Plasma lipoprotein(a) is associated with calcification activity of the thoracic aorta and aortic valve in statin naïve individuals with diabetes mellitus

Cardiovascular disease (CVD) is a leading cause of death worldwide and rapid progression of arterial calcification is associated with increased CVD risk.1 18F-Sodium Fluoride Positron Emission Tomography (18F-NaF PET) is a novel molecular imaging modality for detecting calcification activity, the earliest form of arterial calcification, and can accurately identify patients who are at risk of progressive arterial calcification of structures such as the thoracic aorta and the aortic valve.2, 3 Individuals with diabetes mellitus have a higher burden of arterial calcification activity and carry a disproportionately higher risk of CVD.4, 5 However, predicting those diabetic individuals who will suffer from rapidly progressive arterial calcification is difficult, as the pathophysiology of arterial calcification is complex, likely influenced by many local and systemic mediators. Statin therapies are known to modulate the progression of arterial calcification.6 Lipoprotein(a) (Lp(a)), an LDL-like particle, characterized by the presence of the apolipoprotein(a)(apo(a)) component that is covalently linked to the apoB moiety by a single disulfide bound, is a causal risk factor for CVD, principally coronary artery disease and calcific aortic valve stenosis (CAVS), probably through multiple mechanisms, including pro-inflammatory, pro-atherogenic and pro-calcification effects.7 However, the relationship between Lp(a) and arterial calcification activity remains incompletely understood. In this study, we aimed to determine the relationship between serum Lp(a) levels and calcification activity of arterial structures, as determined by 18F-NaF PET, in statin naïve individuals with diabetes mellitus. Individuals aged 50–80 with type 1 or type 2 diabetes mellitus and no prior clinical coronary heart disease were recruited from the community between 2015 and 2018 as part of a randomized controlled trial of vitamin K and colchicine for arterial calcification activity; The ViKCoVaC trial.8 All individuals were screened with a CT coronary calcium score, and individuals with a CT calcium score ≥10 Agatston units were included in the trial. Ten participants with a CT calcium score of zero were included as part of a natural history cohort.3, 8 Briefly, individuals with symptomatic coronary artery disease, planned or prior coronary artery bypass surgery or percutaneous coronary intervention, chronic kidney disease (eGFR ≤30 mL/min/1.73 m2) or individuals using vitamin K antagonists were excluded. The full trial details, including detailed inclusion and exclusion criteria, have been previously published.3, 8 The study was approved by the Royal Perth Hospital Research Ethics committee (REG14-095), is registered on www.anzctr.org.au (ACTRN12616000024448) and complies with the declaration of Helsinki. All participants provided written informed consent. In the present cross-sectional analysis, participants in the randomized and natural history arm not taking statin therapy (i.e. statin naïve) at time of recruitment (n = 46) were included. After screening, participants underwent a clinical visit including a medical and medication history. Plasma lipid, lipoprotein, glycated haemoglobin A1c (HbA1c) and high sensitivity C-reactive protein (hsCRP) concentrations were measured using standard methods (Architect c16000 Analyser; Abbott Diagnostics, Abbott Laboratories, Abbott Park, IL). LDL-cholesterol was estimated using the Friedewald calculation. Total Lp(a) particle concentration was undertaken using a Roche assay (Tina-quant Lipoprotein (a) Gen.2 on the Cobas pro c503 module). All participants underwent a screening cardiac gated CT coronary artery calcium (CAC) score on a Philips iCT (128 detector row, 256 slice, 120 kV, 40 mAs) or Siemens Definition AS+ (64 detector row, 128 slice, 120 kV, 60mAs) CT scanner. All participants underwent 18F-NaF PET imaging using a Siemens mCT64 PET/CT scanner after intravenous administration of 250 MBq of 18F-NaF and a 1 h resting period. Pre-treatment with beta blocker or ivabradine therapy was undertaken prior to PET imaging, to target a heart rate ≤60.8 Gated 18F-NaF PET scans were reconstructed in the diastolic phase and fused with the CT coronary calcium score scan and regions of interest were drawn around the aortic valve to determine the maximum SUV (SUVmax). Ungated 18F-NaF PET scans were fused with the attenuation correction CT scans and regions of interest were drawn around thoracic aorta. Three adjacent slices centred on the highest SUVmax from the thoracic aorta were averaged to result the most diseased segment (MDS) SUVmax.8 All SUVmax measures were adjusted for blood-pool mean SUV, to result the maximum tissue to background ratio (TBRmax).8 The SUVmax and TBRmax values are both established measures of calcification activity.8 For the purpose of this analysis, only baseline 18F-NaF PET scans performed prior to the commencement of trial therapies, are used. Data were analysed using the Statistical Packages for the Social Sciences (SPSS, version 29.0, USA). Data are presented as mean ± SD unless otherwise indicated. Skewed variables were log-transformed. Associations were evaluated using Pearson two-tailed correlation and linear regression. A multiple variable linear regression model was developed, including clinically relevant covariates that likely influence calcification of the aortic valve and thoracic aorta. Statistical significance is defined as a p-value <.05. Forty-six statin naïve individuals were included in the current analysis (Figure 1). Table 1 describes the clinical and biochemical characteristics and measures of arterial calcification activity in statin naive individuals with diabetes mellitus. They were, on average, middle-aged and overweight. Two participants (4%) had a history of noncoronary vascular disease and 40 participants (87%) had subclinical coronary artery disease (the presence of a nonzero coronary artery calcium score). The prevalence of hypertension, overweight (body mass index >25 kg/m2) and smoking status (current/ex-smokers) was 59%, 91% and 35%, respectively. The median serum Lp(a) level was 22 nmol/L (7.0–68 nmol/L; 25th–75th percentile); 13% of participants had an elevated Lp(a) level (>125 nmol/L). In univariate regression analysis, there were statistically significant associations of serum Lp(a) concentration with thoracic aorta MDS SUVmax (r = 0.390, p = .007; Figure 2A) and aortic valve SUVmax (r = .344, p = .019; Figure 2B). Serum hsCRP concentration was also positively associated with aortic valve SUVmax (r = 0.293, p = .048), but not with thoracic aorta SUVmax (r = .178, p = .237). Lp(a) was not associated with the thoracic aorta MDS TBRmax (r = .265, p = .075) or aortic valve TBRmax (r = .229, p = .126). In multiple regression analysis, including age, male sex and hsCRP levels (Table 2), serum Lp(a) level was independently and significantly correlated to the SUVmax in the thoracic aorta (β coefficient 0.310; SE 0.035, p = .030; adjusted R2 = 31.9%) and aortic valve (β coefficient 0.328; SE 0.047, p = .029; adjusted R2 = 25.1%). Replacing hsCRP concentration with hypertension (yes or no) or smoking status (yes or no) as an independent variable did not alter these associations (data not shown). Our major findings were that serum concentration of Lp(a) was positively associated with the calcification activity in the thoracic aorta and aortic valve using 18F-NaF PET/CT SUVmax. These significant associations were independent of age, sex and serum hsCRP and LDL-cholesterol levels. Previous studies using 18F-NaF PET/CT imaging have examined the association between Lp(a) and arterial calcification activity, but none focus on diabetes mellitus.9-11 Després et al found that individuals with elevated Lp(a) had a higher aortic valve mean TBR compared with those with low Lp(a) levels.9 Zheng et al also reported that elevated Lp(a) was independently associated with increased aortic valve mean MDS TBR in patients with CAVS.10 In another study of patients with CAVS, Kaiser et al found that Lp(a) was not associated with aortic valve mean or maximal SUV or TBR.11 Discrepancies between these studies referred to may be due to differences in subject characteristics (established calcification vs. apparent healthy status), imaging protocols and statin use. We have extended the previous reports by examining the association between Lp(a) and calcification activity not only in aortic valve, but also thoracic aorta, in diabetic individuals with majority having subclinical atherosclerosis. Whilst a meta-analysis of these studies and ours would be worthwhile, it must consider the significant heterogeneity in image acquisition and analysis protocols, endpoints and study populations. We speculate that the pathophysiological role of Lp(a) in promoting arterial calcification activity is likely different between healthy individuals and patients with CAVS, as previously reported.9-11 The mechanisms by which Lp(a) may promote vascular and/or valvular calcification activity remain unclear. In vitro analyses have demonstrated that Lp(a) can promote cell mineralization, as well as the expression of pro-calcific proteins, including bone morphogenetic protein 2 (BMP-2), osteopontin (OPN) and runt-related transcription factor 2 (RUNX2), through upregulation of pro-inflammatory nuclear factor-κB and interleukin-6 (IL-6) expression.12 Lp(a) can also induce osteogenic differentiation of valvular interstitial cells.10 The pro-calcifying properties may partly relate to the oxidized phospholipids (OxPLs) carried by Lp(a),13, 14 with experimental evidence showing that such effects could be alleviated by inactivation of OxPL.10, 12 The complex and possibly indirect association between serum Lp(a) and calcification activity is supported by the modest strength of the relationship observed and, despite adjusting for confounding variables, may represent only part of a systemic pro-calcifying phenotype of patients inherently susceptible to an increased risk of cardiovascular disease. Furthermore, the observed association between serum hsCRP concentration and aortic valve SUVmax also suggests that local and systemic nonspecific inflammation is another likely mediator in the development and progression of aortic valve calcification. The effect of Lp(a) on arterial inflammation, measured using 18F-fluorodexoyglucose (18F-FDG) PET/CT, in patients with diabetes mellitus also merits investigation. We found no significant association between Lp(a) and the thoracic aorta MDS TBRmax or aortic valve TBRmax. Unlike SUVmax, TBRmax is corrected for background blood activity which may increase variability of the quantitative parameters. Hence, TBRmax may not be sufficiently precise and sensitive to quantify calcification activity and may diminish its association with Lp(a). However, this speculation merits further investigation. Our study has several limitations. The cross-sectional design does not allow causal inferences to be drawn. Hence, our results should be viewed as hypothesis-generating only. The small sample size of this study is noteworthy and may reduce the confidence in the observations. However, a small sample size tends to increase the risk of a type II error, and therefore, it is also possible the strength of association may be underestimated in the current study. Indeed, longitudinal studies with larger sample sizes are needed to draw definite causal conclusions and confirm these associations. We only studied diabetic patients with a relatively low range of Lp(a). Whether our principal findings apply to patients with high Lp(a) (such as a concentration of >200 mmol/L) or to nondiabetic population remains to be formally tested. We excluded participants taking statins from this analysis. This approach may introduce a selection bias that warrants consideration when interpreting the results. However, all 46 statin naïve participants were included, and the biological rationale for this approach is to remove the potential confounding effects of statin on Lp(a) concentration and vascular calcification.6 In addition, excluding statin users may also limit the translational capacity of our findings to high CVD risk cohorts, in which many patients are on statin therapy. In conclusion, our study provides new evidence that Lp(a) may play a role in promoting arterial calcification activity in statin naïve individuals with diabetes mellitus. Recent attempts to reduce calcification burden and progression within vascular territories have been unsuccessful. Meanwhile, newer therapies such as antisense inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) or apo(a) have been shown to significantly reduce Lp(a) concentrations.15 Whether such reduction in Lp(a) translates into a reduction in the complications associated with calcification of arterial structures, notably CVD, warrants further demonstration. This work was supported by funding from the Royal Perth Hospital Research Foundation (14-095) and a National Health and Medical Research Council Investigator Grant (APP1194199). During part of this work, Dr Jamie Bellinge was supported by an Australian Government Research Training Program Scholarship at The University of Western Australia. Open access publishing facilitated by The University of Western Australia, as part of the Wiley & The University of Western Australia agreement via the Council of Australian University Librarians. The authors have no relevant conflicts of interest to disclose.