Laurent M Riou
email: <laurent.riou@ujf-grenoble.fr

Alexis Broisat

Julien Dimastromatteo

G Pons

Daniel Fagret

Catherine Ghezzi

Radiopharmaceutiques Biocliniques

Pre-clinical and clinical evaluation of nuclear tracers for the molecular imaging of vulnerable atherosclerosis: an overview

Keywords: Atherosclerosis, diagnosis, immunology, Diagnostic Imaging, methods, Evaluation Studies as Topic, Humans, Nuclear Medicine, methods, Positron-Emission Tomography, methods atherosclerosis, vulnerable coronary plaque, nuclear molecular imaging

Cardiovascular diseases (CVD) are the leading cause of mortality worldwide. Despite major advances in the treatment of CVD, a high proportion of CVD victims die suddenly while being apparently healthy, the great majority of these accidents being due to the rupture or erosion of a vulnerable coronary atherosclerotic plaque. A non-invasive imaging methodology allowing the early detection of vulnerable atherosclerotic plaques in selected individuals prior to the occurrence of any symptom would therefore be of great public health benefit. Nuclear imaging could allow the identification of vulnerable patients by non-invasive in vivo scintigraphic imaging following administration of a radiolabeled tracer. The purpose of this review is to provide an overview of radiotracers that have been recently evaluated for the detection of vulnerable plaques together with the biological rationale that initiated their development. Radiotracers targeted at the inflammatory process seem particularly relevant and promising. Recently, macrophage targeting allowed the experimental in vivo detection of atherosclerosis using either SPECT or PET. A few tracers have also been evaluated clinically. Targeting of apoptosis and macrophage metabolism both allowed the imaging of vulnerable plaques in carotid vessels of patients. However, nuclear imaging of vulnerable plaques at the level of coronary arteries remains challenging, mostly because of their small size and their vicinity with unbound circulating tracer. The experimental and pilot clinical studies reviewed in the present paper represent a fundamental step prior to the evaluation of the efficacy of any selected tracer for the early, non-invasive detection of vulnerable patients.

% [START_REF] Fuster | Atherothrombosis and high-risk plaque: part I: evolving concepts[END_REF] of a coronary event is therefore of great clinical relevance since it would allow the early identification and management of those patients at high cardiovascular risk . [START_REF] Naghavi | From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I[END_REF] Atherosclerosis is a chronic inflammatory disease in which oxidized Low Density Lipoproteins (oxLDL) initiate the inflammatory process at sites of endothelial dysfunction , . Atherosclerotic plaques initially develop excentrically rather than concentrically , [2 4 -6 ] [START_REF] Glagov | Compensatory enlargement of human atherosclerotic coronary arteries[END_REF] and non-or moderately obstructive plaques are responsible for the majority of acute coronary events . An increased risk of coronary [START_REF] Franco | Coronary intravascular ultrasound: implications for understanding the development and potential regression of atherosclerosis[END_REF] event is therefore not exclusively correlated with the degree of stenosis of any given plaque but also with the probability for a non-occlusive lesion to rupture or erode , . Since coronary angiography only provides information about vessel lumen,

[3 9 ]
non-obstructive vulnerable plaques cannot be detected using this technique. Imaging modalities such as intravascular ultrasound or optical coherence tomography are currently being evaluated for vulnerable plaque detection . However, the invasive nature of these [START_REF] Schaar | Current diagnostic modalities for vulnerable plaque detection[END_REF] techniques limits their use to previously identified high-risk patients . There is therefore a clinical need for a non-invasive molecular [START_REF] Naghavi | From vulnerable plaque to vulnerable patient: Part III: Executive summary of the Screening for Heart Attack Prevention and Education (SHAPE) Task Force report[END_REF] imaging modality allowing the screening of a large population for the detection of vulnerable coronary plaques. However, no such technique is currently available for routine clinical practice . Among the various techniques that are currently being evaluated, nuclear [START_REF] Davies | Radionuclide imaging for the detection of inflammation in vulnerable plaques[END_REF] imaging is a highly sensitive non-invasive imaging modality .

[13 ]

The recent development of high-resolution, high-sensitivity small animal dedicated imaging systems has greatly helped for the [START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF] pre-clinical in vivo evaluation of a number of new potential tracers targeted at key steps of the atherogenic process leading to vulnerable plaques. The present paper will provide an overview of radiotracers that have been experimentally evaluated for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) imaging together with the biological rationales that justified their developments, with emphasis on the most promising agents.

Depending on the stage of its development and on its constituents, atherosclerotic plaques are classified into six different categories according the American Heart Association (AHA) classification . However, since the AHA classification was defined before the [15 -17 ] concept of vulnerable plaque emerged, further modifications have been proposed .

[18 ]

Initiation

Atherosclerotic lesions predominantly appear at sites of endothelial dysfunction, a phenomenon which in turn has been described at sites of arterial curvatures and bifurcations where low and oscillatory endothelial shear stress occurs , . Under such conditions [6 19 ] responsive elements are activated, leading to endothelial gene expression responsible for a large spectrum of modifications among which intimal thickening, defined as the proliferation of SMCs in the intima, and decreased nitric oxide (NO) synthesis. Not only is NO a key regulator of vascular tone but it also is a powerful anti-inflammatory, anti-mitotic and anti-thrombotic compound . In addition, [START_REF] Harrison | Endothelial mechanotransduction, nitric oxide and vascular inflammation[END_REF] endothelial dysfunction is characterized by increased reactive oxygen species (ROS) production. The increased endothelial permeability together with ROS production are responsible for the accumulation of immunogenic oxLDL in the subendothelial space. Moreover, activation of the transcriptional nuclear factor NFKB leads to the activation of multiple pro-immunogenic genes whose products are involved in the recruitment of leucocytes . Adhesion molecules, such as the E-selectin and the intercellular and vascular adhesion [START_REF] Collins | NF-kappaB: pivotal mediator or innocent bystander in atherogenesis?[END_REF] molecules ICAM-1 and VCAM-1, as well as the chemokines monocyte chemoattractant protein- Factor (MCSF) induces the differentiation of recruited monocytes into macrophages. These activated macrophages express the scavenger receptors SR-A, CD36 and LOX-1 on their surface . These receptors are involved in the recognition and uptake of oxLDL.

[ 24 -26 ] Ultimately, the intracytoplasmic accumulation of lipid droplets within macrophages results in the formation of foam cells. Moreover, Lectin-like OxLDL receptor-1 (LOX-1) is also expressed by the endothelium, smooth muscle cells (SMC) and platelets, and oxLDL binding to LOX-1 leads to intracellular signaling resulting in ROS-and NFKB-induced endothelial dysfunction as well as in apoptosis

Advanced Atherosclerotic Plaques

Macrophages and foam cells synthetize pro-inflammatory cytokines such as MCP-1. In addition, macrophages interact with T1 and T2 lymphocytes ,

. The interaction between CD40 and its ligand CD40L plays a major role in the interaction between macrophages and [4 29 ] lymphocytes, as well as between those inflammatory cells and SMCs , . As a result, the immune response is greatly amplified. [30 31 ] Among the various cytokines synthesized at this stage of atherosclerosis development, interferon-gamma (IFN ) stimulates the γ proliferation of SMCs already present in areas of intimal thickening. In order for macrophages, lymphocytes and SMCs to migrate within the intima and media, these cells release metalloproteinases (MMPs) that digest the extracellular matrix (collagenase, gelatinases, elastases and stromelysins) , . Those three cell types also produce tissue factor, a small pro-thrombotic compound that plays a major role in [32 33 ] thrombus formation when exposed to the blood stream at the time of plaque rupture . Macrophage-and SMC-derived foam cells [START_REF] Steffel | Tissue factor in cardiovascular diseases: molecular mechanisms and clinical implications[END_REF] eventually undergo apoptosis as a result of intracellular oxLDL toxicity and therefore participate to the formation of an acellular lipidic and necrotic core , . SMCs exert different roles in advanced atherosclerotic plaques. On one hand and as mentioned above, SMCs [ 29 35 ] produce MMPs and ingest oxLDL, thereby participating in plaque destabilisation and to foam cell formation. On the other hand, SMCs synthesize the fibrous cap that covers the lipidic and necrotic core, therefore stabilizing the plaque , . It has become clear that the [36 37 ] cellular and extracellular composition of the plaque represent a primary determinant of plaque stability. Prone-to-rupture vulnerable plaques are characterized by a large lipidic and necrotic core (>40 ) covered by a fine fibrous cap (<65 m) and by intense inflammatory

% μ
processes whereas more stable plaques are characterized by a minimal lipidic and necrotic core and a thick fibrous or calcified cap , .

[3 9 ] Because of arterial wall remodeling, blood diffusion from the arterial lumen or blood supply from the preexisting vaso vasorum is no longer sufficient and angiogenesis is necessary. Neovessels originate from the existing vasa vasorum in the adventitia and extend within the atherosclerotic lesion in the media. Angiogenesis participates in lesion development by extracellular matrix destabilisation as well as by acting as a pathway for LDL diffusion, mononuclear leucocytes recruitment, and red blood cell extravasation .

[ 38 -40 ] AHA type IV lesions are characterized by a large lipidic and necrotic core resulting from the fusion of droplets observed in type III plaques. Type Va lesions are characterized by a large lipidic and necrotic core covered by a fibrous cap. Calcified type V plaques are called Vb whereas type V plaques with minimal lipidic and necrotic core and extensive fibrosis are classified as Vc. Type V plaques might become occlusive and cause unstable angina. There is no specific category for the vulnerable thin cap fibroatheroma. This type of vulnerable plaque belongs either to type IV or V lesion .

[ 15 -17 ] Thrombus Formation

Following plaque rupture, a number of thrombogenic factors influence the magnitude and stability of the thrombus and therefore the development of atherosclerotic lesions and the clinical severity of the coronary syndrome. In order to take into account the key role of thrombosis, the concepts of atherosclerosis and thrombosis have therefore been unified in the term atherothrombosis .

[2 ]

In approximately two third of the cases, thrombus formation occurs at the site of plaque rupture, the remaining thrombotic events being due to erosion of a vulnerable plaque . The combination of weak plaque composition as usually observed at the shoulder of [START_REF] Burke | Coronary risk factors and plaque morphology in men with coronary disease who died suddenly[END_REF] plaques (i.e. thin fibrous cap, intensive macrophages infiltration, matrix degradation and apoptosis) with an increase in the external circumferential and shear stresses are held responsible for the rupture of vulnerable plaques. The amplitude of thrombus formation depends on various factors including the rheology and the concentration of pro-thrombotic factors present within the ruptured exposed plaque as well as in the blood . The thrombus might be occlusive or not, and might cause clinical symptoms. Acute occlusion at the site of plaque [START_REF] Fuster | Atherothrombosis and high-risk plaque: part I: evolving concepts[END_REF] rupture or downstream can be responsible for myocardial infarction.

Complicated Atherosclerotic Plaques

Finally, AHA type VI atherosclerotic lesions result from the complication of type IV or type Va lesions following plaque rupture/erosion or intra-plaque haemorrhage . Following culprit lesion healing and thrombus inclusion, the plaque might then [15 -17 ] evolve into a fibrous and potentially stenotic lesion. Alternatively, new episodes of rupture and thrombus formation can also occur.

Nuclear Imaging of Vulnerable Plaques

The non-invasive imaging modalities currently under evaluation for the detection of vulnerable plaques can be divided into morphologic techniques such as magnetic resonance or computed tomography, and molecular imaging techniques such as nuclear medicine or ultrasound. While exquisite resolution is required for morphologic techniques, the very high sensitivity of molecular nuclear imaging allows to distinguish a vulnerable from a stable plaque based on the detection of a specific molecular target. Potential radiotracers for the molecular imaging of vulnerable atherosclerosis are compounds targeted at key molecules or processes involved in atherogenesis.

Such agents are radiolabeled either with -or -emitters, allowing the non-invasive imaging of their biodistribution using either SPECT

γ β +
or PET cameras, respectively. A brief overview of the radiotracers that have been evaluated so far for the detection of atherosclerotic lesions is provided in . Table 1

Low Density Lipoprotein Imaging

Countless studies indicated that native LDLs and oxLDLs play a critical role in the pathogenesis of unstable atherosclerosis . the carotid, iliac, and femoral vessels of 4/17 patients with atherosclerosis. However, the slow kinetics of LDL accumulation together with the high circulating tracer activity precluded in vivo image acquisition. As oxLDLs are predominantly implicated in the processes leading to vulnerable plaque formation, some authors performed in vitro oxidation of native LDLs with copper and hydrogen peroxide prior to radiolabeling using Tc . Tc -oxLDLs demonstrated better tissue accumulation and more favorable blood kinetics than 99m [START_REF] Iuliano | Preparation and biodistribution of 99m technetium labelled oxidized LDL in man[END_REF] [ 99m ] radiolabeled native LDL. In 7 patients with transient ischemic attacks, Tc -oxLDLs allowed carotid plaque imaging in 10 out of 11

[ 99m ]
lesions with target-to-background ratios of ~1.5 . Hardoff et al. have attempted in vivo imaging of aortic atherosclerosis in Watanabe [START_REF] Iuliano | Preparation and biodistribution of 99m technetium labelled oxidized LDL in man[END_REF] Heritable Hyper-Lipidemic (WHHL) rabbits with a radiolabeled synthetic oligopeptide fragment of apolipoprotein B, SP-4 , . . The authors demonstrated that intravenously injected, radiolabeled oxidation-specific antibodies (Ox-AB), such as the [START_REF] Tsimikas | Radiolabeled MDA2, an oxidation-specific, monoclonal antibody, identifies native atherosclerotic lesions in vivo[END_REF] murine monoclonal antibody (mAb) MDA2, which detects malondialdehyde (MDA) lysine epitopes, have a strong and specific predilection for atherosclerotic lesions but not for normal arteries. Aortic uptake of I -MDA2 was enhanced in lipid-rich lesions of

[ 125 ]
LDLR mice fed a western-type diet whereas it was reduced in the lesions of animals on antioxidant diets .

Tc -labeled MDA2 / -- [54 ] [ 99m ]
allowed the non-invasive imaging of atherosclerotic lesions in 4 out of 7 WHHL rabbits , . However, the slow blood kinetics of [53 55 ] radiolabeled MDA2 required an extensive period of time between tracer injection and image acquisition together with the injection of MDA-LDL in order to minimize the radiolabeled MDA2 circulating activity. The phage display method has also been used to identify human monoclonal antibodies against oxidation-specific epitopes of oxLDL. Various sequences have been described, among which IK17 specifically bound MDA-LDL, intact oxLDL but not native LDL. Intravenous injection of I -IK17 into hypercholesterolemic LDLR conjugation of the soluble dimeric form of the scavenger receptor CD68 to a human Fc-fragment could be used to perform autoradiographic imaging of oxLDL accumulation in atherosclerotic lesions from hypercholestrolemic ApoE mice following / -radiolabeling with the PET-suitable isotope I .

124 [START_REF] Langer | Radionuclide imaging: a molecular key to the atherosclerotic plaque[END_REF] LOX-1 is a scavenger receptor expressed by endothelial cells, monocytes, macrophages, smooth muscle cells, and platelets which displays picomolar affinity for oxLDL. The role of LOX-1 in the initiation, progression, and destabilization of atherosclerotic plaques has been recently described , . In human carotid atherosclerotic lesions, LOX-1 expression has been observed in luminal endothelial [59 26 ] cells from early atherosclerotic lesions as well as in intimal endothelial cells from the neovasculature and in macrophages and smooth muscle cells from advanced atherosclerotic lesions . As shown in , Ishino S. et al recently described the experimental in myocardial infarction prone Watanabe heritable hyperlipidemic WHHLMI rabbits. The authors showed that despite a poor correlation

- [ ]
between tracer activity and LOX-1-expressing macrophages on autoradiographic and immunohistologic images, respectively, Tc

[ 99m ]
-LOX-1-mAb allowed the non-invasive planar imaging of atherosclerosis at 24 hours following intravenous injection with robust differential uptake ratios (DURs). Tc -LOX-1-mAb DURs were 2.4-and 3.2-fold higher than F -FDG DURs in the thoracic and

[ 99m ] [ 18 ]
abdominal aortic segments of WHHL rabbits, respectively , and 5.7-and 3.7-fold higher than Tc -Annexin A5 DURs in the

[62 ] [ 99m ]
thoracic and abdominal aortic segments of WHHLMI rabbits, respectively . However, as usually observed when evaluating

[63 ]
radiolabeled antibodies, high levels of circulating tracer probably accounted for the relatively low lesion-to-blood ratios, which might be improved by evaluating alternative strategies aimed at accelerating Tc -LOX-1-mAb blood clearance.

[ 99m ]

Inflammation Imaging

Monocyte Chemoattractant Protein-1 (MCP-1)

Circulating monocytes are attracted to the site of atherosclerotic plaque development by MCP-1, a chemokine which is produced by endothelial cells, smooth muscle cells and monocytes/macrophages over the course of atherosclerotic plaque formation. MCP-1 binds to the CCR-2 receptor on the cellular membrane of monocytes/macrophages . I-radiolabeled MCP-1 has therefore been evaluated for [START_REF] Kinlay | Endothelial function and coronary artery disease[END_REF] 125 the molecular imaging of atherosclerotic plaque following injection into hypercholesterolemic rabbits with arterial injury-induced atherosclerotic lesions. A fast blood clearance was observed with a halflife of ~10 minutes. Although in vivo imaging was not attempted due to the suboptimal image quality obtained when using I-labeled tracers, autoradiograms revealed I -MCP-1 accumulation in the 125

[ 125 ]
damaged artery wall which was correlated with the number of infiltrated macrophages . MCP-1 has been recently radiolabelled with

[64 ]
Tc and injected into rabbits with atherosclerotic lesion induced by balloon deendothelialization of the abdominal aorta . Ex vivo 99m [START_REF] Hartung | Radiolabeled Monocyte Chemotactic Protein 1 for the detection of inflammation in experimental atherosclerosis[END_REF] imaging indicated that Tc -MCP-1 uptake in atherosclerotic lesions was 4-fold higher than that observed in the vessels of control

[ 99m ]
animals. A strong correlation between Tc -MCP-1 and macrophage density was also observed, and tracer uptake was detected at the imaging of vulnerable atherosclerotic plaques.

[ 18 F -FDG ] Atherosclerotic plaque inflammation was imaged in patients with symptomatic carotid atherosclerosis using the glucose analog F

[ 18 ]
-FDG by Rudd et al.

, and the authors recently demonstrated the reproducibility of the methodology . F -FDG accumulated into

[66 ] [67 ] [ 18 ]
macrophage-rich areas of the plaque, and symptomatic, unstable plaques accumulated more F -FDG than did asymptomatic lesions.

[ 18 ]

Experimental and clinical studies have since then confirmed that the vascular uptake of the tracer is correlated with macrophage content, but not with smooth muscle cells or vascular wall thickness, and that the most likely mechanism for F -FDG accumulation into Based on these studies, F -FDG has been used to evaluate the prevalence of inflammation in documented carotid atherosclerosis increased inflammation, in carotid atherosclerosis. F -FDG has also been successfully used to monitor the effect of lipid-lowering and

[ 18 ]
anti-oxidant treatments on plaque inflammation , . The only controversial study with regards to F -FDG uptake in atheroscletoric

[73 74 ] [ 18 ]
plaque was that of Laurberg et al . The authors did not observe increased aortic and carotid tracer accumulation in [START_REF] Laurberg | Imaging of vulnerable atherosclerotic plaques with FDG-microPET: no FDG accumulation[END_REF] hypercholesterolemic, apoE mice neither by in vivo PET imaging nor by -well counting of excised vessels. The discrepancy between / -γ the results of this study and others remains unexplained . Strong evidence is therefore now available to suggest that F -FDG carotid

[76 ] [ 18 ]
imaging is a sensitive and reproducible method for the detection of plaque inflammation. However, a potential limitation of coronary atherosclerotic plaque imaging with F -FDG is the high myocardial background uptake of the tracer which might hamper the

[ 18 ]
signal-to-background ratio. Positron-sensitive probes are being evaluated in the setting of phantom, ex vivo, or in vivo studies. Although tracer detection would now be performed invasively, initial results suggested the feasibility of intravascular F -FDG detection in vessels

[ 18 ]
1 mm in diameter . transport mechanisms, phosphorylated by choline kinase, metabolized into phosphatidylcholine, and eventually incorporated into the cell membrane. Increased choline uptake has been observed in activated macrophages , and a correlation was observed between F

[83 ] [ 18 ]
-Choline uptake and macrophage accumulation in experimental models of soft tissue infection and acute cerebral radiation injury vivo imaging, F -Choline uptake correlated better with lipid-positive and macrophage-positive areas than C -FDG in

[ 18 ] [ 14 ]
hypercholesterolemic, ApoE mice . However, PET imaging failed to detect F -Choline uptake in atherosclerotic lesions in vivo. Further studies are therefore warranted to clarify the potential of F -Choline for the in vivo imaging of macrophage accumulation in

[ 18 ]
atherosclerotic plaques.

Monocytes/Macrophages Cellular Imaging

Two recent studies focused on monocyte/macrophage cellular imaging. First, Kircher et

Vascular Cell Adhesion Molecule -1 (VCAM-1)

Endothelial cells express specific molecules when subjected to pro-atherogenic conditions . Among those, VCAM-1 plays a major [START_REF] Kinlay | Endothelial function and coronary artery disease[END_REF] role in the recruitment of inflammatory cells such as monocytes and lymphocytes to developing atherosclerotic plaques . In advanced [START_REF] Ley | VCAM-1 is critical in atherosclerosis[END_REF] lesions, VCAM-1 is also expressed at the level of neovessels as well as by SMCs and macrophages from the media .

[38 ]

To date, a few studies have addressed the use of VCAM-1 as a target for the molecular imaging of vulnerable plaques. Using intravascular ultrasound and echogenic immunoliposomes (ELIPSs) targeted at VCAM-1, Hamilton and colleagues demonstrated a significant increase in mean gray scale values on endothelial images of injured carotid arteries from Yucatan miniswines when compared to images obtained with non-specific ELIPs . More recently, in vitro phage display was used by Kelly et al. to select a VCAM-1 [START_REF] Hamilton | Intravascular ultrasound molecular imaging of atheroma components in vivo[END_REF] specific peptide sequence that was used for the production of magnetofluorescent nanoparticles . Although ex vivo images of [START_REF] Kelly | Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle[END_REF] VCAM-1 expression were performed, this agent turned out to be suboptimal for in vivo imaging. Using in vivo phage display in apoE / -mice, the authors then described a peptide sequence highly homologous to the known VCAM-1 ligand VLA-4 which displayed an improved affinity for VCAM-1. This peptide was used for the production of a second-generation agent detectable through MRI and optical imaging which provided very promising results with respect to the in vivo imaging of variable levels of VCAM-1 expression . Finally, [START_REF] Nahrendorf | Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis[END_REF] Kaufmann et al. recently described the feasibility of the in vivo monitoring of VCAM-1 aortic expression of control and hypercholesterolemic mice fed normal or high-fat diets using contrast echocardiography and VCAM-1-targeted microbubbles .

[92 ]

We recently described the ex vivo imaging of VCAM-1 expression in hypercholesterolemic rabbits using a Tc-labeled version of 99m the HLA class I-derived peptide B2702 that was previously shown to bind specifically to VCAM-1 . Moreover, in vivo imaging

[93 ] [93 ]
of VCAM-1 expression in a mouse model of atherosclerotic plaque development induced by left carotid artery ligation in apoE animals / -was performed using a peptidic derivative of Tc -B2702 .

[ 99m ]

[95 ]

Matrix Metalloproteinases (MMPs) Imaging

MMPs are involved in the remodeling of connective tissue. In the setting of atherogenesis, MMPs are secreted by various cell types [ , . Macrophages represent the predominant source of MMPs over the course of vulnerable plaque development. MMP inhibitors 32 33 ] (MMPIs) bind to MMPs with high affinity. Several hydroxamate-derivatives with MMP inhibitory properties in the nanomolar range have been synthetized (HO-CGS 27023A 1a-1i) and radiolabelled using iodine-123 ( I -HO-CGS 27023A 1a-1i) in order to evaluate their

[ 123 ]
potential for the molecular imaging of atherosclerotic plaques . I -HO-CGS 27023A has been injected in an experimental mouse

[96 ] [ 123 ]
model of carotid arterial remodeling induced by carotid artery ligation and characterized by macrophage infiltration and MMP overexpression. I -HO-CGS 27023A in vivo accumulation in the carotid lesion was inhibited by pretreatment with an excess of

[ 123 ]
unlabeled compound and this specific uptake was readily detected by non-invasive planar imaging . CGS 27023A together with

[97 ]
alternative hydroxamate-derivatives were recently labeled with F for PET imaging , .

18

[98 99 ]
Preliminary biodistribution studies indicated that there was no tissue-specific accumulation of F -CGS 27023A 1f and F -CGS

[ 18 ] [ 18 ]
27023A 1e in wild-type mice. The preparation of non-hydroxamate derivatives MMPIs has also been described recently . It is [100 ] expected that on-going studies will quickly determine whether these new agents have potential for the molecular imaging of MMP expression in atherosclerotic plaques.

Apoptosis Imaging

Annexin A5

Kolodgie et al. first demonstrated the feasibility of non-invasive imaging of apoptosis using Tc -Annexin A5 in

[101 ] [ 99m ]
experimental atherosclerotic lesion induced by deendothelialization of the infradiaphragmatic aorta in rabbits fed a high-cholesterol diet.

The rapid blood kinetics of the tracer allowed in vivo planar imaging to be performed 2 hours following injection, and a positive correlation was observed between Tc -Annexin A5 uptake and macrophage burden or apoptosis but not with SMC. The feasibility of

[ 99m ]
in vivo planar imaging of apoptosis with Tc -Annexin A5 was confirmed in an experimental model of genetically

[ 99m ]
hypercholesterolemic rabbits . Johnson et al. provided in vivo SPECT images of Tc -Annexin A5 uptake in injured coronary

[63 ] [102 ] [ 99m ]
arteries of swines also fed a high-cholesterol diet, a model characterized by predominant smooth muscle cell apoptosis. Thirteen out of 22 injured vessels showed focal tracer uptake, and scan-positive vessels had a significantly higher apoptotic index than scan-negative vessels.

Dedicated small animal imaging equipment recently allowed tomographic imaging of aortic atherosclerotic lesions in mouse models of plaque development as demonstrated in . Several experimental studies indicated that variations in the apoptotic status of showed that dietary modification

[ 99m ] [104 ]
and statin therapy decreased Tc -Annexin A5 uptake by ex vivo imaging and -well counting in a rabbit model of atherosclerosis

[ 99m ] γ
induced by deendothelialization of the abdominal aorta. The inhibitory effect of broad and specific caspase inhibition on apoptosis as assessed with Tc -Annexin A5 in vivo SPECT imaging was also recently studied on a similar experimental model . Tc

[ 99m ] [105 ] [ 99m ]
-Annexin A5 and F -FDG were recently compared in an autoradiographic study for the detection of aortic atherosclerosis in ApoE

[ 18 ] / --
mice fed a hypercholesterolemic diet. Although the absolute uptake levels of F -FDG in atherosclerotic lesions were 5-to 6-fold higher,

[ 18 ]
the uptake ratios of advanced-to-early lesions were significantly higher with Tc -Annexin A5 than with F -FDG. Therefore, Tc

[ 99m ] [ 18 ] [ 99m ]
-Annexin A5 might possibly have a greater potential than F -FDG as an indicator of late stage, vulnerable plaques as compared with

[ 18 ]
early lesions . Phase I clinical studies indicated that the biodistribution and dosimetry of Tc -Annexin A5 were suitable for

[106 ] [ 99m ]
apoptosis imaging in the abdominal and thoracic area of patients . In a pilot clinical study, Kietselaer et al. evaluated Tc

[107 ] [108 ] [ 99m ]
-Annexin A5 carotid uptake in patients. The results indicated that tracer uptake was visible in those patients with a recent, but not remote, history of transient ischemic attack, and that Tc -Annexin A5 accumulation correlated with histopathological features of unstable

[ 99m ]
plaques at endarterectomy.

Additional Apoptosis Imaging Agents

Although Tc -Annexin A5 remains the most largely studied tracer of apoptosis, alternative peptidic and non-peptidic agents have

[ 99m ]
been described . The behavior of these new compounds warrant further investigation.

[109 -113 ]

Additional Molecular Targets for Vulnerable Atherosclerosis Imaging

Endothelin Receptors

Since endothelins have been implicated in the pathogenesis of atherosclerosis, endothelin derivatives seemed promising agents for atherosclerosis imaging through binding to endothelin receptors , . However, further characterization of 2 rabbit models of [114 115 ] atherosclerosis demonstrated that neither the density of overall endothelin receptors nor the density of either endothelin receptor subtype was significantly increased in atherosclerotic aortas. Therefore, the authors concluded that endothelin receptor was not a suitable target for atherosclerosis imaging . Therefore, the authors concluded that endothelin receptor was not a suitable target for atherosclerosis [116 ] imaging .

[116 ]

Angiogenesis

As described above, angiogenesis has been recently recognized as a major contributor to vulnerable plaque development . An [START_REF] Moreno | Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability[END_REF] autoradiographic study indicated that the v 3 integrin specific tracer In -RP748 preferentially localized to the atherosclerotic lesion

α β - [ 111 ]
in a hypercholesterolemic mouse model of left carotid artery injury .

[117 ]

The extra-domain B (ED-B) is inserted into the fibronectin molecule during angiogenesis. Matter et al. used the human antibody

[118 ]
L19 specific against ED-B to perform autoradiographic imaging of plaque neoangiogenesis in aortas of hypercholesterolemic mice. The authors also compared the behaviour of I -L19 with that of I -G11, a radiolabeled antibody specific against the C domain of

[ 125 ] [ 125 ]
tenascin-C, which is overexpressed in atherosclerotic plaques and absent from normal adult tissues . Both of these agents seemed to [119 ] have potential for imaging atherosclerosis providing that enough time is allowed for blood clearance, but no in vivo imaging study is yet available.

Antisense Oligonucleotide Strategy

Finally, recent studies have suggested that different signaling pathways and various cell transcription factors are activated during the early stage of the atherosclerotic process, including proto-oncogenes such as c-fos and c-myc , . The activation of [120 121 ] proto-oncogenes over the course of atherosclerosis may result in an increase in corresponding mRNA levels, which might represent a suitable target for imaging using radiolabelled antisense oligonucleotides (ASONs).

Tc -labeled ASONs directed against the

[ 99m ]
proto-oncogene c-myc were therefore injected to New Zealand White rabbits fed an high-cholesterol diet. The accumulation of the tracer in the vascular wall correlated with areas of plaque development and could be detected by in vivo planar imaging . However, the

ASON strategy remains challenging because of the complexity of in vivo probe delivery. This can mainly be attributed to the low amout of target and to the numerous biological barriers to overcome while maintaining the stability of the probe [123 ]

CONCLUSION

Over the last decade several radiotracers have been evaluated for the in vivo molecular imaging of vulnerable atherosclerotic plaques.

Radiotracers targeted at the inflammatory process seem particularly relevant and promising. Tc -MCP-1 , for SPECT imaging

[ 99m ]
[64 65 ] as well as F -FDG for PET imaging both demonstrated the feasibility of in vivo imaging of macrophage recruitment and

[ 18 ] [62 ]
metabolism within atherosclerotic lesions. In addition, a few tracers have been evaluated clinically. Both Tc -Annexin A5 for

[ 99m ] [108 ]
SPECT imaging and F -FDG , for PET imaging successfully identified vulnerable atherosclerotic plaques in the carotid vessels

[ 18 ]
[66 67 ] of patients. However, nuclear imaging of vulnerable plaques at the level of coronary arteries remains a challenging issue because of the small size of the atherosclerotic lesions, of their vicinity with blood and the unbound, circulating tracer activity, and of the motion of the heart. ECG-gated SPECT or PET acquisitions might be employed to partially overcome the limitation associated with cardiac motion. In the future, in addition to the development of new radiotracers with improved pharmacokinetics, new SPECT and PET multi-modality cameras with better resolution and sensitivity might play a major role in vulnerable plaque imaging using radiolabeled tracers.

[124 ]

Expression in Atherosclerotic Plaques Oral communication abdominal aortas of atherosclerotic and control animals represented as mean ID/g SD. Tc -MCP-1 uptake ( ID/g) was significantly

% ± [ 99m ] %
higher in atherosclerotic animals (P<0.0001) compared with that of control animals. Reproduced from with permission from the Society [START_REF] Hartung | Radiolabeled Monocyte Chemotactic Protein 1 for the detection of inflammation in experimental atherosclerosis[END_REF] of Nuclear Medicine. Orange arrows, orange arrowheads, and white arrowheads indicate aortas, kidneys, and livers, respectively. Reproduced from with [START_REF] Ogawa | 18)F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study[END_REF] permission from the Society of Nuclear Medicine. Association.

[ 26 .[27 28 ]

 2628 In addition to scavenger receptors, it has been proposed that another class of immune receptors, the toll-like receptors (TLRs), may play ] a role in the initiation and progression of the atherosclerotic lesions. TLRs recognize a number of pathogens as well as endogenous molecules involved in the onset of atherosclerosis (necrotic cells, heat shock proteins, fibronectin) and their stimulation results in the activation of NFKB and various cytokines , .According to the AHA classification, type I lesions consist in isolated macrophage-derived foam cells that contain lipid droplets whereas type II lesions contain numerous macrophage foam cells. Finally, type III lesions are characterized by extracellular lipid deposit [ . 15-17 ]

[ 4 ]

 4 Different strategies have therefore been used to image LDL accumulation into atherosclerotic lesions. Direct radiolabeling of LDLs was

[51 52 ]

 52 Although I -labeled SP-4 allowed the non-invasive detection of atherosclerotic lesions in 12 out of 14 WHHL animals, radiolabeling of [ 123 ] SP-4 with the more suitable isotope Tc yielded mixed results with only weak focal aortic uptake of Tc -SP-4 in 8 out of 11 99m [ 99m ] hyperchoelsterolemic rabbits. An alternative strategy to image oxLDL accumulation into atherosclerotic plaques was employed by Tsimikas et al.

  mice resulted in specific uptake of the tracer in aortic atherosclerotic lesions , . However, the feasibility of non-invasive imaging of [56 57 ] atherosclerotic lesions using I -IK 17 has not been demonstrated so far. Preliminary data from Langer et al. recently suggested that the [ 125 ]

Fig. ( 1 ) [ 61 ]

 161 Fig. (1)

  [ 99m ] lesion site by non-invasive in vivo imaging as shown in . Tc -labeled MCP-1 is therefore a promising agent for the molecular Fig. (2) [ 99m ]

[ 18 ]

 18 Fig. (3)

71 ,

 71 and Tahara et al. recently suggested that the metabolic syndrome was associated with increased F -FDG uptake, and therefore

.

  Matter et al. have therefore investigated the potential of F -Choline for the molecular imaging of atherosclerotic plaque. Using ex[START_REF] Matter | 18F-choline images murine atherosclerotic plaques ex vivo[END_REF] [ 18 ]

Fig. ( 4 )

 4 Fig. (4)

Fig. ( 5 )

 5 Fig. (5) experimental atherosclerotic lesions could be monitored with Tc -Annexin A5. Hartung et al. showed that dietary modification

[].Fig 1 Fig 2

 12 Fig 1 Non-invasive in vivo planar imaging of lectin-like oxLDL receptor-1 (LOX-1) with Tc -LOX-1-mAb. Non-invasive imaging of [ 99m ] abdominal region with Tc -LOX-1-mAb (A, B, D, and E) and Tc -IgG2a (C and F). Planar images for WHHLMI (B, C, E, and F) and [ 99m ] [ 99m ] control (A and D) rabbits at 10 min (A C) and 24 hours (D F) after injection are shown. Field of view 170 120 mm. Arrows aorta; K --= × = = kidney; L liver; S spleen. Reproduced from with permission from the Society of Nuclear Medicine. = = [61 ]

  [ 99m ]White (NZW) rabbits submited to deendothelialization of the abdominal aorta and fed a high-cholesterol diet (atherosclerotic, A C ) and [ -] control, unmanipulated, NZW animals fed normal rabbit chow (non-atherosclerotic, D F ) rabbits. (A C) Planar -images of atherosclerotic [ -] -γ rabbit. (A) Image obtained immediately after intravenous Tc -MCP-1 administration outlines aortic blood-pool activity (arrows). (B) At 3 [ 99m ] hours after radiotracer administration, significant radiotracer accumulation is evident in abdominal aorta (arrows). (C) Ex vivo image of explanted aorta confirms in vivo evidence of Tc -MCP-1 uptake (arrows). (D F) Planar -images of control rabbit. (D) Aortic blood pool [ 99m ] -γ is seen at time of intravenous Tc -MCP-1 injection (arrows). (E) Image shows lack of Tc -MCP-1 uptake in region of abdominal [ 99m ] [ 99m ] aorta, as denoted by arrows in normal rabbit with no atherosclerotic lesions. (F) Ex vivo aortic image of control animal demonstrates absence of Tc -MCP-1 uptake (arrows). L liver; S spleen; K kidney. (G) Bar graphs show quantitative Tc -MCP-1 uptake within[

Fig 3

 3 Fig 3 Non-invasive in vivo PET imaging of macrophage metabolism using F -FDG. PET images (A and D), CT images (B and E), and [ 18 ]superimposed PET/CT images (C and F) in the sagittal (I) and coronal (II) planes for WHHL (A, B, and C) and control (D, E, and F) rabbits.

Fig 4

 4 Fig 4 Non-invasive in vivo PET imaging of macrophages with a Cu-labeled trireporter nanoparticle Cu -TNP. Cu -TNP facilitates PET-CT 64

Fig 5

 5 Fig 5 Non-invasive in vivo SPECT imaging of apoptosis usingTc -annexin A5. In vivo and ex vivo images of control mice (A) and apoE[ 99m ]

  [START_REF] Virmani | Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions[END_REF] F -Choline was initially introduced as a tracer for brain and prostate cancer imaging . Choline is taken up into cells by specific

	[ 18 F -Choline
	]	[82 ]
	≥	[77 -81 ]
		5 14

] [

  Five days following injection, monocyte recruitment to plaques could be assessed by micro-SPECT/CT imaging and visualized as a focal hotspot in the ascending aorta with good correlation between the in vivo SPECT signal and the monocyte content (R 2 0.87). The inhibition of monocyte recruitment following statin treatment was also evidenced using this methodology. Clinical studies are

			al.	[87 ]	performed in vitro	111	In-Oxine
	monocytes labeling prior to reinjection of radiolabeled cells into apoE or control mice in order to gain insights into the pathogenesis of / --
	atherosclerosis. [ 64 ]
	atherosclerotic plaques 24 hours following injection in ApoE mice	as shown in	.

=

now warranted, especially considering the fact that In-oxine is approved by the Food and Drug Administration for clinical use. 111 Second, based on the concept that macrophages are avid for polysaccharide-containing supramolecular structures such as nanoparticules, a dextranated and DTPA-modified magnetofluorescent 20-nm nanoparticle was recently labeled with the PET tracer Cu ( 64 Cu -TNP) to yield a PET, magnetic resonance, and optically detectable imaging agent which provided nuclear images of aortic / --

[START_REF] Nahrendorf | Nanoparticle PET-CT imaging of macrophages in inflammatory atherosclerosis[END_REF]

Table 1

 1 Summary of previously evaluated radiotracers for the nuclear detection of atherosclerotic lesions. NZW, New Zealand White; WHHL, Watanabe Heritable Hyperlipidemic; WHHLMI, WHHL Myocardial Infarction-prone; der., derivative

	Target	Tracer	Nature of tracer	Model	Lesion location Assessment of tracer uptake	Reference
	LDL	[ 125 I -LDL ]	Lipoprotein	Humans	Carotid	In vivo imaging	[ 42 ]
				NZW Rabbits	Aorta	Ex vivo biodistribtion	[ 48 ]
		[ 99m Tc -LDL ]	Lipoprotein	Humans	Carotid	In vivo imaging	[ 45 ]
				NZW Rabbits	Aorta	In vivo imaging	[ 43 ]
		[ 123 I -LDL ]	Lipoprotein	NZW Rabbits	Aorta	In vivo imaging	[ 49 ]
		[ 111 In -LDL ]	Lipoprotein	NZW Rabbits	Aorta	Ex vivo biodistribution	[ 46 ]
	OxLDL	[ 99m Tc -oxLDL ]	Lipoprotein	Humans	Carotid	In vivo imaging	[ 50 ]
		[ 123 I -SP4 ]	Oligopeptide	WHHL Rabbits	Aorta	In vivo imaging	[ 51 ]
		[ 99m Tc -P199 ]	Oligopeptide	WHHL Rabbits	Aorta/carotid	Ex vivo imaging	[ 52 ]
		[ 125 I -MDA2 ]	Antibody	LDLR mice/WHHL Rabbits / --	Aorta	Ex vivo imaging	[ 54 ]
				LDLR mice / --	Aorta	Ex vivo imaging	[ 55 ]
		[ 99m Tc -MDA2 ]	Antibody	WHHL Rabbits	Aorta	In vivo imaging	[ 53 ]
		[ 125 I -IK17 ]	Antibody	LDLR mice / --	Aorta	Ex vivo imaging	[ 56 ]
		[ 124 I -CD68Fc ]	Peptide	ApoE mice / --	Aorta	Ex vivo imaging	[ 58 ]
	LOX-1	[ 99m Tc -LOX1-mAb ]	Antibody	WHHLMI Rabbits	Aorta	In vivo imaging	[ 61 ]
	Monocyte/macrophage CCR-2	[ 125 I -MCP-1 ]	Peptide	NZW Rabbits	Aorta	Ex vivo imaging	[ 62 ]
		[ 99m Tc -MCP-1 ]	Peptide	NZW Rabbits	Aorta	In vivo imaging	[ 63 ]
	Macrophage metabolism	[ 18 F -FDG ]	Carbohydrate	Humans	Carotid/Aorta	In vivo imaging	[ 64 , 65 ]
				Humans	Carotid	In vivo imaging	[ 68 ]
				Humans	Carotid/Aorta	In vivo imaging	[ 70 -72 ]
				WHHL/WHHLMI/NZW rabbits	Aorta	In vivo imaging	[ 66 , 73 ]
				NZW rabbits	Aorta	In vivo imaging	[ 67 ]
				NZW rabbits	Aorta	In vivo imaging	[ 69 ]
				ApoE mice / --	Aorta	No tracer accumulation in lesions	[ 74 ]
		[ 18 F -Choline ]	Amino alcohol	ApoE mice / --	Aorta	Ex vivo imaging	[ 85 ]

]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits . Eur J Nucl Med Mol Imaging . 2007 ; 34 : 889 -99

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]

[START_REF] Franc | Small animal SPECT and SPECT/CT: important tools for preclinical investigation[END_REF]


Carlos Quiroz
email: cquirozs@umich.edu

Saba Gerami
email: sgerami@umich.edu

Vilma Mesa
email: vmesa@umich.edu

Student utilization schemes of questioning devices in undergraduate mathematics dynamic textbooks

Keywords: Questioning devices in textbooks, instrumental approach, undergraduate students, mathematics textbooks, utilization scheme

We describe how students utilize questioning devices, an interactive feature designed into university textbooks to invite them to read the textbook before the class in which the content will be discussed. This feature has been added to three open-source and open-access dynamic textbooks intended for calculus, linear algebra, and abstract algebra. Using self-reports from 392 students in 23 courses and an instrumental approach, we identified three classes of situations in which the feature was used and three corresponding utilization schemes: Familiarizing with Content Before Class, Studying by Practicing and Doing Homework, and Self-Evaluating Understanding of Content. We suggest further areas of inquiry.

Mathematics digital textbooks offer opportunities for students to engage with mathematics in ways that go beyond the mere act of reading the textbook. In this paper, we focus on what we refer to as dynamic textbooks-a special type of digital textbooks that are open-source and open-access and have embedded interactive features. Because textbook features shape students' opportunities to engage with mathematics, how they are designed to engage students matters (e.g., level of interactivity, presentation, and order of features). In writing these textbooks, authors need to make many design decisions with students in mind as the main textbook users; as such they may not fully anticipate all the ways in which students will use textbooks features or the conditions that motivate their use. In the absence of this understanding, author-designers may add features that in the end might not be as useful for the students. In this study, we contribute by investigating student use of questioning devices that were added to three undergraduate dynamic mathematics textbooks in linear algebra, abstract algebra, and calculus. Specifically, we answer the following research question: What are the students' utilization schemes of these questioning devices?

We build on prior work [START_REF] Mesa | University instructors' use of questioning devices in mathematics textbooks: an instrumental approach[END_REF] in which we defined questioning devices as a form of questioning embedded in textbooks that seek to engage students in thinking about the content on their own before class. Differently from questions used in classrooms or asked in exercises, this other form of questioning in textbooks seeks to establish a personal relationship between the reader, the author, and the mathematics [START_REF] Weinberg | Understanding mathematics textbooks through reader-oriented theory[END_REF]. In the textbooks used in this study readers can type responses to those questions directly into their textbooks, immediately sharing their thoughts with their teacher prior to a lesson. Teachers then become aware of how students might be thinking about the material and adjust their lessons accordingly. In [START_REF] Mesa | University instructors' use of questioning devices in mathematics textbooks: an instrumental approach[END_REF], we found instructors' utilization schemes of these questioning devices. Because instructors' schemes can be related to students' utilization schemes, in this paper we complement our investigation by turning our attention to students. Because we have reviewed the literature on questioning devices in detail in [START_REF] Mesa | University instructors' use of questioning devices in mathematics textbooks: an instrumental approach[END_REF], in this paper we provide an overview of the literature that focuses on students' use of digital and interactive textbooks.

Literature Review

Instructors indicate that reading the textbook is a very important skill relevant not only for mathematics majors [START_REF] Mesa | Textbook mediation of teaching: An example from tertiary mathematics instructors[END_REF] but for all students because it supports mathematics learning. Judging from the extant research on student reading practices in university mathematics (e.g., [START_REF] Wiesner | The roles of textual features, background knowledge, and disciplinary expertise in reading a calculus textbook[END_REF], knowing whether and how students read their textbooks is a key question for teachers and researchers. Research on university student use of digital textbooks are typically case studies at single universities or classrooms and describe use of interactive components (e.g., [START_REF] Johnston | University students' engagement with textbooks in print and ebook formats[END_REF]; relation between student grades and time spent reading (e.g., [START_REF] Allred | Interactive electronic textbook use in higher education: grades, engagement, and student perceptions[END_REF]; skills, emotion, and participation and interaction (e.g., [START_REF] Bikowski | Interactive digital textbooks and engagement: A learning strategies framework[END_REF]; differences in student motivation and perceptions of learning when using interactive textbooks [START_REF] Kondratieva | An interactive text in Precalculus: students' response[END_REF]; or use of digital resources in connection to rules of didactical contract [START_REF] Gueudet | Didactic contract at the beginning of university: A focus on resources and their use[END_REF]. These researchers indicate that undergraduate students see digital textbooks as resources that motivate and facilitate learning and have documented different levels of reading, different uses of features, and different expectations regarding the utility of interactive features. The differences seem to be related to students' prior knowledge and to personal goals regarding their courses (e.g., passing a course). While these studies suggest that students might not use their textbooks as intended, they do not indicate how students interact with specific interactive features.

Theoretical Framing

In Rabardel's instrumental approach [START_REF] Rabardel | People and technology: a cognitive approach to contemporary instruments[END_REF] an instrument is "a whole incorporating an artifact (a human-made or a physical object) and one or more utilization schemes" (p. 65). A utilization scheme encompasses stable and structured manners that a user has for operating an artifact that includes observable behaviors and non-observable goals and rationales. Following Vergnaud (1998) we define schemes as the "invariant organization of behavior for a certain class of situations" (p. 229). This organization includes goals and expectations; rules of action (which can be seen as the "generative part of the schemes"); operational invariants (propositions that are held to be true by the subject when they act, that are used "to infer, from the available and relevant information, appropriate goals and rules); and possibilities of inferences (p. 229). Vergnaud views "situations and schemes [as] essential in the development of knowledge" (p. 237). Instrumental genesis s the process through which a user transforms an artifact into an instrument by creating utilization schemes associated with that artifact. Because pre-existing artifacts are instrumentalized by a user, different subjects can produce different instruments for the same artifact. Instrument production, carried out by the subject, encompasses two subprocesses, instrumentalization (the "emergence and evolution" of the artifact component of the instrument) and instrumentation ("the emergence and evolution of utilization schemes and instrument-mediated action: their constitution, their functioning, their evolution by adaptation, combination coordination, inclusion and reciprocal assimilation, the assimilation of new artifacts to already constituted schemes, etc." Rabardel, 2002, p. 103). The notion of utilization schemes is central to instrumentation. To document student instrumentation of an artifact-questioning devices in dynamic textbooks-we identified the utilization schemes students reported when using questioning devices available in three textbooks designed to increase their interaction with the content.

Methods

This research is part of a larger mixed-method study that seeks to understand how teachers and students in undergraduate levels utilize three open-access open-source dynamic textbooks: Active Calculus (hereafter AC, [START_REF] Boelkins | Active Calculus[END_REF], First Course in Linear Algebra (hereafter FCLA, [START_REF] Beezer | First course in linear algebra[END_REF], and Abstract Algebra: Theory and Applications (hereafter AATA, [START_REF] Judson | Abstract algebra: Theory and applications[END_REF]. The three textbooks are written in PreTeXt (https://pretextbook.org/), a markup language that allows the textbooks to be rendered in various formats (e.g., HTML, PDF, Braille, bounded). With PreTeXt, authors can infuse the textbook with interactive features, such as live computation cells with Python (Sage cells), immediate solution feedback, and collection of student responses to questioning devices for instructor use. The three textbooks were chosen to represent different student audiences. In the United States, calculus is a course that is taken by most students seeking an undergraduate degree in science, technology, engineering, mathematics, business, economics, and health. Linear algebra is a gateway course for mathematics and computing related majors, and in some cases for biology. Abstract algebra is a course usually taken only by mathematics majors (which include future secondary teachers). The questioning devices are called Preview Activities in the calculus textbook and Reading Questions in the linear algebra and abstract algebra textbooks. For this study, we analyzed data from 392 consenting students from 23 teachers across the United States who participated for a semester from Spring 2019 to Spring 2021 (187 used AC, 145 used FCLA, and 60 used AATA). Students (147 female, 142 male, 3 non-binary or trans, 100 no information) had an average age of 20 years, an average GPA of 3.43, and obtained an average grade of 82.58 in their respective courses.

We used data from five student logs (biweekly surveys with 8 to 19 closed-and open-ended questions about student textbook use). We used responses to two questions (see Table 1) intended to identify why and how students used the questioning devices (Q9, and Q5 are open-ended)1 .

Analysis

We used a constant comparative analysis with the answers to the two open-ended questions (Q9 and Q5) to identify classes of situations, goals, and rules of actions. The analysis proceeded in four steps.

First, we identified three classes of situations in which students reported using questioning devices: Before Class Preparation, Study, and Self-Evaluation. Before Class Preparation included situations in which students expressed, they used questioning devices to get ready for an incoming class. Study included situations in which the students expressed using questioning devices for practicing, doing homework, and studying for exams. Self-Evaluation included situations in which students said they used the questioning devices for testing their own understanding of the content. Using these categories, we classified each of the 559 responses (316, Log 1 Q9 + 243, Log 2 Q5) under one of these situations or as "No Class" if the response did not convey enough information to identify a situation (e.g., "yes"). In the second step, we inspected responses within each class to identify goals. We identified and coded eight goals that students associated with the classes of situations: three related to Before Class Preparation, four with Study, and one with Self-Evaluation. In the third step, with the responses organized by class of situation, we identified 10 rules of action. In the fourth step, the first and second author identified the operational invariants, looking for explanations for why students were using the questioning devices. The most frequent reason was that students used the questioning devices because they were required by their teacher (61 out of 511). Other operational invariants appeared but were less frequent.

The first authors coded all the responses; the other two authors independently coded classes of situations, goals, and rules of actions for 10% of the responses. The average Cohen's Kappa for coding for situation, goals, and actions were 0.78, 0.71, and 0.63. These three values are within the range of [0.61 -0.80], which correspond with a substantial agreement.

Findings: Students' Instrumentation of Questioning Devices

We identified three utilization schemes of the questioning devices, one per class of situations, Familiarizing with Content Before Class, Studying by Practicing and Doing Homework, and Self-Evaluating Understanding of Content (see Figure 1). While we characterized the utilization schemes separately to make sense of students' use of the questions devices, students often mentioned multiple goals, rules of actions, and operational invariants. Moreover, although we identified all three utilization schemes across students using the three textbooks in calculus, abstract algebra, and linear algebra, not all goals were mentioned across these groups of students.

In the first utilization scheme, familiarizing with content before class, students said they use questioning devices to get acquainted with the upcoming material in the course:

They have been assigned to us before classes. I look over (rule of action: skimming) the preview questions [questioning device], try to solve the activity (rule of action: answering) and it gives me an idea of what we will be working on in class that day (goal). (30.07, Spring19, AC)2

In this excerpt, a clear goal is stated: to get "an idea" of what will be taught in class. We identified two more goals under this class of situations in the corpus: to better prepare for class and to connect previous material with new content. To accomplish these goals, four actions were mentioned: skimming and answering the questioning devices, reading the chapter associated with the questioning devices, and writing down questions they could ask in class. To justify these actions, students collectively offered four reasons: the questioning devices were assigned (e.g., "My professor has it mandatory to answer the reading questions for each section," 36.20, Fall20, FCLA), the questioning devices addressed important content (e.g., "I usually remember the material in a reading question as what is most important for me to know," 46.05, Spring21, FCLA), the questioning devices helped them with understanding the new material (e.g., "These are very helpful in introducing the concepts of the section," 35.70, Fall20, AC), or because their instructors used the questioning devices to teach in class (e.g., "We have used these in our class to give our initial answers to the reading, and then our professor gives us feedback on our responses," 23.20, Fall20, AATA). This student indicates using the questioning devices as "extra practice" when doing homework because doing so will help gain a "better understanding" of the material. We identified four goals associated with this scheme: to practice (when questioning devices are not assigned as homework), to do homework (when questioning devices are assigned as homework), to prepare for assessments (e.g., "Used them as reference to how class questions on exams and quizzes will look like," 45.53, Spring21, FCLA), and to find out what is important to study in the chapter or section associated with the questioning devices (e.g., "If these are not assigned as homework, I use these questions as reinforcement of the main/big ideas from the chapter," 33.13, Spring20, AATA). We identified four student actions: skimming and answering questioning devices, checking answers to the questioning devices, and taking notes on the questioning devices and their solutions. We identified four operational invariants that students reported as justifying these actions: Students were required by their instructors to work on the questioning devices during class or at home (e.g., "We use them in class and as homework," 30.23, Spring20, AC); using the questioning devices benefited their own learning of the material (e.g., "I take advantage of this completely in order to be successful. i [sic] enjoy how you can reflect and absorb the material this way," 44.03, Spring21, FCLA), problems on their examinations were similar to those in the questioning devices (e.g., "I complete the preview activities for a grade but I think that they help a lot in the initial learning process because it lets students know what questions on a test might look like," 35.57, Fall20, AC), and ideas showcased in the questioning devices were important ideas in the chapter or section (e.g., "I find them [reading questions] helpful because they make sure I understand important parts of the section," 44.06, Spring21, FCLA).

In the third utilization scheme students said they use the questioning devices for self-evaluation of their understanding of the material:

I mostly use this to check for my understanding of the chapter. If i [sic] get to the reading questions and do not understand them I know I have to reread the chapter again. (37.08,Fall19,FCLA) This quote represents the essence of the scheme, that students take the questioning devices as a kind of arbiter of the content of the chapter; not knowing the answers triggers a process of reading other parts of the textbook. Three rules of action were associated with this scheme: attempting the questioning devices to evaluate their understanding before reading a chapter (e.g., "Do them to see where my baseline is for this chapter," 35.65, Fall20, AC); attempting the questioning devices after reading the chapter to check ability to answer them; when unable, students re-read the chapter; and compare their answers to the questioning devices to other resources, such as the solutions provided by their textbook, their classmates, or their instructors (e.g., "Do them and then go over them with classmates," 35.81, Fall20, AC). The operational invariant is inferred as the need to know that their knowledge is correct; this is done either by contrasting responses with the content of the reading questions or with external resources, such as peers or the instructor.

Discussion and Conclusion

These findings are important for textbook designers, as well as instructors using the textbooks, because they confirm that the questioning devices are being used for their intended purpose and more.

The three utilization schemes help us understand what motivates students to use questioning devices and how they use them. For two schemes, (Familiarizing with Content Before Class, Studying by Practicing and Doing Homework) one main motivator was that teachers assigned the questioning devices to the students. This highlights the important role that teachers have in influencing student use of textbooks, supporting prior findings that indicate that when asked, students will use the textbooks as teachers suggest [START_REF] Mali | Instructors' and students' uses of dynamic textbooks: What is new? Joint meeting of the Mathematical Association of America and the American Mathematical Society[END_REF]. However, that was not the only motivator for students to use the questioning devices; students, by motu proprio used the questioning devices for familiarizing themselves with the content, studying by practicing, and more interestingly for evaluating their own understanding of the material. Because the third scheme does not seem to be motivated by the teacher assigning the questioning devices, we wonder whether this particular scheme would emerge without exposure to the questioning devices initiated by the teachers.

The findings show that although designer-authors and instructors may have specific ideas of how a feature should be used, students find ways to exert their agency and decide how to use them. In the case of questioning devices, these features were designed to be completed before a lesson; therefore, they may not be the best fit for students when it comes to studying and self-assessment. Although we encourage students to use their textbooks for their needs, we believe that by learning about the intended purposes and embedded opportunities of textbooks' features students can make better use of their textbooks tailored for specific situations. For example, the WeBWorK exercises embedded within the AC textbook provide immediate feedback to students while questioning devices do not; thus, we expect that these features are better suited for self-assessment than questioning devices.

This study was done to complement our prior work on teacher utilization schemes of questioning devices identified in [START_REF] Mesa | University instructors' use of questioning devices in mathematics textbooks: an instrumental approach[END_REF], namely (1) Instructors complete the questioning devices for lesson pre-planning, (2) Instructors require students to do the questioning devices for the purpose of lesson planning, (3) Instructors use the questioning devices for the purpose of instruction, and (4) Instructors require students to complete the questioning devices for the purpose of assessment. We anticipate that the four teacher utilization schemes are related to the student utilization schemes of the questioning devices we found here. As a next phase in our work, we will determine the connection between these utilization schemes and investigate differences by textbook.

Figure 1 :

 1 Figure 1: Students' utilization schemes of questioning devices

Table 1 : Log questions used in this study and number of responses by textbook

 1

	Question	FCLA AATA AC	Total
	Log 1 Q9: Your textbooks include Reading Questions/Preview Activities in some sections. How have you used these?	130	41	145	316
	Log 2 Q5: What are your priorities (objectives, goals) while using Preview Activities/Reading Questions? Explain how you use them.	86	32	125	243

Across all the textbooks less than 10% of the students stated that they never used these features, indicating high use across the sample.

We identify quotes of a student using the convention "XX.YY, term, textbook" where XX refers to the instructor and YY to the student of that instructor.

Acknowledgment

Funding has been provided by the National Science Foundation through Awards IUSE 1624634 and 1821509. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.