The Association Between Coronary 18F-Sodium Fluoride Uptake With Pro-Atherosclerosis Factors in Patients With Multivessel Coronary Artery Disease: A Mono-Centric Pilot Study


 Purpose: 18F-Sodium fluoride (18F-NaF) positron emission tomography (PET) is a novel approach to detect and quantify microcalcification in atherosclerosis. Peri-coronary adipose tissue (PCAT) is associated with vascular inflammation and high-risk atherosclerotic plaque. We aimed to assess the association between coronary 18F-NaF uptake with pro-atherosclerosis factors in patients with multivessel coronary artery disease (CAD) and to explore the systematic vascular osteogenesis in the coronary artery and aorta in these patients. Methods: Patients with multivessel CAD prospectively underwent cardiac computed tomography (CT) and 18F-NaF PET/CT. PCAT density was measured in the coronary artery and the average PCAT value was calculated from the three coronary arteries in each patient. 18F-NaF tissue-to-blood ratios (TBR) in the coronary artery (TBRCoronary) and aorta (TBRAorta) were calculated. Correlations between coronary 18F-NaF uptake with PCAT density, coronary artery calcium (CAC) burden, CAD risk factors, serum biomarkers, and aortic 18F-NaF uptake were evaluated, respectively. Patients were categorized by a median of TBRCoronary 2.49. Results: 100 multivessel CAD patients (64.00 [57.00 - 67.75] years; 76 men) were prospectively recruited. 6010 active aortic segments (TBR ≥ 1.6) were identified. TBRCoronary was significantly associated with the PCAT density (r = 0.56, p < 0.001) and CAC score (r = 0.45, p < 0.001). TBRCoronary was also significantly associated with the TBRAorta (r = 0.42, p < 0.001). In addition, patients with higher TBRCoronary showed elevated PCAT density (-75.89[-79.07 - -70.06] vs -84.54[-90.21 - -79.46]; p < 0.001) and CAC score (1495.20[619.80 - 2225.40] vs 273.75[116.73 - 1198.18]; p < 0.001) in comparsion patients with lower TBRCoronary. TBRCoronary was correlated with the age (r = 0.24, p = 0.019) and the serum troponin I levels (r = 0.22, p = 0.039). There were no significant correlations between TBRCoronary with other conventional CAD risk factors and other serum biomarkers.Conclusion: Coronary 18F-NaF uptake was correlated with the PCAT density. A significant correlation between 18F-NaF uptake in the coronary artery and aorta might indicate a systematic vascular osteogenesis in patients with multivessel CAD.


Introduction
Coronary atherosclerotic plaque rupture is the principal cause of acute coronary syndrome and a signi cant cause of sudden cardiac death and its prevention is a crucial adjective [1,2]. During atherosclerosis progression, macrophage-derived cytokines induce osteogenic differentiation and mineralization of vascular cells, which suggests that pro-in ammatory molecules could promote atherosclerotic osteogenesis by regulating the differentiation of calcifying vascular cells [3]. Active microcalci cations in the atherosclerotic plaque is considered as a marker of cell death and in ammation and carries an increased risk of plaque rupture and associated complications [4]. 18 F-sodium uoride ( 18 F-NaF) has been used for bone positron emission tomography (PET) imaging to de ne osteogenic activity and its feasibility for identifying increased intraplaque osteogenic activity in vivo was appreciated [5,6]. By providing molecular information vascular microcalci cation, 18 F-NaF PET/computed tomography (CT) is potentially capable to identify high-risk atherosclerotic plaques in patients with multivessel coronary artery disease (CAD). Additionally, in complex cardiovascular diseases, the relevance of systemic causes of atherosclerosis development and progression is widely recognized, 18 F-NaF PET coud be a novel approach to visualize and quantify biochemical activity in systematic vasculature with high sensitivity.
Peri-coronary adipose tissue (PCAT) is a part of epicardial adipose tissue depot with brown and beige features, which is a source of some in ammatory mediators and pro-atherogenic mediators [7,8]. Its closely near to the coronary artery tree has been implied to be potentially relevant for the development and progression of atherosclerosis by local in ammation and paracrine mechanisms [9, 10]. Increased density of PCAT plays an important role in the development of vascular in ammation and coronary atherosclerosis through bidirectional communication with the vessel wall at a cellular level [11,12]. A recent large cohort study demonstrated that high PCAT density predicted all-cause and cardiac mortality and could enhance cardiac risk prediction and risk strati cation by providing a quantitative measurement of coronary in ammation [13]. In addition, new-onset or rapid coronary calci cation progression is associated with an enhanced risk for future CAD events and cardiovascular risk prediction can be improved by examining the coronary artery calcium (CAC) burden.
In the present study, we aimed to analyze the association between coronary artery osteogenic activity and conventional pro-atherosclerosis factors, including PCAT density, CAC burden, CAD risk factors, and serum biomarkers in patients with multivessel CAD. In addition, we also evaluated the systematic vascular osteogenesis in the coronary artery and aorta in these patients.

Patient population
This observational cross-sectional study was a mono-centric pilot study to a prospective trial registered with the Chinese Clinical Trial Registry (ChiCTR1900022527). A total of 457 consecutive patients with CAD were prospectively recruited in Beijing Anzhen Hospital between February 2018 and April 2021. Inclusion in this study required angiographically con rmed multivessel CAD, de ned as having at least 2 of 3 epicardial vessels with a stenosis ≥70% or left the main stenosis ≥50%. Patients were excluded if: 1) a recent myocardial infarction (< 4 weeks), 2) history of malignancy, acute or chronic in ammatory and autoimmune disease, 3) history of cardiovascular surgery or cardiac transplantation. Finally, a total of 100 multivessel CAD patients were recruited in our current study. The study ow chart is shown in Figure  1. The project was approved by the Medicine Ethics Committee of Beijing Anzhen Hospital (2018055X) and adhered to the principles laid out in the Declaration of Helsinki. Baseline characteristics of study population are listed in Table 1.
Analysis of CAC burden and PCAT on CT All cardias CT scans were conducted using electrocardiography-gated cardiac CT using a 128-slice multidetector computed tomography scanner (Biograph mCT, Siemens Healthcare, Erlangen, Germany). The scan parameters were: 128 x 0.6 mm collimation; tube voltage, 120 kV; gantry rotation time, 330ms; and tube current, 770-850 mAs. Coronary calcium was quanti ed on both a per-patient and per-segment level by an experienced observer (WW) using volume analysis software (Cascoring Siemens Healthcare, mCT).
The CAC score was derived using the Agatston method [14]. To quantify the PCAT density, a CT attenuation threshold of -190 to -30 Houns eld Units was used to isolate adipose tissue by Mimics Medical software (version 21.0; Materialise, Leuven, Belgium) [15], and the PCAT density was de ned as the mean attenuation within such contamination-free volumes of interest and was measured in the reference region of the proximal left anterior descending (LAD), proximal left circum ex (LCX), and midright coronary artery (RCA) on axial CT images. For each coronary artery, ve of regions of interest (ROIs, each ROI area = 3 mm 2 ) were manually placed on the region of distance the outer coronary artery wall equal in width to the vessel diameter [16]. The PCAT density of the LAD (PCAT LAD ), LCX (PCAT LCX ) and RCA (PCAT RCA ) was calculated by the average PCAT value from the value of ve ROIs in LAD, LCX, and RCA, respectively. The PCAT density in each patient was calculated as the average PCAT value from three main coronary arteries (LAD, LCX, and RCA). PCAT density measurement by cardiac CT was performed by two experienced nuclear cardiologists (MJ and WW), who were blinded to the quantitative analysis data as well as 18 F-NaF PET/CT image analysis.
Cardiac 18 F-NaF PET/CT and image analysis All patients were administered a target dose of 18 F-NaF (3.7 MBq/kg) intravenously and subsequently rested in a quiet environment for a 120-min uptake period, an electrocardiogram-gated cardiac 18 F-NaF PET/CT imaging (Biograph mCT, Siemens Medical Systems, Erlangen, Germany) was performed. A lowdose attenuation correction CT scan (120 kV, 50 mAs) was then acquired. The PET data were reconstructed using a point spread function + time of ight algorithm (time of ight + TrueX, Siemens Ultra-HD), with 5 iterations and 21 subsets. Due to the small size of the vulnerable plaques, an in-plane pixel size of 2 mm with a corresponding reconstructed image matrix size of 400×400 was used to achieve a high spatial resolution.
To evaluate the coronary 18 F-NaF uptake, the maximum standardized uptake value (SUV max ) (a validated measure of tissue radiotracer uptake) of LAD, LCX and RCA were quanti ed from ROIs by delimiting threedimensional regions, respectively. The tissue-to-background ratios (TBR) in the LAD (TBR LAD ), LCX (TBR LCX ), and RCA (TBR RCA ) were then calculated by correction for background blood pool activity using the right atrium (mean SUV using cylindrical volumes-of-interest [radius: 10 mm; thickness: 5 mm] at the level of the RCA ostium). The TBR in the coronary artery (TBR Coronary ) was calculated as the average TBR value from three main coronary arteries (LAD, LCX, and RCA) in each patient.
The aortic (ascending aorta, aortic arch, descending aorta) 18 F-NaF uptake was determined by manually placing oval ROIs on the equatorial plane of these major arteries to avoid artifacts from the accumulation of 18 F-NaF in the vertebral body [17]. The SUV max of 18 F-NaF avid focus more than 1.6 times the mean SUV of the right atrium blood pool was considered an abnormal aorta lesion. The number of lesions and SUV max of each lesion in the aorta were recorded and measured. The TBR in the aorta (TBR Aorta ) was calculated by the average of lesions SUV max in the aorta corrected by the mean of SUV in the right atrium.

Statistical analysis
All statistical analyses were performed using SPSS software (version 25, SPSS, Inc., Chicago, IL). Continuous variables were tested for normality using Shapiro-Wilk test and were presented as mean ± standard deviation or median (interquartile range) dependent on the distribution. Patients were divided dichotomously by the median TBR Coronary value into group 1 (TBR Coronary ≥ 2.49, n = 50) and group 2 (TBR Coronary < 2.49, n = 50). Data were compared by using two-sample t-test or Mann-Whitney U tests.
Categorical variables were summarized using frequencies and percentages and were compared by using a chi-squared test (with a Yates correction or a Fisher exact test for smaller sample sizes). Spearman's correlation analyses and multiple linear regression analyses were used to assess the correlations between the coronary 18 F-NaF uptake with the PCAT density, CAC burden, CAD risk factors, serum biomarkers, and aortic 18 F-NaF uptake, respectively. Bland-Altman analyses were employed to assess the repeatability of the PCAT density and coronary 18 F-NaF uptake (Additional le: Figure S1 and Figure S2). A 2-sided p-value < 0.05 was regarded as signi cant.
Per patient, we found that PCAT density was independently associated with the TBR Coronary (Beta = 0.489; 95% con dence interval [CI]: 0.032 -0.067; p < 0.001) by multiple linear regression analyses (Demographics as covariates) (Table 3). In addition, the PCAT density was elevated in patients in group 1 in comparison with in group 2 (p < 0.001) (Supplemental le: Table S1).
There was a signi cant association between the TBR Coronary and the CAC score (r = 0.45, p < 0.001) ( Table 2). The CAC score was signi cantly higher in group 1 compared with that in group 2 (p < 0.001) (Supplemental le: Table S1).
Correlation between coronary 18 F-NaF uptake and aortic 18 F-NaF uptake On image analysis of aortic PET, we identi ed 6010 active segments in aorta. The TBR Coronary was signi cantly correlated with the TBR Aorta in all individuals (r = 0.42, p < 0.001) ( Table 2). Representative patients presenting in groups 1 and 2 are illustrated in Figures 3 and 4, respectively. Moreover, after adjustment confounding factors (age, gender, body mass index), we observed that the TBR Aorta (Beta = 0.409; 95% CI: 0.215 -0.619; p < 0.001) were independently associated with the TBR Coronary by multiple linear regression analyses (Table 3). The TBR Aorta in group 1 was signi cantly higher than that in group 2 (p = 0.001) (Supplemental le: Table S1).
Serum troponin I level in all individuals was correlated with TBR Coronary (r = 0.22, p = 0.039) ( Table 2).
There was no signi cant correlation between traditional CAD risk factors (eg. diabetes, hyperlipidemia, hypertension, smoker, family history of CAD, high-density lipoprotein, low-density lipoprotein, highsensitivity C-reactive protein, interleukin-6, tumor necrosis factor alpha, and creatinine clearance rate) with neither TBR Coronary (Table 2) and TBR Aorta (Table 4).

Discussion
In this present study, we investigated the correlations between coronary artery and aorta osteogenic activity with pro-atherosclerotic factors, including PCAT density, CAC score, and CAD risk factors in patients with multivessel CAD. We found that coronary 18 F-NaF uptake was signi cantly correlated with the PCAT density as well as the CAC score. Furthermore, a systematic osteogenesis activation in coronary artery and aorta was appreciated.
Atherosclerosis is a fundamental pathogenic process in many diseases, including cerebrovascular and cardiovascular diseases, aortic aneurysm/dissection, and arteriosclerosis obliterans. Plaque is known to be the major characteristics of atherosclerosis and various pathophysiologic processes are involved in the formation and progression of atherosclerotic plaque, including in ammation, apoptosis, and mineralization [18,19]. In ammation mainly mediated by macrophages is involved at the beginning of the formation of plaque. Macrophages promote the proin ammatory milieu and send speci c signals to vascular wall cells to initiate osteogenic differentiation. Once equilibrium in the arterial wall shifts toward calci cation, deposition of hydroxyapatite could progress quickly, and gives rise to microcalci cation, which is coalesce and ultimately pervade into the atherosclerotic plaque [20]. Microcalci cation, which represents a speci c phase in the evolution of an atheroma, is a key feature of atherosclerotic plaque rupture, that is embedded in the brous cap of atherosclerotic plaques and, then lead to considerable stress accumulation in the brous cap and destabilize the structural integrity of the brous cap [21]. 18 F-NaF is a radiotracer that preferentially identi es microcalci cation in arteries by binding to hydroxyapatite. Therefore, vascular 18 F-NaF PET may identify high-risk atherosclerotic plaque lesions and enable the quanti cation of osteogenic activity before therapeutic interventions, thereby providing a powerful tool for improving patient risk strati cation.
PCAT is an ectopic thoracic fat tissue located between the visceral layer of the pericardium and the myocardium, surrounding the coronary artery tree [7, 8, 10]. A large body of evidence, including experimental and clinical studies, has demonstrated that PCAT is a recognized source of proin ammatory mediators in high-risk cardiac patients, which can directly modulate the coronary artery through the mechanism of paracrine and autocrine [9,22]. PCAT exhibits a broadly pathogenic mRNA pro le, and it is associated with the presence and incidence of cardiovascular and cerebrovascular events independent of traditional risk factors [23]. Moreover, several studies have indicated that the relationship of adipose tissue and the vascular wall is a complex interaction, PCAT releases a wide range of bioactive molecules that exert endocrine and paracrine effects on the vascular lipid metabolism and vascular in ammation [10,24]. 18 F-NaF PET/CT has emerged as a noninvasive quantitative imaging modality and is able to measure the microcalci cation activity in the vasculature [4,25]. In this study, we found a signi cant correlation between coronary 18 F-NaF activity and PCAT density, which was concordant with ndings by Kwecinski et al [26,27], who demonstrated an association of increased PCAT CT attenuation with higher 18 F-NaF PET activity in patients with high-risk plaques. In contrast to previous studies, we conducted an observational cross-sectional study including 100 multivessel CAD patients and performed a delay PET scans (120-min) with potentially improved imaging contrast. We observed that PCAT density was signi cantly increased in patients with higher coronary 18 F-NaF uptake, and it was independently associated with the coronary 18 F-NaF uptake after adjustment for confounding factors.
Pioneering studies demonstrated that the coronary 18 F-NaF uptake was signi cantly correlated with the CAC score and the progression of coronary calci cation [17,28]. Increased coronary 18 F-NaF uptake was associated with more rapid progression of coronary calci cation at one year in patients with clinically stable multivessel CAD [28]. And intriguingly, we also found that coronary 18 F-NaF uptake was correlated with the calcium burden in the coronary artery assessed by cardiac CT. These results may indicate that the underlying correlation between the accumulation of 18 F-NaF and the incremental change in calci ed plaque progression. Moreover, McKenney-Drake et al demonstrated that 18 F-NaF uptake in all vascular segments was signi cantly correlated with age in patients with chest pain syndromes [29]. In our observation, increased coronary and aortic 18 F-NaF uptake were also presented in older patients, which might raise a intriguing possibility that intense hydroxyapatite deposition was developed in older patients.
Cardiac troponin I was used to detect myocardial necrosis as the preferred biomarker in the diagnostic of myocardial infarction [30]. Joshi et al reported an association between increased coronary 18 F-NaF uptake and higher plasma high-sensitivity cardiac troponin I concentrations in patients with stable CAD [31]. In this study, we also observed that serum troponin I level was associated with coronary 18 F-NaF uptake in multivessel CAD patients. In fact, silent plaque rupture and subclinical plaque thrombus formation are frequent incidental post-mortem ndings in patients with multivessel CAD. These results suggests that coronary 18 F-NaF uptake may identify high risk plaques which might be associated with thrombus formation and subclinical myocardial injury from microemboli.

Study Limitations
This study had several limitations. First, this was a single-center study given limited number of observations, and bias in patient selection was possible; however, adjustments were made for the confounding effects of risk factors for the association of PCAT density and coronary 18 F-NaF activity. Second, partial volume effects and cardiac motion could have affected the PET quanti cation in coronary artery lesions. Third, CT angiography is not performed in this study cohort. Finally, the patient outcome assessment is lacking from the current study.

Conclusion
In multivessel CAD patients, increased coronary 18 F-NaF uptake was signi cantly associated with the classic pro-atherosclerosis factors, including PCAT density and CAC score. We also observed an 18 F-NaF uptake cross-talk between the coronary artery and aorta. Patients' clinical research to validate that such a pro-atherosclerosis axis translates into a better outcome is warranted.       Representative case showing the relationship between coronary TBR with aortic TBR and PCAT in patients with prominent 18F-NaF uptake. Patient (male; 64y; TBRCoronary: 4.55; TBRAorta: 4.85; PCAT: -71.53; Coronary artery calcium score: 2995.50) suffered multivessel lesions presenting intense focal 18F-NaF uptake in left anterior descending artery overlying existing extensive coronary calcium (ABC), coupled with increased 18F-NaF uptake in aortic arch (DEF) and descending aorta (GHI), and with intense PCAT density (JKL). Epicardial adipose area (green) for placing ve regions of interest (3 mm2) and measuring PCAT density. 18F-NaF = 18F-sodium uoride; PCAT = peri-coronary adipose tissue; TBR = tissue-to-background ratio Figure 4 Representative case showing the relationship between coronary TBR and aortic TBR in patients with negative 18F-NaF uptake. Patient (male; 53y; TBRCoronary: 2.46; TBRAorta: 2.20; PCAT: -86.33; Coronary