Significance of Determining Inflammatory Markers and Carotid Artery Intima Media Thickness Values in the Detection of Coronary Heart Disease in Asymptomatic Patients with Type 2 Diabetes

Malesevic G, Abraham KH, Jamila MM and Almaz AA

Published on: 2025-05-15

Abstract

Introduction: Increased levels of high-sensitivity C - reactive protein (hs-CRP) and Interleukin 6 (IL-6) accelerates atherosclerosis and occurrence of cardiovascular complications in patients with Type 2 Diabetes (T2D). Intima-Media Thickness (IMT) is a surrogate marker of atherosclerosis in patients with T2D. The aim of the study was to evaluate the relation inflammatory markers IL-6, hs-CRP and carotid atherosclerosis with the presence of Coronary Heart Disease (CHD) in asymptomatic patients with T2D.

Methods: The study included 169 patients with T2D, without any symptoms and signs of CHD. Exercise stress testing proved or ruled out the presence of silent CHD. Carotid ultrasonography was performed and IMT was measured. The levels of hs-CRP and IL-6 were determined by ELISA.

Results: The presence of silent CHD was proven in 52 subjects using ergometric testing, while 117 subjects were without CHD. Higher values of IL-6, hs-CRP and IMT were correlated with silent CHD.

Age, HbA1c, LDL cholesterol and albuminuria values were significant predictors of silent CHD (p<0.05). Other analysed risk factors: gender, smoking, duration of T2D, BMI, hypertension, total cholesterol and triglycerides were not related to the presence of CHD.

Conclusion: It was proven that there was a greater possibility of the presence of silent CHD in asymptomatic patients with T2D with higher values of IMT (p<0.05), IL-6 (p<0.05) and hs-CRP (p<0.001).

Keywords

Diabetes mellitus; Coronary heart disease; Inflammatory markers; Intima-media complex

Introduction

Cardiovascular Disease (CVD) is the leading cause of morbidity and mortality in patients with T2D. Understanding how novel (e.g., inflammatory) factors as well as subclinical atherosclerosis can add to the prediction of CVD beyond traditional risk factors has been an important focus of research in the field [1]. Traditional risk factors for CVD, such as high LDL cholesterol, low HDL cholesterol, hypertension and smoking explain only a part of cardiovascular risk in T2D patients [2]. Therefore, it is crucial to determine other risk factors for CVD occurrence such as progressive inflammatory tissue response to continuous deposition and modification of lipoproteins in the vascular wall [3]. At present, atherosclerosis is considered an inflammatory disease, given the key role of inflammation in all stages of the occurrence and development of atherosclerotic process, and the inflammatory nature of atherosclerosis is manifested by correlation of inflammatory marker levels in blood with its occurrence and progression [4, 5]. Acute hyperglycaemia has also been proven to induce proinflammatory cytokine genes in T cells as well as inflammatory cytokines, especially hs-CRP and IL-6 [6, 7]. Recent research has shown that hs-CRP is a more powerful tool for predicting the risk of CVD occurrence than LDL cholesterol [8]. Since atherosclerotic process occurs simultaneously in the carotid, cerebral and coronary arteries, detection of atherosclerotic changes is focused on the carotid arteries, due to much more suitable and less invasive techniques such as measuring the values of Intima-Media Thickness (IMT) of carotid arteries, which is simultaneously a predictor of occurrence of cardiovascular events same as traditional risk factors in patients with T2D [9]. The aim of the study was to evaluate the significance of determining inflammatory markers IL-6 and hs-CRP and values of IMT of carotid arteries, as atherosclerosis markers, during screening for presence of CHD in asymptomatic patients with T2D.

Patients And Methods

The study has been conducted as a cross-sectional study at the Republic of Srpska University Clinical Centre, Banja Luka, and it included 169 persons with T2D without previously known or newly diagnosed CHD, aged 40 to 70. The subjects underwent ergometric testing, and based on the obtained results, they were divided into two groups. The first group consisted of 117 individuals with T2D without the presence of CHD, proven by the absence of symptoms and a negative ergometric test. The second group consisted of 52 individuals with T2D and presence of silent ischaemia of the heart proven by a positive stress test. All subjects underwent an anamnestic interview and physical examination, after which they all gave written consent to participate in the research. Calculation of Body Mass Index (BMI) for assessment and monitoring of nutritional status was performed according to Quetelet's formula: BMI = body weight in kg/square of body height in meters (kg/m2). Subjects with T2D with CHD, with a history of cerebrovascular, peripheral vascular and malignant diseases were excluded from the study, as well as subjects who had an acute or chronic infection or who received corticosteroids or immunosuppressants as part of their therapy.

The study was conducted in compliance with the Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects. Ethics Committee of the Republic of Srpska University Clinical Centre in Banja Luka gave their consent for approval of the research protocol.

CHD Diagnosis

Ergometric testing was performed on a General Electric treadmill type T-2100. Testing was performed according to the standard Bruce protocol. The test was evaluated as positive in subjects with horizontal or descending ST-segment depression equal to or >1mm for 60-80 ms after the J-point, at least in three successive QRS complexes, as well as in patients who experienced ST-segment elevation during the stress test that was characterized as pathological if it occurred with the same characteristics, as well as ST-segment depression (>1mm, lasting longer than 60-80 ms). The test was defined as positive and negative, and the patients in whom it was described as inconclusive were not considered [10].

Evaluation of Carotid Artery Atherosclerosis

The IMT values of carotid arteries were measured by B-mode ultrasound using a LOGIQ IM 7 PRO machine, with electronic linear probe with a frequency of 8 MHz. The patients were examined in a supine position with their head turned 45 degrees contralaterally from the scanned side, according to the so far standardised method by Pignoli et al. The IMT value is defined as the distance between the leading edges of two echogenic lines, where the first line represents the boundary between the blood vessel lumen and the tunica intima, and the second one the boundary between the tunica media and the tunica adventitia. The IMT value was measured three times, 1 cm distally from the bulb of the common carotid artery, and the mean value was taken for analysis. The IMT values in healthy middle-aged people are 0.6-0.7 mm and they increase at a rate of 0.005 to 0.010 mm/year. The IMT values > 0.9 mm are defined as carotid artery atherosclerosis [11].

Laboratory Analysis

Biochemical blood tests for laboratory processing were taken in the morning after a 12-hour overnight fasting. The total cholesterol, HDL cholesterol, LDL cholesterol and serum triglycerides were measured directly, by homogeneous enzymatic procedure on INTEGRA® 400 plus analyser, manufactured by Roche, and HbA1c and urine albumin concentration in a 24-hour urine by a turbidimetric assay method. Determination of the levels of inflammatory markers hs-CRP and IL-6 was performed using ELISA (R&D Systems, Inc., Minneapolis, USA). It is a quantitative sandwich enzyme-linked immunosorbent assay technique. Blood serum was used for this test. The blood was then centrifuged at 3000 rpm at 4°C for 15 minutes, and aliquots were stored at -70°C. A commercial calibrator was used for calibration. Subjects with hs-CRP values above 10 mg/L were excluded from the study because such hs-CRP values indicate the presence of acute inflammatory disease. An hs-CRP value of 1 mg/L indicates a low risk for CVD; from 1-3mg/L=moderate risk; from 3-10 mg/L = high risk [12]. The lowest level of IL-6 detectability in the serum was 1.5 pg/ml [12]. The coefficients of variation of the test were 5%. Calibrations of the testing instrument were performed as recommended by the manufacturer within the given specifications.

Statistical Analysis

The data were analysed using a commercially available statistical programme (SPSS 17.0 for Windows; SPSS, Chicago, IL, USA). Continuous variables are summarized as mean ± SD or as a percentage of frequency. Categorical variables are expressed as proportions (percentage), Student’s t-test (for continuous variables) or Chi-square proportion test (for categorical variables) were used. Appropriate descriptive and analytical methods (absolute and relative numbers, t test, Wilcoxon test, Mann-Whitney U test) were also used. Multiple logistic regression was applied to predict and evaluate one variable based on the value of the other variable or multiple variables. The significance level was less than 0.05 [13].

Results

Ergometric screening tests for presence of CHD were performed in 169 asymptomatic patients with T2D without a history of any CVD. The presence of silent CHD was proven in 52 subjects using ergometric testing, while 117 subjects were without CHD. We examined whether there were differences in cardiovascular risk factors between the study groups.

Table 1: Demographic and anthropometric characteristics of the T2D patients with and without silent CHD.

Characteristics

CHD + n=52

CHD - n= 117

p value

Gender (male vs female)

24/28

47/70

ns

Smoking , n (%)

32 (38.5)

16 (13.7)

<0.05

Age (years)

58.71 ± 6.76

54.98 ± 6.69

<0.05

Duration of DM (years)

10.52 ± 4.60

7.08± 3.19

<0.05

BMI, kg/m2

27.6 ±1.58

27.2±1.46

ns

Systolic BP (mm Hg)

139.90 ± 11.7

128.16 ± 10.72

<0.05

Diastolic BP (mm Hg)

88.56 ± 9.3

81.03± 7.27

<0.05

HbA1c, (%)

9.16 ±1.91

7.43± 1.08

<0.05

Total cholesterol, mmol/l

6.07± 1.33

5.37± 1.11

<0.05

LDL cholesterol, mmol/l

3.97±1.11

3.33 ±0.86

<0.05

HDL cholesterol, mmol/

1.17 5±0.2

1.14 ±0.37

ns

Triglyceride, mmol/l

2.35±1.1

2.06 ± 1.37

<0.05

Microalbuminuria, n (%)

47(90.4)

20(17.1)

<0.001

DM=diabetes mellitus; CHD=coronary heart disease; BMI= Body Mass Index; BP=blood pressure; HbA1c= Glycosylated haemoglobin; HDL=High Density Lipoprotein; LDL=Low Density Lipoprotein.

Table 1 shows a comparison of demographic and risk factors between subjects with CHD and without CHD. Subjects did not differ significantly by gender. The patients with silent CHD were older with a longer duration of diabetes and a higher incidence of smokers compared to the patients without CHD (p <0.05). The difference in BMI between the study groups with and without CHD was not statistically significant. Prevalence of hypertension as well as HbA1c values were statistically significantly higher in subjects with silent CHD compared to the subjects without CHD (p<0.05). Regarding the lipid parameters, total LDL cholesterol as well as triglycerides, were significantly higher in the group of subjects with silent CHD (p<0.05), whereas the values of HDL cholesterol did not differ significantly between the study groups. In subjects with silent CHD, microalbuminuria was present in 47 subjects (90.4%). In subjects in whom CHD was not detected, microalbuminuria was present in 20 subjects (17.1%), which was statistically significantly different (p <0.001).

The use of therapy in the examined groups was also analysed (Table 2). The prevalence of insulin therapy did not differ significantly between the study groups. The use of GLP 1-RA was significantly higher in subjects without silent CHD (p <0.05). Analyzing the prevalence of oral antidiabetic drugs, the results of our study showed that the use of sulfonylurea was more significant in subjects without CHD (p <0.05), as well as the use of metformin, DPP4 inhibitors and SGLT-2 inhibitors (p <0.001). Statin use was significantly higher in subjects without silent CHD (p <0.001), while the prevalence of antihypertensive use was also significantly higher in subjects without silent CHD (p <0.005).

Table 2: Baseline therapies of the T2D patients with and without silent CHD.

 

CHD + N= 117

CHD - N=52

p

Insulin

25 (44.6%)

31 (55.4%)

ns

GLP-1 RA

7 (24.1%)

22 (75.9%)

<0.05

Sulfonylurea

13 (30.2%)

30 (69.8%)

<0.05

Metformin

42 (33.1%)

85 (66.9%)

<0.001

DPP-4i

7 (22.6%)

24 (77.4%)

<0.001

SGLT-2i

-

3 (100.0%)

<0.001

Statins

32 (31.7%)

69 (68.3%)

<0.001

Antihypertensives

30 (35.3%)

55 (64.7%)

<0.05

T2D=type 2 diabetes; CHD=coronary heart disease; GLP-1 RA=glucagon-like peptide 1 receptor agonist; DPP-4i = dipeptidyl peptidase 4 inhibitor; SGLT-2i=sodium-glucose cotransporter 2 inhibitor.

Using the regression analysis model, we concluded that age, HbA1c, LDL cholesterol and albuminuria values were significant predictors of silent CHD proven by ergometric testing, i.e. with their increase the probability of positive ergometric testing increased too (p<0.05). Other analysed risk factors: gender, smoking, duration of T2D, BMI, hypertension, total cholesterol, HDL cholesterol, and triglycerides did not show statistical significance in the prediction of CHD.

Table 3: Multivariate logistic regression analysis of risk factors for prediction of silent CHD amongst the study population.

Variables

B

S.E.

p

OR

95% CI. for OR

Lower

Upper

Gender (male vs female)

-0.878

0.65

0.177

0.416

0.116

1.486

Smoking status

1.06

0.625

0.09

2.886

0.848

9.82

Age

0.112

0.055

0.043

1.118

1.003

1.247

Diabetes duration

0.101

0.086

0.244

1.106

0.934

1.31

BMI

0.072

0.119

0.545

1.075

0.851

1.357

Systolic blood pressure

0.027

0.04

0.505

1.027

0.949

1.112

Diastolic blood pressure

0.038

0.051

0.449

1.039

0.941

1.147

Glycosylated haemoglobin

0.792

0.235

0.001

2.208

1.393

3.499

Total cholesterol

-0.773

0.618

0.211

0.461

0.137

1.551

Triglyceride

0.179

0.321

0.578

1.196

0.637

2.246

LDL cholesterol

1.606

0.682

0.019

4.982

1.308

18.975

HDL cholesterol

1.073

0.853

0.208

2.923

0.549

15.555

Microalbuminuria

0.018

0.005

0.000

1.018

1.008

1.027

Constant

-27.255

6.447

0.000

0.000

   

BMI= Body Mass Index; LDL: Low Density Lipoprotein; HDL: High Density Lipoprotein; OR: odds ratio; CI: confidence interval.

Simultaneously, we found that IMT was significantly higher in patients with CHD (1.10±0.12 mm) compared to patients without CHD (0.85±0.09 mm), which is highly significantly different (p<0.001).

Figure 1A: IMT values in patients with and without CHD proven by ergometric testing.

When we analysed inflammatory markers (IL-6 and hs CRP) we found that IL-6 values was higher in patients with CHD (6.83±1.99 pg/mL) compared to those without CHD (3.04±1.39 pg/mL), which differs significantly (p<0.001).

Figure 1B: IL-6 value in patients with and without CHD proven by ergometric testing.

Similarly, we also found that hs-CRP values in patients with CHD was significantly higher in comparison to patients without CHD (6.37±2.25 vs1.67±1.41 mg/L; p <0.001).

Figure 1C: hs-CRP value in patients with and without CHD proven by ergometric testing.

We also examined the influence of investigated inflammatory markers (hs-CRP and IL-6) and IMT values of carotid arteries in prediction of ergometric test outcomes in asymptomatic patients with T2D by using logistic regression model. (Table 4.) The results of our study showed that higher values of IL-6, hs-CRP and IMT were correlated with silent CHD proven by ergometric testing, whereas the patients without CHD had statistically significantly lower values of these parameters. The patients with higher IL-6 values were 1.5 times more likely to have positive ergometric tests than those with lower IL-6 (p<0.05), whereas subjects with elevated hs-CRP values were 1.9 times significantly more likely to have positive ergometric tests (p<0.001).

Table 4: Multivariate logistic regression analysis to predict the significance of IL-6, hs-CRP and IMT for silent CHD amongst the study population.

Variables

B

S.E.

p

OR

95% C.I. for OR

Lower

Upper

IL-6

0.376

0.181

0.037

1.457

1.022

2.076

hs-CRP

0.638

0.162

0.000

1.892

1.377

2.599

IMT mean

17.809

0.154

0.000

2.29

1.987

2.593

Constant

-4.859

0.708

0.000

0.008

 

 

IL-6= interleukin 6; hs-CRP= high-sensitivity C-reactive protein; ITM: intima-media thickness; OR: odds ratio; CI: confidence interval.

In the above stated results (Table 4), we have demonstrated that the prediction of ergometric testing findings, based on IMT values, proved that the subjects with higher IMT values were more likely to have silent CHD, i.e. positive ergometric tests, compared to the subjects with negative ergometric tests. Elevated IMT values increase the probability for 2.3 times that the subjects will have silent CHD, compared to the subjects with lower IMT values, which is statistically significant (p<0.01).

Discussion

The results within our study show that: (1) a large percentage of patients with T2D have silent CHD and that (2) elevated levels of inflammatory markers (IL6 and CRP), as well as the presence of IMT > 0.9 mm represent a strong marker for presence of silent CHD. Previous research has shown that silent CHD in people with diabetes varied and that there was a need to define the degree of cardiovascular risk in people with silent CHD who could benefit from screening [14, 15]. The prevalence of silent myocardial ischaemia in our study was 29%, which is mostly consistent with previously published literature.

T2D is a large and independent risk factor for occurrence of CVD, coexistence of traditional cardiovascular risk factors significantly increases the risk for occurrence of silent CHD in T2D patients. The results of our study showed that the patients with silent CHD were older, with longer duration of diabetes, higher prevalence of hypertension, poorer glucose regulation, and higher values of total cholesterol, LDL cholesterol and triglycerides and higher prevalence of albuminuria compared to subjects without CHD. BMI and HDL cholesterol levels had no statistical significance between the study groups (Table 1). Our results are consistent with the evidence published in previous studies [16, 17]. Analysis of predictive value of risk factors in occurrence of silent CHD showed that age, HbA1c, LDL cholesterol and presence of microalbuminuria did have statistical significance, while other analysed risk factors: gender, smoking, duration of T2D, BMI, hypertension, total cholesterol, HDL cholesterol and triglycerides showed no statistical significance in CHD prediction (Table 3).

Guidelines recommend population based screening algorithms that include Framingham Risk Score, Systematic Coronary Risk Evaluation (SCORE), and Reynolds Score [18, 19]. However, these models are less useful for assessing individual risk, which is why the focus of the study is to identify biomarkers such as hs-CRP, IL-6, and IMT to predict individual risk. In recent years, IMT has been shown as an independent predictor of CV risk and the presence of carotid plaque as a strong predictor of CV events and mortality [20]. Our study shows that the average IMT in subjects with silent CHD was significantly higher compared to the patients without CHD. (Figure 1A). The results obtained match with previous studies that reported that the IMT value was an early marker of atherosclerosis, and that its elevated value was significantly correlated with an increased risk of CHD [21]. A meta-analysis of 28 randomised clinical trials with 15,598 patients showed that smaller progression of carotid IMT value over time was correlated with smaller probability of nonfatal myocardial infarction [22]. Conclusions of previous studies in the assessment of asymptomatic patients with T2D indicated that with increasing the value of IMT, the risk of CVD occurrence increased and that adding measurements of carotid artery IMT values to traditional risk factors would improve risk stratification for CVD [23, 24]. Increased value of IMT of carotid arteries increases the risk (fitted to match age and gender) of CVD occurrence, but it is also a predictor of cardiovascular events same as traditional risk factors [25]. This is supported by the results of our study, which showed the significance of IMT values in prediction of silent CHD i.e. that with increasing the value of IMT, the probability of silent CHD existence increased (Table 4).

In this study, we also analysed the significance of inflammatory risk markers (CRP, IL-6) for the appearance of silent CHD in patients with T2D. In subjects with silent CHD, there was a direct correlation with IL-6 and hs-CRP values that were significantly higher compared to the subjects without CHD (Figure 1B and 1C). Moreover, analysis of prediction clearly shown that patients with higher IL-6 values were 1.457 more likely, while patients with elevated hs-CRP values were 1.892 times more likely to have silent CHD detected by ergometric tests (Table 4). This is supported by the results of previous research which showed that hs-CRP was detected in the intima of an atherosclerotic lesion as well as in atherosclerotic plaque macrophages where hs-CRP was produced by macrophages through CRP mRNA. In addition to stimulating endothelial dysfunction and progression of atherosclerosis in the early stages, it also plays an important role in plaque destabilisation and CVD occurrence [26-28]. Previous research has shown that hs-CRP was a predictor of CVD, even after adjusting to traditional risk factors indicating that hs-CRP may provide additional significant prognostic information in cardiovascular risk assessment [29]. Elevated levels of IL-6 in the serum are positively correlated with the development of CAD, and IL-6 is an important cytokine in the detection of atherosclerosis in people with T2D. IL-6 has become an important cytokine in the assessment of atherosclerosis in individuals with T2D due to its positive correlation with the development of CAD [30-32]. It has been also suggested that elevated IL-6 values were correlated with an increased risk of future myocardial infarction even after adjustment in initial differences in total cholesterol, HDL-cholesterol, BMI, blood pressure, diabetes mellitus, family medical history, alcohol consumption and doing physical activity [33, 34]. Also, elevated levels of IL-6 can play a predictive role in occurrence of CVD, thus providing a potential prognostic means in detection of CVD [25].

Treatment of hyperglycaemia alone in T2D may have limitations in preventing cardiovascular events occurrence, which is why comprehensive and aggressive early-stage T2D treatment and multifocal interventions to control individual cardiovascular risk factors must be approached in order to prevent or slow down occurrence of CVD in patients with T2D [35-37]. CVD management in T2D patients has been the focus of research in recent years. Several clinical trials have demonstrated the cardiovascular safety of antidiabetic medicines, while other studies: EMPA-REG OUTCOME, LEADER®, SUSTAIN-6 and IRIS - have shown benefits in application of empagliflozin, liraglutide, semaglutide, and pioglitazone on cardiovascular outcomes [38]. In our study, there was no significant difference in use of insulin therapy between the investigated groups, while usage of GLP1-RA, sulphonylurea, metformin, DPP4 inhibitors and SGLT2 inhibitors were more often in subjects without CHD.

Application of antihypertensives and statins is well known, not only as a therapy for hypertension and dyslipidaemia but also as a therapy in prevention of CVD, which is why it is recommended as a pharmacological therapy in patients with T2D [39]. Consistent with the results of previous studies, application of antihypertensives and statins was more significantly represented in subjects without CHD compared to the subjects with silent CHD. Our study showed that elevated values of hs-CRP, IL-6 and IMT are strong markers of the presence of silent CHD in asymptomatic patients with T2D. Traditional CVD risk factors explain only part of cardiovascular risk in T2D patients, and current screening recommendations are based on their use, it would be important to include determination of inflammatory markers and IMT values to improve cardiovascular risk stratification in asymptomatic T2D patients.

The limitations of our study are that the study was a single central trial with a relatively small number of subjects and was cross-sectional, without proper follow-up, so it could not show long-term the incidence of silent CHD or the influence of the investigated markers on the future occurrence of CHD. This could be the main reason to extend the research to a larger number of subjects and a longer follow-up in the future in order to obtain stronger results.

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