VC resembles bone mineralization and presents into 2 types:

(1) Atherosclerotic-related calcification: this is associated with intimal artery calcification and the early stages of this process are characterized by the development of micro-calcification. Calcium phosphate hydroxyapatite crystals are deposited into the extracellular matrix of atherosclerotic lesions, and their accumulation varies between patients. Spotty distribution of bone mineralization within atherosclerotic plaques has been associated with clinical manifestations, such as acute coronary events and cardiovascular death in the long term [19]. The dynamic process of microcalcification is indicative of the risk of plaque rupture and unfavorable clinical outcomes [20]. On the other hand, macro-calcification of the atherosclerotic plaques, characterized by the deposition of large amounts of calcium, gradually decreases the lumen patency and creates a more stable phenotype [21]. This is a dual-edged sword, because heavily calcified plaques are stiff and less amenable to transcutaneous revascularization, but they appear with a low propensity to rupture. Perhaps, the inhomogeneous texture of the plaques leads to variable resistance to the hemodynamic forces in the bloodstream, which increases their vulnerability. Statins may change the localization of calcium microdeposits enhancing their deposition around the necrotic core of the atherosclerotic plaque which stabilizes the lesions [22].

(2) Medial calcification (MScl): It is usually circumferential, located in the medial layer. Most frequently is observed in diabetes mellitus and chronic kidney disease and its clinical significance is the subject of debate [9, 23]. The clinical repercussions are rare because the reduction of the lumen is minimal, unless it is overlapped by an atherogenic process, where the clinical manifestations become more evident [24, 25]. This is a bone-like morphology of the artery wall. It is believed that the lesion is produced by the fatty degeneration of the VSMCs of the middle layer, forming a mass that undergoes hyaline degeneration which then becomes calcified. MScl is not related to atherosclerosis, and its link with dyslipidemia is not established. Statins cannot alter MScl [26] and there are no current data regarding the impact of statins on arterial calcification. For that reason, our review focused primarily on the intimal artery calcification which relates to dyslipidemia and statins exert significant effects.

Inflammation plays a critical role in the advancement of atherosclerosis and contributes to approximately 20% to 30% of the remaining risk for adverse cardiovascular events, often linked to the rupture of unstable coronary plaques. This relationship is supported by various studies [27, 28]. Systemic inflammatory disorders are associated with an enhanced risk of adverse events and early ASCVDs [29, 30]. In the early stage of atherosclerosis, inflammation is the predominant pathophysiological mechanism that promotes plaque progression and calcification [31]. The repeated cycles of inflammatory damage and repair ultimately lead to calcification of the initial atherosclerotic plaques, whose progress provides an important estimate of clinical prognosis [12]. Inflammation also plays an important role in the calcification process, while macrophages, neutrophils, and T cells promote extracellular matrix remodeling,osteogenic differentiation and apoptosis of VSMCs [22, 32]. A recent experimental study showed that statin therapy is related to increased coronary artery calcification by stimulating inflammasome and macrophages, leading to nuclear factor-k$\beta$ (NF-k$\beta$) activation and the secretion of IL-1$\beta$ mRNA and Rac1-depended IL-1$\beta$ protein [33, 34]. Racs are small GTPases and signal inflammatory transducers affecting the expression of growth factors and cytokines. The anti-inflammatory actions of statins have long been proven [35]. Statins disrupt Rac1 isoprenylation by inhibiting geranylgeranyl diphosphate synthesis and promote plaque Rac-1 activation and expression of osteogenic markers, alkaline phosphate (ALP) and runt-related transcription factor 2 (RUNX2) [34]. RUNX2 promotes the differentiation of VSMCs to osteoblast-like cells by upregulating receptor activator of NF-k$\beta$ ligand (RANKL) and creating a microenvironment suitable for capture of phosphates for mineralization [36]. Moreover, statin therapy enhances the healing process against atherosclerotic plaque inflammation by activating M2 (anti-inflammatory phenotype) macrophages, resulting in increased plaque calcification across its volume regression [37].

Until recently, it was commonly believed that VC was a passive and degenerative process. A variety of factors are involved in the active ossification process of the arterial wall, which either act protectively by inhibiting the calcification of the arterial wall: fetuin-A, Matrix GLa Protein (MGP), osteopontin (OPN), osteoprotegerin (OPG), inorganic pyrophosphate (PPi), or by precipitating calcium and phosphorus deposition through the regulation of bone metabolism: vitamin D, parathormone (PTH), PTH related peptide (PTHrP), Receptor Activator of Nuclear factor $\kappa$B (RANK), RANK Ligand (RANKL), bone morphogenetic proteins 2 and 7 (BMP2 and BMP7) [22], involved at different stages of VC process [38, 39]. OPG, a member of the tumor necrosis factor (TNF) family, is known for its ability to inhibit the formation of bone-resorbing cells called osteoclasts [40]. Recent research has revealed that OPG is produced in various tissues, including the heart and arteries [41, 42]. The loss of other local and circulating calcification inhibitors, like fetuin-A, osteopontin, and matrix Gla protein (MGP) may also contribute to VC formation [33]. Those inhibitors behave as decoy ligands for both minerals and calcification proteins. Though osteoclasts stimulation those inhibitors suppress the VC progression rather than actively decalcify the already existing lesions [34]. Both clinical and animal studies have demonstrated a connection between OPG and atherosclerotic cardiovascular diseases (ASCVDs). Moreover, investigations have been conducted to examine the association between OPG, other VC inhibitors (e.g. OPN) and plaque characteristics [43, 44, 45].

Limited data from genetic polymorphisms of some of the aforementioned factors have been found to be involved in 40-50% cases of coronary arterial calcification [46, 47]. Although the data from genetic analysis are limited, they make more robust the evidence for the contribution of bone metabolism to VC. For example, the CD73 gene deficiency generates a loss of its activity and triggers the tissue-nonspecific alkaline phosphatase, a key protein for bone formation and the main conductor of the medial VC [48].

The medial layer of the vessel wall is composed of smooth muscle cells and elastin-rich extracellular matrix. One of the major mechanisms of arterial calcification includes the differentiation of VSMCs into osteoblast-like cells. This is regulated by the protein Cfba1/Runx2 (core-binding factor subunit 1$\alpha$/runt-related transcription factor 2) [49, 50], high extracellular phosphate levels [51] and BMPs. Additional factors such as oxidative stress, oxidized lipids, and inflammatory cytokines trigger the differentiation of VSMCs and thereby increase the sources of calcium deposition [52]. Under pathological conditions, like calcium overload, high pro-inflammatory milieu, and atherosclerosis, the osteoblast-like VSMCs release matrix vesicles (MVs) in the extracellular matrix leading to mineral deposition forming foci of metallic calcium nuclei [53]. Extracellular MVs are tiny nanoparticles enclosed in phospholipids carrying calcium, phosphate, and matrix proteins. Those MVs either promote or hinder mineralization, depending on their content of inhibitory proteins, such as matrix $\gamma$-carboxyglutamic acid protein (MGP). The latter belongs to a family of vitamin K-dependent proteins and contains $\gamma$-carboxylated glutamate residues [54]. Among others, ALP, produced by cells resembling osteoblasts, plays a role in matrix mineralization and the deposition of hydroxyapatite. It accomplishes this by breaking down inorganic pyrophosphate, a major inhibitor of calcium phosphate nucleation [55]. In addition to differentiation, damaged VSMCs may release apoptotic bodies, which further contribute to VC by accumulating calcium [56]. Experimental studies examining the impact of statins on the VC process have yielded inconsistent findings. For instance, in a model of human VSMCs involving inflammatory VC, statins demonstrated a dose-dependent inhibition of calcification [57]. Conversely, in an experiment where VSMCs from the aortas of Wistar rats were incubated in a specialized calcification medium, atorvastatin showed a dose-dependent stimulation of calcification. Furthermore, atorvastatin also induced significant apoptosis of the VSMCs [58]. In vitro cell culture studies have also indicated that statins stimulate calcium deposition in VSMCs and mesenchymal stem cells [58, 59].

Matrix metalloproteinases (MMPs) have been long validated as important contributors of atherosclerosis development, progression, and destabilization, while preliminary data implicate their role in VC [60]. Statins may act as a stabilizing factor by inhibiting the secretion of MMPs from VSMCs and inflammatory cells, with still unknown impact on VC [61, 62].

Okuyama et al. [63] declared that despite the current belief that cholesterol reduction with statins decreases atherosclerosis, simultaneously statins lead to coronary artery calcification as mitochondrial toxins impair muscle function in the heart and blood vessels through the depletion of coenzyme Q10 and ‘heme A’, and thereby ATP generation. Statins possess a mechanism that inhibits the synthesis of vitamin K${}_2$, which is the co-factor for matrix Gla-protein activation, which in turn protects arteries from calcification [63]. An experimental study examined the effects of pravastatin on VC in mice and showed that pravastatin-treated groups had a larger number of alkaline phosphate (ALP) positive cells, suggesting a possible increase in osteoblast-like cells in those mice [19]. The pathophysiologic mechanisms of atherosclerotic calcification are summarized in Fig. 1.

Fig. 1.

Pathophysiologic mechanisms of statins on vascular calcification. (A) Anti-inflammatory effect and vascular calcification promotion. (B) Direct vascular calcification promotion. OPN, osteopontin; OPG, osteoprotegerin; NF-$\kappa$B, nuclear factor kappa light chain enhancer of activated B cells; IL-1$\beta$, interleukin-1$\beta$; RUNX2, runt-related transcription factor 2; RANKL, receptor activator of nuclear factor kappa-$\beta$ ligand; ALP, alkaline phosphatase.

Standard single energy and standard dual energy chest x-rays is a low cost, low radiation diagnostic modality that can detect arterial calcifications. The chest x-ray is an already ordered procedure in almost all patients, and advanced processing has been created to enable the detection of coronary arteries calcium [64]. Also, aortic arch and peripheral artery calcification can be detected readily and reproducibly using x-rays and its presence is an independent predictor of arterial and atherosclerotic calcification, indicating CAD severity [65]. Furthermore, breast arterial calcification has been detected during mammogram screening and has been correlated with arterial calcification in the extremities and in other arterial beds, being a predictor of ASCVDs [21]. VC seen in x-rays may implicate an increased atherosclerotic calcification which cannot be distinguished from MScl.

It continues to be the most reliable and sensitive non-invasive tool for coronary and peripheral artery calcification. Since the 1940s, calcium found in the coronary arteries has served as a surrogate marker of CAD. Its presence and progression have been linked to a higher cardiovascular risk [66]. With the advances in imaging modalities, we can now detect and quantify more accurately coronary artery calcification (CAC) for cardiovascular risk assessment. For CAC quantification there are three semi-quantitative scores available: the mass equivalent score, the volume score, and the commonly utilized Agatston score. Those scoring methods exhibit a strong correlation with each other [67]. Among them, the Agatston scoring method calculates the CAC value by multiplying the area of calcified plaque by the density score. It is a widely known, valid, approach and has been endorsed by all recent international guidelines for major cardiovascular risk assessment [68]. CAC can be identified through electron beam computed tomography (EBCT) [69] and multi-slice CT [70]. These methods offer rapid scanning and processing times, taking approximately a few minutes. Additionally, the radiation dose involved is low, around 1 mSv, and there is no requirement for a contrast agent. Magnetic resonance angiography has also been employed in medical settings. However, assessment of the coronary arteries remains a challenge due to several factors such as the small size of the vessels, extended acquisition time, and the intrinsic motions caused by cardiac contractions and respiration [71]. On the other hand, CT quantification of peripheral arteries has not been clinically applicable and there is less robust evidence with scarce data. In this case, a more qualitative approach is performed in clinical practice.

It is considered a radiation free, cost effective and easily repeatable technique [72]. It is well known that there is a strong association of carotid plaque echolucency with the histologic content of vulnerable carotid plaques and the subsequent risk of a cerebrovascular event [73]. Plaque echogenicity is directly associated with the degree of calcification and fibrous tissue, and inversely associated with the lipid content of the plaque. Several scores have been proposed. In the Gray Weale-Nicolaides (GWN) classification lipid-rich plaques appear echolucent, while those with fibrous and calcific content appear echogenic [74, 75]. The Gray Scale Median (GSM) score constitutes the quantified measurement of plaque echogenicity and an important, objective, valid, marker of carotid plaque vulnerability [76] associated with increased cardiovascular mortality [77]. The GSM represents the median of the frequency distribution of tones of pixels included in the plaque areas. In other words, GSM is a median value of pixel brightness of the plaque, ignoring focal variability of the atherosclerotic lesion [76]. There are a number of factors which may influence the GSM score calculation, including the physical distance from the transducer and the consistency of the atherosclerotic plaque [73].

Also, intravascular ultrasound (IVUS) is an invasive diagnostic modality for detecting coronary calcification and stratifying plaque stability [42]. A high frequency ultrasound transducer–containing catheter is placed within the coronary arterial lumen, which detects calcium as significantly echogenic areas and provides detailed information about the distribution and nature of plaque burden. The sensitivity and specificity of IVUS detecting coronary calcification are 90% and 100%, respectively [32]. However, calcium measurement with IVUS is semi-quantitative technique limited by ultrasound’s inability to penetrate the calcium deposits [19].

Recently, there has been a significant focus on utilizing 18F-NaF positron emission tomography (PET) for the examination of VC in various arteries, including the coronary arteries, the aorta, and the carotid arteries. This technique gained considerable recognition, but its usage remains limited in clinical routine for the understanding of the underlying processes of plaque formation [78, 79].

Statins, also known as 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors, have been shown to reduce the risk of ASCVDs in numerous studies [121, 122]. Statins with “pleiotropic” anti-inflammatory actions effectively reduce cardiovascular events, presumably through plaque stabilization [123]. However, these drugs have also been associated with an increase in the progression of coronary and aortic calcification, which is known to be linked to an elevated risk of cardiovascular events [124, 125, 126]. Recent findings by Hanai et al. [127] suggest that lipophilic statins may have a negative impact on kidney function. The use of statins has been linked to a high CAC score, indicating a potential promotion of VC in predisposed individuals. The observed correlation between statin use and increased CAC score in the study by Li et al. [128] may be due to the phenomenon of “confounding by indication”, as statin users were older, had a higher body mass index (BMI), and had a greater burden of diabetes and cardiovascular disease. Although some studies attribute this link to confounding factors, randomized controlled trials have failed to demonstrate a survival advantage of statins in dialysis patients, leaving open the possibility that statins may not prevent or even contribute to arterial calcification. Importantly, even after adjusting for age, diabetes, BMI, and inflammation, the connection between statin use and increased CAC score persisted [129, 130, 131].

Puri et al. [126] found that although statins reduced atheroma volume, they promoted calcification in coronary atheroma. In addition to reducing the lipid-rich core of atherosclerotic plaques, statins may also increase the density of calcification [132] as part of a healing process which could result in plaque stabilization and eventually reduce the incidence of cardiovascular events. In a recent study involving 3483 participants, statin intake was linked to a 31% higher progression of coronary calcification, even after adjustment for cardiovascular risk factors [113]. Based on an old meta-analysis of controlled trials assessing the impact of statins on CAC and coronary stenoses, Henein et al. [133] concluded that the precipitated progression of coronary calcification (CAC growth rate) was due to increased transformation of noncalcified coronary atherosclerotic plaques to calcified plaques. The same authors re-analyzed data from two clinical trials to further investigate the time and dose dependent effects of statins on CAC and whether progression is accompanied by a higher incidence of cardiovascular events [112]. The included trials had the following characteristics: (1) St. Francis Heart Study (SFHS): 419 and 432 patients treated with placebo and 20 mg atorvastatin daily, respectively; CAC assessment at baseline, 2 years and 4–6 years follow-up. (2) EBEAT Study: 164 and 179 patients treated with 10 mg and 80 mg atorvastatin daily, respectively; CAC score assessment at baseline and 12 months. The accumulated data showed a similar CAC increase in the short-term follow-up (12–24 months) between placebo and low-dose atorvastatin, while 80 mg/daily atorvastatin further increased CAC by 12–14% over placebo. In the long term, a high dose of atorvastatin considerably increased the CAC score in both studies, however, that effect was not accompanied by an increase in cardiovascular events. In the SFHS trial patients experiencing cardiovascular events after the second CT scan had less-frequently prescribed statins while they had higher progression of CAC. The authors concluded that statins-induced CAC progression was not an independent predictor of cardiovascular events occurrence indicating probably plaque stabilization rather than plaque expansion [112].

In a recent multinational observational registry titled the Progression of Atherosclerotic Plaque Determined by Computed Tomographic Angiography Imaging (PARADIGM), the researchers collected data from patients who underwent serial coronary computed tomography angiography (CCTA) [134]. Statin administration actually stimulated the calcification of coronary arteries. Surprisingly, this increased calcification was associated with a reduced risk of adverse cardiac events, in agreement with previous reports [114]. In the absence of statin therapy, an increase in the CAC score indicated progression in both previously noncalcified and already calcified plaques. In contrast, the statin-related increase in CAC score indicated “calcification progression” only in previously calcified plaques [122]. In line with the findings from the PARADIGM study, Scott et al. [109] and other researchers found that statin treatment, as a primary prevention measure, correlated to a slower progression of overall coronary atherosclerosis volume, reduction of high-risk plaque features, and increased plaque calcification burden [135]. Participants with higher baseline inflammation experienced a significant increase in coronary calcification over 2 years of statin treatment, illustrating the association between inflammation, microcalcification, and the enhancing effect of statin treatment on coronary calcification. However, CAC scores did not differ between high versus low hs-CRP groups over 2 years. This further confirmed that although the CAC score is a good measure of overall plaque burden and stable end-stage macroscopic calcification, it cannot identify unstable atherosclerotic plaques [109, 112, 136]. In addition, those findings question the link of CAC score with long-term clinical outcomes [137].

Additional clinical studies shed more light on the effects of statins on plaque vulnerability. A previous meta-analysis of nine studies (a total 830 individuals) investigated the possible effect of statin therapy on the composition of coronary atherosclerotic plaques using virtual histology intravascular ultrasound (VH-IVUS) [122]. It was evident that statin administration reduced plaque volume and simultaneously increased dense calcium volume [122]. That increase has been negatively correlated with vessel remodeling in a number of studies, which may be interpreted as a stabilization of atherosclerosis [138, 139]. A recent observational study reported similar findings after the comparison of high-intensity statin treatment with standard medical treatment in patients with CAD regarding changes in plaque morphology [140]. In particular, dense calcium area increased in the high-intensity statin group compared to controls along with smoothing of atherosclerotic plaques and less shear stress and thereby less predisposition to rupture. In parallel to imaging modalities, studies assessing biomarkers have documented a positive effect of statins of VC mediators. In patients with newly diagnosed CAD, 6-months simvastatin therapy significantly reduced VC inhibitors, such as OPG, OPN and fetuin-A [141].

The possible protective effects of statins on patients with PAD has been supported by numerous old studies. The Heart Protection Study included a large number of participants from the UK, a subgroup of whom had PAD and were randomly allocated to simvastatin or placebo [142]. In this sub-group of participants, statin use was associated with a 24% lower probability of major vascular event in comparison with the placebo group (relative risk (RR): 0.76, 95% CI: 0.72, 0.81). Another large observation study of 155647 individuals with incident PAD investigated how statin use may affect amputation and mortality [143]. The results indicated a 33% lower risk of amputation for high-intensity statin users in comparison to antiplatelet-only users (HR: 0.67, 95% CI: 0.61, 0.74). A protective effect was also observed in the low to moderate intensity statin group, but to a lesser extent (HR: 0.81, 95% CI: 0.75, 0.86). After that robust evidence, statins’ administration is highly recommended (level of evidence: IA) in all patients with PAD by both the American Heart Association/American College of Cardiology and the European Society of Cardiology [144, 145]. A population-based cohort study in Spain included 5480 adults with ABI 0.95 and without known ASCVD [146]. They were divided into two groups, based on the use of statin or not and followed up for a median time of 3.6 years. Major cardiovascular events were more common in the non-statin group (24.7 events per 1000 person-years vs 19.7 respectively), since their counterparts receiving statins had a 20% lower probability of major cardiovascular events (HR: 0.80, 95% CI: 0.66, 0.97) and 19% lower probability of overall mortality (HR: 0.81, 95% CI: 0.68, 0.97).

There are limited data regarding the effect of statin use on carotid atherosclerosis and in particular through calcification. A number of studies have demonstrated the stabilizing impact of statins on carotid atherosclerotic plaques [147]. The Rotterdam study, a prospective cohort study of 1740 participants with carotid atherosclerosis undergoing carotid MRI angiography [109]. Statin users had a 73% higher probability of having intra-plaque calcification in comparison to individuals who had never taken statins (OR: 1.73, 95% CI:1.22, 2.44). The duration of statin therapy was an important factor for plaque calcification, since only statin therapy lasting more than 48 months seemed to be associated with calcification with a pronounced protective effect (OR: 1.74, 95% CI: 1.09, 2.77). However, three small (number of participants range: 26–33), observational studies reported no impact of statins on plaque calcification. During 3 years [148], 2 years [149] and 6 months follow-up [150], the administration of either low- or high-dose statins did not significantly alter either the absolute or the percentage of calcification within carotid plaques. However, the inability to reach statistical significance may be due to the study design. Interestingly, the percentage change of calcification between baseline and 6 months correlated with percentage plaque volume change in the last study. Using ultrasound, statin therapy may increase plaque echogenicity, GSM score and hence its stability [75]. In patients with established carotid atherosclerosis (carotid stenosis $>$40%) but without indication for intervention of 6 months atorvastatin therapy (dose range: 10–80 mg) targeting LDL 100 mg/dL improved lipid profile, reduced inflammatory biomarkers along with VC inhibitors OPN and OPG [151]. Most importantly, intensive lipid-lowering enhanced carotid plaque echogenicity (GSM score elevation) outlining a beneficial impact on plaque stability, in peri-procedural period for carotid revascularization [152]. Among elderly people, the Thoracic Aorta Calcium Score was associated with a two-fold higher risk of stroke [153]. It is not yet clarified how the prescription of statins could contribute to reducing this risk. A summary of the results of statins on atherosclerotic calcification in clinical studies is presented in Table 1, Ref. [73, 109, 114, 115, 116, 117, 121, 122, 124, 125, 126, 131, 132, 133, 134, 135, 151, 152].

Some studies have attempted to explain this paradoxical effect by suggesting that the statins-induced progression of calcification may occur in a way that simultaneously reduces cardiovascular risk, such as by altering the size or density of calcium deposits. For example, a reduction in mineral surface area resulting from the coalescence of small deposits and/or decreased porosity may lower the risk of debonding and subsequent plaque rupture, which could contribute to the reduction in cardiovascular risk observed with statin therapy [117, 154]. To explore this possibility, F-NaF micro-positron emission tomography (µPET) imaging, which can detect fluoride adsorption on the surfaces of actively mineralizing apatite mineral deposits, may serve as a useful tool to quantify the surface area of cardiovascular calcium deposits [155].