INFLAMMATORY CARDIOVASCULAR RISK MARKERS AND SILENT MYOCARDIAL ISCHEMIA IN TYPE 2 DIABETIC PATIENTS INFLAMATORNI KARDIOVASKULARNI MARKERI RIZIKA I NIJEMA ISHEMIJA MIOKARDA KOD PACIJENATA SA TIPOM 2 DIJABETESA

Background/Aim. A special feature of Coronary Heart Disease (CHD) in patients with type 2 diabetes (T2D) is that it is often asymptomatic and occurs as a consecuence of cardiovascular auotonomic neuropathy. Dysregulation of the autonomic nervous system is associated with elevated values of inflammatory markers such as highly sensitive C-reactive protein (hs-CRP) and interleukin 6 (IL-6) which accelerate atherosclerosis and the occurrence of cardiovascular complications in patients with T2D. The aim of the study was to evaluate the importance of determining inflammatory cardiovascular risk markers IL-6 and hs-CRP in screening for the presence of CHD in asymptomatic patients with T2D. Methods: The study included 169 patients with T2D, without any symptoms and signs of CHD. Ergometric testing proved or ruled out the presence of silent CHD. The levels of hs-CRP and IL-6 were determined by ELISA. Results : IL6 values were significantly higher in patients with positive ergometric test (6.83±1.99 pg/mL) compared to patients with negative ergometric test (3.04±1.39 pg/mL) (p<0.001). We also found that hs-CRP values in patients with positive ergometric test was significantly higher in comparison to patients with negative ergometric test (6.37±2.25 vs 1.67±1.41 mg/L; p <0.001). Combinations of IL-6 and hs-CRP with age, HbA1c values and duration of diabetes, presented through three binary logistic regression models, are significant predictors of silent CHD proven by ergometric testing, i.e. with their increase in the probability of positive ergometric testing increased too (p <0.01). The sensitivity of the associated finding of elevated IL-6 and hs-CRP values in the detection of silent CHD by testing 90% and specificity was 86%. Conclusion: with T2D.


Introduction
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in three out of four type 2 diabetes (T2D) patients. A special feature of CVD in patients with diabetes is that it is often asymptomatic and occurs as a consequence of cardiovascular autonomic neuropathy. The absence of pain during ischemic myocardial episodes, atypical and mild symptoms of acute myocardial infarction delay the start of treatment, causing increased morbidity and mortality in patients. In patients with T2D, autonomic dysfunction is thought to lead to the development of silent episodes of ischemia and silent infarction 1,2 .
Recent studies have shown that autonomic nervous system dysregulation is associated with elevated inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) and their determination could reflect the severity of atherosclerosis as well as the risk of developing future cardiovascular events in T2D patients 3,4 .
In order to identify presence of coronary heart disease (CHD) in asymptomatic patients with diabetes, only an approach based on detailed assessment of traditional cardiovascular risk factors has still been recommended for the time being. Predicting the risk of cardiovascular events occurrence and progression of atherosclerosis and correlation of inflammatory agents in its progression has increasingly been the focus of research 5,6 .
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 7,8 .
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 9,10 . Vascular complications arise primarily as a consequence of endothelial dysfunction and inflammatory processes that play a role not only in initiation but also in the progression of atherosclerosis. 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 11,12 .
The aim of the study was to evaluate the significance of determining inflammatory markers IL-6 and hs-CRP as atherosclerosis markers, during screening for presence of CHD in asymptomatic patients with T2D.

Patients and methods
Our examination was conducted at the Republic of Srpska University as a cross-sectional study ,that included 169 type 2 diabetic patients, men (n=71) and women (n=98). All subjects underwent ergometric testing, and based on the obtained results, they were divided into two groups. The first group consisted of 117 type 2 diabetic patients without the presence of CHD, proven by the absence of symptoms and a negative ergometric test. The second group consisted of 52 type 2 diabetic patinets with silent ishemic heart disease, proven by a positive stres test.
All subjects underwent an anamnestic interview after which they all gave their written consent to participate in the study. After that, a physical examination was performed defining the anthropometric measures. Calculation of Body Mass Index (BMI) for the 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/m 2 ). Subjects with T2D with CHD, with a history of cerebrovascular, peripheral vascular and malignant diseases were excluded from the study. Also, all the subjects who had an acute or chronic infection or who have been receiving corticosteroids or immunosuppressants within their therapy were excluded from our study.

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, lasts 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 13 .

Laboratory analysis
Biochemical blood tests for laboratory processing were taken in the morning after a 12hour 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 14 . The lowest level of IL-6 detectability in the serum was 1.5 pg/ml 15 . 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.

Results
Screening test for presence of CHD was performed in 169 asymptomatic patients mean age 58.71 ± 6.76 ; range from 40 to 70 years 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 shows a comparison of demographic and risk factors between subjects with positive or negative ergometric test result. Subjects did not differ significantly by gender.
The patients with positive ergometric test (silent CHD) were older with a longer duration of diabetes and a higher incidence of smokers compared to the patients with negative ergometric test result (p <0.05). The difference in BMI between the study groups with positive or negative ergometric test was not statistically significant. Prevalence of hypertension as well as HbA1c values were statistically significantly higher in subjects with positive ergometric test compared to the patients with negative ergometric test (p<0.05). Regarding the lipid parameters, total LDL cholesterol as well as triglycerides, were significantly higher in the group of subjects with positive ergometric test (p<0.05), whereas the values of HDL cholesterol did not differ significantly between the study groups. In subjects with with positive ergometric test, microalbuminuria was present in 47 subjects (90.4%). In subjects in whom CHD was not detected (negative ergometric test ), microalbuminuria was present in 20 subjects (17.1%), which was statistically significantly different (p <0.001).
When we analysed inflammatory markers (IL-6 and hs-CRP) we found that IL-6 values significantly higher in patients with positive ergometric test (p<0.001) (Table1). Similarly, we also found that hs-CRP values in patients with positive ergometric test was significantly higher in comparison to patients with negative ergometric test ( Table 1) We examined the significance of the associated risk of increased hs-CRP and IL-6 values in the detection of silent CHD proven by ergometric testing. In patients with T2D in whom silent CHD was detected, 47 (90%) subjects had a finding of associated risk, while in 5 (10%) there was no associated risk. In patients with T2D without CHD, there was an associated risk in 16 (14%), while in 101 (86%) subjects it did not exist. This difference in frequency distribution is statistically significant (p <0.001). (Figure 3).

Discussion
Our study demonstrated that a large percentage of patients with T2D have silent CHD and that elevated levels of inflammatory markers (IL6 and CRP) 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 16 . In the detection of ischemia in asymptomatic diabetics (DIAD) study were randomly assigned to either stress testing and 5-year clinical follow-up or to follow-up only. A total of 22% of patients had silent ischemia 17 . The prevalence of silent myocardial ischaemia in our study was 29%, which is mostly consistent with previously published literature. Due to all the above, coronary artery disease in patients with diabetes is a diagnostic and therapeutic challenge.
Although T2D alone 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 Multiple Risk Factor Intervention Trial showed that multiple risk factors in the same patient significantly increased the overall cardiovascular risk 18 . Gaede et al. reported that an intervention directed at multiple risk factors significantly improves cardiovascular prognosis 19 . This is supported by the results of our study which showed that the patients with silent CHD were older, with longer duration of diabetes, higher prevalence of hypertension, poorer glucose regulation, higher values of total cholesterol, LDL cholesterol and triglycerides as well as higher prevalence of albuminuria compared to subjects who did not have CHD, while HDL cholesterol levels and BMI had no statistical significance between the study groups.
Since the inflammatory process is an integral part of the evolution of atherosclerosis, the use of CRP as a biomarker becomes very useful in combination with the control of classic risk factors such as lipid levels, changes in eating habits, weight loss, regular physical activity, glycemic control and smoking cessation. The interrelation of these risk factors for CVD are strategies to reduce cardiovascular events in primary and secondary prevention 20 .
Due to the relationship between high CRP plasma levels and cardiovascular mortality and morbidity risk it is important to establish a primary care line to decrease CVDs. For this, it is essential the evaluation of cardiovascular risk factors to stop their progression. Several prospective studies having CRP as a central target have shown the benefits of primary prevention. In 1999, the MONICA-Augsburg study performed in a sample of 936 asymptomatic men, concluded that the increase in hs-CRP leads to a 19% increased risk of fatal and non-fatal coronary events 21 . In the same way, the PREVEND study in 8139 asymptomatic men and women observed a relationship between hs-CRP and angiographic characteristics and consequently clinical instability of the atherosclerotic plaque 22 .
A six-year follow-up study of healthy middle-aged men showed that baseline IL-6 levels greater than 2.28 pg / ml were associated with a 2.3-fold higher risk of future myocardial infarction, which is why IL-6 was also identified as a significant predictor risk of cardiovascular events 23 . Recent research has reported the importance of inflammation in the development and progression of atherosclerosis as well as the possibility of using inflammatory markers to assess cardiovascular risk. Among the several biomarkers proposed in cardiovascular risk stratification is CRP, which would be used in the identification of individuals at risk for developing CVD, but this is not yet recommended in the guidelines 24,25,26 . Prospective clinical case-control studies Physician's Health Study and Women Health Study, have identified CRP as a strong, independent risk factor for CHD 27,28 . 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 . Also, elevated levels of IL-6 in serum is correlated with the development of CAD, which is why IL-6 has become an important cytokine in assessment of atherosclerosis in people with T2D, as evidenced by two large genetic studies reporting correlation between IL-6 receptor signalling and CVD 30,31,32 . 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 35 .
The results of our research also support these studies. We concluded that the combined finding of increased values of IL-6 and hs-CRP posed a high risk of the presence of silent CHD in T2D patients.
In this study, we also analysed the significance of inflammatory cardiovascular 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. The results of our study showed that IL-6 and hs-CRP are significant predictors of silent CHD and showed that the association of IL-6 and hs-CRP with age, HbA1c values and duration of diabetes are significant predictors of silent CHD proven by ergometric testing.
In conclusion, our study showed that a large percentage of T2D patients had silent CHD.
Elevated levels of inflammatory cardiovascular risk markers hs-CRP and IL-6 value are strong markers of the presence of silent CHD in asymptomatic T2D patients. Given that traditional risk factors for CVD explain only a part of cardiovascular risk in T2D patients, and current screening recommendations are based on their use, it would be important to include the determination of inflammatory cardiovascular risk markers in order to improve cardiovascular risk stratification in asymptomatic patients with T2D. By doing so, we would be able to reduce the incidence of cardiovascular complications occurrence and apply appropriate treatment modalities in a timely manner, whether it was a conservative or invasive treatment.
Ultimately, there are some limitations of our study. First, the study was single centre trial assay with relatively small number of subjects. Second, this study was cross sectional, without appropriate follow up, so our study could not demonstrate, in the long term, the incidence of silent CHD or the influence of investigated markers to the future appearance of CHD. This could be main reason to extended the investigation to a larger number of subjects and a longer follow-up in the future in order to get stronger results.