*Review* **Clinical Significance of Carotid Intima-Media Complex and Carotid Plaque Assessment by Ultrasound for the Prediction of Adverse Cardiovascular Events in Primary and Secondary Care Patients**

**Anna Kabłak-Ziembicka 1,2,\* and Tadeusz Przewłocki 3,4**


**Abstract:** Recently published recommendations from the American Society of Echocardiography on 'Carotid Arterial Plaque Assessment by Ultrasound for the Characterization of Atherosclerosis and Evaluation of Cardiovascular Risk' provoked discussion once more on the potential clinical applications of carotid intima-media complex thickness (CIMT) and carotid plaque assessment in the context of cardiovascular risk in both primary and secondary care patients. This review paper addresses key issues and milestones regarding indications, assessment, technical aspects, recommendations, and interpretations of CIMT and carotid plaque findings. We discuss lacks of evidence, limitations, and possible future directions.

**Keywords:** cardiovascular risk; carotid intima-media complex; carotid plaque; major adverse cardiac and cerebral events; prevention; scores

#### **1. Introduction**

Cardiovascular disease (CVD) is a leading global problem [1]. An estimated 17.9 million people died from CVDs in 2019, representing 32% of all global deaths [1]. Of these deaths, 85% were due to major adverse cardiac and cerebral events (MACCE) [1]. Atherosclerosis and its complications, i.e., MACCE, heart failure, disability, vascular dementia, renal failure, lower limb ischemia, etc. are responsible for more than 50% of all deaths in westernized societies [2]. According to the WHO targets, it is important to detect CVD as early as possible so that management with counseling and medicine can begin [1].

As atherosclerosis is a generalized disease affecting many arterial beds at the same time, assessment of carotid arteries theoretically creates a unique opportunity to mirror and track atherosclerotic disease [2–7].

Recently published recommendations from the American Society of Echocardiography on 'Carotid Arterial Plaque Assessment by Ultrasound for the Characterization of Atherosclerosis and Evaluation of Cardiovascular Risk' [8], once more opened discussion on the potential clinical applications of carotid intima-media complex thickness (CIMT) and carotid plaque assessments in the context of cardiovascular (CV) risk in both primary and secondary care patients.

CIMT assessment, with or without carotid plaque inclusion, was considered a surrogate measure of atherosclerosis to provide information on the CV outcome in asymptomatic patients with CV risk factors and patients with known atherosclerotic disease, or to measure the effect of medical therapy [8–42].

**Citation:** Kabłak-Ziembicka, A.; Przewłocki, T. Clinical Significance of Carotid Intima-Media Complex and Carotid Plaque Assessment by Ultrasound for the Prediction of Adverse Cardiovascular Events in Primary and Secondary Care Patients. *J. Clin. Med.* **2021**, *10*, 4628. https:// doi.org/10.3390/jcm10204628

Academic Editor: Osama Soliman

Received: 15 September 2021 Accepted: 5 October 2021 Published: 9 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In epidemiological studies in asymptomatic individuals, increased CIMT values indicate higher risk of stroke, myocardial infarction (MI), or CV mortality [10]. The identification of carotid plaque even enhances this risk [8,11]. Carotid plaque presence, its number and size, volume, surface, echogenicity, or vascularization are all possible measures of MACCE [12–20]. Additionally, in secondary prevention for high-risk patients who already suffered from MI or stroke, the carotid atherosclerosis burden may have a role in the prediction of recurrent MACCE [21–25].

Despite so many advantages, the main flaw of CIMT and carotid plaque assessments is the heterogeneous techniques used for taking measurements, data reproducibility, and the limitations on the plaque composition judgements.

Therefore, this review paper addresses key issues and milestones regarding the indications, assessment, technical aspects, and interpretation of CIMT and carotid plaque findings. We discuss lacks of evidence, limitations, and possible future directions.

#### **2. Historical Rationale for CIMT Assessment**

The conception of CIMT and carotid plaque assessment dates back to the late years of the 20th century [43,44]. Its introduction was closely related to the development of highresolution ultrasound techniques that allowed for the imaging of a double-layer carotid wall structure (intima-media and adventitia) [43,44].

The first report showed a difference in CIMT values between patients with hyperlipidemia as compared to healthy age-matched individuals [44]. In result, ultrasound images of the carotid artery at the level of the common carotid (CIMT-CCA), the carotid bifurcation (CIMT-CB), or the proximal segment of internal carotid artery (CIMT-ICA) were quickly adopted as a surrogate measure for atherosclerosis by epidemiological studies, i.e., the Kuopio Heart Study, the Atherosclerosis Risk in Communities Study, and the Cardiovascular Health Study [45–47].

Later, CIMT was recognized as the equivalent of "subclinical" CVD in asymptomatic patients before an individual develops symptoms such as angina, ischemic stroke (IS), or limb ischemia and was validated to predict CV outcomes [12–20].

#### **3. Normal vs. Abnormal CIMT Value: Carotid Plaque Definitions**

CIMT values are age and sex specific, and males have higher CIMT on average compared to females [48–52]. Therefore, normative absolute CIMT values are obsolete, and previous normative CIMT cut-off values such as the 0.9 mm mentioned in the European Society of Cardiology guideline should not be used [53]. Several studies have investigated normal CIMT value ranges, and they are summarized in Table 1.





lipoprotein cholesterol; ICA, internal carotid artery; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides.

A CIMT value over the 75th percentile, according to American Society of Echocardiography (ASE), should be considered abnormal [54]. In 2002, the National Cholesterol Education Program (NCEP) Adult Treatment Panel III stated that an elevated CIMT (above 75th percentile for age and sex) could elevate a person with multiple risk factors at higher risk category [55].

In a research study enrolling 24 medical centers, the 75th percentile of the CIMT-CCA distribution was established at 0.5 and 0.51 mm in female and male below 25 years of age, while it was established at 0.80 and 0.83 mm in healthy individuals over 80 years of age, respectively (Table 1) [51]. Later, Randrianarisoa et al. performed an update on normal values for CIMT, including traditional as well as novel cardiovascular risk factors of atherosclerosis progression, like the body fat distribution, metabolic syndrome, subclinical inflammation, insulin resistance, and disturbances in glucose metabolism [52].

#### **4. Techniques for CIMT and Carotid Plaque Assessment: Strengths and Weaknesses**

B-mode high-resolution ultrasound is a noninvasive technique that provides one of the best methods for the detection of early stages of atherosclerotic disease [10]. Many studies have successfully applied CIMT as a technique to monitor arterial wall alterations based upon its association with CV risk factors, the incident CVD, and the outcome [9–20]. CIMT and carotid plaque measurements including mean-maximal or mean-mean CIMT, plaque thickness, area and volume, and plaque score were all used as imaging outcomes [9–20,56–59].

Unfortunately, diverse approaches for measuring CIMT and plaque as well as different cut-offs and acquisition techniques have caused confusion for the interpretation of CIMT and plaque findings [9–20]. Thus, technique, the number of segments of the carotid artery tree, the near or far wall, and the use of contrast-enhanced agents are all points for discussion [56].

Another, but not less important issue, is whether measurements can be manual, (semi) automated, or if computer-assisted analysis software should be used to automatically track the intima-media layer [10,54,59]. Semi-automated edge detection is more often applied in the setting where only the CCA is examined, while manual edge detection is usually applied in the setting where the CB and the ICA are also measured [58].

For example, in the Carotid Atherosclerosis Progression Study (CAPS), CIMT was expressed as the mean CCA at the far wall and in the Cardiovascular Health Study (CHS) as the mean of the maximum at the near and far CCA and ICA, while in the Malmö Diet and Cancer Study (MDCS) and the Kuopio Ischemic Heart Disease Study (KIHD), it was expressed as the maximum CIMT-CCA at the far wall [12–16]. The Rotterdam study presented results as the maximum value from the near and far CCA [17], while the Atherosclerosis Risk in Communities Study (ARIC) measured CIMT as the mean of means at the right and left CCA, CB, and ICA [18,19].

The lack of methodological standardization of CIMT resulted in contraindication (Class III) for CIMT assessment as CV risk modifier from the 2021 ESC Guidelines on CVD prevention in clinical practice [60].

Thus, to overcome methodological flaws, guidelines for obtaining CIMT and carotid plaque measurements were published with the intention to reduce measurement variability, a key parameter for a high-quality study, statistical power, and sample size determination (Table 2, Figure 1).



**Figure 1.** CIMT and carotid plaque assessment. (**A**) CIMT is a double-line pattern on both walls of the common carotid artery (CCA), the carotid bulb (CB), the internal carotid artery (ICA), and the external carotid artery (ECA) in a B-Mode 2D longitudinal image. Two parallel lines are the lumen-intima and media-adventitia interfaces. (**B**) According to the Mannheim IMT consensus [59], CIMT should be measured within a region free of plaque with a clearly identified double-line pattern, preferably on the far wall of the CCA at least 5 mm below its end. If the CIMT-CB and the CIMT-ICA are measured, the results should be reported separately from CIMT-CCA. According to the Mannheim [59], limiting CIMT measurements to the far wall of the CCA and distal 1 cm of each CCA is the preferred strategy. (**C**) Diffuse-type plaques (Grades II or III, and CIMT ≥ 1.5 mm). (**C–E**) In line with the ASE consensus, the maximal plaque height should be measured from the side in which a plaque is detected (unilateral) or from the right and left carotid arterial segments (bilateral), using a caliper placed at the adventitial plane and extending into the center of the lumen to the vessel wall. For the purposes of standardization, this measurement should be taken from the long (**C**) and short axis (**D**,**E**) of the carotid artery. Arrows indicate regions of taking CIMT, plaque measurements.

> The 2008 ASE consensus statement for CIMT is based on the concept of identifying asymptomatic patients at high risk who might be candidates for more intensive, evidencebased medical interventions that reduce CVD risk (Table 2) [54]. The 2008 ASE guidelines recommend measuring CIMT and identifying carotid plaque by ultrasound for refining CVD risk assessment in patients at intermediate CVD risk (FRS 6%–20%) without established coronary heart disease (CHD), peripheral arterial disease (PAD), cerebrovascular disease, diabetes, or abdominal aortic aneurysm. Patients with the following clinical circumstances also might be considered for CIMT and carotid plaque measurement: (1) family history of premature CVD in a first degree relative (men < 55 years old, women <

65 years old); (2) individuals younger than 60 years old with severe abnormalities in a single risk factor (e.g., genetic dyslipidemia) who otherwise would not be candidates for pharmacotherapy; or (3) women younger than 60 years old with at least two CV risk factors.

The Mannheim IMT Consensus [59] recommended CIMT assessment for the initial detection of CHD risk in (1) asymptomatic patients at intermediate risk, (2) in the setting of two or more NCEP risk factors, (3) with metabolic syndrome, (4) with a family history of premature CHD, or (5) with a known coronary artery calcium (CAC) score of zero and FRS 11%–20%. According to Mannheim IMT consensus, measurements may include the CCA, the ICA, or the CB segments. Whereas nearly all patients have their CIMT-CCA imaged, successful imaging of the CIMT-ICA and of the CIMT-CB depends both upon the anatomical topography of the patient and on sonographer's expertise. Thus, the Mannheim IMT consensus advises rather for CIMT-CCA at far wall measurements than from the whole carotid artery tree. The overview of the Mannheim recommendations is presented in Table 2. The Mannheim definition of plaque was adopted by the 2021 ESC Guidelines on CVD prevention in clinical practice as possible CV risk modifier (Class II-b) [60].

The 2020 ASE recommendations for carotid plaque ultrasound suggested a stepwise approach to CV risk stratification using plaque grading via a focused carotid vascular ultrasound and subsequent 2D or 3D plaque quantification in the assessment of asymptomatic patients at risk (Table 2). In patients presented with symptoms suggestive of CHD but normal non-invasive tests (e.g., stress electrocardiogram, stress echocardiography, stress MRI, and nuclear imaging), patients with atherosclerotic plaques in the carotid artery may benefit from more aggressive medical treatment. In contrast, patients without plaque have an excellent CV prognosis [8]. The 2020 ASE consensus did not focus on CIMT, referring to 2008 ASE recommendations for CIMT assessment [8,54].

#### **5. Simultaneity of Atherosclerotic Burden across Arterial Beds**

Multi-site steno-occlusive arterial disease is invariably associated with worse clinical outcomes [4], accounting for 51% MACCE incidence rate in patients with arterial disease (at least 50% lumen reduction) in either coronary, carotid, and renal and lower extremity arterial territories as compared to 27%, 18%, and 9% in patients with 3-site, 2-site, and 1-site arterial disease at 4 years follow-up, respectively [4]. Furthermore, studies are ongoing about which patients with multi-site atherosclerotic occlusive disease would have decreased risk of MACCE following revascularization, and who would benefit more from medical treatment [61].

In the general population of individuals aged 30–79 years, the global prevalence of increased CIMT and carotid plaque is estimated to be 27.6% (16.9% to 41.3%) and 21.1% (13.2% to 31.5%), respectively [62,63]. Multisite arterial disease is common in patients with atherosclerotic involvement in one vascular bed, ranging from 10% to 15% in patients with CAD to 60% to 70% in patients with severe carotid stenosis or PAD [64,65].

At large, screening for asymptomatic disease in additional vascular sites has not been proved to improve prognosis. Nevertheless, the mean-max CIMT values from the CCA, the CB, and the ICA with a cut-off value of 1.30 mm nicely distinguish patients with no steno-occlusive arterial disease or stenosis limited to one arterial territory from individuals with larger arterial territory involvements (odds ratio, OR, 35.9, 95% Confidence Interval, 95% CI, 20 to 65) with a sensitivity of 81.6%, specificity of 88.8%, and positive and negative predictive value of 85.1% and 86.3%, respectively [66].

A variety of studies evaluated the relationship between CIMT and presence of atherosclerotic abnormalities in the other territories of the arterial system [67–77]. Most studies demonstrated associations between increasing CIMT value and presence and severity of a significant arterial disease (defined as at least 50% or more lumen reduction): CAD, renal artery stenosis, lower and upper extremity athero-oclussive disease, or the abdominal aorta [67–77]. However, correlations between CIMT with incidence and severity of lesions in the other arterial sites are modest, especially when only CIMT-CCA is reported [78–80]. Rohani et al. demonstrated a correlation between the extent of CAD and the mean CIMT-CCA of r = 0.44; Adams et al. demonstrated a correlation between 0.23 and 0.29, while Azarkish et al. demonstrated a correlation between 0.36 and 0.47 [78–80]. Interestingly, CIMT can rule out significant CAD in women and patients with degenerative aortic stenosis, e.g., a mean-maximum CIMT value of greater than 1.2 mm was predictive (sensitivity, 73.5%; specificity, 72.7%) of concomitant CAD in patients with aortic stenosis [70,73].

A recent meta-analysis of 89 studies showed moderate correlation between CIMT and severity of CAD (r = 0.60, *p* < 0.001) and the number of diseased vessels (r = 0.49, *p* < 0.001) [67]. Additionally, carotid plaque presence and calcification were less, and lipid-rich necrotic core was highly prevalent in nonsignificant versus significant CAD (*p* < 0.001, *p* = 0.03, *p* < 0.001, respectively) [67]. In another large meta-analysis, including 22 studies, the diagnostic sensitivity and specificity of CIMT for CAD were 0.68 and 0.70, respectively [81].

#### **6. CIMT and Carotid Plaque in the Context of Cardiovascular Risk Factors**

Various risk factors influence CIMT and carotid plaque, including age, gender, diabetes, dyslipidemia, hypertension, cigarette smoking, genetics, and inflammation [82–84]. Song et al. performed a systematic review and meta-analysis of the 75 articles on CIMT, carotid plaque, and carotid stenosis [62]. The influence of CV risk factors for increased CIMT and carotid plaque were 2.71 and 1.79 for age per 10-year increase, 0.49 and 0.55 for female sex, 1.76 and 1.70 for current smoking, 2.23 and 1.45 for diabetes, and 1.55 and 1.75 for hypertension, respectively (Table 3) [62]. Another meta-analysis by Ji et al. of 76 cross-sectional studies that evaluated 11 risk factors showed a pooled OR and 95% CI for the probability of the carotid plaque incidence (the Mannheim definition) to be associated with hypertension, diabetes, dyslipidemia, current smoking, hypertriglyceridemia, LDL-C, hypertriglyceridemia, hyperuricemia, hyperhomocysteinemia, and metabolic syndrome (Table 3) [85].

**Song et al. 2020 [62] Ji et al. 2019 [85] Risk Factor Pooled Data OR (95% CI) Pooled Data OR (95% CI)** Increased CIMT (>1.0 mm) Age Female sex Current smoking Diabetes Hypertension Dyslipidemia 2.71 (2.07–3.55) 0.49 (0.38–0.64) 1.76 (1.34–2.30) 2.23 (1.48–3.36) N/A N/A N/A N/A 1.55 (1.03–2.34) 2.60 (1.33–5.08) 0.90 (0.65–1.25) N/A Carotid plaque Age Female sex Current smoking Diabetes Hypertension Dyslipidemia HDL-C LDL-C 1.79 (0.93–3.43) 0.55 (0.33–0.94) - - 1.70 (1.41–2.04) 1.45 (1.12–1.90) 1.75 (1.44–2.13) - 0.46 (0.21–0.99) - 1.41 (1.08–1.87) 1.31 (1.13–1.53) 1.81 (1.55–2.13) 1.20 (0.80–1.82) 1.28 (0.99–1.67) 1.11 (1.08–1.13)

> - - - -

1.33 (1.14–1.55) 1.57 (1.11–2.22) 1.88 (1.19–2.95) 1.71 (1.10–2.66)

**Table 3.** Meta-analyses on the increased CIMT and carotid plaque incidence with cardiovascular risk factors.

Hypertriglyceridemia Hyperuricemia Hyperhomocysteinemia Metabolic syndrome

In the NOMAS study that assessed 2D carotid plaque area in 1730 primary care individuals above 39 years old, the associations between carotid plaque and age, smoking, systolic blood pressure, diabetes, LDL-C:HDL-C ratio, and homocysteine levels were found, with respective contributions of 13.5%, 2.8%, 1.1%, 0.8%, 0.7%, and 0.7% [86].

There is much confusion with regard to lipoproteins and CIMT. Single-center studies indicate the relationship between higher CIMT and higher levels of total cholesterol (TC), LDL-C, lipoprotein (a), and non-HDL-cholesterol, as well as inverse associations with HDL-C; however, meta-analyses fail to show associations [62,85–89]. For example, in a study by Stamler et al. in a group of men aged 18 to 39 years, those with TC levels ≥ 6.21 mmol/L had a greater risk of CHD (2.15 to 3.63 times) and CV mortality (2.10 to 2.87 times) in comparison to individuals with TC < 5.17 mmol/L [90]. In this study, LDL-C, which is a classical atherogenic lipid, had a lower predictive value for the presence of carotid plaque than TC. The problem of lipids and atherosclerosis is much more complex, as there are many different fractions of lipoproteins that are atherogenic (i.e., very lowdensity lipoprotein cholesterol, intermediate-density lipoprotein cholesterol, or lipoprotein (a)) [90].

Of note, the accumulation of many various risk factors impacts overall CIMT and plaque parameters [91]. Many systemic inflammatory and thrombotic biomarkers are associated with increased CIMT and asymptomatic and symptomatic plaque incidence. Associations were proven for CIMT and interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), Apolipoprotein E (ApoE), white blood cell (WBC) count, T lymphocytes, fibrin, and adiponectin [21,90]. Carotid plaque presence was associated with intercellular adhesion molecule 1 (ICAM-1), L-selectin, E-selectin, IL-1β, tumor necrosis factor α (TNF-α), lipoprotein phospholipase A2 (Lp-PLA2), WBC count, and mi-RNAs [90]. While transformation of asymptomatic into symptomatic carotid plaque was associated with levels of high-sensitivity C-reactive protein (hs-CRP), serum amyloid-A Protein (SAA), TNF-α, plasma-soluble urokinase plasminogen activator receptor (suPAR), matrix metalloproteinases (MPO-1, -2, -7, -9), tissue inhibitors of metalloproteinase (TIMP), ApoE, ApoA-I, Lp-PLA2, and miRNAs [92].

#### **7. Additive Value of CIMT and Carotid Plaque to the Traditional Cardiovascular Risk Scoring Systems**

As CIMT and carotid plaque are measures of atherosclerosis, it seems reasonable to combine established traditional risk scores with carotid imaging [93,94]. In a study by Elaid et al., 127 (37.8%) out of 336 initially 'low -risk' primary care patients (FRS event risk < 5% in 10 years) were re-classified as higher risk (>5%) when high CIMT (CIMT ≥ 75th percentile adjusted for age, gender, race, and presence of plaque) was found on ultrasound [95]. Plaque exceeding 1.5 mm was present in 17.3% of low-risk patients [95].

The risk calculators may integrate CIMT measurement with CV risk factors [86,93–102]. The ARIC study published an adjusted FRS calculator incorporating mean-maximum CIMT from six carotid segments and plaque assessment to determine the probability of MI or death from CHD within 10 years [96]. In that study including 13,145 individuals, approximately 23% were reclassified by adding CIMT and plaque information [96]. The addition of CIMT and plaque together to the traditional risk factors improved the prediction model from 0.742 for traditional risk factors to 0.755 for the CV risk factors, CIMT and plaque [96].

Recently, STRATEGY study assessed three scoring systems: the FRS, the Prospective Cardiovascular Münster Study Score (PROCAM), and the European Society of Cardiology SCORE in the context of possible additive CIMT value [85,92,95,96]. All scores correlated significantly with CIMT, but this correlation was only moderate [87]. The FRS correlated most strongly and predicted 27% of the CIMT variance in men and 20% in women [87].

The IMPROVE study, in a group of 3703 primary care patients aged 54–79 years with at least three CV risk factors, but free of any CV events prior to enrolment, evaluated the independence of carotid plaque thickness and the mean CIMT (measured in plaque-free areas bilaterally in the CCA, the CB, and the ICA) in CV risk stratification

at 3 years follow-up [99]. In this study, both plaque and CIMT occurred as independent predictors of MACCE, with values of 1.98 (1.47 to 2.67) and 1.68 (1.23 to 2.29), respectively, as well as cerebrovascular events. However, only plaque was an independent predictor of coronary events like MI, sudden cardiac death (SCD), angina pectoris, and coronary revascularization [99]. The authors concluded that in reclassification analyses, CIMT and plaque significantly add to the FRS score [99]. In line, in the study by Gaibazzi et al., carotid plaques (not CIMT) and echocardiographic cardiac calcium were significant predictors of angiographic CAD in patients without prior CHD but with signs or symptoms suspect of CAD, again incrementally correlated to FRS [102].

Mitu et al. found a relationship between CIMT and arterial stiffness with SCORE, FRS, QRISK, and PROCAM in an asymptomatic population [100,101]. The SCORE risk correlated better with CIMT, while the FRS and QRISK seemed more specific for increased arterial stiffness parameters [100]. Of note, arterial stiffness proved its clinical value for MACCE in various clinical scenarios, e.g., in patients with aortic valve stenosis [103].

#### **8. Primary and Secondary Care Population and MACCE**

There is much evidence that higher CIMT corresponds to higher likelihood of MACCE. At least six primary care large-cohort prospective studies examined the predictive value of CIMT (without plaque) on MACCE (Table 4). In general, CIMT values in the highest range are associated with a 1.4- to 3.2-fold risk increase for MI, a 1.4- to 3.5-fold risk increase for IS, a 2.3- to 2.9-fold risk increase for CV death, and a 1.75-fold risk increase for SCD (Table 4) [11,12,15–19,104–108]. This was evidenced regardless of method used for CIMT calculation (mean-mean, mean-maximum, the CCA only, or combined CCA-CB-ICA) [12,13,15,104]. Importantly, in most studies, the ability of CIMT to predict future MACCE was independent of traditional risk factors [11,12,15–19,104–108].

There is much evidence that plaque presence is stronger predictor of MACCE than CIMT alone (Table 4) [11,59,94,108–120]. In a study comparing results of ARIC and CHS studies, the presence of plaque was associated with over 30% increased risk of SCD: 1.37 in the ARIC and 1.32 in the CHS [105]. In the Manhattan Study, carotid plaque thickness exceeding 1.9 mm was associated with a MACCE incidence risk of 2.8 (2.03 to 3.84), as compared to individuals with no plaque at all [110].

Further improvement in risk estimation may be gained by considering not only the largest identified plaque, but also the total plaque burden, plaque area, plaque score (a sum of all plaques heights), or a number of segments containing plaque in both carotid arteries (Table 4) [109,111–118,121]. According to some authors, the average of all the CIMTmax observed in each carotid segment (CIMTmean-max), is the variable that best describes the total plaque profile, and which has the best predictive power [21,118]. Additionally, carotid plaque burden measured by 3D ultrasound is highly correlated with CAC scores and predictive of MACCE (CV death, MI, and stroke) [111]. The High-Risk Plaque BioImage Study compared CIMT, carotid plaque burden, and maximum carotid plaque thickness in nearly 6000 individuals [111]. Both carotid plaque burden and carotid plaque thickness were predictive of primary and secondary MACCE, whereas CIMT was not [111].

**Table 4.** Overview of studies on the relationship between CIMT, carotid plaque, and MACCE in primary and secondary cardiovascular risk prevention individuals.



**Table 4.** *Cont.*

AIS, acute ischemic stroke; ASCVD, atherosclerotic cardiovascular disease; CLI, critical limb ischemia; CV, cardiovascular; F, female gender; HF, heart failure; IS, ischemic stroke; M, male gender; MI, myocardial infarction; PC, primary care subjects; SCD, sudden cardiac death; SSSS, symptomatic subclavian steal syndrome.

> The estimated added predictive value of carotid plaque thickness in comparison to traditional risk factors accounts respectively for 0.73 vs. 0.72 in the CHS study [116], 0.72 vs. 0.67 in Stork et al.'s study [113], and 0.90 vs. 0.88 in Prati et al.'s study [115], as well as 0.75 vs. 0.74 in the Tromso, ARIC, and Xie et al. studies [105,108,112]. The addition of the carotid plaque score to the established risk factors can significantly improve risk discrimination (C-index 0.746 vs. 0.726; *p* = 0.017) [114].

> Importantly, plaque echogenicity, surface, angiogenesis, and size (volume and area) are all among risk factors for both cerebrovascular and cardiac events [121,123–127]. There is strong association between an increased risk of IS and plaques that are low echogenic (echolucent), ulcerated, with neovascularization, or containing mobile fragments with estimated respective risk of IS (HR, 95% CI): 3.99 (3.06 to 5.19), 3.58 (1.66 to 7.71), 9.68 (3.14 to 123.2), and 1.57 (1.02 to 2.41), respectively [125].

> In contrast to primary prevention studies, there are only few studies that address role of CIMT/plaque assessment to calculate risk of MACCE recurrence. Although the issue is clinically relevant, the assessment of CV risk in patients with already known atherooclussive disease at any arterial site (coronary, carotid, or other), or after index CV event (primary MI, IS, critical limb ischemia (CLI)) is not supported by the guidelines [55,125]. This attitude seems justified with regard to CIMT assessment (with exclusion of plaque parameters) and evidenced by studies of Yoon et al. and Tada et al. (Table 4) [23,122]. In contrast, in the study of Yoon et al. performed in 479 patients with index acute IS,

carotid plaque (not mean CIMT-CCA) was associated with risk of secondary CV event (Table 4) [23].

In the study including 652 patients with angiographic stenosis ≥ 50% in at least one arterial territory (coronary, supra-aortic, renal, and/or lower extremity), who underwent a revascularization procedure for index lesion, a mean-max CIMT (plaque included) exceeding ≥ 1.25 mm (HR, 2.52; 95% CI, 1.5 to 4.24; *p* = 0.001) was associated with increased risk of MACCE, abdominal aortic aneurysm rupture, or development of new symptomatic lesions requiring revascularization [4]. In this study, inclusion of CIMT and plaque into the stratification model significantly improved the prediction of CV event risk [4]. Incremental value of mean-max CIMT plus plaque, TNF-α, and hs-CRP to traditional risk factors in risk stratification was also found in another study of patients with confirmed atherosclerosis [21]. Yet, the study by Tada et al. showed a combined risk for all-cause death, CV death, MI, IS, revascularization, heart failure of 3.38 (95% CI, 1.82 to 6.27) among 2035 patients diagnosed with atherosclerotic CVD [122].

#### **9. Follow-Up of Changes in CIMT and Carotid Plaque Thickness with Multiple Assessments—Is It Worth It?**

Serial assessment of CIMT change over time is considered a good method to monitor the natural progression of atherosclerosis in epidemiological studies and/or to assess the average response to treatment in clinical trials [27,40,41,44,116,121,122,128–131]. A major advantage of measuring carotid plaque burden is that progression/regression of plaque can be measured in clinically relevant time frames. The spatial resolution of carotid ultrasound is approximately 0.3 mm, and on average, CIMT changes by only 0.015 mm/year [128]. It is therefore not possible to reliably measure change in CIMT within an individual over short period of time. The consensus sample size for studies of effects of therapy on CIMT is 200 to 300 patients per group, followed for 2 years [27]. Thus, an appropriate time span is required between individual CIMT and plaque size assessments.

In the Malmo Diet and Cancer Study (MDCS) including 3426 primary care middleaged Swedish patients, there was a cumulative relationship between traditional CV risk factors and CIMT progression rates during the 16-year follow-up period. The ORs of a high CIMT-CCA progression rate (>75th percentile) were 1.0 (reference), 1.4 (95% CI: 1.1 to 1.7), 1.7 (95% CI: 1.3 to 2.2), and 2.1 (95% CI: 1.4 to 3.1), respectively, for individuals with none, one, two, and three risk factors [44]. Favorable changes in systolic blood pressure, LDL-C, and HDL-C during over 15 years of follow-up decreased the CIMT progression rate in the CCA [44]. Interestingly, averaged CIMT progression rates were lower in the CCA (0.011 mm/year for men and 0.010 mm/year for women) but greater in the CB (0.036 mm/year for men and 0.030 mm/year for women) [44].

In a prospective study of a primary care population in the Cholesterol-Lowering Atherosclerosis Study trial, during an 8.8-year observation, Hodis et al. showed that the risk of coronary events was increased with the rate of CIMT progression (Table 5). The researchers observed ORs of coronary events of 1.0 (reference), 1.6, 2.3, and 2.8 for CIMT progression rates of less than 0.011, 0.011 to 0.017, 0.018 to 0.033, and greater than 0.033 mm/year, respectively [40]. In another study, CIMT progression predicted CV events in patients with type 2 diabetes [41].

*J. Clin. Med.* **2021**, *10*, 4628


**Table 5.** The relationship between CIMT or carotid plaque progression over time and MACCE.

ACS, acute coronary syndrome; ASCVD, atherosclerotic cardiovascular disease, defined as lesions in at least one major arterial territory including coronary, carotid, renal, or lower extremity 50% lumen reduction; CRF, cardiovascular risk factors; IS, ischemic stroke; M, month; MI, myocardial infarction; TPA, total plaque area; TPV, total plaque volume; UA, unstable angina.

A novel approach was recently proposed by the IMPROVE study to assess carotid CIMT progression [118]. In this study, the greatest value among the progressions of CIMTmax observed in the whole carotid tree identified focal increases of CIMT and was associated with cardiovascular risk (Table 5) [118].

In a secondary prevention population of 108 patients who had stent-supported angioplasty for symptomatic subclavian steal syndrome, followed for a mean of 4.8 years, Wrotniak et al. found CIMT progression of 0.060 mm/year to increase risk of MACCE and lesion progression by 22% (OR, 1.22; 95% CI, 1.02 to 1.46; *p* = 0.033) with a sensitivity of 75.0% and specificity of 61.8% [24]. In this study, despite medical treatment adhering to guidelines, atherosclerosis progression was found in 53 (49%), no change in 10 (9.3%), and regression in 45 (41.7%) patients [24].

Gaco ´n et al. demonstrated in a group of 215 patients admitted with acute coronary event, that patients with MACCE at follow-up, compared to MACCE-free subjects, had a greater annual CIMT progression rate either at first (0.024 ± 0.12 vs. 0.009 ± 0.16 mm/year; *p* < 0.001) or at subsequent follow-up ultrasound visits (0.050 ± 0.1 vs. 0.001 ± 0.1 mm/year; *p* < 0.001) [130]. Of note, initial CIMT values were similar in MACCE vs. MACCE-free patients (1.43 ± 0.40 vs. 1.45 ± 0.44 mm; *p* = 0.486) [130].

In Hirano et al.'s study including 240 patients with CHD confirmed on angiography, the average number of carotid plaques (≥1.1 mm of CIMTmax) at baseline was 2.5 ± 1.8 in a patient [128]. The change in plaque-IMTmax over 6 months ranged from −0.85 to 0.97 mm (mean, −0.006 ± 0.319 mm). The study showed that progression of carotid plaque-IMTmax over 6 months despite anti-atherosclerotic therapy was an independent predictor of future coronary events in CHD patients (Table 5) [133].

It is extremely important to understand that CIMT and plaque progression rate is nonlinear [42,130]. Among that innumerous studies that were published, a study by Olmastroni et al. deserves particular attention as it is a large primary care cohort (1175 participants), with participants initially at low and intermediate CV risk with a prospective follow-up of 12 years, with the use of individual CIMT growth curve modeling [42]. Participants completed four visits with ultrasound examination, which proved that the rate of change in CIMT over time is a sign of the development of atherosclerosis, with periods of rapid and attenuated CIMT growth, which cannot be a priori assumed as linear [42]. In that study, the fastest mean and max CIMT growth was observed in patients between 50 and 70 years old. Of 966 subjects free from carotid atherosclerosis at baseline, 31.8% developed multifocal carotid atherosclerosis and 11.8% developed focal carotid atherosclerosis [42].

The non-linear response of atherosclerosis to so-called optimal medical treatment was also reported by study of Gaco ´n et al., including 466 secondary care patients [134]. In this study, regression of the mean-max CIMT (with inclusion of plaque thickness when present) was observed in 37.1% of the study group at the first ultrasound re-examination between month 12 and 24, and it went down to 26.6% at the second re-examination between month 24 and 36 [134]. The attenuated CIMT/plaque progression was independently associated with a reduced risk of MACCE (HR, 0.25; 95% CI, 0.15 to 0.42), MI (HR, 0.32; 95% CI, 0.20 to 0.51), IS (HR, 0.29; 95% CI, 0.18 to 0.45), and CV death (HR, 0.24; 95% CI, 0.15 to 0.40) [134]. In contrast, a carotid atherosclerosis progression of >0.056 mm/year was associated with increased risk of MACCE, however, with only moderate sensitivity and specificity of 53.2% and 72.2%, respectively (Table 5). Thus, achieving regression in CIMT and plaque thickness may constitute a better measure of treatment efficacy.

#### **10. CIMT and Carotid Plaque Changes in Response to Medical Treatment**

CIMT was used in randomized clinical trials (RCT) to measure the effect of medical intervention, targeted at CV risk factor control, and the carotid atherosclerosis progression or regression, as possible modifiers of adverse CV outcomes [12,26–36]. Positive response to the medical intervention was defined as a measurable decrease in CIMT and carotid plaque values of the treated group compared to patients' group, with treatment failure defined as CIMT or carotid plaque increase despite treatment in the context of future MACCE [37–42].

As effective interventions targeting pre-existing CVD, lifestyle and diet may reduce the risk of carotid atherosclerosis [85]. Huang et al. suggested that antihypertensive medication use may be the strongest modifiable predictor of slowing CIMT progression over time, especially when measurements are taken at the CB [135]. Overall, 8 out of 10 analyzed statin RCTs showed that conventional statins therapy are efficient and safe to decrease the rate of carotid atherosclerosis progression in the long term, and aggressive statins may provide superior efficacy for carotid atherosclerosis regression [34]. According to Wannanong et al., for assessment of response to anti-atherosclerotic therapy, measurement of total plaque volume is superior to both CIMT and total plaque area measurements [128]. This finding is in line with meta-analysis of seven studies including 361 patients receiving statin therapy, in which there was significant decrease in lipid-rich necrotic-core volume at >12 months (−9.9 mm3, 95% CI −8.9, −2.3); however, no significant reduction in carotid wall volume was seen on high-resolution carotid plaque MRI [136].

Conversely, according to the SAIP research group, the scientific bases for monitoring changes in single individuals are still not convincing [137]. First, Goldberger suggested caution in using CIMT as a surrogate endpoint of outcome in trials with statins, focused on CIMT progression/regression and MACCE incidence, although a smaller rate of change in CIMT was associated with a reduced MI incidence 0.82 (95% CI, 0.69 to 0.96; *p* = 0.018) [35]. Alas, there was no significant relationship between mean change in CIMT and nonfatal MI in RCTs [35]. Another large meta-analysis of 16 prospective studies performed by the PROG-IMT collaboration revealed a positive association between the mean CIMT-CCA and a 16% increase in CV risk, but no association between CIMT progression and CV events [132]. However, in this meta-analysis, the reproducibility between first and the second CIMT measurement was surprisingly low (correlation coefficient < 0.10), resulting in huge bias for data interpretation [138]. As consequence, the conclusion from metaanalyses of RCTs was that CIMT changes (regression or progression) did not correlate with changes in the incidence of MACCE induced by several drug treatments in different categories of subjects at intermediate to high CV risk [29].

This lack of associations between CIMT changes and clinical outcomes is surprising, as active medical treatment with either statins, calcium channel blockers, angiotensinconverting enzymes, or sartans was associated both with MACCE rate reduction as well as CIMT decrease in comparison to placebo groups [12,26–38].

To overcome existing confusion, a meta-analysis of 119 clinical trials involving 100,667 patients done by Willeit et al. shed some light on this puzzle [132]. Data from individual RCTs were systematized (Table 5). CIMT was assessed as the mean value at the CCA; if unavailable, the maximum value at the CCA or other CIMT measures were utilized. The primary outcome was a combined CVD endpoint defined as MI, stroke, revascularization procedures, or CV death. Authors estimated intervention effects on CIMT progression and incident CVD for each trial, before relating the two using a Bayesian meta-regression approach. This meticulous work resulted in conclusion that medical interventions reducing CIMT progression by only 0.01, 0.02, 0.03, or 0.04 mm/year would decrease the relative risks for CVD of 0.84 (95% CI, 0.75 to 0.93), 0.76 (95% CI, 0.67 to 0.85), 0.69 (95% CI, 0.59 to 0.79), or 0.63 (95% CI, 0.52 to 0.74), respectively [132].

#### **11. Important Limitation for Comprehensive Data Analysis and Results Interpretation**

Based on the experience in previous large-scale trials, there is a number of aspects that one may consider in designing a trial with CIMT as primary outcome parameter. For example, a major flaw in CIMT and carotid plaque measurements is the inter-observer and intra-observer reproducibility of measures. Although many recent studies demonstrated good agreements between intra-observer (between 91% and 97%) and inter-observer (between 88% and 91%) reliability of CIMT [120,122,139], data reproducibility must be assured.

An important limitation for comprehensive data analysis and results interpretation is the different methodology of CIMT and plaque measurements used in individual studies. That issue was clarified in the dedicated ASE and Mannheim recommendations [8,54,59].

Information on plaque changes in time carries useful information on treatment efficacy. Patients who are responders to medical treatment in terms of attenuation of carotid atherosclerosis growth have a decreased risk of MACCE. Therefore, last but not least, it is important to perform serial repeated carotid atherosclerotic burden measurements with appropriate period intervals between measurements, as atherosclerosis changes can fluctuate with periods of rapid and slow growth or regression. The single re-assessment of CIMT and plaque is a shortcoming.

#### **12. Perspectives for CIMT and Carotid Plaque on Ultrasound**

The 2020 ASE recommendations for carotid plaque ultrasound suggested a stepwise approach to CV risk stratification adopted from Greenland et al. [140] and Piepoli et al. [124]. At baseline, carotid vascular ultrasound and subsequent 2D or 3D plaque quantification would be performed in the assessment of asymptomatic patients at low or intermediate risk according to an FRS and European SCORE. Patients with no plaque or carotid plaque thickness less than 1.5 mm would be considered as low risk. Patients with plaques thickness (CIMT) between 1.5 and 2.4 mm would be allocated to the intermediate risk class, while whose with plaques exceeding 2.4 mm would be considered in the high CV risk class with subsequent assessment of patient and plaque vulnerability (neovascularization and echolucency) [8].

In conclusion, CIMT and carotid plaque reflects atherosclerosis burden in the whole arterial tree. Incidence and severity of CV risk factors (both traditional and non-traditional) have an impact on CIMT thickness and plaque burden, and more importantly, they are responsible for the rate of carotid atherosclerosis progression. CIMT and carotid plaque may play an additive role in scoring systems evaluating CV risk. Thus, it appears reasonable to combine established risk scores with CIMT and plaque imaging. Both in the primary and the secondary care populations of patients, baseline parameters of CIMT and plaque thickness are associated with risk of future CV events. Aggressive medical treatment focused on CV risk factors' elimination is associated with lesser progression of carotid atherosclerosis. However, whether medical interventions have an impact on the decreased risk of CV events through the reduction of CIMT and carotid plaque burden remains a matter of debate and needs further studies.

The authors of this review believe that averaged value of maximum CIMT with inclusion of maximum plaque thickness (when applicable) assessed at both the CCA, the CB, and the ICA is the best way to display atherosclerosis burden, and it well stratifies the CV risk and adverse events incidence both as baseline values and as a serial assessment. However, its clinical appliance should be matter of further investigations.

**Author Contributions:** Conceptualization, A.K.-Z.; methodology, A.K.-Z. and T.P.; writing—original draft preparation, A.K.-Z. and T.P.; writing—review and editing, A.K.-Z.; visualization, A.K.-Z. and T.P.; supervision, T.P.; project administration, A.K.-Z.; funding acquisition, A.K.-Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Jagiellonian University Medical College, grant number N41/DBS/000752. This article was supported by the science fund of the John Paul II Hospital, Cracow, Poland (no. FN/22/2021 to A.K.-Z.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Small Differences in Vitamin D Levels between Male Cardiac Patients in Different Stages of Coronary Artery Disease**

**Ewelina A. Dziedzic 1,\*, William B. Grant 2, Izabela Sowi ´nska 3, Marek D ˛abrowski <sup>4</sup> and Piotr Jankowski 5,6**


**Abstract:** Cardiovascular diseases are the main cause of mortality in males older than 65 years of age. The prevalent vitamin D deficiency in the worldwide population may have multiple effects on the cardiovascular system. This study sought to determine the association between serum levels of 25-hydroxyvitamin D (25(OH)D) and the stage of coronary artery disease (CAD) in Polish male subjects. Additionally, subjects with a history of myocardial infarction (MI) were analyzed for potential differences in 25(OH)D levels in comparison with those diagnosed with stable CAD. The study was conducted prospectively in a group of 669 male patients subjected to coronarography examination. CAD stage was defined using the Coronary Artery Surgery Study Score. Patients without significant coronary lesions had significantly higher 25(OH)D levels than patients with single-, double-, or triple-vessel disease (median, 17 vs. 15 ng/mL; *p* < 0.01). Significantly lower levels of 25(OH)D were apparent when MI was identified as the cause of the then-current hospitalization in comparison with stable CAD, as well as in patients with a history of MI; all of these cases had lower levels of 25(OH)D in comparison with patients with no such history. Male patients with single-, double-, or triple-vessel CAD, acute coronary syndrome, or a history of MI presented lower serum 25(OH)D.

**Keywords:** vitamin D; coronary artery disease; myocardial infarction; males; Coronary Artery Surgery Study Score

#### **1. Introduction**

The aging of the worldwide population is being observed in recent decades. Consequently, the subpopulation of male individuals older than 65 years in Poland has gradually increased [1–4]. In this group of patients, cardiovascular diseases account for more than half of all deaths [5]. Despite the observed improvement concerning mortality rates, a large difference between the average male and female lifespans persisted (73 and 81 years, respectively) [5]. Elderly men should be considered a unique group of patients liable to major cardiovascular events. Considering exceedingly high rates of the risk of death, cardiovascular diseases (CVDs) continue to prompt the need for the relentless investigation of their risk factors and new therapies [6]. As a result of advancements in treatment, including non-pharmacological prevention, the risk of death from CVDs has dropped by 20% over the last few decades [7]. Despite these improvements, CVD remains responsible for about 18 billion deaths per year, the majority of which are a consequence of myocardial infarction or stroke [8]. Appropriate physical activity and dietary interventions both reduce the cardiovascular risk [9,10] and affect the level of 25-hydroxyvitamin D. Notably, calcitriol

Sowi ´nska, I.; D ˛abrowski, M.; Jankowski, P. Small Differences in Vitamin D Levels between Male Cardiac Patients in Different Stages of Coronary Artery Disease. *J. Clin. Med.* **2022**, *11*, 779. https://doi.org/ 10.3390/jcm11030779

**Citation:** Dziedzic, E.A.; Grant, W.B.;

Academic Editor: Anna Kabłak-Ziembicka

Received: 30 December 2021 Accepted: 28 January 2022 Published: 31 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

deficiency is identified as one of the new cardiovascular disease risk factors and has been proven to be prevalent in human populations worldwide [11–13].

The discovery of the biological mechanisms underlying vitamin D effects justifies studies of the association between its deficiency and the risk of cardiovascular diseases. The 1,25-dihydroxyvitamin D (calcitriol) receptor is present in numerous cells of the cardiovascular system [14]. Studies conducted on an animal model have shown an adverse effect of vitamin D deficiency on the functions of endothelial cells, vascular smooth muscle, and cardiomyocytes [14]. Due to the presence of the enzyme 1-α hydroxylase, these cells are capable of autocrine calcitriol synthesis [15,16]. This hormone is a negative regulator of the axis of the renin–angiotensin–aldosterone system, the increased activity of which leads to the development of arterial hypertension and myocardial hypertrophy [17–19]. The relationship between calcitriol deficiency and individual stages of plaque formation and destabilization, as well as documented risk factors for coronary artery disease, has been documented [13,20].

To date, the results of studies do not offer an explicit agreement on how vitamin D affects the cardiovascular system [21]. However, some reports suggest that 25(OH)D levels below the reference range may increase the risk of CVD [21], whereas optimum levels may exert a protective effect on both musculoskeletal and cardiovascular systems [21–23]. The main objective of this study was to assess the potential association between serum 25(OH)D levels and the stage of coronary artery disease (CAD) in Polish males.

#### **2. Materials and Methods**

#### *2.1. Population*

This study is part of a research project focused on the relationship between the level of vitamin D and the severity of coronary artery atherosclerosis in Polish cardiac patients. Results of the analysis of this association among 637 patients are presented in previously published articles, in which details of the study population and measurements (diabetes diagnosis, acute coronary syndrome (ACS) diagnosis, interview questionnaire, body mass index (BMI), concentration of total cholesterol (TC) and/or triglycerides (TG), systolic and diastolic blood pressure, coronary angiography, and total 25(OH)D in participant serum and plasma) were described [24–26]. Abnormal serum levels of phosphate, calcium and parathyroid hormone (PTH) treatments, or supplementation containing vitamin D or calcium served as exclusion criteria. Patients with stages III-V chronic kidney disease, active malignancy, and elevated inflammatory markers or fever were also excluded from the study. New patients were continuously examined, and final pooled data of Polish patients hospitalized in the Cardiology Department who underwent diagnostic catheter angiography for the evaluation of coronary artery disease in the years 2013 to 2017 were presented in the most previous study [27]. This study presents data for male patients.

The serum level of 25(OH)D was determined with the Vitamin D total assay by Roche Diagnostics, certified by VDSP. The Roche Diagnostics Vitamin D total assay is a competitive electrochemiluminescence protein-binding assay intended for the quantitative determination of the total 25-OH vitamin D in human serum and plasma. The assay utilizes a vitamin-D-binding protein (VDBP) as the capture protein, which binds to both 25-OH D3 and 25-OH D2 [28].

The status of vitamin D levels was classified according to the Endocrine Society Clinical Practice Guidelines for Vitamin D Deficiency [29]: 25(OH)D level <10 ng/mL was considered as a severe deficiency; ≥10 to <20 ng/mL as a moderate deficiency; ≥20 to <30 ng/mL as a mild deficiency; ≥30 ng/mL as optimal.

Coronary atherosclerosis was assessed by Coronary Artery Surgery Study Score (CASSS) according to the following rules [30]. Stenosis ≥50% of the left main coronary artery (LMCA) was scored at 2 points. Stenosis >70% in any of the large epicardial coronary arteries (anterior descending branch, LAD; circumflex branch, LCx; right coronary artery, RCA) was scored at 1 point. The sum of all points equaled the score, which may indicate, respectively a one-, two-, or three-vessel CAD [30].

The study project was approved by the University Bioethical Committee (KB/124/2014) and followed the rules and principles of the Helsinki Declaration.

#### *2.2. Statistics*

The Shapiro–Wilk test was used to evaluate the normal distribution of data. The Poisson regression analysis was used to assess the relationship between 25(OH)D levels and selected variables. To compare the results of continuous variables between the two groups, the Mann–Whitney test or *t*-test was used. For comparisons of three or more independent groups, the Kruskal–Wallis test or one-way analysis of variance were used, depending on the presence of a Gaussian distribution (Shapiro–Wilk normality test). Pearson's chisquared test or Fisher's exact test was used to determine differences between prevalence in selected groups. Statistical analyses were performed with a significance level of 5% (*p* value < 0.05). The statistical analysis was carried out with STATISTICA 12.5 software.

#### **3. Results**

#### *3.1. Characteristics of the Study Group*

The study was conducted on 1345 patients admitted to the Department of Cardiology in the Bielanski Hospital in Warsaw (Poland) for coronarographic examination as a result of suspected CAD as identified in an outpatient setting. Final statistical analysis was carried out in 1043 patients (669 males, 374 females). The data of 302 subjects were excluded, as they met the study exclusion criteria (for details, see [27]).

#### *3.2. Comparisons between Female and Male Subpopulations*

Statistically significant differences were observed between females and males with regard to age, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels. Statistically significant disproportions were observed between the female and male subpopulations with regard to smoking status, history of myocardial infarction, and the number of arteries presenting with significant stenosis (Table 1).


**Table 1.** Results between female and male subpopulations.


**Table 1.** *Cont.*

25(OH)D = 25-hydroxyvitamin D; BMI, body mass index; CASSS, Coronary Artery Surgery Study Score; HDL, high-density lipoprotein; LDL, low-density lipoprotein. † BMI class 1, <25; class 2, 25–30; class 3, >30; ‡ 0, stable coronary artery disease; 1, myocardial infarction; \*\* 1: <10 ng/mL severe deficiency, 2: ≥10 to <20 ng/mL moderate deficiency, 3: ≥20 to <30 ng/mL mild deficiency, 4: ≥30 ng/mL optimal.

Statistically significant differences were observed between females and males with regard to age, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels. Statistically significant disproportions were observed between the female and male subpopulations with regard to smoking status, history of myocardial infarction, and the number of arteries presenting with significant stenosis.

*3.3. Analysis of Male Subpopulation, Correlation between 25(OH)D Levels or Other Parameters, and the CASSS Stage of Coronary Artery Disease in Male Subpopulation*

Among male patients divided into individual CASSS severity groups (Table 2), analysis of variance revealed statistically significant differences in age, total cholesterol levels, HDL cholesterol levels, and LDL cholesterol levels.

**Table 2.** Characteristics of the examined group divided according to degree of coronary atherosclerosis (the Coronary Artery Surgery Study Score (CASSS)) into four subgroups.


25(OH)D = 25-hydroxyvitamin D; BMI, body mass index; CASSS, Coronary Artery Surgery Study Score; HDL, high-density lipoprotein; LDL, low-density lipoprotein. † BMI class 1: <25; class 2: 25–30; class: 3 >30; ‡ 0, stable coronary artery disease; 1, myocardial infarction; \*\* 1: <10 ng/mL severe deficiency, 2: ≥10 to <20 ng/mL moderate deficiency, 3: ≥20 to <30 ng/mL mild deficiency, 4: ≥30 ng/mL optimal.

Post hoc analyses in unequal subgroup populations revealed that CASSS 3 patients were significantly older than patients in all other CASSS groups (*p* < 0.01 for all comparisons). Individual subgroups of patients with different CASSS scores were characterized by statistically significant differences in the prevalence of diabetes, hypertension, smoking, history of myocardial infarction, or the cause of the then ongoing hospitalization.

No significant differences were observed in 25(OH)D levels, BMI values, or triglyceride levels between patients across all CASSS groups. Neither could significant differences be observed in the prevalence of hyperlipidemia or the season of examination between individual CASSS groups.

Poisson distribution and multiple regression analysis were used to identify factors that significantly determined the CASSS in the male population. Factors/determinants in the analysis included serum 25(OH)D levels, age, BMI, smoking status, hypertension, concomitant diabetes, hyperlipidemia, history of myocardial infarction, cause of the then-current hospitalization, and season during the examination. Factors significantly determining the CASSS value included age (*p* < 0.05), cause of ongoing hospitalization (*p* < 0.001), smoking status (*p* < 0.05), and history of myocardial infarction (*p* < 0.001).

#### *3.4. Male Patients without Significant Arterial Stenosis (CASSS 0) in Comparison with Patients with Significant Arterial Stenosis (CASSS 1–3)*

The group of patients without significant changes within the coronary arteries (CASSS 0) presented with higher 25(OH)D levels than patients with single-, double-, or triple-vessel disease (CASSS 1, 2, or 3, respectively). Considering the above, we carried out further analyses with the study population divided into two subgroups: the CASSS 0 subgroup and the CASSS 1–3 subgroup (Table 3).

**Table 3.** Characteristics of the examined group divided according to degree of coronary atherosclerosis (CASSS) into two subgroups.


† BMI class 1 < 25; class 2 25–30; class 3 > 30; ‡ 0, stable coronary artery disease; 1, myocardial infarction; \*\* 1: <10 ng/mL severe deficiency, 2: ≥10 to <20 ng/mL moderate deficiency, 3: ≥20 to <30 ng/mL mild deficiency, 4: ≥30 ng/mL optimal.

Statistically significant differences were observed between male patients with and without significant coronary stenosis with regard to serum 25(OH)D, BMI values, total cholesterol levels, and HDL cholesterol levels.

Statistically significant disproportions were observed between subgroups with regard to arterial hypertension status, smoking status, history of myocardial infarction, and cause of the then ongoing hospitalization.

Only 13 patients from the CASSS 0 subgroup were hospitalized for myocardial infarction; the remaining 113 subjects were hospitalized as a result of stable CAD. In addition, 11 patients in the CASSS 0 subgroup had a history of myocardial infarction. About half of the patients from the CASSS 1–3 subgroup were hospitalized for myocardial infarction; about half also had a history of myocardial infarction.

Factors significantly determining the CASSS value in the entire male subpopulation included age (*p* < 0.05), cause of the then-current hospitalization (*p* < 0.001), smoking status (*p* < 0.05), and history of myocardial infarction (*p* < 0.001). Among the CASSS 1–3 subgroup of male patients, larger percentages of patients had arterial hypertension, were smokers, had a history of myocardial infarction, and reported to the department because of an acute coronary syndrome rather than stable CAD.

#### *3.5. Determinants of Serum 25(OH)D Levels in Male Cardiac Patients*

In the presented study group, significant determinants of serum 25(OH)D levels included the season of the year (*p* < 0.001) and hyperlipidemia (*p* < 0.01). Lower serum 25(OH)D levels were presented independently by patients with hyperlipidemia and those examined between October and April.

#### *3.6. Identifying the Group with the Lowest 25(OH)D Levels among Male Cardiac Patients*

Factors significantly determining the CASSS value included age (*p* < 0.05), cause of the then-ongoing hospitalization (*p* < 0.001), smoking status (*p* < 0.05), and history of myocardial infarction (*p* < 0.001). The lowest serum 25(OH)D levels were measured in elderly male cardiac patients hospitalized for an acute coronary syndrome, presenting with a history of myocardial infarction, positive smoking status, and diagnosis of hyperlipidemia, as well as undergoing examination between October and April.

#### **4. Discussion**

Cardiovascular diseases are ranked as the main cause of death in male patients aged over 65 years old, remaining the second in younger individuals [8]. Mortality rates due to CVD for males are higher than those for females [31]. To date, numerous studies suggested a correlation between low 25(OH)D levels and increased risk of death from cardiovascular causes was suggested [32,33]. The aim of our research was to assess the association between vitamin D serum levels the severity of CAD.

The findings of this research are consistent with the results of previous studies examining the association between serum 25(OH)D levels and CVD [12]. However, only a few studies thus far were conducted in male subpopulations only; most were carried out in mixed-sex populations [34–38]. In our study, male patients without significant coronary lesions (CASSS 0) presented with statistically higher serum vitamin D levels than patients with significant stenosis of coronary arteries (CASSS 1–3); however, the nominal difference was negligible. We showed that calcitriol serum levels were significantly lower in patients with a history of MI. Elderly patients with hyperlipidemia, actively smoking, hospitalized for an ACS, with a history of MI, were a subgroup presenting with the lowest 25(OH)D serum levels.

The impact of serum vitamin D level on the established cardiovascular risk factors (i.e., development of type 2 diabetes, metabolic syndrome) was repeatedly proven in the literature [39,40].

Both nuclear vitamin D receptor (VDR) and the enzyme 25-hydroxyvitamin D3-1αhydroxylase have been identified in various cells of the cardiovascular system, indicating a direct involvement of this hormone group in the initiation and progression of CVD [41]. Importantly, in patients with heart failure, atrial fibrillation, or coronary artery disease vitamin D deficiency was associated with a worse prognosis [42–44]. Moreover, hypovitaminosis D was proven to affect the established cardiovascular risk factors such as arterial hypertension [45], type 2 diabetes [44], or dyslipidemia [44]. Calcitriol inhibits the reninangiotensin–aldosterone system (RAAS) and the secretion of natriuretic peptides, thus having a hypotensive effect [46]. Activation of the VDR receptor has a protective effect on the excess of angiotensin II by inhibiting fibrosis and exerting anti-inflammatory and antiproliferative effects [47]. Mediated by cells of the immune system, calcitriol modulates the secretion of miR-106b-5p and inhibits the secretion of renin by the glomerular apparatus [48]. The impact of vitamin D on various stages of atherosclerosis is currently being a subject of some studies [13]. Previous studies demonstrated that vitamin D affects atherosclerotic plaque formation in numerous ways, including reducing the inflammatory response, inhibiting the NF-κB pathway [49], and suppressing the post-infarction scar formation [50]. Proper serum 25(OH)D level was proven to reduce the activity of the metalloproteinases, which degrade the fibrous cap of the atherosclerotic plaque [51]. In addition, vitamin D also inhibits the activity of vascular endothelial growth factors, preventing the formation of new vessels within an already formed plaque, thus contributing to its better stability [52]. After plaque rupture, vitamin D exerts an antithrombotic effect by increasing the production of thrombomodulin and reducing the expression of platelet tissue factors. Hence, it inhibits the adhesion of platelets to vascular endothelial cells [53]. This process may be a way of vitamin D contributing to the prevention of ACS.

A study based on an analysis of more than 1000 Polish patients confirmed the already reported low vitamin D levels in the Polish population [54,55], as well as the higher 25(OH)D levels in males than in females [26]. On the other hand, Verdoia emphasizes the importance of higher 25(OH)D levels noted in men, compared with women [56]. The results of that study provided a stimulus to expand the research and reassess how 25(OH)D levels affect the stage of CAD and incidence of MI in the entirely male cohort. The results of the above-mentioned studies stimulated us to evaluate the impact of 25(OH)D levels on the stage of coronary artery disease and the incidence of MI in an all-male cohort. The influence of vitamin D deficiency on episodes of MI in men is supported by several cohort studies [57,58]. Patients with 25(OH)D levels of ≤15 ng/mL were proven to have a more than 1.5-fold increase in the risk of adverse cardiovascular events (i.e., MI, angina pectoris, stroke, TIA, and heart failure) [57] and a twofold increase in the risk of ACS [58]. Moreover, low vitamin D levels and a history of MI were associated with a significant increase in the risk of further major adverse cardiovascular events (MACE), including reoccurrence of MI [37]. In addition, serum 25 (OH) D levels above 7.3 ng/mL were associated with a 40% reduction in the risk of non-fatal MACE in patients with ACS.

To date, several studies examining vitamin D supplementation in patients with CVD have been conducted. Although none of the large cohort studies showed a favorable cardiovascular outcome, individual experiments have proven that six-month calcitriol supplementation significantly reduced the inflammation of coronary arteries [59] and declined SYNTAX score (67). However, poor bioavailability and large intervals between consecutive doses of cholecalciferol should be underlined as potentially resolvable issues [59]. Another possible mechanism by which vitamin D may affect the degree of progression of coronary disease is its effect on the metabolism of sex hormones. In the MESA study, lower 25(OH)D levels were found to be associated with lower sex-hormone-binding globulin concentrations and higher levels of free testosterone, which are important in the course of coronary artery disease [60].

Our research suffers from several limitations. The study group consisted of residents of only central Poland, most residing in urban areas. Expanding the study group to include residents of other provinces would facilitate the translation of the results to the entire Polish population. CAD staging was classified based on the results of coronary angiography using the CASSS. The classification of the severity of atherosclerosis based on the SYNTAX might change our results.

The results of the observational studies carried out so far have shown that the endocrine system of vitamin D, in addition to its documented effect on the skeletal system, exerts a wide spectrum of extra-skeletal activity [61,62]. In these studies, low vitamin D levels were found to be associated with an increased risk of cardiovascular diseases, including hypertension, congestive heart failure, as well as adverse cardiovascular events (MACE, heart attacks, and strokes). In a meta-analysis involving nearly 850,000 people, low serum 25(OH)D levels were associated with a 1.42 times higher risk of developing MACE, compared with patients with higher levels of vitamin D [63]. On the other hand, the results of randomized clinical trials (VITAL, ViDa, D2d), which included over 30,000 participants, showed that supplementation with vitamin D does not prevent cardiovascular events or the progression of type 2 diabetes [64–66]. It should be emphasized that the initial serum level of 25 (OH) D in the respondents of the above-mentioned studies fluctuated above 50 nmol/L, and post hoc analysis suggested some extra-skeletal benefits in the vitamin D deficiency group. The causal association between calcitriol and cardiovascular mortality continues to be the subject of much debate. New information was provided by the recently presented results of the non-linear MR analysis carried out at UK Biobank [67]. The authors of the cited study presented the association between the genetically predicted serum 25 (OH) D levels and the risk of cardiovascular diseases to be L shaped. This research seems to confirm the results of observational and interventional studies and determines a specific range of vitamin D levels within which vitamin D supplementation may have a beneficial effect in short- and long-term observations. At the same time, it explains why supplementing people rich in vitamin D does not generate overall health benefits, and correction of a severe deficiency of this hormone may be necessary. At present, the opinions of scientists around the world unanimously recommend the correction of vitamin D (25 (OH) D serum deficiency <30 nmol/L), and most scientific societies recommend a target level of >50 nmol/L as optimal for bone health. In our opinion, vitamin D deficiency may also be an easily modifiable risk factor of the acute coronary syndrome in men, which should undoubtedly be the subject of further research. Perhaps, well-designed and conducted social campaigns in the field of proper exposure to solar radiation, food fortification, or pharmacological supplementation of vitamin D could considerably contribute to the prevention of CAD and its complications.

#### **5. Conclusions**

In conclusion, we demonstrated that male patients with a history of ACS and MI presented reduced serum calcitriol levels. Patients with advanced CAD presented with significantly lower levels of 25(OH)D than those without significant atherosclerotic lesions; however, the difference should be considered as clinically negligible. Further studies should be undertaken in specific subgroups, to assess the potential beneficial effects of vitamin D supplementation in this group of patients.

**Author Contributions:** Conceptualization, E.A.D.; methodology, E.A.D.; formal analysis, E.A.D.; investigation, E.A.D.; resources, E.A.D.; data curation, E.A.D.; writing—original draft preparation, E.A.D., W.B.G., I.S., M.D. and P.J.; writing—review and editing, E.A.D., W.B.G., I.S., M.D. and P.J.; funding acquisition, E.A.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partly supported by a statutory grant to the Cardiology Clinic of Physiotherapy Division from the Second Faculty of Medicine, Medical University of Warsaw, Poland Grant Number 2F5/PM2/16. The APC was funded by the Medical University of Warsaw.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Bioethical Committee of Medical University (KB/124/2014).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data can be provided by the authors upon reasonable request.

**Conflicts of Interest:** William B. Grant receives funding from Bio-Tech Pharmacal, Inc. (Fayetteville, AR, USA). Other authors declare no conflict of interest.

#### **References**


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