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.
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.
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.
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 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.
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.
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).
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.
Conflicts of interest
There are no conflicts of interest.
1. Catanzariti L, Faulks K, Moon L, Waters AM, Flack J, Craig ME.Australia’s national trends in the incidence of type 1 diabetes in 0–14-year-olds, 2000–2006.Diabet Med2009;26:596–601.
2. Patterson CC, Dahlquist GG, Gyürüs E, Green A, Solte´sz G.Incidence trends for childhood type 1 diabetes in Europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study.Lancet2009;373:2027–2033.
3. Dursun H, Bayazit AK, Cengiz N, Seydaoglu G, Buyukcelik M, Soran M, et al..Ambulatory blood pressure monitoring and renal functions in children with a solitary kidney.Pediatr Nephrol2007;22:559–564.
4. Gallego PH, Craig ME, Hing S, Donaghue KC.Role of blood pressure in development of early retinopathy in adolescents with type 1 diabetes: prospective cohort study.BMJ2008;337:a918.
5. Seckin D, Ilhan N, Ilhan N, Ertugrul S.Glycaemic control, markers of endothelial cell activation and oxidative stress in children with type 1 diabetes mellitus.Diabetes Res Clin Pract2006;73:191–197.
6. Downie E, Craig ME, Hing S, Cusumano J, Chan AK, Donaghue KC.Continued reduction in the prevalence of retinopathy in adolescents with type 1 diabetes: role of insulin therapy and glycemic control.Diabetes Care2011;34:2368–2373.
7. Kollias A, Antonodimitrakis P, Grammatikos E, Chatziantonakis N, Grammatikos EE, Stergiou GS.Trends in high blood pressure prevalence in Greek adolescents.J Hum Hypertens2009;23:385–390.
8. Palta M, LeCaire T.Managing type 1 diabetes: trends and outcomes over 20 years in the Wisconsin Diabetes Registry cohort.WMJ2009;108:231–235.
9. Trigona B, Aggoun Y, Maggio A, Martin XE, Marchand LM, Beghetti M, et al..Preclinical noninvasive markers of atherosclerosis in children and adolescents with type 1 diabetes are influenced by physical activity.J Pediatr2010;157:533–539.
10. Glowinska B, Urban M, Peczynska J, Florys B.Soluble adhesion molecules (sICAM-1, sVCAM-1) and selectins (sE-selectin, sP-selectin, sL-selectin) levels in children and adolescents with obesity, hypertension, and diabetes.Metabolism2005;54:1020–1026.
11. Maggio AB, Farpour-Lambert NJ, Montecucco F, Pelli G, Marchand LM, Schwitzgebel V, et al..Elevated E-selectin and diastolic blood pressure in diabetic children.Eur J Clin Invest2012;42:303–309.
12. El-Mesallamy HO, Hamdy NM, Ibrahim SM.Adiponectin and pro-inflammatory cytokines in obese diabetic boys.Indian Pediatr2011;48:815–816.
13. Velarde MS, Del R Carrizo T, Prado MM, Díaz EI, Fonio MC, Bazán MC, Abregu AV.Inflammation markers and endothelial dysfunction in children with type 1 diabetes.Medicina (B Aires)2010;70:44–48.
14. .WHO child growth standards: length/height-forage, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: methods and development.WHO Child Growth Standards2006.Geneva, Switzerland:World Health Organisation;312Available at: http://www.who.int/childgrowth/standards/Technical_report.pdf
. [Accessed 11 August 2011].
15. Wuhl E, Witte K, Soergel M, Mehls O, Schaefer F.Distribution of 24-h ambulatory blood pressure in children: normalized reference values and role of body dimensions.J Hypertens2002;20:1995–2007.
16. Weykamp CW, Penders TJ, Muskeit FAJ, van der Silk W.Glycohemoglobin: comparison of 12 analytical methods, applied to lyophilized hemolysates by 101 laboratories in an external quality control programmer.Ann Clin Biochem1993;30:169–174.
17. Pham MN, Hawa MI, Roden M, Schernthaner G, Pozzilli P, Buzzetti R, et al..Increased serum concentrations of adhesion molecules but not of chemokines in patients with type 2 diabetes compared with patients with type 1 diabetes and latent autoimmune diabetes in adult age: action LADA 5.Diabet Med2012;29:470–478.
18. Showell PJ, Matters DJ, Long JM, Carr Smith HD, Bradwell AR.Evaluation of latex-enhanced nephelometric reagents for measuring free immunoglobulin light-chains on a modified MININEPHTM.Clin Chem2002;48SupplA22, E.
19. Dudley WN, Benuzillo JG, Carrico MS.SPSS and SAS programming for the testing of mediationmodels.Nurs Res2004;53:59–62.
20. De Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH.Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes.J Am Soc Nephrol2001;12:993–1000.
21. Elhadd TA, Neary R, Abdu TA, Kennedy G, Hill A, McLaren M, Akber M.Influence of the hormonal changes during the normal menstrual cycle in healthy young women on soluble adhesion molecules, plasma homocysteine, free radical markers and lipoprotein fractions.Int Angiol2003;22:222–228.
22. Marcovecchio ML, Chiarelli F.Microvascular disease in children and adolescents with type 1 diabetes and obesity.Pediatr Nephrol2011;26:365–375.
23. Cominacini L, Fratta Pasini A, Garbin U, Campagnola M, Davoli A, Rigoni A, et al..E-selectin plasma concentration is influenced by glycaemic control in NIDDM patients: possible role of oxidative stress.Diabetologia1997;40:584–589.
24. Albertini JP, Valensi P, Lormeau B, Aurousseau MH, Ferrière F, Attali JR, Gattegno L.Elevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment.Diabetes Care1998;21:1008–1013.
25. Ferri C, Desideri G, Valenti M, Bellini C, Pasin M, Santucci A, De Mattia G.Early upregulation of endothelial adhesion molecules in obese hypertensive men.Hypertension1999;34Pt 1568–573.
26. Mishra M, Kumar H, Bajpai S, Singh RK, Tripathi K.Level of serum IL-12 and its correlation with endothelial dysfunction, insulin resistance, proinflammatory cytokines and lipid profile in newly diagnosed type 2 diabetes.Diabetes Res Clin Pract2011;94:255–261.
27. Valle Jiménez M, Estepa RM, Camacho RM, Estrada RC, Luna FG, Guitarte FB.Endothelial dysfunction is related to insulin resistance and inflammatory biomarker levels in obese prepubertal children.Eur J Endocrinol2007;156:497–502.
28. Soedamah-Muthu SS, Chaturvedi N, Schalkwijk CG, Stehouwer CD, Ebeling P, Fuller JH.Soluble vascular cell adhesion molecule-1 and soluble E-selectin are associated with micro- and macrovascular complications in type 1 diabetic patients.J Diabetes Complications2006;20:188–195.
29. Gearing AJ, Newman W.Circulating adhesion molecules in disease.Immunol Today1993;14:506–512.
30. Mehta JL.Inflammatory and infectious basis of atherosclerosis2001.Basel, Switzerland:Birkhauser Verlag;280.
31. Slim IB.Cardiovascular risk in type 1 diabetes mellitus.Indian J Endocrinol Metab2013;17Suppl 1S7–S13.
32. Orchard TJ, Forrest KY, Kuller LH, Becker DJ.Lipid and blood pressure treatment goals for type 1 diabetes: 10-year incidence data from the Pittsburgh Epidemiology of Diabetes Complications Study.Diabetes Care2001;24:1053–1059.