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ORIGINAL ARTICLES

E-selectin and vascular complications in children with type 1 diabetes mellitus

El Wakeel, Maged A.a; El-Kassas, Ghada M.a; Amer, Ahmed F.a; Elbatal, Wael H.a; Sabry, Rania N.a; EL-Ghaffar Mohammed, Nagwa Abdb

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doi: 10.1097/01.MJX.0000446937.40653.3d
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Abstract

Introduction

In recent years, a marked increase in type 1 diabetes mellitus (T1DM) incidence has been reported for the pediatric population 1,2. Along with increased incidence of diabetes, the risk of development of diabetic microvascular complications such as albuminuria and retinopathy, as well as the percentage of patients with arterial hypertension, has also increased 3,4. Many studies have demonstrated that diabetic microvascular complications are the major cause of morbidity and early mortality in diabetes 5. The development of such complications is related to the duration of diabetes and the degree of glycemic control 6. According to various estimations, 1–5% of children have hypertension 7. It has also been shown that elevated blood pressure is two to three times more frequent in diabetic patients as compared with the general population 8.

In children with T1DM, impaired endothelial function, increased arterial intima-media thickness, and changes in elastic properties of the arteries have been demonstrated 9. Some biochemical markers (E-selectin and intercellular adhesion molecule) have also been shown to be abnormal in this population compared with healthy children 10; however, studies are scarce.

Soluble E-selectin is a type of cell adhesion molecule exclusively expressed by the activated or dysfunctional vascular endothelium. Therefore, it can be used as a specific and early marker of endothelial dysfunction 11. Several studies observed elevated E-selectin concentrations in diabetic children 12,13.

The aim of the study was to evaluate the concentration of E-selectin (as a marker of endothelial activation) in children and adolescents with T1DM and to assess its relationship with glycemic control in these patients, as well as to find out the associations between E-selectin and vascular complications of diabetes such as hypertension and albuminuria.

Participants and methods

This was a case–control study including 30 children and adolescents with T1DM and 30 matched healthy individuals aged 6–16 years. Patients with a diagnosis of T1DM were recruited from Endocrinology and Diabetology Unit, Pediatric Hospital, Ain Shams University. During the same period, healthy children were recruited from peers of diabetic patients and from local schools and were matched with T1DM patients. Participants were excluded for any of the following reasons: presence of other chronic or inflammatory disease; medications or hormones (other than insulin); presence of known renal disease; and systemic disease and acute infection at the time of testing. The study was approved by the local ethics committee, and written informed consent was obtained from both parents. T1DM patients were further subdivided according to HbA1c into group I with HbA1C of 8% or less (well-controlled T1DM) and group II with HbA1c greater than 8% (poorly controlled T1DM). Full history was taken from the parents, including the onset of diabetes. Clinical examination and anthropometric measurements were performed. Height was measured by Harpenden stadiometer in centimeters. Weight was recorded in kilograms using an electronic weight scale. BMI was calculated by weight in kg/(height in meters)2. BMI Z-score was assessed using the new WHO reference 14. Blood pressure (BP) was measured by a sphygmomanometer (Accoson, London, UK) as the mean of two measurements. Z-scores were determined using the German references 15.

Three milliliter of fasting (8 h) venous blood samples were taken from each child participating in the study and divided into two parts: the first part was added to tube containing EDTA for glycosylated hemoglobin determination by cation-exchange resin 16 and the second part was put in a serum separator tube. The separated serum was stored at −20°C for determination of E-selectin, total cholesterol, and triglyceride (TG). Total cholesterol and TG were determined using colorimetric techniques on Synchron Cx7 (Beckman Instruments Inc., Fullerton city, California, USA). The determination of serum E-selectin was carried out using quantitative sandwich enzyme immunoassay technique 17, and the kit was supplied from R&D Systems Inc. (614 McKinley place; Minneapolis, Minnesota, USA). For random urinary albumin measurement, we used early-morning midstream samples. Urine samples were centrifuged before use and clear supernatant was stored at −20°C until analysis. Albumin concentrations were measured in urine using a Minineph microalbumin kit based on nephlometry method on Minineph-nephelometer (AD200) (The Binding Site, Birmingham, UK) 18.

Statistical analyses

Data entry was carried out on excel sheet, and statistical analysis was performed using SPSS software program version 18.0 (IBM, Chicago, Illinois, USA) 19. Data were analyzed and characteristics of the sample were presented; mean and SD was estimated with respect to age, BMI Z-score, systolic and diastolic BP Z-score, level of HbA1C, cholesterol, TG, and level of E-selectin. In contrast, numbers and percentages were calculated with respect to sex distribution and presence of albuminuria. The χ2-test was performed for comparison of qualitative data, which were presented by numbers and percentages; the t-test was performed for comparison between two means and Pearson correlation was performed for determining relationship between two quantitative variables. Linear regression analysis was performed to predict risk factors significantly associated with increased level of E-selectin. P value was considered statistically significant when it was less than 0.05 and was considered statistically highly significant when its value was less than 0.01.

Results

A total of 60 children were included in the study: 30 T1DM patients and 30 controls. Table 1 shows the characteristics of the study group.

Table 1
Table 1:
Characteristics and metabolic variables of patients with type 1 diabetes mellitus and healthy children

The two study groups did not differ in age or sex (Table 1). Diabetic children had higher BMI Z-score, diastolic blood pressure (DBP) Z-score, and as expected HbA1c. The mean levels of serum E-selectin, cholesterol, and TG were significantly elevated in T1DM children compared with healthy ones. The mean duration of diabetes among T1DM was 7.4 year and 23% of them had albuminuria.

Uncontrolled T1DM patients had higher DBP Z-score and serum E-selectin levels compared with the well-controlled diabetic group. In all, 29% of poorly controlled diabetic patients had albuminuria, whereas albuminuria was found in only 11% of patients in the well-controlled diabetic group Table 2.

Table 2
Table 2:
Comparison among patients with respect to level of HbA1C

Table 3 shows that mean levels of serum E-selectin, cholesterol, and TG were significantly higher in diabetic patients with albuminuria compared with nonalbuminuric patients, whereas there was no statistically significant difference in HbA1C levels regarding presence of albuminuria.

Table 3
Table 3:
Comparison among patients with respect to presence of albuminuria

Correlation analysis between serum E-selectin and BMI, lipid levels, blood pressure Z-score, HbA1c, and duration of diabetes in diabetic patients showed significant positive correlation between E-selectin and HbA1c (r=0.9, P<0.001) (Fig. 1), DBP Z-score (r=0.5, P<0.01) (Fig. 2), cholesterol (r=0.7, P<0.001), TG (r=0.6, P<0.001), and duration of diabetes (r=0.4, P<0.03). There was no significant correlation between serum E-selectin and age and systolic blood pressure Z-score. Positive correlations between DBP Z-score and duration of diabetes (r=0.4, P=0.04), HbA1c (r=0.5, P<0.01) and TG (r=0.3, P=0.01) were also found.

Fig. 1
Fig. 1:
Correlation between serum E-selectin and HbA1C.
Fig. 2
Fig. 2:
Correlation between serum E-selectin and diastolic blood pressureZ-score.

When applying linear regression to detect factors affecting serum levels of E-selectin, we found that HbA1c was the most effective factor predicting the level of serum E-selectin followed by serum cholesterol and DBP (Table 4).

Table 4
Table 4:
Predictive factors for increased level of E-selectin in the studied diabetic patients as estimated by linear regression

Discussion

Microvascular and macrovascular complications are the most important cause of increased morbidity and mortality in types 1 and 2 diabetic patients 20.

In our study, serum E-selectin levels were significantly higher in type 1 diabetic patients compared with controls (P<0.0001). There was also significant correlation between HbA1c and serum E-selectin (P<0.0001) and on applying linear regression, HbA1c was the most effective factor controlling serum E-selectin level. Elhadd et al.21 showed, as in our study, an increased level of serum E-selectin and ICAM-1 in children with diabetes type 1, with an absence of the clinical features of angiopathy.

Hyperglycemia is the dominant pathophysiological hallmark of diabetes and may exert toxic pathological effects on the endothelium through accelerated formation and accumulation of advanced glycation end-products, thereby providing a link with atherosclerosis 22. The mechanisms involved in the endothelial cell activation are probably complex. Hyperglycemia per se is important for the expression of adhesion molecules in diabetes. Cominacini et al.23 reported that levels of adhesion molecules are influenced by glycemic control, a finding confirmed recently by Albertini et al.24 who found a high correlation between E-selectin and glycated hemoglobin and a marked reduction in E-selectin after improvement in glycemic control. As a consequence, elevated E-selectin may affect endothelial cell activity, and subsequently the development of microvascular and macrovascular complications 24.

Our results showed a significant correlation between the parameters of lipid metabolism and serum E-selectin. The latter correlated significantly with the serum cholesterol and TG concentration. Regression analysis showed significant association between serum E-selectin and serum cholesterol. Therefore, it would seem that abnormal lipid metabolism may lead to activation of the endothelium in young people. Our study also demonstrated a significant association between BMI and serum E-selectin. Ferri et al.25 showed an increased concentration of E-selectin in obese patients compared with healthy ones, with significant decrease in concentration of adhesion molecules 12 weeks after a low-calorie diet, with a resultant weight loss. This link between obesity and endothelial dysfunction might result from an association between obesity and insulin resistance, which has been confirmed by recent studies 26,27.

An interesting and important observation in our study is that serum E-selectin level was significantly higher in diabetic patients with albuminuria compared with nonalbuminuric ones. This was in agreement with small cross-sectional studies in type 1 diabetic patients, which found that increased plasma/serum levels of E-selectin are related to albuminuria in diabetic patients 10,28. It is not clear how exactly the adhesion molecules could lead to microvascular and macrovascular complications, but possible pathophysiological pathways have been suggested. Increased plasma/serum levels of E-selectin and vascular cell adhesion molecule-1 indicate increased expression of these molecules on the endothelial and smooth muscle cells and macrophages, which could reflect progressive formation of atherosclerotic lesions 29. Alternatively, modified lipoproteins, established risk factors, and, possibly, diabetes itself could lead to inflammation, causing raised levels of cytokines, which increase the secretion of acute-phase proteins and adhesion molecules, which could cause the atherosclerotic plaque to rupture and subsequent development of vascular disease 30.

Increased BP is a common complication of T1DM and is associated with increased mortality 31. DBP is implicated in the development of fibrous plaques and is an important determinant of lower-extremity peripheral arterial diseases and neuropathy, nephropathy, and proliferative retinopathy 32. In our young cohort, we found that DBP was in the normal range but was already higher than in healthy children. It was correlated with disease duration and HbA1c levels. These findings highlight the impact of deregulated glucose metabolism on the premature development of hypertension. We found an association between serum E-selectin and both DBP and serum TG. These findings confirm previously published data showing E-selectin and TG level associations 11. TGs may activate the endothelial cell and induce a release of E-selectin. Elevated BP may be the result of the conjugated effect of endothelial activation and formation of atherosclerotic plaques but could also be itself an activator of endothelial cell by its mechanical action on the vascular wall.

We conclude that children with type 1 diabetes show impairment of arterial vasoreactivity in association with elevated biological markers of endothelial cell activation, such as serum E-selectin and higher DBP, indicating early development of CVDs. Although it is difficult to establish direct relationships between these clinical parameters, we may hypothesize that the presence of circulating vascular biomarkers such as E-selectin may be related to vascular complications of DM, such as diastolic hypertension and microalbuminuria; in addition, these markers may be altered by good glycemic control. Normative references must be established to use these markers in a clinical setting to detect children with T1DM at risk for vascular complications.

Table
Table:
No title available.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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