In a European population of patients with low prevalence of obstructive CAD, the present study demonstrated that higher levels of circulating PCSK9, while being related with moderately higher levels of total and LDL cholesterol, were not associated with obstructive coronary disease. On the other hand, the novel finding of this study was that lower PCSK9 was associated with specific metabolic abnormalities and diffuse coronary atherosclerosis, beyond LDL cholesterol levels and statin use. In particular patients with lower PCSK9 had low HDL cholesterol, a higher CTA score (an integrated measurement of coronary atherosclerosis and risk) and a higher number of non-obstructive and mixed coronary atherosclerotic plaques. At multivariable analyses, lower PCSK9 levels were an independent determinant of the CTA score, and HDL cholesterol significantly contributed to this association.
PCSK9 and coronary atherosclerotic disease
The relationship between circulating PCSK9 levels and CAD has been extensively explored. PCSK9 plasma levels have been associated with obstructive coronary lesions in patients with acute coronary syndrome and myocardial infarction [
6‐
10] as well as with subclinical coronary atherosclerosis and imaging markers of atherosclerotic risk even if in some reports this relationship is not so clear [
11‐
18]. The association of PCSK9 with coronary atherosclerosis in contemporary populations may be influenced by a number of factors. There is a shift in the clinical presentation of atherosclerosis from acute coronary syndromes to chronic atherosclerotic disease, which implies a possible change in the underlying mechanisms and prevents a simple extension of the results obtained by association studies in high risk patients to lower risk populations [
19]. The diffusion of treatments with lipid lowering drugs have altered the relationship between lipid profiles and coronary disease [
19,
20] and may uncover other emerging disease determinants, such as obesity, metabolic syndrome and diabetes. The present study was specifically designed to explore the relationships between circulating levels of PCSK9, comprehensive circulating determinants of the atherosclerotic risk and precise characterization of coronary artery disease in contemporary patient populations with suspected CAD. We did not find an association of PCSK9 levels with obstructive CAD but we documented a clear inverse correlation between PCSK9 and global coronary atherosclerotic burden as described by CTA score. These somewhat surprising results could be explained tacking into account some peculiarity of the EVINCI study. Firstly, the population was a contemporary European multicentric group of patients with stable angina, receiving optimal medical treatment and with a lower prevalence of obstructive CAD (35% of patients with CTA stenosis > 50% and 11% with stenosis > 70%) than usually found either in patients with acute coronary syndromes [
7,
8] or referred to coronary catheterization in whom circulating PCSK9 had been previously measured [
9,
10]. Secondly, using a comprehensive CTA score, we were able to assess the global coronary atherosclerotic burden at the patient level [
27,
28]. In fact, this score, incorporating number and characteristics of all coronary plaques, is a better marker of the presence and extent of coronary atherosclerosis than estimates of CAD presence and severity only based on “hemodynamically significant” lesions. Thus, the CTA score takes into account the possible effects of multiple atherogenic processes on the final coronary phenotype and is a comprehensive predictor of coronary outcome [
28]. This was particularly relevant considering that the present study group had a peculiar metabolic profile linked to diffuse coronary atherosclerosis in the absence of high LDL cholesterol levels usually associated with obstructive CAD.
In our population circulating PCSK9 was still correlated with total and LDL cholesterol [together with Apo B and Lipoprotein (a)] but showed also a strong correlation with HDL cholesterol levels (together with Apo A1). The relationship of PCSK9, total or LDL cholesterol and Lipoprotein (a) has been demonstrated in many different populations and in large study cohorts [
11,
34‐
37]. On the other hand, the relationship of PCSK9 levels and HDL cholesterol is less well established, and a clear involvement of PCSK9 in HDL metabolism is indeed uncertain [
12,
38‐
42]. Interestingly, we found that the relationships of PCSK9 with LDL cholesterol was affected and, as matter of fact, obscured by statin treatment, while that with HDL cholesterol was not (Fig.
4). This is consistent with previous observations studying the effects of statin treatment on PCSK9 levels [
43‐
45] showing that the direct correlation between PCSK9 and LDL cholesterol is lost after treatment [
43]. Current results add evidence that statins do not affect the strong relationship of PCSK9 with HDL cholesterol.
We found a relationship of low PCSK9 levels with insulin resistance or diabetes, obesity or metabolic syndrome. The association of PCSK9 with glucose metabolism has been extensively studied at epidemiological, genetic, clinical and animal levels [
46‐
50]. The possible interaction of PCSK9, glucose metabolism and treatment has also been investigated. Some antidiabetic drugs have been shown to decrease PCSK9 levels [
51] while treatment with PCSK9 inhibitors was not associated with increased incidence of diabetes [
52]. Even if most findings suggest a direct correlation of PCSK9 with abnormal glucose metabolism and insulin resistance, some studies suggest an opposite relation similar to that found in the present work.
In particular, patients with specific loss-of-function PCSK9 mutations or polymorphisms had increased prevalence in pre-diabetes and diabetes [
53] as well as higher HOMA-IR index and insulin levels [
54]. Interestingly, the direct relationship between PCSK9 and Lipoprotein (a) observed in the present study is in keeping with the association of low PCSK9 levels with insulin resistance and diabetes. In fact, in a large population study and metanalysis Paige et al. demonstrated the association of low Lipoprotein (a) concentrations and higher risk of type-2 diabetes [
55].
Similarly, the inverse correlation of circulating PCSK9 with BMI, consistent with the direct correlation with adiponectin, observed in the present study, was not a completely expected result [
46,
56]. Actually, an inverse association between PCSK9 and BMI had been already observed in some low cardiovascular risk populations such as healthy subjects [
11], patients with an altered metabolic liver function, such as those with an increased hepatic fat [
57], children and adolescents after adjustment for age, glucose, insulin, and adiponectin [
58]. However, the mechanism of these associations is still unknown.
Similarly, clinical, genetic, and experimental evidence linked PCSK9 with metabolic syndrome [
46]. In the present study, we found no association between PCSK9 and some components of the metabolic syndrome such as triglycerides and hypertension, while we found a very strong association between low PCSK9 levels and HDL cholesterol levels, independent of statin use. The association between lower PCSK9 levels and lower levels of adiponectin further underlines the link of PCSK9 with a specific metabolic phenotype [
56]. As matter of fact, these results add some complexity to the known relation between PCSK9 and cholesterol. In fact, at least in this population, diabetes, obesity and the metabolic syndrome seem to override the influence of PCSK9 on LDL metabolism, as demonstrated by the loss of the correlation between PCSK9 and LDL cholesterol in patients with altered metabolic phenotype (Fig.
3).
Moreover, we also observed a tight relationship between circulating PCSK9 and liver enzymes, classically considered as markers of liver cellular damage. More recently liver enzymes have been also considered as surrogate biomarkers of liver metabolic function [
59]. In the present population liver enzymes where in the normal range and the observed correlation with PCSK9 might express variable liver metabolic function (Additional file
1: Tables S6, S7). This observation needs further validation and the molecular mechanisms could be explored.
Given the peculiar metabolic patterns associated with low PCSK9 levels and the inverse relationship observed between PCSK9 and the CTA score, the present results suggest a possible pathophysiologic link between low circulating PCSK9, a metabolic phenotype characterized in particular by low HDL cholesterol and diffuse coronary atherosclerosis in a population at low prevalence of obstructive CAD. In a previous study performed in this population, we demonstrated that low plasma HDL cholesterol was an independent predictor of the CTA score together with age, gender, leptin and IL6 [
23]. In the present study, low PCSK9 was an independent predictor of the CTA score in a multivariable model including the same variables and outperforming a wide variety of risk factors and bio-humoral markers. However, when HDL cholesterol levels were added to the analysis, the association of PCSK9 with the CTA score lost significance, strongly suggesting a contribution of HDL cholesterol to PCSK9-associated atherosclerotic risk. It has been very recently reported that individuals who reach low levels of LDL cholesterol may be persistently at high risk of atherosclerotic disease when total/HDL cholesterol ratio is discordantly higher [
60]. Patients with such discordance had a greater atherogenic clinical profile with high levels of triglycerides, high BMI and a high prevalence of risk factors, including diabetes [
60]. Interestingly, a similar metabolic pattern was recognized in the present study in patients with low PCSK9 levels who also showed low HDL and high total/HDL cholesterol ratio (Table
2).