The leukocyte adhesion and their transendothelial migration play an important role in the initial phase of atherogenesis [1]. Processes are regulated by various types of adhesion molecules, such as platelet endothelial cell adhesion molecule 1 (PECAM-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The plasma level of adhesion molecules is elevated in individuals with atherosclerosis [2-4].
Platelet endothelial cell adhesion molecule 1, also called CD31, is a 130 kD member of the immunoglobulin superfamily, consisting of six extracellular immunoglo-bulin-like domains, one transmembrane domain, and one cytoplasmic domain. Their expression takes place on the surface of circulating platelets, monocytes, neutrophils and selected T cells [5,6]. The PECAM-1 is a signaling molecule that plays diverse roles in vascular biology, including modulation of platelet function [7,8], angiogenesis [9], vasculogenesis [10], integrin regulation [11], T-cell and B-cell activation [12] and mediation of leukocyte migration across the endothelium [13].
The
The interaction or activation of the PECAM-1 take place
Platelet endothelial cell adhesion molecule 1 is important in the detection of mechanoreception (mechanical shear force) and mechanotransduction (conversion into chemical signals) by the endothelium [22,23]. Atherosclerotic lesion development occurs at sites of the vessel where flow and shear stress conditions are disturbed [24]. Pulsatile or oscillatory shear stresses induce pro inflammatory gene expression [25]. Using the mouse model, the effect of PECAM-1 deficiency (double knock-out mice model without the presence of the
This study included 595 consecutive subjects with T2DM, admitted to the diabetes outpatient clinics of the general hospitals at Murska Sobota and Slovenj Gradec, Slovenia, and from the outpatient department at the Medical Center Medicor, Ljubljana, Slovenia. The inclusion criteria for the control group was the absence of T2DM, and consisted of employees of the General Hospital Murska Sobota, Slovenia. Another inclusion criteria for the subjects with T2DM and for the subjects in the control group was the age from 40 to 70. The exclusion criteria for subjects with T2DM and for the subjects in the control group was a history of either myocardial infarction (MI) or ischemic stroke. The study protocol was approved by the Slovene Medical Ethics Committee (98/08/10). The patients and control subjects were enrolled and followed in the period from 2008 to 2014.
Patients were classified as having T2DM according to the current report of the American Diabetes Association [26]. After informed consent was obtained from the patients, a detailed interview was conducted concerning smoking habits, the duration and treatment of diabetes, arterial hypertension, and hyperlipidemia. Patients were asked whether they were smokers at the time of recruitment (current smoker). Subjects with T2DM with systolic blood pressure ≥140.0 mm Hg or diastolic blood pressure ≥85.0 mm Hg and/or subjects who were taking anti hypertensive drugs were considered to be hypertensive.
All ultrasound examinations were performed by two experienced doctors blinded to the participants’ diabetes status. The carotid intima-media thickness (CIMT), defined as the distance from the leading edge of the lumen-intima interface to the leading edge of the media-adventitia interface, was measured as previously described [27]. Plaques were defined as a focal intima-media thickening, and divided into five types according to their echogenic/ echolucent characteristics, as previously described [27]. The interobserver reliability for carotid plaque characterization was found to be substantial (κ = 0.64,
After the patients with T2DM and subjects without T2DM (control group) were enrolled, they were prospectively followed-up for a few years. From the group with T2DM, 426 responded and participated in the control ultrasound examination of the neck artery, whereas, 132 re-sponded from the group of subjects without T2DM; 3.8 ± 0.5 years passed between the first and the control ultrasound examination.
Fasting blood samples for biochemical analysis were collected twice patients: upon enrollment and upon follow-up after a few years. Analyses were made at the hospital accredited laboratory. The following parameters were determined: total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood sugar and high-sensitive C-reactive protein (hsCRP).
Genomic DNA was extracted from 100 μL of whole blood using a
Continuous variables that were normally distributed, were reported in the form of mean ± standard deviation (SD). Variables that were not normally distributed, were presented in the form of median (inter-quartile range). The normality of distribution of continuous variables was examined using the Kolmogorov-Smirnov test. We used the Student’s
The Pearson analyses was performed to examine the correlation between the independent variables. The results showed a high degree of correlation between the serum levels of total and LDL cholesterol (
The change in the value of ultrasound markers of carotid artery atherosclerosis was calculated by deducting the values measured at two ultrasound examinations. The criteria for a statistically significant difference was a
The clinical characteristics of subjects with T2DM and control subjects are shown in Table 1. Patients with T2DM had a greater waist circumference and there were more smokers compared to the control group. There were no statistically significant differences between patients with T2DM and controls in other clinical characteristics [age, body mass index (BMI), systolic and diastolic pressure]. A biochemical examination of patients with T2DM showed statistically significant higher levels of fasting glucose, Hb A1c, total cholesterol, HDL, LDL, triglyceride and hsCRP compared with the control group (Table 1). Moreover, higher CIMT was found in patients with T2DM in comparison with subjects without T2DM (Table 1).
Initial clinical and biochemical characteristics of patients with type 2 diabetes mellitus and the control group.
Parameters | Patients with T2DM ( | Control Group ( | |
---|---|---|---|
Age (mean ± SD) | 61.38 ± 9.65 | 60.07 ± 9.18 | 0.07 |
Sex: M (%); F (%) | M: 338 (56.8); F: 191 (43.2) | M: 92 (46.0); F: 108 (54.0) | 0.008 |
Duration of T2DM | 11.5 ± 7.88 | - | - |
Smokers: M (%); F (%) | M: 30 (8.9); F: 23 (12.0) | M: 19 (20.6); F: 15 (13.9) | 0.002 |
Waist circumference (cm) | 108.65 ± 12.88 | 93.31 ± 13.18 | <0.001 |
BMI (kg/m2) | 30.96 ± 4.74 | 27.90 ± 4.42 | 0.16 |
Systolic pressure (mm Hg) | 146.98 ± 19.98 | 143.30 ± 16.60 | 0.86 |
Diastolic pressure (mm Hg) | 85.75 ± 11.62 | 84.70 ± 11.60 | 0.19 |
Fasting glucose (mmol/L) | 8.04 ± 2.57 | 5.27 ± 0.87 | <0.001 |
Hb A1c (%) | 7.89 ± 3.56 | 4.79 ± 0.29 | <0.001 |
Total cholesterol (nmol/L) | 4.70 ± 1.19 | 5.36 ± 1.08 | <0.001 |
HDL cholesterol (mmol/L) | 1.19 ± 0.35 | 1.43 ± 0.37 | <0.001 |
LDL cholesterol (mmol/L) | 2.63 ± 0.94 | 3.24 ± 0.98 | <0.001 |
Triglycerides (mmol/L) | 1.90 (1.20-2.70) | 1.30 (0.90-1.90) | <0.001 |
High sensitivity CRP (mg/L) | 2.20 (1.00-4.30) | 1.30 (0.80-2.70) | <0.001 |
CIMT (μm) | 1013.00 ± 208.00 | 979.00 ± 141.00 | 0.030 |
T2DM: type 2 diabetes mellitus; BMI: body mass index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; CRP: C-reactive protein; CIMT: carotid intima-media thickness.
The ultrasound examination of the carotid artery was performed at the time of enrollment in the study, and 3.8 ± 0.5 years after the initial examination. Changes in the progression of atherosclerotic markers (change in annual CIMT increase, change in the number of plaque segments and change in the sum of the plaque thickness) between subjects with T2DM and the control group are shown in Table 2. Statistically significantly faster progression of the atherosclerotic markers was shown in subjects with T2DM in comparison with the control group (Table 2).
Changes in ultrasound markers of atherosclerosis at carotid artery in patients with type 2 diabetes mellitus and the control group between the initial and control ultrasound examinations.
Parameters | Patients with T2DM ( | Control Group ( | 0.50 |
---|---|---|---|
Annual CIMT increment (μm/year) | 20.33 (11.74-29.86) | 12.83 (8.82-20.66) | 0.00 |
Δ number of plaque segments | 2.00 (1.00-3.00) | 1.50 (0.70-2.20) | 0.00 |
Δ sum of the plaque thickness (mm) | 5.40 (2.40-7.05) | 3.64 (2.88-5.48) | 0.02 |
CIMT: carotid intima-media thickness; Δ: variable value changes during the observation period, expressed as a percentage of baseline values.
The distribution of genotypes in the population of patients with T2DM was in Hardy-Weinberg equilibrium (T2DM: χ2 = 0.45;
Comparison of ultrasound markers of the carotid artery atherosclerosis in patients with type 2 diabetes mellitus according to the
Parameters | CC Genotype | CG Genotype | GG Genotype | |
---|---|---|---|---|
CIMT (μm) | 1041.00 ± 198.00 | 990.00 ± 187.00 | 988.00 ± 211.00 | 0.19 |
Number of segments with plaques | 2.56 ± 1.65 | 2.39 ± 1.72 | 2.51 ± 1.54 | 0.36 |
Sum of the plaque thickness (mm) | 8.14 ± 4.77 | 7.99 ± 5.23 | 7.66 ± 4.02 | 0.16 |
Presence of plaques: [+] | 149.00 (85.60) | 256.00 (84.50) | 94.00 (79.70) | 0.36 |
Presence of unstable plaques: [+] | 86.00 (57.70) | 144.00 (56.30) | 58.00 (61.70) | 0.66 |
CIMT: carotid intima-media thickness.
Changes in ultrasound markers of carotid artery atherosclerosis in patients with type 2 diabetes mellitus between the first and the second ultrasound examination of the carotid arteries according to the
Parameters | CC Genotype | CG Genotype | GG Genotype | |
---|---|---|---|---|
Annual CIMT increment (μm/year) | 20.34 (11.64-28.04) | 20.69 (7.14-32.28) | 14.28 (10.71-35.97) | 0.71 |
Δ number of plaque segments | 2.00 (0.50-2.50) | 2.00 (1.00-3.00) | 2.00 (2.00-3.00) | 0.74 |
Δ sum of the plaque thickness (mm) | 7.30 (3.85-8.70) | 5.45 (2.30-9.20) | 4.40 (1.40-8.42) | 0.38 |
CIMT: carotid intima-media thickness; Δ: variable value changes during the observation period, expressed as a percentage of baseline values.
Table 5 shows the relation between the rs668 and the incidence of either plaques or unstable plaques in subjects with T2DM. When adjusted to other risk factors, the rs668 GG genotype was associated with an increased risk of carotid plaques in subjects with T2DM (Table 5).
Relationship of the
Presence of Plaques | Presence of Unstable Plaques | |||
---|---|---|---|---|
Parameters | OR (95% CI) | OR (95% CI) | ||
Hypertension (0 = no; 1 = yes) | 1.88 | 0.35 | 1.38 | 0.52 |
Systolic pressure (mm Hg) | 0.24 | 0.68 | 0.26 | 0.32 |
Serum LDL (mmol/L) | 1.41 | 0.41 | 1.34 | 0.25 |
Serum HDL (mmol/L) | 0.11 | 0.02 | 0.31 | 0.34 |
Hb A1c (%) | 0.89 | 0.03 | 1.23 | 0.43 |
CG allele | 1.03 | 0.49 | 0.72 | 0.17 |
GG allele | 1.18 | 0.03 | 0.68 | 0.51 |
OR (95% CI): odds ratio (95% confidence interval); LDL: low-density lipoprotein; HDL: high-density lipoprotien; all models were adjusted by age, sex, smoking habits and treatment with statins. The reference group are homozygotes for the C allele (the CC genotype).
In the present study, we demonstrated an association between the rs668 GG genotype and carotid artery plaque incidence in patients with T2DM when adjusted to other risk factors. On the other hand, we did not find any effect of the rs688 genotype on the progression of atherosclerosis in patients with T2DM.
Our study is the first demonstrating an association between the rs668 PECAM-1 and the presence of carotid plaques in patients with T2DM. In our study, we observed a greater number of plaques in subjects with the GG rs668 genotype. The importance of the
An association between the rs668 PECAM-1 and car-diovascular disorders was reported several times, but not in all studies [29-33]. Similarly, an association between the rs668
Similarly, Fang
In a recently published meta-analysis of 15 studies, including 7636 subjects, no association between the rs668 PECAM-1 and cardiovascular diseases was demonstrated [35]. Moreover, the progression of subclinical markers of carotid atherosclerosis was statistically significantly faster in subjects with T2DM in comparison with subjects without T2DM. This finding is in accordance with expectations of other researchers as well [32,33].
Our study has some limitations due to its actual design (cross-sectional design at the enrollment of subjects with T2DM and control subjects) and relatively small sample size
In conclusion, our study demonstrated a minor effect of the rs668 PECAM-1 on the markers of carotid atherosclerosis in subjects with T2DM, as the GG genotype of the rs668 PECAM-1 was associated with a higher incidence of carotid plaques in subjects with T2DM. With that kind of associations established in genetic studies, we presumed that we might predict the genetic risk of carotid atherosclerosis in subjects with T2DM.