fracture leading to the formation of nodular calcification thus compromising the continuity of the endothelial lining and underlying collagen matrix [
      • Otsuka F.
      • Sakakura K.
      • Yahagi K.
      • Joner M.
      • Virmani R.
      Has our understanding of calcification in human coronary atherosclerosis progressed? Arterioscler.
      ]. Sugiyama and colleagues have shown that superficial calcification is a prevalent type of macrocalcification in acute coronary syndrome and is associated with greater post-intervention myocardial damage [
      • Sugiyama T.
      • Yamamoto E.
      • Fracassi F.
      • Lee H.
      • Yonetsu T.
      • Kakuta T.
      • et al.
      Calcified plaques in patients with acute coronary syndromes.
      ].
      The biological processes underpinning the transformation from micro-deposits of calcium to more organised stable deposits of macrocalcification are closely linked and, to a degree, regulated by the underling inflammatory process within atherosclerotic plaque.

      2.1 Role of inflammation

      Vascular inflammation appears to proceed and to trigger the calcification process [
      • Legein B.
      • Temmerman L.
      • Biessen E.A.L.
      • Lutgens E.
      Inflammation and immune system interactions in atherosclerosis, Cell.
      ]. Initial calcium deposition in response to pro-inflammatory stimuli results in the formation of granular calcification (“microcalcification”). A positive feed-back loop further stimulates macrophage activation and mineralisation produces foci of calcification which induce further inflammation [
      • Nadra I.
      • Mason J.C.
      • Philippidis P.
      • Florey O.
      • Smythe C.D.W.
      • McCarthy G.M.
      • et al.
      Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?.
      ]. Pre-clinical animal studies suggest in the early stages of the atherosclerotic process, vascular inflammation and osteogenesis progressed in close proximity to, and increased in parallel with, plaque progression [
      • Boström K.
      Proinflammatory vascular calcification.
      ]. Paradoxically, advanced atherosclerotic lesions demonstrated an inverse relation between inflammation and calcification.
      In the early stages of atherosclerosis, the M1 subtype of macrophages predominate, and pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) promote early phases of osteogenic differentiation of VSMC and vesicle-mediated calcification as the result of apoptosis of macrophages and VSMCs themselves [
      • Stöger J.L.
      • Gijbels M.J.J.
      • van der Velden S.
      • Manca M.
      • van der Loos C.M.
      • Biessen E.A.L.
      • et al.
      Distribution of macrophage polarization markers in human atherosclerosis.
      ]. It is postulated that this microcalcification represents the early stages of the process and occurs as part of the healing response to intense inflammation within the necrotic core and may in fact prompt a vicious circle of inflammation and calcium deposition [
      • Aikawa E.
      Optical molecular imaging of inflammation and calcification in atherosclerosis.
      ]. The presence of microcalcification within plaques is associated with larger lipid burden, thinner fibrous cap and higher frequency of microchannels which predispose to plaque rupture [
      • Kataoka Y.
      • Puri R.
      • Hammadah M.
      • Duggal B.
      • Uno K.
      • Kapadia S.R.
      • et al.
      Spotty calcification and plaque vulnerability in vivo: frequency-domain optical coherence tomography analysis.
      ].
      As plaques stabilise, the M2 subtype of macrophages predominate [
      • Stöger J.L.
      • Gijbels M.J.J.
      • van der Velden S.
      • Manca M.
      • van der Loos C.M.
      • Biessen E.A.L.
      • et al.
      Distribution of macrophage polarization markers in human atherosclerosis.
      ]. M2 contribute to inflammation resolution and plaque remodelling. They secrete a number of anti-inflammatory cytokines, such interleukins (e.g. interleukin-10), which promotes osteoblastic differentiation and maturation of VSMC, which facilitate macrocalcification [
      • Peled M.
      • Fisher E.A.
      Dynamic aspects of macrophage polarization during atherosclerosis progression and regression.
      ]. In contrast to the aforementioned microcalcfication, macrocalcification represents the stable stages of more advanced atherosclerosis with the formation of homogeneous or sheet-like calcification which effectively walls off the inflamed necrotic core, and stabilises the plaque by serving as a barrier to inflammation and rupture.

      2.2 Molecular proteins, osteoregulation and calcification

      Calcification within atherosclerotic lesions has features similar to resorptive and remodelling sites in trabecular bone. The mechanisms by which bone-regulatory proteins influence the pathophysiology of atherosclerotic calcification is the subject of intense interest.
      Bone-related proteins - bone morphogenetic protein (BMP)-1 and BMP-4 [
      • Hong O.-K.
      • Yoo S.-J.
      • Son J.-W.
      • Kim M.-K.
      • Baek K.-H.
      • Song K.-H.
      • et al.
      High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells.
      ], osteocalcin, matrix Gla protein, osteonectin, osteopontin [
      • Hirota S.
      • Imakita M.
      • Kohri K.
      • Ito A.
      • Morii E.
      • Adachi S.
      • et al.
      Expression of osteopontin messenger RNA by macrophages in atherosclerotic plaques. A possible association with calcification.
      ], and osteoprotegerin – have been identified within the vessel wall, regulating the deposition of vascular calcium [
      • Shanahan C.M.
      • Cary N.R.
      • Metcalfe J.C.
      • Weissberg P.L.
      High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.
      ]. Functional matrix Gla protein (MGP), a tissue-derived vitamin K dependent protein, is primarily secreted by vascular smooth muscle cells (VSMCs) in the arterial medial layer and is thought to be a potent inhibitor of bone morphogenetic protein and therefore vascular calcification [
      • Price P.A.
      • Otsuka A.A.
      • Poser J.W.
      • Kristaponis J.
      • Raman N.
      Characterization of a gamma-carboxyglutamic acid-containing protein from bone.
      ]. Lack of MGP activates BMP signalling throughout the vascular wall and is associated with ectopic osteochondrogenic differentiation, vascular calcification, and endothelial-mesenchymal transitions (EndMTs); a process by which endothelial cells acquire a mesenchymal phenotype and stem-cell like characteristics [
      • Bardeesi A.S.A.
      • Gao J.
      • Zhang K.
      • Yu S.
      • Wei M.
      • Liu P.
      • et al.
      A novel role of cellular interactions in vascular calcification.
      ].
      Osteopontin, calcium-binding glycophosphoprotein, expressed by macrophages as well as smooth muscle and endothelial cells within plaque also appears to inhibit calcification [
      • Steitz S.A.
      • Speer M.Y.
      • McKee M.D.
      • Liaw L.
      • Almeida M.
      • Yang H.
      • et al.
      Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification.
      ]. Plasma osteopontin concentrations are higher in patients with coronary artery disease [
      • Ohmori R.
      • Momiyama Y.
      • Taniguchi H.
      • Takahashi R.
      • Kusuhara M.
      • Nakamura H.
      • et al.
      Plasma osteopontin levels are associated with the presence and extent of coronary artery disease.
      ] but were not independently associated with coronary calcification. Histological studies have confirmed the presence of osteopontin, and matrix Gla protein at sites of microcalcification early on in the disease process [
      • Roijers R.B.
      • Debernardi N.
      • Cleutjens J.P.M.
      • Schurgers L.J.
      • Mutsaers P.H.A.
      • van der Vusse G.J.
      Microcalcifications in early intimal lesions of atherosclerotic human coronary arteries.
      ]. Meanwhile, fibrocalcific plaques have been found to contain BMP-2, BMP-4, osteopontin, and osteonectin [
      • Dhore C.R.
      • Cleutjens J.P.
      • Lutgens E.
      • Cleutjens K.B.
      • Geusens P.P.
      • Kitslaar P.J.
      • et al.
      Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.
      ].
      The osteoprotegerin (OPG), the receptor activator of NF-κB ligand (RANKL) and the receptor activator of NF-κB (RANK) cytokine network regulates balance between bone formation (osteoblasts) and bone resorption (osteoclasts) [
      • Lacey D.L.
      • Timms E.
      • Tan H.L.
      • Kelley M.J.
      • Dunstan C.R.
      • Burgess T.
      • et al.
      Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.
      ]. Osteoprotegerin (OPG) prevents osteoclast differentiation and bone resorption and exerts its effect through binding and neutralizing RANKL with strong osteoclast-inducing activity [
      • O'Brien E.A.
      • Williams J.H.
      • Marshall M.J.
      Osteoprotegerin ligand regulates osteoclast adherence to the bone surface in mouse calvaria.
      ]. In addition to its osteoregulatory role, OPG is also able to bind and to neutralise the pro-apoptotic actions of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expressed by VSMC. It may in fact play an important role in plaque stability [
      • Shaker O.G.
      • El-Shehaby A.
      • Nabih M.
      Possible role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand as markers of plaque instability in coronary artery disease.
      ].
      Osteoprotegerin (OPG), which has been shown to be present in atherosclerotic plaques [
      • Lieb W.
      • Gona P.
      • Larson M.G.
      • Massaro J.M.
      • Lipinska I.
      • Keaney J.F.
      • et al.
      Biomarkers of the osteoprotegerin pathway: clinical correlates, subclinical disease, incident cardiovascular disease, and mortality.
      ], appears to act as an autocrine or paracrine regulator of vascular calcification and may be a useful serum marker of vascular disease [
      • Mogelvang R.
      • Pedersen S.H.
      • Flyvbjerg A.
      • Bjerre M.
      • Iversen A.Z.
      • Galatius S.
      • et al.
      Comparison of osteoprotegerin to traditional atherosclerotic risk factors and high-sensitivity C-reactive protein for diagnosis of atherosclerosis.
      ]. Clinical studies demonstrate increased serum OPG concentrations in association with vascular calcification [
      • Lieb W.
      • Gona P.
      • Larson M.G.
      • Massaro J.M.
      • Lipinska I.
      • Keaney J.F.
      • et al.
      Biomarkers of the osteoprotegerin pathway: clinical correlates, subclinical disease, incident cardiovascular disease, and mortality.
      ], coronary artery disease [
      • Kiechl S.
      • Schett G.
      • Wenning G.
      • Redlich K.
      • Oberhollenzer M.
      • Mayr A.
      • et al.
      Osteoprotegerin is a risk factor for progressive atherosclerosis and cardiovascular disease.
      ], cerebrovascular disease and future cardiovascular risk [
      • Lieb W.
      • Gona P.
      • Larson M.G.
      • Massaro J.M.
      • Lipinska I.
      • Keaney J.F.
      • et al.
      Biomarkers of the osteoprotegerin pathway: clinical correlates, subclinical disease, incident cardiovascular disease, and mortality.
      ]. Some early data suggest that serum OPG concentrations may be an indicator of subclinical atherosclerosis [
      • Nybo M.
      • Rasmussen L.M.
      The capability of plasma osteoprotegerin as a predictor of cardiovascular disease: a systematic literature review.
      ]. In patients with established atherosclerotic lesions [
      • Yang Q.
      • Lu S.
      • Chen Y.
      • Song X.
      • Jin Z.
      • Yuan F.
      • et al.
      Plasma osteoprotegerin levels and long-term prognosis in patients with intermediate coronary artery lesions.
      ] and angina pectoris [
      • Pedersen E.R.
      • Ueland T.
      • Seifert R.
      • Aukrust P.
      • Schartum-Hansen H.
      • Ebbing M.
      • et al.
      Serum osteoprotegerin levels and long-term prognosis in patients with stable angina pectoris.
      ], serum OPG appears to predict long-term prognosis.
      Current studies are somewhat conflicting, and the role of each of these molecular proteins in the diverse morphological manifestations of disease progression is not yet clearly understood. Further prospective trials are needed to confirm this association between osteoregulatory mechanisms and cardiovascular outcome. Drugs that inhibit the RANK/RANKL and bone resorption interaction have yet to demonstrate any cardiovascular benefit.

      2.3 Calcification and mechanical stress

      Mesenchymal cells are maintained in a quiescent state by the surrounding extracellular matrix and regulated by multiple microenvironmental cues. Mechanical stiffness is capable of governing cell differentiation through activation of intracellular signalling mediators [
      • Engler A.J.
      • Sen S.
      • Sweeney H.L.
      • Discher D.E.
      Matrix elasticity directs stem cell lineage specification.
      ], which enhance the osteogenic potential of mesenchymal cells. The resultant calcium deposits can themselves weaken vasomotor responses and alter atherosclerotic plaque stability, depending on the size and distribution of deposits. The shape of a microcalcification is determined by the relationship with collagen and extracellular vesicles. Irregular microcalcification inflicts higher stress than spherical microcalcifications. Large deposits reduce circumferential stress in adjacent plaque [
      • Huang H.
      • Virmani R.
      • Younis H.
      • Burke A.P.
      • Kamm R.D.
      • Lee R.T.
      The impact of calcification on the biomechanical stability of atherosclerotic plaques.
      ] and small deposits increase stress at their edges [
      • Vengrenyuk Y.
      • Carlier S.
      • Xanthos S.
      • Cardoso L.
      • Ganatos P.
      • Virmani R.
      • et al.
      A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps.
      ]. This is reflected in the different clinical manifestation of coronary calcification: in unstable disease, atherosclerotic lesions demonstrate multiple deposits of “spotty calcification” [
      • Ehara S.
      • Kobayashi Y.
      • Yoshiyama M.
      • Shimada K.
      • Shimada Y.
      • Fukuda D.
      • et al.
      Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction: an intravascular ultrasound study.
      ] whereas stable disease is associated with macrocalcification and large calcium deposits [
      • Shemesh J.
      • Stroh C.I.
      • Tenenbaum A.
      • Hod H.
      • Boyko V.
      • Fisman E.Z.
      • et al.
      Comparison of coronary calcium in stable angina pectoris and in first acute myocardial infarction utilizing double helical computerized tomography.
      ]. It has been suggested that spotty calcification within the fibrous cap will increase biomechanical plaque stress and increase the risk of plaque rupture [
      • Bluestein D.
      • Alemu Y.
      • Avrahami I.
      • Gharib M.
      • Dumont K.
      • Ricotta J.J.
      • et al.
      Influence of microcalcifications on vulnerable plaque mechanics using FSI modeling.
      ,
      • Hutcheson J.D.
      • Maldonado N.
      • Aikawa E.
      Small entities with large impact: microcalcifications and atherosclerotic plaque vulnerability.
      ].

      3. Imaging of coronary calcification

      Microcalcification and inflammation play a key role in plaque rupture, therefore representing important potential imaging targets. With modern advances in imaging technology, we now have multiple different techniques to image various aspects of atherosclerotic plaque across different vascular beds. These techniques include invasive imaging using optical coherence tomography (OCT) and intravascular ultrasound (IVUS), and non-invasive imaging with computed tomography, and positron emission tomography, with each modality offering different advantages and disadvantages. Below we discuss how these different imaging approaches can provide a comprehensive non-invasive assessment of atherosclerotic calcification, informing about disease burden, plaque morphology and disease activity.

      3.1 Invasive imaging of atherosclerotic calcification

      Initial attempts to assess plaque composition and morphology were based around invasive imaging strategies (Fig. 1), predominantly optical coherence tomography (OCT) and intravascular ultrasound (IVUS), and more recently near-infrared spectroscopy: a novel technique to quantitatively and qualitatively assess lipid cores.
      Fig. 1
      Fig. 118 F-Sodium fluoride uptake in coronary artery atherosclerotic plaque.
      Micro-positron emission tomography (PET) and computed tomography (CT) of 18F-sodium fluoride uptake in a coronary artery plaque (A and C), which colocalizes with active calcification demonstrated by alizarin (red) staining (B and D).
      Intravascular ultrasound is an invasive catheter-based technique which uses high-frequency sound waves to generate greyscale cross-sectional images of the arterial wall. It allows the detection and quantification of calcium within a plaque. IVUS appears to provide an accurate quantification of plaque burden, acting as a powerful predictor of disease progression and adverse clinical outcomes [
      • Nicholls S.J.
      • Hsu A.
      • Wolski K.
      • Hu B.
      • Bayturan O.
      • Lavoie A.
      • et al.
      Intravascular ultrasound-derived measures of coronary atherosclerotic plaque burden and clinical outcome.
      ] and has been used in trials to measure the effect of medical therapies on atherosclerosis [
      • Nissen S.E.
      • Tuzcu E.M.
      • Schoenhagen P.
      • Brown B.G.
      • Ganz P.
      • Vogel R.A.
      • et al.
      Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis: a randomized controlled trial.
      ]. IVUS-identified spotty calcification, defined as calcium deposits within an arc of <90°, is most frequently observed in unstable compared to stable plaques (51% vs 30%; p < 0.001) [
      • Ehara S.
      • Kobayashi Y.
      • Yoshiyama M.
      • Shimada K.
      • Shimada Y.
      • Fukuda D.
      • et al.
      Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction: an intravascular ultrasound study.
      ]. Furthermore, the presence of spotty calcification, is associated with other features of plaque vulnerability namely positive remodelling and fibrofatty plaque [
      • Ehara S.
      • Kobayashi Y.
      • Yoshiyama M.
      • Shimada K.
      • Shimada Y.
      • Fukuda D.
      • et al.
      Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction: an intravascular ultrasound study.
      ]. Virtual histology (VH) IVUS uses spectral analysis of ultrasound backscatter to categorise plaque constituents into fibrous, fibrolipidic, calcific, and necrotic tissue in real time [
      • Nair A.
      • Kuban B.D.
      • Tuzcu E.M.
      • Schoenhagen P.
      • Nissen S.E.
      • Vince D.G.
      Coronary plaque classification with intravascular ultrasound radiofrequency data analysis.
      ]. The ability of virtual histology IVUS (VH-IVUS) to detect adverse plaques and then to predict outcomes was investigated in the prospective natural-history study of coronary atherosclerosis (PROSPECT) trial [
      • Stone G.W.
      • Maehara A.
      • Lansky A.J.
      • de Bruyne B.
      • Cristea E.
      • Mintz G.S.
      • et al.
      A prospective natural-history study of coronary atherosclerosis.
      ].
      OCT works on similar principles to IVUS but uses light with a wavelength of about 1-300 nm rather than ultrasound. It has emerged as an insightful intracoronary imaging technology with a higher resolution (10–20 μm) than IVUS (100–200 μm). Unlike IVUS, OCT can penetrate calcium and assess its thickness, area, and volume, thus having the potential to provide microstructural detail. It can potentially identify features associated with increased vulnerability such as the presence of macrophages, neovascularization [
      • Ichibori Y.
      • Ohtani T.
      • Nakatani D.
      • Tachibana K.
      • Yamaguchi O.
      • Toda K.
      • et al.
      Optical coherence tomography and intravascular ultrasound evaluation of cardiac allograft vasculopathy with and without intimal neovascularization.
      ], and microcalcifications [
      • Ijichi T.
      • Nakazawa G.
      • Torii S.
      • Nakano M.
      • Yoshikawa A.
      • Morino Y.
      • et al.
      Evaluation of coronary arterial calcification - ex-vivo assessment by optical frequency domain imaging.
      ]. The co-localisation of apparent macrophages and microcalcifications in the same plaque is associated with increased plaque vulnerability [
      • Burgmaier M.
      • Milzi A.
      • Dettori R.
      • Burgmaier K.
      • Marx N.
      • Reith S.
      Co-localization of plaque macrophages with calcification is associated with a more vulnerable plaque phenotype and a greater calcification burden in coronary target segments as determined by OCT.
      ]. In acute coronary syndrome patients, OCT assessment of the culprit vessel highlights a higher prevalence of spotty calcifications at the site of plaque rupture [
      • Sakaguchi M.
      • Hasegawa T.
      • Ehara S.
      • Matsumoto K.
      • Mizutani K.
      • Iguchi T.
      • et al.
      New insights into spotty calcification and plaque rupture in acute coronary syndrome: an optical coherence tomography study.
      ], demonstrating the relationship between microcalcification and plaque stability.
      Invasive vascular imaging is associated with a risk of complications: 1.6% of the patients in PROSPECT had a complication attributed to IVUS imaging [
      • Stone G.W.
      • Maehara A.
      • Lansky A.J.
      • de Bruyne B.
      • Cristea E.
      • Mintz G.S.
      • et al.
      A prospective natural-history study of coronary atherosclerosis.
      ]. Furthermore, intra-coronary imaging is unable to image the entirety of the coronary tree, and whilst some basic correlation can be assumed, it cannot assess the overall total plaque burden. Conversely, non-invasive imaging of plaque characteristics across the entire coronary vasculature appears to hold greater clinical potential as a method for identifying patients at increased cardiovascular risk. This is based upon the rationale that patients with a propensity to develop adverse plaque characteristics will do so at multiple sites over time. The vast majority of high-risk thin-capped fibroatheromatous plaques do not result in clinical events [
      • Stone G.W.
      • Maehara A.
      • Lansky A.J.
      • de Bruyne B.
      • Cristea E.
      • Mintz G.S.
      • et al.
      A prospective natural-history study of coronary atherosclerosis.
      ]. The risk of one such plaque rupturing at an inopportune moment and causing a future clinical event is therefore potentially increased when considered at the level of the patient. Atherosclerosis imaging is accordingly evolving to include not only anatomical but also metabolic imaging, thus providing insight into the underlying vascular biology of patient.

      3.2 Coronary calcium scoring

      Coronary artery calcium score (CAC) measures macroscopic calcification in the coronary arteries and provides an efficient and non-invasive means of assessing and monitoring plaque burden in the totality of the coronary arterial bed. It has repeatedly been shown to correlate with clinical outcomes [
      • Greenland P.
      • LaBree L.
      • Azen S.P.
      • Doherty T.M.
      • Detrano R.C.
      Coronary artery calcium score combined with Framingham score for risk prediction in asymptomatic individuals.
      ]. In fact, when added to traditional risk score, CAC has the ability to provide incremental risk prediction and appropriately re-classify individuals into higher or lower risk groups [
      • Silverman M.G.
      • Blaha M.J.
      • Krumholz H.M.
      • Budoff M.J.
      • Blankstein R.
      • Sibley C.T.
      • et al.
      Impact of coronary artery calcium on coronary heart disease events in individuals at the extremes of traditional risk factor burden: the Multi-Ethnic Study of Atherosclerosis.
      ]. In asymptomatic patients, a calcium score of zero, has a negative predictive value of 95–99% [
      • Gottlieb I.
      • Miller J.M.
      • Arbab-Zadeh A.
      • Dewey M.
      • Clouse M.E.
      • Sara L.
      • et al.
      The absence of coronary calcification does not exclude obstructive coronary artery disease or the need for revascularization in patients referred for conventional coronary angiography.
      ]. In these patients, the absence of calcium reliably excludes obstructive coronary artery stenosis.
      Coupled with its non-invasive nature, minimal radiation exposure and no requirement for patient preparation, its powerful predictive ability makes CAC scoring an attractive option for population screening. Current Guidelines recommend CAC scoring in selected patients with a cardiovascular disease risk between 5 and 20% in the context of shared decision-making [
      • Hecht H.S.
      • Cronin P.
      • Blaha M.J.
      • Budoff M.J.
      • Kazerooni E.A.
      • Narula J.
      • et al.
      2016 SCCT/STR guidelines for coronary artery calcium scoring of noncontrast noncardiac chest CT scans: a report of the Society of Cardiovascular Computed Tomography and Society of Thoracic Radiology.
      ]. Coronary artery calcium scoring should also be considered in patients with cardiovascular disease risk <5% who have a family history of premature coronary heart disease [
      • Hecht H.S.
      • Cronin P.
      • Blaha M.J.
      • Budoff M.J.
      • Kazerooni E.A.
      • Narula J.
      • et al.
      2016 SCCT/STR guidelines for coronary artery calcium scoring of noncontrast noncardiac chest CT scans: a report of the Society of Cardiovascular Computed Tomography and Society of Thoracic Radiology.
      ]. A coronary artery calcium score >300 Agatston units (AU) is associated with a four-fold higher risk of cardiovascular events compared to a calcium score of zero [
      • Shanahan C.M.
      • Cary N.R.
      • Metcalfe J.C.
      • Weissberg P.L.
      High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.
      ]. On this basis, the 2013 ACC/AHA Guideline on the Management of High Cholesterol [
      • Stone N.J.
      • Robinson J.G.
      • Lichtenstein A.H.
      • Bairey Merz C.N.
      • Blum C.B.
      • Eckel R.H.
      • et al.
      2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.
      ] recommended that a CAC score of >300 AU be used as a modifier to justify statin therapy for primary prevention in adults between 40 and 75 years old without diabetes and with a serum low-density lipoprotein cholesterol concentration 70–189 mg/dL.
      In addition to the traditional Agatston score, routine CAC scoring can also provide information about the density, volume and mass of calcified plaques. There is growing evidence to support the prognostic benefit of coronary density [
      • Criqui M.H.
      • Denenberg J.O.
      • Ix J.H.
      • McClelland R.L.
      • Wassel C.L.
      • Rifkin D.E.
      • et al.
      Calcium density of coronary artery plaque and risk of incident cardiovascular events.
      ], especially in symptomatic patients where it is a stronger predictor of adverse events compared to Agatston score [
      • Lo-Kioeng-Shioe M.S.
      • Vavere A.L.
      • Arbab-Zadeh A.
      • Schuijf J.D.
      • Rochitte C.E.
      • Chen M.Y.
      • et al.
      Coronary calcium characteristics as predictors of major adverse cardiac events in symptomatic patients: insights from the CORE 320 multinational study.
      ]. Whilst traditional calcium scoring remains one of the most power prognostic tools, the Agatston score fails to incorporate information about the number and size of calcified lesions and is weighted for increasing calcium with higher calcium density. This is contrary to histological data suggesting that plaques with high calcium density have smaller lipid cores, whilst plaques with low calcium density have large lipid cores and positive remodelling. This highlights an important limitation of CT calcium scoring: namely this approach is actually targeting a more stable form of plaque that itself is less prone to rupture or cause clinical events. This may explain why in symptomatic patients, the presence of coronary calcification correlates poorly with the degree of coronary stenosis [
      • Gottlieb I.
      • Miller J.M.
      • Arbab-Zadeh A.
      • Dewey M.
      • Clouse M.E.
      • Sara L.
      • et al.
      The absence of coronary calcification does not exclude obstructive coronary artery disease or the need for revascularization in patients referred for conventional coronary angiography.
      ]. On this basis, the most recent National Institute of Clinical Excellence (NICE) chest pain guidelines recommend coronary CT angiography rather than CAC scoring in symptomatic patients [
      Recently updated guidelines from the national Institute for health and care excellence (NICE) and the royal colleges.
      ]. It is important to consider not only how much plaque a patient has but also what kind of plaque they have and whether the disease process in that area is active or not.

      3.3 CT assessment of plaque morphology

      The last few years have seen rapid growth in the clinical use CT coronary angiography in the diagnosis of coronary artery disease. Our traditional approach to the diagnosis and treatment of coronary disease is centred around the assessment of luminal stenosis. However, there are growing data to support the prognostic power of non-obstructive coronary artery disease [
      • Douglas P.S.
      • Hoffmann U.
      • Patel M.R.
      • Mark D.B.
      • Al-Khalidi H.R.
      • Cavanaugh B.
      • et al.
      Outcomes of anatomical versus functional testing for coronary artery disease.
      ,
      • SCOT-HEART investigators
      CT coronary angiography in patients with suspected angina due to coronary heart disease (SCOT-HEART): an open-label, parallel-group, multicentre trial.
      ], which is associated with similar event rates to localised obstructive disease [
      • Bittencourt M.S.
      • Hulten E.A.
      • Murthy V.L.
      • Cheezum M.
      • Rochitte C.E.
      • Carli M.F.D.
      • et al.
      Clinical outcomes after evaluation of stable chest pain by coronary computed tomographic angiography versus usual care.
      ]. This is consistent with the finding that percutaneous coronary intervention does not reduce the risk of myocardial infarction despite effective relief of obstructive disease and consequent ischaemia [
      • Boden W.E.
      • O'Rourke R.A.
      • Teo K.K.
      Optimal medical therapy with or without PCI for stable coronary disease.
      ,
      • BARI 2D Study Group
      • Frye R.L.
      • August P.
      • Brooks M.M.
      • Hardison R.M.
      • Kelsey S.F.
      • et al.
      A randomized trial of therapies for type 2 diabetes and coronary artery disease.
      ]. Accordingly, there has been growing interest in alternative imaging strategies targeting different aspects of the atherosclerotic disease process.
      The sub-millimeter spatial resolution of coronary CT angiography enables imaging of the lumen, as well as the coronary artery wall. At the very least, it is able to differentiate between calcific, partially calcified (mixed) and non-calcified coronary plaque, thereby potentially overcoming an important limitation of CAC scoring [
      • Plank F.
      • Friedrich G.
      • Dichtl W.
      • Klauser A.
      • Jaschke W.
      • Franz W.-M.
      • et al.
      The diagnostic and prognostic value of coronary CT angiography in asymptomatic high-risk patients: a cohort study.
      ]. Non-calcified coronary plaques identified by coronary CT angiography confer a poorer prognosis [
      • Hou Z.-H.
      • Lu B.
      • Gao Y.
      • Jiang S.-L.
      • Wang Y.
      • Li W.
      • et al.
      Prognostic value of coronary CT angiography and calcium score for major adverse cardiac events in outpatients.
      ,
      • Hulten E.A.
      • Carbonaro S.
      • Petrillo S.P.
      • Mitchell J.D.
      • Villines T.C.
      Prognostic value of cardiac computed tomography angiography: a systematic review and meta-analysis.
      ]. These findings are in line with histopathological studies which have reported that lesions associated with acute coronary events are often not heavily calcified [
      • Virmani R.
      • Kolodgie F.D.
      • Burke A.P.
      • Farb A.
      • Schwartz S.M.
      Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.
      ,
      • Virmani R.
      • Burke A.P.
      • Kolodgie F.D.
      • Farb A.
      Pathology of the thin-cap fibroatheroma: a type of vulnerable plaque.
      ].
      There are several classic CT coronary angiography features of high risk plaque (Fig. 2) which reflect the underlying pathological changes: low-attenuation (<30 Hounsfield Units), positive remodelling (defined as a remodelling index >1.1), spotty calcification (defined as a calcified plaque component <3 mm with a >130 HU density) and the napkin-ring sign (low-attenuation plaque core with a rim of higher attenuation). There is a large body of non-randomised evidence demonstrating the prognostic value of these findings [
      • Halon D.A.
      • Lavi I.
      • Barnett-Griness O.
      • Rubinshtein R.
      • Zafrir B.
      • Azencot M.
      • et al.
      Plaque morphology as predictor of late plaque events in patients with asymptomatic type 2 diabetes: a long-term observational study.
      ,
      • Motoyama S.
      • Ito H.
      • Sarai M.
      • Kondo T.
      • Kawai H.
      • Nagahara Y.
      • et al.
      Plaque characterization by coronary computed tomography angiography and the likelihood of acute coronary events in mid-term follow-up.
      ,
      • Chang H.-J.
      • Lin F.Y.
      • Lee S.-E.
      • Andreini D.
      • Bax J.
      • Cademartiri F.
      • et al.
      Coronary atherosclerotic precursors of acute coronary syndromes.
      ]. Recent analyses from the two largest randomised trials of CT coronary angiography in symptomatic patients with suspected stable coronary artery – the Prospective Multicenter Imaging Study for Evaluation of Chest Pain (PROMISE) [
      • Ferencik M.
      • Mayrhofer T.
      • Bittner D.O.
      • Emami H.
      • Puchner S.B.
      • Lu M.T.
      • et al.
      Use of high-risk coronary atherosclerotic plaque detection for risk stratification of patients with stable chest pain: a secondary analysis of the PROMISE randomized clinical trial.
      ] and Scottish Computed Tomography of the Heart (SCOT-HEART) [
      • Williams M.C.
      • Moss A.J.
      • Dweck M.
      • Adamson P.D.
      • Alam S.
      • Hunter A.
      • et al.
      Coronary artery plaque characteristics associated with adverse outcomes in the SCOT-heart study.
      ] trials – have added further weight to the prognostic power of CT coronary angiography assessments of vulnerable plaque. Importantly, approximately half of patients with subsequent adverse events did not have obstructive coronary artery disease [
      • Hoffmann U.
      • Ferencik M.
      • Udelson J.E.
      • Picard M.H.
      • Truong Q.A.
      • Patel M.R.
      • et al.
      Prognostic value of noninvasive cardiovascular testing in patients with stable chest pain: insights from the PROMISE trial (prospective multicenter imaging study for evaluation of chest pain).
      ,
      • SCOT-HEART investigators
      • Newby D.E.
      • Adamson P.D.
      • Berry C.
      • Boon N.A.
      • Dweck M.R.
      • et al.
      Coronary CT angiography and 5-year risk of myocardial infarction.
      ].
      Fig. 2
      Fig. 2Mixed types of coronary atherosclerotic plaque on computed tomography coronary angiography. Computed tomography coronary angiogram demonstrating areas of macrocalcification (blue arrow) in the proximal vessel with a further atherosclerotic plaque in the mid vessel with spotty calcification (green arrow) and associated non-calcific positive remodelling (yellow arrow) on the opposing wall.
      Motoyama and colleagues demonstrated that the presence of spotty calcification was significantly more frequent in the ACS lesions (63% vs. 21%, p = 0.0005) [
      • Motoyama S.
      • Kondo T.
      • Sarai M.
      • Sugiura A.
      • Harigaya H.
      • Sato T.
      • et al.
      Multislice computed tomographic characteristics of coronary lesions in acute coronary syndromes.
      ] and that the presence of high-risk plaque was predictive of future events [
      • Motoyama S.
      • Ito H.
      • Sarai M.
      • Kondo T.
      • Kawai H.
      • Nagahara Y.
      • et al.
      Plaque characterization by coronary computed tomography angiography and the likelihood of acute coronary events in mid-term follow-up.
      ]. In stable patients, CT angiography defined spotty calcification is also associated with adverse outcome (hazard ratio 1.89, 95% confidence interval 1.07–3.33, p = 0.0292) [
      • Yamamoto H.
      • Kihara Y.
      • Kitagawa T.
      • Ohashi N.
      • Kunita E.
      • Iwanaga Y.
      • et al.
      Coronary plaque characteristics in computed tomography and 2-year outcomes: the PREDICT study.
      ]. This is in contradiction to a recent prospective study of 245 patients with non-obstructive coronary artery disease on CT angiography which showed that whilst presence of at least two adverse plaque features was associated with a statistically higher rate of cardiac death or acute coronary syndrome (hazard ratio 7.54, 95% confidence interval 2.43–23.34, p = 0.0002), spotty calcification alone was not in fact predictive of acute coronary syndrome, all-cause and cardiac death, or very late elective revascularization [
      • Conte E.
      • Annoni A.
      • Pontone G.
      • Mushtaq S.
      • Guglielmo M.
      • Baggiano A.
      • et al.
      Evaluation of coronary plaque characteristics with coronary computed tomography angiography in patients with non-obstructive coronary artery disease: a long-term follow-up study.
      ].
      The discrepancy in the result of these trials may be caused by differences in patient backgrounds, as well as difficulty in assessment of spotty calcification on CT angiography. Moreover, the limited resolution of coronary CT means it is only able to detect coronary calcifications with minimal diameter of 215 μm [
      • Kristanto W.
      • van Ooijen P.M.A.
      • Groen J.M.
      • Vliegenthart R.
      • Oudkerk M.
      Small calcified coronary atherosclerotic plaque simulation model: minimal size and attenuation detectable by 64-MDCT and MicroCT.
      ]. A promising technique for the identification of microcalcifications beyond the resolution limits of CT angiography is 18F-sodium fluoride positron emission tomography imaging.

      3.4 Positron emission tomography

      Advances in hybrid scanners now allow combined non-invasive measurement of both disease activity by positron emission tomography (PET) alongside the anatomical detail provided by CT. Targeted PET radiotracers are injected intravenously and accumulate in areas where the disease process of interest is active. The radiation that they emit can then be detected and localised by the PET scanner before being fused with the anatomical data sets.
      18F-Sodium fluoride (18F–NaF) is an established radiotracer originally used for the detection of bony metastases and has now found a potential application in hybrid cardiac imaging. It has been used to study vascular calcification activity in a range of conditions including aortic stenosis [
      • Dweck M.R.
      • Jenkins W.S.A.
      • Vesey A.T.
      • Pringle M.A.H.
      • Chin C.W.L.
      • Malley T.S.
      • et al.
      18F-sodium fluoride uptake is a marker of active calcification and disease progression in patients with aortic stenosis.
      ], abdominal aortic aneurysm disease [
      • Forsythe R.O.
      • Dweck M.R.
      • McBride O.M.B.
      • Vesey A.T.
      • Semple S.I.
      • Shah A.S.V.
      • et al.
      18F–Sodium fluoride uptake in abdominal aortic aneurysms: the SoFIA3 study.
      ] and both carotid and coronary atherosclerosis [
      • Joshi N.V.
      • Vesey A.T.
      • Williams M.C.
      • Shah A.S.V.
      • Calvert P.A.
      • Craighead F.H.M.
      • et al.
      18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial.
      ]. 18F–NaF binds to hydroxyapatite through an exchange of fluoride ions with hydroxyl groups where binding is proportional to the surface area of exposed hydroxyapatite [
      • Creager M.D.
      • Hohl T.
      • Hutcheson J.D.
      • Moss A.J.
      • Schlotter F.
      • Blaser M.C.
      • et al.
      18F-Fluoride signal amplification identifies microcalcifications associated with atherosclerotic plaque instability in positron emission tomography/computed tomography images.
      ]. This allows 18F–NaF to detect active microcalcification area beyond the resolution of CT scan [
      • Irkle A.
      • Vesey A.T.
      • Lewis D.Y.
      • Skepper J.N.
      • Bird J.L.E.
      • Dweck M.R.
      • et al.
      Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography.
      ]. The increased surface area to volume ratio of micro-calcification relative to macro-calcification results in both increased and concentrated 18F tracer uptake in the former (Fig. 3). Recent evidence demonstrates an inverse correlation between plaque calcium density and tracer uptake, with lesions at the lower end of the Hounsfield unit coefficient exhibiting greater radioisotope accumulation whilst denser plaque with high calcium score had relatively lower 18F–NaF uptake [
      • Fiz F.
      • Morbelli S.
      • Piccardo A.
      • Bauckneht M.
      • Ferrarazzo G.
      • Pestarino E.
      • et al.
      1⁸F-NaF uptake by atherosclerotic plaque on PET/CT imaging: inverse correlation between calcification density and mineral metabolic activity.
      ]. This may explain the lack of correlation between 18F–NaF atherosclerotic plaque uptake and CAC score observed in high-risk individuals [
      • de Oliveira-Santos M.
      • Castelo-Branco M.
      • Silva R.
      • Gomes A.
      • Chichorro N.
      • Abrunhosa A.
      • et al.
      Atherosclerotic plaque metabolism in high cardiovascular risk subjects - a subclinical atherosclerosis imaging study with 18F-NaF PET-CT.
      ]. This suggests that computed tomography evidence of calcification and positron emission tomography evidence of 18F-sodium fluoride uptake represent two different markers of atherosclerosis. The former appears to be a surrogate marker of total plaque burden whilst the later may represent an active disease process and denote increased vulnerability.
      Fig. 3
      Fig. 3Culprit plaque imaging in a patient with anterior myocardial infarction.
      Invasive coronary angiography showing a culprit stenosis of the left anterior descending coronary artery (red arrow; A) in a patient with an anterior myocardial infarction. Intense focal 18 F-sodium fluoride uptake is observed at the site of the culprit plaque on the combined positron emission and computed tomography coronary angiogram (B).
      18F-Sodium fluoride has the potential to act as a marker of disease activity in the coronary vasculature. Several clinical studies have demonstrated uptake to be associated with culprit and high-risk coronary plaque as defined by invasive angiography (Fig. 4), intravascular ultrasound and CT coronary angiography [
      • Joshi N.V.
      • Vesey A.T.
      • Williams M.C.
      • Shah A.S.V.
      • Calvert P.A.
      • Craighead F.H.M.
      • et al.
      18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial.
      ,
      • Creager M.D.
      • Hohl T.
      • Hutcheson J.D.
      • Moss A.J.
      • Schlotter F.
      • Blaser M.C.
      • et al.
      18F-Fluoride signal amplification identifies microcalcifications associated with atherosclerotic plaque instability in positron emission tomography/computed tomography images.
      ,
      • Aikawa E.
      • Nahrendorf M.
      • Figueiredo J.-L.
      • Swirski F.K.
      • Shtatland T.
      • Kohler R.H.
      • et al.
      Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo.
      ]. Autoradiography of carotid endarterectomy specimens confirms localisation of 18F–NaF to the site of macroscopic plaque rupture [
      • Joshi N.V.
      • Vesey A.T.
      • Williams M.C.
      • Shah A.S.V.
      • Calvert P.A.
      • Craighead F.H.M.
      • et al.
      18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial.
      ]. Following acute myocardial infarction, increased 18F–NaF uptake was observed within the culprit plaque [
      • Joshi N.V.
      • Vesey A.T.
      • Williams M.C.
      • Shah A.S.V.
      • Calvert P.A.
      • Craighead F.H.M.
      • et al.
      18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial.
      ], a finding supported by subsequent smaller studies [
      • Marchesseau S.
      • Seneviratna A.
      • Sjöholm A.T.
      • Qin D.L.
      • Ho J.X.M.
      • Hausenloy D.J.
      • et al.
      Hybrid PET/CT and PET/MRI imaging of vulnerable coronary plaque and myocardial scar tissue in acute myocardial infarction.
      ]. In patients with stable disease, increased 18F–NaF activity localises to plaque with multiple adverse characteristics – including spotty calcification – on intra-vascular ultrasound [
      • Joshi N.V.
      • Vesey A.T.
      • Williams M.C.
      • Shah A.S.V.
      • Calvert P.A.
      • Craighead F.H.M.
      • et al.
      18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial.
      ,
      • Kitagawa T.
      • Yamamoto H.
      • Nakamoto Y.
      • Sasaki K.
      • Toshimitsu S.
      • Tatsugami F.
      • et al.
      Predictive value of 18F-sodium fluoride positron emission tomography in detecting high-risk coronary artery disease in combination with computed tomography.
      ]. In peripheral arterial disease, both inflammation, as measured by 18F-fluorodeoxyglucose uptake, and 18F–NaF uptake appear to predict subsequent restenosis following angioplasty [
      • Chowdhury M.M.
      • Tarkin J.M.
      • Albaghdadi M.S.
      • Evans N.R.
      • Le E.P.V.
      • Berrett T.B.
      • et al.
      Vascular positron emission tomography and restenosis in symptomatic peripheral arterial disease: a prospective clinical study.
      ]. Recent data suggest that coronary 18F–NaF uptake may also predict progression of coronary calcification in patient with established stable multivessel coronary artery disease.
      Fig. 4
      Fig. 4Multi-modality imaging of atherosclerotic plaque.
      Invasive coronary angiography demonstrating a moderate stenosis on the left anterior descending artery (blue arrow; A) with corresponding computed tomography coronary angiography confirming the presence of a heavily calcified plaque in the proximal left anterior descending coronary artery (B and C). Optical coherence tomography (D) demonstrating a sharp luminal border low-signal area (*) indicating an eccentric calcified plaque and a near-infrared spectroscopy-intravascular ultrasound (E) of the same vessel demonstrating a ring of calcium (red arrow) with signal drop out and corresponding chemogram demonstrating a lipid-rich core burden (yellow arc).
      These data suggest that 18F–NaF positron emission tomography-computed tomography is a potentially valuable tool in cardiovascular risk stratification. The question that remains to be answered is: can 18F–NaF signals provide additional risk prediction beyond clinical risk factor scores, blood biomarkers, and anatomic imaging? This is currently being addressed by the ongoing perspective PRE18FFIR trial (NCT02278211).

      4. Calcification as a therapeutic target

      Statins, an established preventative treatment strategy for coronary artery disease [
      • Heart Protection Study Collaborative Group
      MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial.
      ], appear to increase not decrease the CT calcium score [
      • Houslay E.S.
      • Cowell S.J.
      • Prescott R.J.
      • Reid J.
      • Burton J.
      • Northridge D.B.
      • et al.
      Progressive coronary calcification despite intensive lipid-lowering treatment: a randomised controlled trial.
      ,
      • Dykun I.
      • Lehmann N.
      • Kälsch H.
      • Möhlenkamp S.
      • Moebus S.
      • Budde T.
      • et al.
      Statin medication enhances progression of coronary artery calcification: the heinz nixdorf recall study.
      ]. The beneficial effects of statin are attributed to their effect on plaque stabilisation and slowing of plaque progression [
      • Nicholls S.J.
      • Hsu A.
      • Wolski K.
      • Hu B.
      • Bayturan O.
      • Lavoie A.
      • et al.
      Intravascular ultrasound-derived measures of coronary atherosclerotic plaque burden and clinical outcome.
      ,
      • Nissen S.E.
      • Nicholls S.J.
      • Sipahi I.
      • Libby P.
      • Raichlen J.S.
      • Ballantyne C.M.
      • et al.
      Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: the ASTEROID trial.
      ]. This is thought to be partly driven by the pro-calcific effects of statin therapy on coronary atheroma that is independent of their plaque-regressive effect [
      • Puri R.
      • Nicholls S.J.
      • Shao M.
      • Kataoka Y.
      • Uno K.
      • Kapadia S.R.
      • et al.
      Impact of statins on serial coronary calcification during atheroma progression and regression.
      ]. Coronary CT angiography studies have shown that initiation of statin therapy reduces progression of noncalcified plaque volume [
      • Hoffmann H.
      • Frieler K.
      • Schlattmann P.
      • Hamm B.
      • Dewey M.
      Influence of statin treatment on coronary atherosclerosis visualised using multidetector computed tomography.
      ,
      • Li Z.
      • Hou Z.
      • Yin W.
      • Liu K.
      • Gao Y.
      • Xu H.
      • et al.
      Effects of statin therapy on progression of mild noncalcified coronary plaque assessed by serial coronary computed tomography angiography: a multicenter prospective study.
      ]. This may reflect a healing response to statins and highlights a key limitation of CAC scoring, which appears to target a stable form of plaque that itself is not prone to rupture and is unlikely to trigger clinical events. Statins appear to reduce cardiovascular events in conjunction with a reduction in inflammatory markers such as circulating C-reactive protein and pro-inflammatory cytokines [
      • Asher J.
      • Houston M.
      Statins and C-reactive protein levels.
      ].
      In chronic inflammatory conditions, such as psoriasis and rheumatoid arthritis, higher CAC scores are associated with increased clinical and biochemical markers of active inflammation [
      • Wahlin B.
      • Meedt T.
      • Jonsson F.
      • Henein M.Y.
      • Wållberg-Jonsson S.
      Coronary artery calcification is related to inflammation in rheumatoid arthritis: a long-term follow-up study.
      ]. It may be reasonable therefore to assume that drugs targeting inflammation, such as tumour necrosis factor alpha antagonists, would result in decreased cardiovascular events. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) has recently shown that interleukin-1β inhibition confers a reduced risk of athero-thrombotic events [
      • Ridker P.M.
      • Thuren T.
      • Zalewski A.
      • Libby P.
      Interleukin-1β inhibition and the prevention of recurrent cardiovascular events: rationale and design of the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS).
      ]. It is unclear whether these cardiovascular benefits are due to an increase in stable coronary plaque calcifications over time.
      Warfarin may accelerate coronary plaque calcification, but unlike statin, it may shift atherosclerotic plaques toward a vulnerable phenotype as demonstrated by intimal microcalcifications[
      • Schurgers L.J.
      • Joosen I.A.
      • Laufer E.M.
      • Chatrou M.L.L.
      • Herfs M.
      • Winkens M.H.M.
      • et al.
      Vitamin K-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype.
      ]. In addition to its effects on vitamin K–dependent coagulation factors, animal studies show that warfarin also inhibits vitamin K–dependent extrahepatic proteins, such as vascular smooth muscle cell–derived matrix Gla-protein (MGP) which normally suppresses calcification of arteries [
      • Schurgers L.J.
      • Uitto J.
      • Reutelingsperger C.P.
      Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization.
      ,
      • Roumeliotis S.
      • Dounousi E.
      • Eleftheriadis T.
      • Liakopoulos V.
      Association of the inactive circulating matrix Gla protein with vitamin K intake, calcification, mortality, and cardiovascular disease: a review.
      ]. Large clinical trials are needed to assess the effects of long-term warfarin use on clinical events in patients with coronary heart disease and assess its impact on vascular calcification. Similarly, preliminary data suggest that vitamin K supplementation may slow the progression of CAC [
      • Shea M.K.
      • O'Donnell C.J.
      • Hoffmann U.
      • Dallal G.E.
      • Dawson-Hughes B.
      • Ordovás J.M.
      • et al.
      Vitamin K supplementation and progression of coronary artery calcium in older men and women.
      ]. However, further research is needed to explore the potential preventative role of vitamin K supplementation in atherosclerotic disease and specifically atherosclerotic calcification.
      No study of high-risk plaque identification has yet demonstrated incremental prognostic benefit over and above the total calcium score [
      • Williams M.C.
      • Moss A.J.
      • Dweck M.
      • Adamson P.D.
      • Alam S.
      • Hunter A.
      • et al.
      Coronary artery plaque characteristics associated with adverse outcomes in the SCOT-heart study.
      ] which remains the most powerful predictor of risk to date. However, evidence relating to the effect of routine medical therapy highlight the calcium paradox and demonstrate that coronary calcium measurements may not accurately reflect the progression of atherosclerotic disease. As previously discussed, the clinical benefit of preventative therapy with statins surpasses their lipid lowering effects and is in contradiction to their impact on the overall calcium score. This raises question regarding whether attenuation of coronary artery calcification progression is a useful therapeutic goal. Can coronary calcification predicts plaque instability or is merely a marker of plaque burden?
      Coronary artery calcification may in fact be protective and may impede further progression of high-risk, low-density plaque. Are current treatment strategies targeting the correct type of calcium? Halting progression of coronary calcification with more intensive modification therapy is perhaps not the most cost-effective way to improve outcomes. Imaging biomarkers targeting the early biopathological steps in atherosclerotic calcification and quantifying active microcalcifications may more accurately reflect the “vulnerable” stage of plaque progression. Large prospective clinical trials comparing their prognostic benefit with established measures of coronary calcification, namely the coronary calcium score, may provide some answers.

      5. Conclusion

      The presence of calcium has long been seen as pathognomonic of atherosclerosis and is a well-established predictor of cardiac risk. Early detection of coronary calcification in younger subjects has important prognostic impact on cardiovascular risk prediction [
      • Carr J.J.
      • Jacobs D.R.
      • Terry J.G.
      • Shay C.M.
      • Sidney S.
      • Liu K.
      • et al.
      Association of coronary artery calcium in adults aged 32 to 46 Years with incident coronary heart disease and death.
      ]. Rapid advances in non-invasive cardiovascular imaging now allow assessment of the morphological coronary calcification as well as the underlying early biological changes and activity of calcification (Fig. 5). While this has provided important pathophysiological insights, further research is now required to investigate whether these novel approaches provide any incremental clinical information beyond standard patient assessments, and what the impact would be on interfering with the calcific process.
      Fig. 5
      Fig. 5Atherosclerotic vascular calcification.
      The relationship between the morphological transformation of coronary calcification and plaque progression highlighting the role of current and emerging imaging modalities at each stage in assess different aspects of coronary calcification. OCT – optical coherence tomography, IVUS - intravascular ultrasound, VH-IVUS – virtual histology intravascular imaging, 18F–NaF-PET – 18F-sodium fluoride positron emission tomography, CTA – CT angiography, CAC- coronary artery calcium score, AIT-adaptive intimal thickening, PIT-pathological intimal thickening.

      Declaration of competing interest

      The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

      Acknowledgements

      MD and DEN are supported by a Wellcome Trust Senior Investigator Award ( WT103782AIA ). DEN is supported by the British Heart Foundation ( CH/09/002 , RG/16/10/32375 , RE/18/5/34216 ).

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