Atherosclerosis xxx (xxxx) xxx

Contents lists available at ScienceDirect

Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Rosuvastatin effect on atherosclerotic plaque metabolism: A subclinical

atherosclerosis imaging study with 18F–NaF PET-CT
Manuel Oliveira-Santos a, b, c, d, *, João Borges-Rosa a, Rodolfo Silva b, c, d, Luís Paixão c,
Cláudio Espírito Santo c, Antero Abrunhosa b, c, d, Miguel Castelo-Branco b, c, d, Piotr J. Slomka f,
Lino Gonçalves a, c, e, Maria João Ferreira a, b, c, d
a
Cardiology Department, Unidade Local de Saúde de Coimbra, Centro Hospitalar e Universitário de Coimbra, Praceta Prof. Mota Pinto, 3000-075, Coimbra, Portugal
b
Institute of Nuclear Sciences Applied to Health - University of Coimbra, Azinhaga de Santa Comba, 3000-548, Coimbra, Portugal
c
Faculty of Medicine, University of Coimbra, Rua Larga, 3004-504, Coimbra, Portugal
d
Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
e
Coimbra Institute for Clinical and Biomedical Research (iCBR), Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
f
Division of Artificil Inteligence in Medicine, Department of Medicine, Cedars-Sinai, Los Angeles, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Background and aims: Atherosclerotic plaque fluorine-18 sodium fluoride (18F–NaF) uptake on positron emission
Atherosclerosis tomography with computed tomography (PET-CT) identifies active microcalcification and has been shown to
Cardiovascular diseases correlate with clinical instability in patients with cardiovascular (CV) disease. Statin therapy promotes coronary
Molecular imaging
macrocalcification over time. Our aim was to investigate rosuvastatin effect on atheroma 18F–NaF uptake.
Positron-emission tomography
Methods: Subjects with high CV risk but without CV events underwent 18F–NaF-PET-CT in a single-centre. Those
Sodium fluoride
Statins with subclinical atherosclerosis and significant 18F–NaF plaque uptake were included in a single-arm clinical
trial, treated with rosuvastatin 20 mg/daily for six months, and re-evaluated by 18F–NaF-PET-CT. Primary
endpoint was reduction in maximum atheroma 18F–NaF uptake in the coronary, aortic or carotid arteries,
assessed by the tissue-to-background ratio (TBR). The secondary endpoint was corrected uptake per lesion (CUL)
variation.
Results: Forty individuals were enrolled and 38 included in the pharmacological trial; mean age was 64 years,
two-thirds were male and most were diabetic. The 10-year expected CV risk was 9.5% (6.0–15.3) for SCORE2 and
31.7 ± 18.7% for ASCVD systems. After six months of rosuvastatin treatment (n = 34), low-density lipoprotein
cholesterol lowered from 133.6 ± 33.8 to 58.8 ± 20.7 mg dL− 1 (60% relative reduction, p < 0.01). There was a
significant 19% reduction in maximum plaque 18F–NaF uptake after treatment, from 1.96 (1.78–2.22) to 1.53
(1.40–2.10), p < 0.001 (primary endpoint analysis). The secondary endpoint CUL was reduced by 23% (p =
0.003).
Conclusion: In a single-centre non-randomized clinical trial of high CV risk individuals with subclinical athero­
sclerosis, the maximum atherosclerotic plaque 18F–NaF uptake was significantly reduced after six months of
high-intensity statin.

1. Introduction Fluorine-18 sodium fluoride (18F–NaF) atherosclerotic plaque uptake

Cardiovascular (CV) diseases are the main cause of mortality in many
countries. Healthy lifestyle recommendations should be universal for CV
disease prevention, and some individuals require tailored risk factor
modification based on accurate risk estimation from clinical, biochem­
ical and imaging variables.
in positron emission tomography with computed tomography (PET-CT)
identifies active microcalcification, a recognized marker of clinical
instability [1]. 18F–NaF specifically adsorbs to hydroxyapatite crystals,
which are largely exposed in microcalcification areas, unlike dense
calcium plaques [2]. There is a positive association between 18F–NaF
uptake in the major arteries and the burden of risk factors in patients

* Corresponding author. Cardiology Department, Unidade Local de Saúde de Coimbra, Centro Hospitalar e Universitário de Coimbra, Praceta Prof. Mota Pinto,
3000-075, Coimbra, Portugal.
E-mail address: oliveirasantos@uc.pt (M. Oliveira-Santos).

https://doi.org/10.1016/j.atherosclerosis.2024.117481
Received 1 December 2023; Received in revised form 6 February 2024; Accepted 15 February 2024
Available online 29 February 2024
0021-9150/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: Manuel Oliveira-Santos et al., Atherosclerosis, https://doi.org/10.1016/j.atherosclerosis.2024.117481
M. Oliveira-Santos et al.
with high CV risk [3]. Culprit plaques/rupture sites in coronary and
carotid arteries have higher 18F–NaF uptake, suggesting atheroma
instability [4]. In a multicentric prospective study, microcalcification
detection through 18F–NaF PET-CT improved risk prediction beyond
standard coronary calcium score determination [5].
Statin therapy is a cornerstone in primary and secondary CV disease
prevention, due to its cholesterol-lowering effect and positive pleio­
tropic properties [6]. Rosuvastatin diminishes mortality and vascular
events when used for secondary prevention. The JUPITER trial also
established reductions in these endpoints in individuals with elevated
inflammatory markers but without CV events [7]. Early trials showed
that statins do not halt calcification [8,9]. In fact, serial coronary
intravascular ultrasound studies demonstrated that statins decrease
atheroma volume but promote calcifications [10]. The clinical effect of
statins in microcalcification activity (18F–NaF uptake) is not known. The
Rosuvastatin Effect on Atherosclerotic Plaque Metabolism - a Subclinical
Atherosclerosis Imaging Study With 18F–NaF PET-CT (ROPPET-NAF) was a
phase IV trial that tested whether rosuvastatin therapy reduces vascular
plaque 18F–NaF uptake in patients at high CV risk but without CV events.
2. Patients and methods
2.1. Trial design and oversight
The Rosuvastatin Effect on Atherosclerotic Plaque Metabolism - a Sub­
clinical Atherosclerosis Imaging Study With 18F–NaF PET-CT (ROPPET-
NAF, NCT 03233243, EudraCT 2018-003837-13) was a single-arm,
single-centre phase IV clinical trial designed to study the effect of
rosuvastatin in 18F–NaF uptake in coronary, aortic and carotid vascular
beds in a primary prevention setting. The trial protocol was designed by
an academic research team, written according to Good Clinical Practice
and the Declaration of Helsinki, and approved by Portuguese author­
ities: Ethics Committee of Faculty of Medicine of the University of
Coimbra (CE-047/2015), Autoridade Nacional do Medicamento e Produtos
de Saúde (INFARMED) and National Ethics Committee for Clinical
Research (CEIC – 2020-RP-05-08). ROPPET-NAF was sponsored by
Centro Hospitalar e Universitário de Coimbra, a tertiary university hospi­
tal, with the financial support of a medical company, AstraZeneca-Pro­
dutos Farmacêuticos, Lda (PET-CT exams and rosuvastatin tablets).
2.2. Trial population
Subjects were eligible for participation in the trial if they were fol­
lowed at the Arterial Hypertension Clinic of Centro Hospitalar e Uni­
versitário de Coimbra, were older than 40 years, provided informed
consent and were considered to be at high or very high CV risk according
to the European Society of Cardiology guidelines [11], with any of the
following criteria: predicted fatal CV event at 10 years ≥ 5% (Systematic
Coronary Risk Evaluation -SCORE - tables for low-risk countries);
chronic kidney disease with estimated glomerular filtration rate (eGFR)
under 60 mL/min (Modification of Diet in Renal Disease equation –
MDRD), diabetes mellitus (type 1 or 2) or markedly elevated single risk
factors. Key exclusion criteria were previous CV events (acute coronary
syndrome, stroke, arterial revascularization procedure), severe renal
Fig. 1. Trial design.
2
Atherosclerosis xxx (xxxx) xxx
impairment with eGFR less than 30 mL min− 1.1.73 m− 2, known hepatic
dysfunction, myopathy and statin hypersensitivity (see protocol in the
Supplementary Materials for full inclusion and exclusion criteria).
After signing the informed consent, patients previously treated with
statins stopped treatment for two weeks before inclusion to ensure a more
accurate assessment at baseline, following recommendations from pre­
viously published work [12]. Baseline assessment included a clinical
questionnaire, physical examination, standard blood analyses and CV risk
estimation according to SCORE 2 equations [13] and the American Heart
Association atherosclerotic cardiovascular disease (ASCVD) calculator.
Risk estimation by SCORE was not reported in results section because it is
outdated, and no patient was included with that sole criterion.
2.3. Microcalcification activity
Participants underwent whole-body PET-CT for identification of
coronary, carotid and aortic atheroma and quantification of 18F–NaF
uptake. The protocol consisted in the administration of 185 Mega­
Becquerel (MBq) 18F–NaF intravenously, followed by an attenuation
correction CT scan and PET imaging after 60 min. Until November 2021,
a Gemini GXL Philips 16 PET/CT system (Philips, Eindhoven, The
Netherlands) was used. Afterwards the exams were performed in a
Siemens Vision 600 machine (Siemens Healthineers, Erlangen, Ger­
many). Coronary images were reconstructed according to the usual
protocol of PET-CT, using the software Syngo. via (Siemens Healthineers,
Erlangen, Germany). The scan was visually inspected for the presence of
plaques, and two-dimensional regions of interest (ROI) were drawn
around all major epicardial coronary vessels and around the major
vessels on axial slices (4 mm for Philips scanner, 1.65 mm for Siemens).
The main measure was tissue-to-background ratio (TBR), determined by
the ratio of the maximum standard uptake value (SUV) in the ROI (the
decay corrected tissue concentration of the tracer divided by the injected
dose per bodyweight) and baseline blood pool activity. Baseline blood
activity was measured in the atria (contemporary method) and the su­
perior vena cava (pre-specified in the protocol). Scans were reviewed
and analysed by two experienced observers blinded to the clinical
diagnosis. In case of disagreement, a third doctor would be consulted.
Corrected uptake per lesion (CUL) was employed as an alternative
method for vascular microcalcification activity quantification. It was
measured in the same fashion as TBR, but instead of ratio, the difference
between the maximum SUV in the ROI and blood pool activity was
calculated. CUL analysis was pre-specified in the original protocol due to
its superior correlation to clinical variables in the pilot study population
[3,14]. We performed a reliability analysis of two independent observers
for radiotracer uptake using intraclass correlation coefficient (Cron­
bach’s Alpha) in the pilot study, which was 0.99 (95% confidence in­
terval 0.99–0.99) for radiotracer uptake [3].
2.4. Statin treatment
Subjects without significant 18F–NaF uptake were excluded from the
pharmacological intervention study. Those with significant 18F–NaF up­
take (coronary, aortic or carotid plaques with TBR >1.5) were treated
with 20 mg of rosuvastatin (crestor®) daily for six months (Fig. 1). A
M. Oliveira-Santos et al. Atherosclerosis xxx (xxxx) xxx

second visit took place eight weeks after therapy initiation to monitor
compliance and adverse events report, including creatine kinase and liver
function tests determination. After six months, a second 18F–NaF PET-CT
was performed. Care was taken so that each patient was re-evaluated in
the same PET-CT scanner. The primary endpoint was reduction in
maximum 18F–NaF plaque uptake (TBR) in any territory (coronary, aortic
and carotid plaques) and the secondary endpoint was CUL reduction.
2.5. Statistical analysis
3. Results
3.1. Participants
From June 2020 to June 2022 a total of 40 participants were enrolled
in the study, and 38 were included in the statin trial. Baseline charac­
teristics are shown in Table I. Mean age was 64 years, two-thirds were
male and most were diabetic (95%). The 10-year expected CV risk was
9.5% (6.0–15.3) for SCORE2 and 31.7 ± 18.7% for ASCVD systems.

Continuous data were described using mean ± standard deviation or 3.2. Statin therapy
median (interquartile range), according to the normality of the distri­
bution. Categorical data were represented by frequency and proportion. Two subjects did not progress to statin therapy due to insignificant
18
The minimum sample size to detect significant variation in 18F–NaF F–NaF uptake. After rosuvastatin therapy, mean LDL significantly
after statin, with paired t-Student test, was calculated for several effect lowered from 133.6 ± 33.8 to 58.8 ± 20.7 mg dL− 1, with a median 60%
sizes and different measures correlations (80% power and significance relative reduction, p < 0.01 (Fig. 2 and Table 2). All patients attained the
level 0.05). The estimation was performed using G × Power 3.1.9.2 target dose of 20 mg rosuvastatin daily. Two patients (2/38) reported
software (Erdfelder, Faul, & Buchner, 1996), based on the data of 25 minor side effects (myalgia and nausea), both resolved with temporary
individuals and the maximum TBR in the three locations, assuming
equal standard deviation between paired groups (pilot study) [3,14].
Assuming a correlation of 80% and 10% difference in TBR, the sample
size was 32. Considering the expected proportion of significant 18F–NaF
plaque uptake (21 out of 25 in the pilot), the trial was projected to
include 44 patients, expecting 36 patients’ re-evaluation after pharma­
cological intervention, with a drop-out margin of 4 subjects. The sta­
tistical analysis plan was pre-registered at ClinicalTrials.gov (U.S.
National Library of Medicine).
The variation in maximum 18F–NaF uptake in major vessels and
other continuous variables, before and after rosuvastatin treatment,
were analysed using paired t-Student or Wilcoxon tests, according to
normality, at a significance level of 0.05. Correlations were computed
with Pearson or Spearman correlation tests (R). Baseline predictors of
variation in 18F–NaF uptake were explored with linear regression
equations.
All analyses were performed with IBM SPSS Statistics for Mac soft­ Fig. 2. Low-density lipoprotein (LDL) cholesterol reduction following rosu­
ware, version 29.0. vastatin therapy.

Table 1
Baseline characteristics (n = 38).
Demographics Medical history – no. (%)

Age (years) 64.0 ± 9.4 Type 2 diabetes 36 (94.7)

Male sex – no. (%) 25 (65.8) Hyperlipidaemia 29 (76.3)
Caucasian – no. (%) 38 (100.0) Hypertension 35 (92.1)
Height (cm) 165 (162–172) Current smoker 0 (0.0)
Weight (kg) 86.1 ± 16.1 Former smoker 10 (26.3)
Body mass index (kg m− 2) 31.8 (28–33) Family history of CHD 2 (5.3)
Waist circumference (cm) 109.6 ± 12.4 Metabolic syndrome (IDF) 36 (94.7)
Systolic blood pressure (mm Hg) 148.5 (136–156) Abdominal obesity (>94/80 cm) 37 (97.4)
Diastolic blood pressure (mm Hg) 85.0 (80–91) Obesity (BMI> 30 kg m2) 24 (63.2)
Heart rate (beats per minute) 71.2 ± 9.8 Chronic kidney disease 6 (15.8)

Medication – no. (%) Biochemistry parameters

ACE inhibitor 20 (52.6) Total cholesterol (mg.dL− 1) 200.4 ± 37.7

Angiotensin-receptor blocker 12 (31.6) Low-density lipoprotein cholesterol (mg.dL− 1) 134.0 ± 33.6
Beta-blocker 10 (26.3) High-density lipoprotein cholesterol (mg.dL− 1) 45.2 ± 10.4
Calcium-channel blocker 16 (42.1) Triglycerides (mg.dL− 1) 136.0 (107–216)
Diuretic 22 (57.9) Fasting glucose (mg.dL− 1) 100.5 (89–132)
Aldosterone antagonist 7 (18.4) Glycated haemoglobin (%) 6.6 ± 0.7
Statin 19 (50.0) High-sensitivity C-reactive protein (mg.dL− 1) 0.25 (0.12–0.45)
Fibrate 5 (13.2) Creatinine (mg.dL− 1) 0.79 (0.73–0.94)
Metformin 32 (84.2) Glomerular filtration rate (mL.min− 1) 87.9 ± 20.9
Insulin 3 (7.9) Creatine kinase (U.L− 1) 98.6 ± 45.0
DPP-4 inhibitors 12 (31.6) Alanine amino-transferase (U.L− 1) 27.5 (18.0–42.0)
Antithrombotic 5 (13.2) Gamma-glutamyl transpeptidase (U.L− 1) 26.5 (18.0–42.0)
Haemoglobin (g.dL− 1) 14.8 (13.5–15.6)

Data is presented as mean (± standard deviation) and median (interquartile range) according to normality. Categorical data were represented by frequency and
proportion.
Angiotensin-converting-enzyme inhibitors – ACE inhibitors, Coronary heart disease – CHD, Dipeptidyl peptidase 4 DPP-4, International Diabetes Federation - IDF.

3
M. Oliveira-Santos et al. Atherosclerosis xxx (xxxx) xxx

dose reduction. There was no clinically significant increase in median

creatine kinase, from 94.5 (66–128) to 98.5 (77–155) U.L− 1 (p = 0.11),

<0.001

<0.001

<0.001
p value

<0.01
0.003

0.79
and a slight reduction in alanine aminotransferase was noted, from 27.5
(18–42) to 22.0 [18–28] U.L− 1, p = 0.03. There were no serious side
effects and no significant elevations of creatine kinase (>5 upper limit of
normal) nor liver function tests (>3 upper limit of normal).

39.86 (− 44.51 to − 26.58)

60.00 (− 64.93 to - 47.97)
− 19.22 (− 27.21 to − 5.04)

31.13 (− 49.32 to − 7.47)

3.3. Microcalcification activity data

3.81 (− 8.33 to 7.40)

Percent change (%)

From the 38 individuals with significant 18F–NaF uptake included in

− 22.97 ± 34.75

the pharmacological intervention arm, four did not repeat 18F–NaF PET-
CT (two lost to follow-up and two withdrew consent). Thirty-one
matched PET-CT exams were analysed, because three datasets were
damaged and unretrievable due to hardware problems. Maximum pla­
−
−
−
−
que 18F–NaF uptake was detected mostly in the aorta: abdominal aorta
(18 individuals), descending aorta and aortic arch (n = 5 for each) and
carotid arteries (n = 3).
There was a significant 19.22% reduction in the primary endpoint of
80.50 (− 100.00 to − 51.00)

maximum plaque 18F–NaF uptake following rosuvastatin treatment

46.50 (− 97.00 to − 9.00)

(Table II). Maximum TBR (maximum lesion SUV/baseline atrial SUV) in

− 0.35 (− 0.59 to − 0.08)

− 0.23 (− 0.41 to 0.02)

1.50 (− 4.00 to 2.00)

any location reduced from 1.96 (1.78–2.22) to 1.53 (1.40–2.10), p <

0.001 (Fig. 3). The reduction in TBR was similar if superior vena cava
74.45 ± 37.61

activity was used instead of atria: 1.76 (1.53–2.03) to 1.51 (1.30–1.78),

p = 0.003. The secondary endpoint - CUL variation – was reduced by
Change

22.97% (p = 0.003). The variation in TBR was similar in individuals

with previous statin therapy (− 0.28 ± 0.39) and statin-naïve (− 0.32 ±
−
−
−
−

0.43), p = 0.75.
Some examples of matched comparisons are shown in Fig. 4. Two
individuals had an increase in TBR, despite having a significant LDL
reduction.
97.50 (85.00–123.00)

The change in microcalcification activity discriminated by vascular

territories shown in Table 3.
1.53 (1.40–2.10)

0.72 (0.43–1.06)

125.70 ± 27.78

45.56 ± 12.38

There was a moderate correlation between baseline maximum TBR

58.8 ± 20.7

Data is presented in mean (± standard deviation) and median (interquartile range) according to normality.

and CV risk estimators: R = 0.38 for ASCVD (p = 0.04) and R = 0.44 for
6 months

SCORE 2 (p = 0.01). Subjects with ASCVD >25% and SCORE 2 > 10%
had higher TBR, 2.11 (1.87–2.47) versus 1.78 (1.56–1.98) and 2.17
(1.93–2.49) versus 1.83 (1.56–2.02), respectively (p = 0.02). This
observation is depicted in Fig. 5. There was no significant association
between baseline 18F–NaF uptake and any other clinical/laboratorial
variables. In addition, there were no significant predictors of 18F–NaF
138.50 (108.00–206.00)

variation following statin treatment.

1.96 (1.78–2.22)

1.04 (0.83–1.28)

199.63 ± 37.60

4. Discussion
45.33 ± 10.26
133.6 ± 33.8
Baseline

Atherosclerotic plaque 18F–NaF uptake was reduced by one-fifth

after six months of high intensity statin in individuals with subclinical
atherosclerosis. This is the first human report about statins’ effect on
microcalcification activity, assessed by 18F–NaF PET-CT (Fig. 6).
Baseline and follow-up PET-CT and lipid analysis.

Maximum plaque tissue-to-background ratio (TBR)

High-density lipoprotein cholesterol (mg.dL− 1)

Low-density lipoprotein cholesterol (mg.dL− 1)
Corrected uptake per lesion (CUL)
Microcalcification activity

Total cholesterol (mg.dL− 1)

Triglycerides (mg.dL− 1)
Secondary endpoint
Primary endpoint

Lipid profile
Table 2

Fig. 3. Maximum plaque 18F–NaF uptake before and after high intensity statin.

4
M. Oliveira-Santos et al. Atherosclerosis xxx (xxxx) xxx

Fig. 4. Fusion 18F–NaF PET-CT images before (upper panel) and after (lower panel) rosuvastatin treatment, showing reduction in maximum plaque uptake in the
coronary arteries (A, B), carotid arteries (C, D) and aortic arch (E, F).

Table 3
Baseline and follow-up microcalcification by vascular territory.
Baseline 6 months Change Percent change (%) p value

Maximum plaque tissue-to-background ratio (TBR)

Aorta
Ascending Aorta 1.65 (1.43–1.78) 1.48 (1.36–1.60) − 0.12 (− 0.33 to 0.01) − 8.19 (− 17.76 to 0.52) 0.007
Aortic arch 1.68 ± 0.25 1.51 (1.39–1.67) − 0.10 ± 0.33 − 4.38 ± 19.35 0.05
Descending thoracic Aorta 1.63 (1.51–1.84) 1.46 (1.38–1.57) − 0.14 (− 0.33 to − 0.06) − 9.02 (− 20.95 to − 3.36) <0.001
Abdominal Aorta 1.84 (1.56–2.21) 1.65 (1.42–2.04) − 0.21 ± 0.43 − 9.76 ± 20.76 0.011
Carotid arteries 1.46 (0.54–1.90) 1.30 (0.48–1.47) − 0.25 ± 0.37 − 12.89 ± 18.77 0.034
Coronary arteries 1.29 (0.00–1.48) 0.84 ± 0.63 − 0.25 ± 0.17 − 16.84 ± 11.07 0.237

Data is presented in mean (± standard deviation) and median (interquartile range) according to normality.

Atherosclerotic vascular calcification occurs in the intima layer,
resulting from the crystallization of calcium/phosphate in the form of
hydroxyapatite in the extracellular matrix. Calcification is histologically
classified in microcalcification (0.5–15 μm), punctate (15 μm - 1 mm),
fragment (≥1 mm) and sheet calcification (>3 mm). Fragment and sheet
calcifications are frequently observed in healed and stable fibrocalcific
plaques, easily apparent in CT and intravascular imaging modalities. In
contrast, plaque rupture, thin cap fibroatheroma and plaque erosion
show little histological calcification [15]. It has been proposed that
microcalcifications (<10 μm) of apoptotic macrophages or smooth
muscle cells in thin fibrous cap can cause local stress and facilitate
plaque rupture [16]. Molecular imaging with 18F–NaF PET-CT can
differentiate macrocalcifications (CT study) from microcalcification
areas (18F–NaF), the latter being associated with clinical instability [17].
Coronary microcalcification activity predicts increased coronary calci­
fication at the same segment after one year, and coronaries without
18
F–NaF do not show macrocalcification progression [18]. As micro­
calcification analysis provides valuable insight on atherosclerosis dis­
ease activity, it can be a useful surrogate endpoint in drug trials aiming
disease modification [18,19].
High-intensity statin therapy with rosuvastatin has been shown to
reduce coronary plaque volume, coronary plaque lipid content and

18
Fig. 5. Heat map of maximum F–NaF uptake distribution according to baseline cardiovascular risk quartiles.

5
M. Oliveira-Santos et al.
Fig. 6. Graphical abstract.
Rosuvastatin effect on vascular microcalcification activity. TBR – tissue to background ratio;
carotid intima-media thickness, assessed respectively by intravascular
ultrasound [20,21], near-infrared spectroscopy [22] and ultrasound
[23] trials. Higher rosuvastatin doses (40 mg versus 5 mg) resulted in
more pronounced reductions in necrotic core volume after 12 months
(intracoronary imaging with virtual histology), without significant
change on fibrous tissue nor dense calcium [24]. Statin therapy has been
shown to decrease arterial wall inflammation in molecular imaging
studies with 18F- fluro-deoxy-glucose (FDG) PET-CT (25). The first
report came from Japan, from an oncological population randomized to
simvastatin and placebo. Aortic and carotid 18F-FDG uptake was
significantly reduced by simvastatin after 12 weeks [26]. In a trial of
stable high CV risk patients, with and without previous atherosclerotic
events, atorvastatin 80 mg was more effective than atorvastatin 10 mg in
reducing aortic and carotid 18F-FDG uptake after 12 weeks [27]. Using
the same imaging datasets, Singh and co-authors showed that the statin
effect was greater in coronary plaques with high-risk features [28].
Considering these studies in which statins demonstrated a positive effect
in several markers of atheroma instability (inflammatory activity, lipid
content and necrotic core volume), we hypothesised that rosuvastatin
therapy reduces vascular plaque 18F–NaF uptake, the gold-standard
non-invasive marker of microcalcification activity.
Recently, Zhang and colleagues reported the effect of atorvastatin
treatment in atherosclerotic rabbit models. In this animal study, ator­
vastatin reduced inflammatory activity, lipid levels and increased
macrocalcification, but reduced 18F–NaF uptake in PET-CT, which was
co-localized with microcalcification areas in histology analysis [29].
This experimental data is in accordance with our findings. The authors
propose a link between the atherosclerotic calcification process and
inflammation. Inflammatory cells secrete pro-osteogenic cytokines that
can induce cellular populations of the vessel wall to differentiate into
osteoblast-like cells, which promote the formation of microcalcifications
[30]. As anti-inflammatory agents, statins may therefore reduce
microcalcification activity detected by PET-CT (29). A longitudinal work
in diabetics by Reijrink et al. showed that baseline arterial wall 18F-FDG
uptake was a significant predictor of 18F–NaF activity after five years of
follow-up, suggesting that arterial inflammation is a precursor of arterial
microcalcification [31]. These findings potentially represent another
6
Atherosclerosis xxx (xxxx) xxx
18
F–NaF: fluorine-18 sodium fluoride.
mechanism of the “so-called” pleiotropic effects, that even though sta­
tins promote macrocalcification over time, probably there is a reduction
in microcalcification activity, protecting against clinical instability.
The quest for the vulnerable plaque and its treatment with invasive
methods is not over, however molecular imaging studies show more
potential to quantify disease activity and identify the vulnerable patient
[32]. As demonstrated previously [33], in this relatively small sample of
high CV risk individuals there was a positive and significant correlation
between 18F–NaF uptake and CV risk estimation with SCORE 2 and
ASCVD. PET-CT with 18F–NaF offers a comprehensive study of the in­
dividual’s atherosclerotic burden, coupled with metabolic information
on plaque activity, therefore more in line with the evolving concept of
CV risk stratification, transitioning from the “vulnerable plaque” to the
“vulnerable patient” concept [34].
4.1. Study limitations
This was a single-arm trial without placebo, as we considered the
absence of statin therapy unethical in this high CV risk population with
atheroma documentation. This design cannot exclude a regression to the
mean phenomenon. The lack of fusion with CT coronary angiography
limits coronary plaque assessment in this trial, not allowing the obser­
vation of possible changes in the characteristics/volume of the plaques,
information that could be of added value to the tracer uptake. ROPPET-
NAF included asymptomatic participants, so its results cannot be
extrapolated to patients with acute atherosclerotic events. Higher risk
patients with greater atheroma volume usually experience less disease
progression with statins [35], so hypothetically a superior effect could
be expected in such populations. The sample size for the matched
analysis was slightly lower than anticipated (n = 31 instead of 32). A
post-hoc power analysis was conducted using the Wilcoxon signed-rank
test, yielding a power (1-β) of 90%, therefore adequate for the scienti­
fic question. However, these findings and further clinical correlations
should be confirmed in larger populations.
M. Oliveira-Santos et al.
4.2. Conclusion
In a single centre non-randomized clinical trial of high CV risk in­
dividuals with subclinical atherosclerosis, maximum atherosclerotic
plaque 18F–NaF uptake was reduced after high-intensity statin treatment
for six months. This is the first human report about statins’ effect on
microcalcification activity, assessed by 18F–NaF PET-CT. These findings
add valuable information to our current knowledge on statin therapy in
CV diseases prevention, especially considering the established concept
of macrocalcification intensification after prolonged statin use.
Furthermore, it shows the potential of molecular imaging methods to
study relevant surrogate endpoints in drug trials.
Financial support
The trial was funded by a research grant from AstraZeneca-Produtos
Farmacêuticos, Lda.
CRediT authorship contribution statement
MOS, MCB and MJF designed the study, undertook experiments,
analysed results and interpreted the data. MJF is the chief investigator.
JR, LP and CES participated in study design and patient recruitment. JR
and RS collected data and analysed it. RS, PS, AA and MJF interpreted
the data. MOS drafted the first and subsequent versions of this report
with key input form MJF, LG and PS, and revisions from all other au­
thors, who reviewed and approved the final submitted report.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.atherosclerosis.2024.117481.
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