PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus

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 PECAM-1 gene is located at the end of the long arm of the chromosome 17 (17q23). Previous studies have reported the existence of 11 different single nucleotide polymorphisms (SNPs) of the PECAM-1 gene. Three of them have been described that encode amino acid substitutions in the PECAM-1 molecule. A mutation in the PECAM-1 gene in exon 3 at position +373 involves a C>G substitution, causing a leucine to valine substitution at position 125 (rs668) [14].

The interaction or activation of the PECAM-1 take place via homophilic binding with its first extracellular Ig-like domains [15,16]. This polymorphism might affect the homophilic binding capability and influence individual susceptibility to the development of atherosclerosis. The association between the rs668 PECAM-1 polymorphism and cardiovascular disease was studied in Caucasians [17-19], Japanese [20] and Chinese [21], but no clear answer on the association between the rs668 polymorphism of PECAM-1 and the development of cardiovascular diseases could be provided.

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 PECAM-1 gene) on the development of atherosclerosis. They reported reduced atherosclerotic lesions in double knock-out mice models [21,25]. The purpose of this study was to investigate an association between the rs668 (+373C/G) polymorphism of the PECAM-1 gene and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM).

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, p <0.001).

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.

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 A_1c, 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).

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).

The distribution of genotypes in the population of patients with T2DM was in Hardy-Weinberg equilibrium (T2DM: χ² = 0.45; p = 0.50; control group: χ² = 1.46; p = 0.23). Table 3 outlines differences in the ultrasound markers of carotid artery atherosclerosis (initial ultrasound examination) in patients with T2DM with regard to the rs668 genotypes. The differences were not statistically significant with regard to rs668 genotypes (Table 3). We did not demonstrate statistically significant differences in the markers of subclinical carotid atherosclerosis between different genotypes in subjects with T2DM (Table 4).

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).

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 PECAM-1 gene in the pathogenesis of atherosclerosis was demonstrated [21,25]. The decrease in areas with atherosclerotic lesion in PECAM-1 double knock-out mice was reported [21,25].

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 PECAM-1 gene and either the increased levels of soluble PECAM-1 or ischemic stroke was found in the Chinese population [21]. An association between the rs668 PECAM-1 gene and increased levels of soluble PECAM-1 was confirmed in the setting of patients with acute MI [28].

Similarly, Fang et al. [29] demonstrated an association between the rs668 PECAM-1 gene and either increased levels of soluble PECAM-1 or the severity of coronary artery disease in the Asian Indian population in Singapore. Reschner et al. [17] reported an association between the PECAM-1 rs668 (the CC genotype) and MI in Caucasians with T2DM. Finally, the effect of several polymorphisms of the PECAM-1 gene on cardiovascular disease was confirmed by Listi et al. [30] in the Northern Italian population. These findings were not confirmed in the general population from Germany, as rs688 was not found to be an independent risk factor for coronary artery disease (CAD) [31].

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 (i.e., less than 1000 subjects with T2DM), however the study was appropriately powered to detect the differences in subclinical markers of carotid atherosclerosis upon enrollment as well as during follow-up. The lack of the effect of rs688 on the progression of atherosclerosis might be due to a rather short interval between the first and control examinations (3.8 ± 0.5 years). Moreover, it is impossible to exclude the impact of interactions with other potentially relevant variables on the development of atherosclerosis. The second limitation of the study is the lack of an intermediate phenotype, i.e., serum PECAM levels. We presume that the effect of rs668 is via increased serum PECAM levels.

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.