Genetics of adrenergic signaling drives coronary artery calcification

1. Genetics and coronary artery calcification

Cardiovascular diseases hold primacy as a leading cause of morbidity and mortality worldwide, making especially engaging the research towards the identification of the potential underlying mechanisms as well as the prognostic factors and predictors of the disease. Despite being extensively studied, atherogenesis and its progression remain among the most fascinating phenomena, with various interrogatives still open. The atherosclerotic process is multifactorial, involves different cells, and generates new cellular phenotypes. Briefly, various triggers, including smoking, hypertensive peaks, and oxidative stress, induce endothelial injury leading to a hyperactive and pro-inflammatory phenotype of endothelial cells [1]. The changes in the pattern of membrane protein expression by endothelial cells facilitate the recruitment of circulating inflammatory cells, their infiltration into the intima of the vessel, and differentiation in macrophages, able to incorporate oxidized-LDL. The new phenotype of intimal macrophages, known as “foam cells”, facilitates the accumulation of cholesterol and lipids, generating the lipid core of atheroma. The highly inflammatory microenvironment increases the expression of interferon-β and PDGF, triggering proliferation and migration of vascular smooth muscle cells (VSMCs) and their switch from contractile to secretory phenotype [2].

The above mentioned events are recognized as leading steps and have been extensively characterized. However, other mechanisms are involved in the progression of atherosclerosis. In this context, the calcification of the atheroma is an intriguing phenomenon contributing to plaque degeneration. Indeed, coronary artery calcification (CAC) has been traditionally used as a valuable marker of coronary atherosclerosis and recent technological improvements have allowed an objective measurement of calcification density and extension, producing an accurate calcium score, widely employed to predict the risk of coronary events [3,4]. Albeit the highly predictive value of CAC has been ascertained, the exact molecular mechanisms leading to coronary calcification are not fully known. Several non-mutually exclusive mechanisms have been proposed to be involved in plaque calcification: i) the death of inflammatory cells within the atheroma and the subsequent release of apoptotic/necrotic bodies could act as a nucleating core for the formation of crystals; ii) the release of matrix vesicles could offer substrates for the nucleation of crystal complexes; iii) differentiation of pericytes or VSMCs in osteoblasts could contribute to the generation of bone-like structures. Each of these events could be further exacerbated by the reduced expression of mineralization inhibitors. Overall these mechanisms orchestrate the origin and progression of calcification in a patient-dependent fashion, as a high inter-individual variability has been reported when evaluating these processes.

2. Is genetics of sympathoadrenergic system pulling a few strings in CAC progression?

A genetically and heritable determination of CAC progression has been suggested, but these aspects have been underinvestigated. In this scenario, the study published by Klenke and colleagues in this issue of Atherosclerosis [5] responds to the desperate need for new alleles with a significant predictor value for CAC progression. In their elegant investigation, the authors use a hypothesis-driven approach to evaluate the contribution of specific single nucleotide polymorphisms (SNPs) in CAC progression. Despite being obviously less unbiased than genome-wide association approaches, the hypothesis-driven studies allow to select a priori specific pathways potentially important for the disease; the upstream gene selection facilitates the identification of SNPs with a mechanistic contribution to disease-risk. As potential risk alleles, Klenke and collaborators [5] selected 3 SNPs affecting the G protein transduction pathway, specifically, ADRB2, GNAS, and GNB3 genes, which encode β2 adrenergic receptor (β2-AR), Gs_α, and G Protein Subunit Beta 3, respectively. They show that the presence of these alleles is associated with an increased CAC progression by 30%, which is significantly higher than the genetic-risk emerging from previous research [6]. The study population composed of 3018 participants from the Heinz Nixdorf Recall study and the use of two different algorithms for the calculation of CAC progression make the data consistent, strongly supporting the idea that the presence of these SNPs cannot be neglectable in the calculation of the individual risk-score.

3. Mechanistic role of β2-AR signaling in determining the genetic risk of CAC progression

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and their encoding genes represent ~5% of the human genome [7]. Since GPCRs are involved in a plethora of signals and biological responses, their function has been extensively studied in several fields of human medicine, also becoming among the first therapeutic targets in the treatment, for example, of asthma and hypertension [8]. β2-AR is a GPCR with a fundamental role in human physiology and disease [9,10]. Its signal is involved in hypertension, myocardial infarction, and coronary artery disease (CAD) [11,12]. Genetic variants of the β2-AR gene (ADRB2) or of the genes encoding coupled G proteins (GNAS and GNB3) have been associated with atherosclerotic pheno-types: specifically with a higher incidence of cardiovascular events in CAD patients, a higher risk of ischemic stroke, and overall reduced survival [13,14]. Hence, to unveil the determinants of genetic heritability in CAC progression, the variants associated with β2AR and G proteins were sound candidates. As expected, the authors show that the different genetic conformations of these genes strongly affects the CAC progression, indicating the crucial function of this pathway in the development and evolution of CAC.

4. Direct involvement of β2-AR signaling in CAC progression

Importantly, the influence of CAC progression by SNPs within the β2AR signal pathway was confirmed after adjustment for other risk factors, such as cholesterol levels, diabetes, systolic blood pressure, and age5. Nevertheless, the Authors did not fully delve into the potential molecular mechanisms mediated by β2AR and G proteins able to affect CAC progression. Here we propose a recent new pathway identified for β2-AR, which could be directly implicated in vessel calcification, enlightening the impact of genetic variations of this pathway in CAC progression. Indeed, recent evidence indicates that β2-AR plays a crucial role in bone-deposition; specifically, the activation of this receptor on osteoblasts, the recruitment of Gsα protein, and the increased levels of intracellular cAMP lead to osteoclastogenesis [15]. Consistent with these findings, a clinical study published in Circulation demonstrates that β2-AR signaling is implicated in aortic valve calcification [16]. Specifically, the sympatho-adrenoceptors stimulation is shown to be involved in the osteogenic differentiation of human valve cells [16]. Although not experimentally proved, it is reasonable to speculate that the participation of the β-AR signaling pathway reported in valve calcification could be translated to the CAC process. For instance, β2-AR and G protein pathways could be implicated in osteogenic differentiation of VSMCs or pericytes during artery calcification, supporting the idea that SNPs associated with this pathway are independent risk factors for CAC progression (Fig. 1).

Fig. 1. β2-AR signaling and CAC progression: impact on genetic risk.

Coronary artery calcification (CAC) is an atherosclerotic phenotype strongly associated to coronary events. Its formation starts with the death of inflammatory cells in the lipid core, causing the formation of a necrotic core. The environment ensuing from these processes within the plaque triggers the differentiation of vascular smooth muscle cells (VSMCs) in osteoblast-like cells, able to produce hydroxyapatite. The progressive deposition of hydroxyapatite around the necrotic core eventually leads to plaque calcification. The molecular mechanisms underlying this phenomenon are not fully elucidated. The Ί2-AR signaling pathway could be involved in VSMCs-osteoblast differentiation, explaining the impact of its variation on CAC progression. Indeed, CAC progression does not seem to be dependent from other common cardiovascular risk factors, and the genetic contribution seems therefore to be very relevant. SNPs in genes related to Ί2-AR signal could determine a different signal transduction, resulting in distinctive entities in CAC progression.

Dedicated projects are needed to investigate these possible mechanism. Hence, the study proposed by Klenke et al. [5] may open a vast unexplored field, providing new compelling insights on the genetic impact on CAC progression and on the pivotal role of β2-AR signaling.

5. Evaluation of other potential candidates

Other molecules known to regulate GPCR-mediated signaling could be involved in this scenario, representing new candidates in contributing to the genetic risk of CAC progression. For instance, GPCR kinases (GRKs) and β-arrestins are fundamental in β2-AR transduction signal, and these molecules are known for their decisive role in modulating cardiovascular functions [17-19]. Specifically, GRK2 is implicated in the phosphorylation-dependent desensitization of β2-AR, which results in dissociation G protein from the receptor and its subsequent internalization mediated by β-arrestins [17]. Both level and activity of GRK2 have been associated with cardiovascular diseases [20-25]. Investigating variants in those genes and their association with CAC could be useful to finally fill the longstanding knowledge gap in the hereditability of CAC progression.