Atherosclerosis 238 (2015) 33e37

Contents lists available at ScienceDirect

Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Inflammation in childhood type 1 diabetes; influence of glycemic control Martin Heier a, b, c, *, Hanna Dis Margeirsdottir b, c, d, Cathrine Brunborg e, Kristian F. Hanssen b, c, f, Knut Dahl-Jørgensen a, b, c, Ingebjørg Seljeflot b, g a

Pediatric Department Ullevål, Oslo University Hospital, Oslo, Norway Faculty of Medicine, University of Oslo, Oslo, Norway Oslo Diabetes Research Centre, Oslo, Norway d Akershus University Hospital, Lørenskog, Norway e Department of Biostatistics and Epidemiology, Oslo University Hospital, Oslo, Norway f Department of Endocrinology, Oslo University Hospital, Oslo, Norway g Center for Clinical Heart Research and Department of Cardiology, Oslo University Hospital Ullevål, Oslo, Norway b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 May 2014 Received in revised form 13 October 2014 Accepted 18 November 2014 Available online 20 November 2014

Objective: Patients with type 1 diabetes have increased mortality from cardiovascular disease, and inflammation is important in the development of atherosclerosis. Our aim was to evaluate the extent of inflammation and the influence of glycemic control in the early phases of atherosclerosis in childhood type 1 diabetes. Materials and methods: A population based cohort representative of all children with type 1 diabetes in Norway was studied. Diabetes patients (n ¼ 314) were compared to healthy controls (n ¼ 120), aged 8e18 years. Circulating levels of VCAM-1, ICAM-1, E-selectin, P-selectin, TNFa, IL-6, CRP, MCP-1, IL-18, MMP-9 and TIMP-1 were measured by immunoassays. Results: The diabetes patients had a mean age of 13.7 (SD ¼ 2.8) years, disease duration of 5.5 (SD ¼ 3.4) years and HbA1c of 8.4 (SD ¼ 1.2) % (68 mmol/mol, SD ¼ 13.1). The levels of most of the measured markers were significantly increased in the diabetes group compared to controls. In the diabetes group, all except MCP-1 and MMP-9 were significantly correlated to HbA1c, albeit the relation to VCAM-1 was inverse. There were no significant correlations in the control group. The measured markers were only to a limited degree associated with traditional risk factors. CRP showed the most pronounced difference between diabetes patients and controls and the strongest correlation with HbA1c. The use of oral contraceptives profoundly increased CRP levels, independent of the presence of diabetes. Conclusions: Our results indicate that inflammation may play an important role in the accelerated atherosclerosis in early type 1 diabetes, and that this process seems primarily driven by hyperglycemia. © 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Inflammation Type 1 diabetes CRP Children

1. Introduction Well known consequences of type 1 diabetes are accelerated atherosclerosis and increased cardiovascular mortality [1,2]. It is currently acknowledged that atherosclerosis is a chronic inflammatory condition [3]. Although the atherosclerotic process starts early in life, only a few studies have investigated the relationship between diabetes, atherosclerosis and inflammation in children and adolescents [4e9]. Except for Snell-Bergeon's study from 2010

* Corresponding author. Oslo University Hospital, Pediatric Department Ullevål, Postboks 4950 Nydalen, 0424 Oslo, Norway. E-mail address: [email protected] (M. Heier). http://dx.doi.org/10.1016/j.atherosclerosis.2014.11.018 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.

[9], these previous studies included a limited number of both patients and markers of inflammation. Thus, further investigations are warranted, as several inflammatory biomarkers are involved in different stages of the atherosclerotic process. Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin and monocyte chemoattractant protein-1 (MCP-1) are all involved in the recruitment of inflammatory cells from the blood stream into the vessel wall, and circulating levels have been associated with atherosclerosis [10e14]. Tumor necrosis factor alpha (TNFa) is a proinflammatory molecule, and circulating levels are associated with degrees of early atherosclerosis [15]. Elevated levels of interleukin6 (IL-6) have been associated with an increased risk of

34

M. Heier et al. / Atherosclerosis 238 (2015) 33e37

cardiovascular death [16]. It is also an important stimulant for production of C-reactive protein (CRP), the most extensively studied marker of inflammation. CRP, as well as the proinflammatory cytokine interleukin-18 (IL-18), have been shown to be powerful predictors of cardiovascular disease [17e19]. Matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), are involved in remodeling the extracellular matrix of the arterial wall, and circulating MMP-9 levels predict cardiovascular mortality in patients with coronary artery disease [20]. In combination, these inflammatory biomarkers reflect several aspects of inflammation in atherosclerosis, and have not previously been studied collectively. The aim of this study was to investigate the influence of childhood type 1 diabetes and glycemic control on a comprehensive selection of markers of inflammation reflecting the early stages of atherosclerosis.

detect associations between categorical independent variables. CRP was polytomized according to the 25%, 50%, and 75% quartiles. The association and gradient effect was estimated using the ManteleHaenzel test of linear trend. Univariate linear regression analysis was performed to study the association between traditional risk factors as exposure variables with markers of inflammation as outcome variables. CRP was loge-transformed to achieve normally distributed residuals. To identify possible confounders, we studied all variables that could influence the markers of inflammation. Only variables with significant relationships with both the exposure and the outcome variables were considered as possible confounders and included in a multivariate analysis. Adjustment for multiple confounding factors was done using multivariate linear regression analysis with a manual backward elimination procedure. A significance level of 5% was used. All statistical analyses were performed using the SPSS software package for Mac, version 19.0 (SPSS, Chicago, IL).

2. Materials and methods 2.1. Study population The study cohort includes 314 patients and 120 healthy controls, aged 8e18 years. It is population based and representative of all children with type 1 diabetes in Norway. The control subjects were primarily classmates of the diabetes patients. The details of the cohort have previously been described [21]. The study was conducted according to the Declaration of Helsinki, and the protocol was approved by the Norwegian Regional Committee for Research Ethics. All participants and their parents gave their written informed consent. 2.2. Laboratory methods Overnight fasting blood samples were collected between 7.30 and 10 am. This was followed by a clinical examination. Within 1 h after venipuncture, blood samples were separated by sentrifugation at 2500  g for 10 min and kept frozen at 80  C until analysis. All markers of inflammation were measured in serum. CRP was determined using an enzyme-linked immunosorbent assay (DRG Instruments GmbH, Germany) with a detection limit of 0.1 mg/L. The enzyme-linked immunosorbent assay method from Medical Biological Laboratories (Naku-ku, Nagoya, Japan) was used for analysis of IL-18. ICAM-1, VCAM-1, E-selectin, P-selectin, TNFa, IL-6, MCP-1, MMP-9 and TIMP-1 were measured by enzyme immunoassays from R&D Systems Europe (Abingdon, Oxon, UK). In our laboratory, the inter-assay coefficients of variation (CV) were: CRP < 5%, IL-18 6.5%, ICAM-1 6.6%, VCAM-1 5.3%, E-selectin 5.2%, Pselectin 7.2%, TNFa 8.5%, IL-6 10.7%, MCP-1 9.2%, MMP-9 7.3% and TIMP-1 4.4%. HbA1c was measured at a DCCT-standardized laboratory using high performance liquid chromatography (Variant; Bio-Rad, Richmond, CA, USA), CV < 3%. Other routine laboratory analyses were performed by conventional methods. 2.3. Statistical analysis Demographic and clinical data are presented as either proportions, means with their standard deviations (SD) or medians with the 25th and 75th percentile. Differences in continuous variables between groups were tested with the Student t-test for normally distributed data and the ManneWhitney U-test for nonnormally distributed data. Correlation analyses between continuous variables were performed using Pearson's correlation coefficient (r) and Spearman's rho (r) when appropriate. The c2-test for contingency tables with different degrees of freedom was used to

3. Results Almost all (97%) of the diabetes patients were on intensive insulin treatment with more than four daily insulin injections or using pumps (60%). The patients had a mean duration of diabetes of 5.5 years (SD ¼ 3.4) and a mean HbA1c of 8.4% (SD ¼ 1.2) (68 mmol/ mol, SD ¼ 13.1). The clinical and metabolic characteristics of the participants are shown in Table 1. The included patients constituted a representative sample of the young type 1 diabetes population in Norway regarding HbA1c, lipid status, blood pressure, gender and stage of puberty [21]. Only patients above the age of 8 years were included in the study, however, so they were slightly older, with marginally longer diabetes duration, higher body mass index (BMI) and were more frequently pump users than the rest of the diabetic children and adolescents in Norway. Compared to the control subjects, the diabetes patients were taller, heavier and had higher BMI and waist circumference. They also had increased systolic and diastolic blood pressure, total cholesterol, HDL-cholesterol, LDL cholesterol, apolipoprotein B and apolipoprotein A1 (Table 1).

Table 1 Clinical and metabolic characteristics. Diabetes patients Controls n Age (years) Girls, n (%) Height (cm) Weight (kg) BMI (kg/m2) Waist circumference (cm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Smokers, n (%) HbA1c (%) [mmol/mol, SD] Total Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L)a Apolipoprotein B (g/L) Apolipoprotein A1 (g/L) ApoB/ApoA1 Urine Albumin/Creatinine (mg/ mmol)a

314 13.7 (2.8) 158 (50.3) 160.4 (14.4) 54.9 (16.7) 20.8 (3.9) 71.2 (10.0) 101.0 (10.1) 60.5 (8.3) 11 (3.5) 8.4 (1.2) [68, 13.1] 4.6 (0.8) 1.8 (0.4) 2.5 (0.7) 0.7 (0.5, 0.9) 0.74 (0.19) 1.55 (0.28) 0.5 (0.2) 0.70 (0.40, 1.33)

Mean values (SD). a Median (25th and 75th percentile).

120 13.2 (2.6) 68 (56.7) 157.0 (13.5) 48.0 (13.4) 19.1 (3.1) 66.6 (6.6) 98.1 (10.2) 58.3 (7.5) 2 (1.7) 5.3 (0.3) [34, 3.3] 4.3 (0.8) 1.7 (0.4) 2.3 (0.7) 0.6 (0.5, 0.9) 0.67 (0.17) 1.44 (0.32) 0.5 (0.3) 0.61 (0.37, 1.35)

pvalue NS NS .019 10 mg/L, the results were essentially the same (data not shown). 4. Discussion The main findings in this study were that compared to healthy controls, several markers of inflammation are increased in young patients with diabetes of relatively short duration, and that this is related to glycemic control. These results indicate an accelerated atherosclerotic process in these patients. The significance of these findings is reinforced by the broad spectrum of markers of inflammation included in this population based study. Our observations are in agreement with a previous report showing increased levels of VCAM-1, ICAM-1 and E-selectin in similar patients, probably due to increased endothelial activation [22]. Although increased levels of P-selectin, as shown in the diabetes patients in our study, are normally attributed to activated platelets, it may also reflect activated endothelium [23]. In the Stansislas study, biological determinants of soluble adhesion molecules in healthy children were explored, and ApoA1 was found to be associated with ICAM-1 [24]. This could not be confirmed in our study, as we did not find any associations between traditional cardiovascular risk factors and circulating adhesion molecules in our control group. In the diabetes group, however, we found a positive association between HbA1c and ICAM-1 and an inverse relationship between HDL cholesterol and E-selectin. The pro-inflammatory markers TNFa and IL-6 were both significantly correlated to HbA1c, but the levels were not significantly increased in the diabetes group compared to controls. In accordance with the Stanislas study [25], IL-6 was negatively associated with HDL-cholesterol in the control group. In our cohort, CRP was the marker with the most pronounced difference between patients and controls and the strongest correlation with HbA1c. CRP was positively associated with BMI in both groups and triglycerides in the diabetes group. This is in accordance with previous reports from smaller studies [7,26]. Higher levels of CRP among girls have been observed by others (7), but the underlying mechanism has not been clarified. Our findings indicate that the use of oral contraceptives and the presence of diabetes account for the entire difference in CRP levels between diabetic girls and controls. CRP was remarkably increased in girls using oral contraceptives, independent of the presence of diabetes. The effect of oral contraceptives on CRP has previously been shown in a large study with healthy adolescent subjects [27]. It has also been discussed that this is due to IL-6 independent hepatic synthesis of CRP [28]. Whether these elevated CRP-values during adolescence result in increased cardiovascular mortality is unknown. Chatzigeorgiou et al. [29] have discussed a possible role for MCP-1 in the destruction of pancreatic beta cells in newly diagnosed type 1 diabetes patients. Elevated levels were not shown, however, when diabetes duration surpassed six months, in line with another study [30]. In contrast to this, we found significantly increased levels of MCP-1 after five years of disease duration in the diabetes group compared to controls, but no correlation with HbA1c or association with traditional cardiovascular risk factors. IL-18 has been shown to be an independent predictor of cardiovascular events in elderly men with the metabolic syndrome, and this effect was potentiated by hyperglycemia [19]. In children,

elevated levels of IL-18 have been demonstrated in patients with the metabolic syndrome [31], and also in newly diagnosed type 1 diabetes [32,33]. This is in line with our observation of significantly increased levels in the young patients with type 1 diabetes. We also found a significant correlation with HbA1c, supporting the assumption that hyperglycemia is an important factor in IL-18 regulation. IL-18 was associated with triglycerides and apolipoprotein B in the control group. A recent study showed increased levels of MMP-9 in tear samples of pediatric type 1 diabetes patients, without any correlation with HbA1c [34]. We found increased levels of both MMP-9 and TIMP-1 in our diabetes patients, the latter significantly correlated with HbA1c. Levels of TIMP-1 may be elevated as a compensatory response to increased MMP-9, but there is also emerging evidence for MMP-independent actions of TIMP-1 [35]. The significant associations between MMP-9 and diastolic blood pressure and smoking, and between TIMP-1 and LDL-cholesterol in the diabetes group, as well as MMP-9 and BMI in the control group, indicate that matrix remodeling might have a more prominent role in early diabetes than previously suspected. Our study has several limitations, one being that most of the significant coefficients of correlation between the markers of inflammation and HbA1c are relatively low, which could imply limited clinical relevance. However, it could also be due to the fact that the diabetes patients were young and had a relatively short duration of disease. Furthermore, the cross-sectional design of the study makes it impossible to prove any direction of causality. The study's strengths, on the other hand, are the relatively large population based cohort of children and adolescents with type 1 diabetes and a broad panel of markers of inflammation reflecting both endothelial activation, systemic inflammation and extracellular remodeling. In conclusion, we have shown increased levels of several markers of inflammation in children and adolescents with type 1 diabetes compared to controls, most of them significantly correlated with HbA1c. The elevated levels were largely independent of traditional cardiovascular risk factors. CRP was remarkably high in girls using oral contraceptives, independent of diabetes. The evidence of matrix remodeling in diabetes at this age was persuasive. This all indicates that inflammation is an important part of the accelerated atherosclerosis in early type 1 diabetes. Conflict of interest None. Acknowlegdements The work was supported by Stein Erik Hagen Foundation for Clinical Heart Research, Oslo, Norway, and by EXTRA funds from the Norwegian Foundation for Health and Rehabilitation. We thank Vibeke Bratseth and Sissel Åkra for excellent laboratory assistance and Eva B. Lindseth for her invaluable work in including and examining patients and managing data. The study was funded by the University of Oslo. There are no conflicts of interest. References [1] A.S. Krolewski, E.J. Kosinski, J.H. Warram, et al., Magnitude and determinants of coronary artery disease in juvenile-onset, insulin-dependent diabetes mellitus, Am. J. Cardiol. 59 (8) (1987) 750e755. [2] S.P. Laing, A.J. Swerdlow, S.D. Slater, et al., Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes, Diabetologia 46 (6) (2003) 760e765. [3] A.J. Lusis, Atherosclerosis, Nature 407 (6801) (2000) 233e241.

M. Heier et al. / Atherosclerosis 238 (2015) 33e37 [4] B. Glowinska-Olszewska, M. Moniuszko, A. Hryniewicz, et al., Relationship between circulating endothelial progenitor cells and endothelial dysfunction in children with type 1 diabetes: a novel paradigm of early atherosclerosis in high-risk young patients, Eur. J. Endocrinol. 168 (2) (2013) 153e161. [5] A. Galler, A. Heitmann, W. Siekmeyer, et al., Increased arterial stiffness in children and adolescents with type 1 diabetes: no association between arterial stiffness and serum levels of adiponectin, Pediatr. Diabetes 11 (1) (2010) 38e46. [6] K. Heilman, M. Zilmer, K. Zilmer, et al., Elevated plasma adiponectin and decreased plasma homocysteine and asymmetric dimethylarginine in children with type 1 diabetes, Scand. J. Clin. Lab. Invest 69 (1) (2009) 85e91. [7] K.E. MacKenzie, E.J. Wiltshire, A.S. Pena, et al., Hs-CRP is associated with weight, BMI, and female sex but not with endothelial function in children with type 1 diabetes, Pediatr. Diabetes 10 (1) (2009) 44e51. [8] H. Mangge, K. Schauenstein, L. Stroedter, et al., Low grade inflammation in juvenile obesity and type 1 diabetes associated with early signs of atherosclerosis, Exp. Clin. Endocrinol. Diabetes 112 (7) (2004) 378e382. [9] J.K. Snell-Bergeon, N.A. West, E.J. Mayer-Davis, et al., Inflammatory markers are increased in youth with type 1 diabetes: the SEARCH case-control study, J. Clin. Endocrinol. Metab. 95 (6) (2010) 2868e2876. [10] K. Ley, C. Laudanna, M.I. Cybulsky, et al., Getting to the site of inflammation: the leukocyte adhesion cascade updated, Nat. Rev. Immunol. 7 (9) (2007) 678e689. [11] E. Galkina, K. Ley, Vascular adhesion molecules in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 27 (11) (2007) 2292e2301. [12] Z.M. Dong, S.M. Chapman, A.A. Brown, et al., The combined role of P- and Eselectins in atherosclerosis, J. Clin. Invest 102 (1) (1998) 145e152. [13] K.D. O'Brien, T.O. McDonald, A. Chait, et al., Neovascular expression of Eselectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content, Circulation 93 (4) (1996) 672e682. [14] V. Braunersreuther, F. Mach, S. Steffens, The specific role of chemokines in atherosclerosis, Thromb. Haemost. 97 (5) (2007) 714e721. [15] T. Skoog, W. Dichtl, S. Boquist, et al., Plasma tumour necrosis factor-alpha and early carotid atherosclerosis in healthy middle-aged men, Eur. Heart J. 23 (5) (2002) 376e383. [16] T.B. Harris, L. Ferrucci, R.P. Tracy, et al., Associations of elevated interleukin-6 and C-reactive protein levels with mortality in the elderly, Am. J. Med. 106 (5) (1999) 506e512. [17] S. Verma, P.E. Szmitko, P.M. Ridker, C-reactive protein comes of age, Nat. Clin. Pract. Cardiovasc Med. 2 (1) (2005) 29e36 quiz 58. [18] B.J. Jefferis, O. Papacosta, C.G. Owen, et al., Interleukin 18 and coronary heart disease: prospective study and systematic review, Atherosclerosis 217 (1) (2011) 227e233. [19] M. Troseid, I. Seljeflot, E.M. Hjerkinn, et al., Interleukin-18 is a strong predictor of cardiovascular events in elderly men with the metabolic syndrome: synergistic effect of inflammation and hyperglycemia, Diabetes Care 32 (3) (2009) 486e492. [20] S. Blankenberg, H.J. Rupprecht, O. Poirier, et al., Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease, Circulation 107 (12) (2003) 1579e1585.

37

[21] H.D. Margeirsdottir, K.H. Stensaeth, J.R. Larsen, et al., Early signs of atherosclerosis in diabetic children on intensive insulin treatment: a populationbased study (1935-5548 (Electronic))(0149-5992 (Linking)), Diabetes Care 33 (9) (2010) 2043e2048. [22] B. Glowinska, M. Urban, J. Peczynska, et al., 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, Metabolism 54 (8) (2005) 1020e1026. [23] V. Gebuhrer, J.F. Murphy, J.C. Bordet, et al., Oxidized low-density lipoprotein induces the expression of P-selectin (GMP140/PADGEM/CD62) on human endothelial cells, Biochem. J. 306 (Pt 1) (1995) 293e298. [24] A. Ponthieux, B. Herbeth, S. Droesch, et al., Biological determinants of serum ICAM-1, E-selectin, P-selectin and L-selectin levels in healthy subjects: the Stanislas study, Atherosclerosis 172 (2) (2004) 299e308. [25] N. Haddy, C. Sass, S. Droesch, et al., IL-6, TNF-alpha and atherosclerosis risk indicators in a healthy family population: the STANISLAS cohort, Atherosclerosis 170 (2) (2003) 277e283. [26] B. Glowinska-Olszewska, M. Urban, J. Peczynska, et al., hsCRP protein in children and adolescents with diabetes type 1, Pediatr. Endocrinol. Diabetes Metab. 13 (2) (2007) 79e84. [27] Y. Du, B.M. Rosner, H. Knopf, et al., Hormonal contraceptive use among adolescent girls in Germany in relation to health behavior and biological cardiovascular risk factors, J. Adolesc. Health 48 (4) (2011) 331e337. [28] M. van Rooijen, L.O. Hansson, J. Frostegard, et al., Treatment with combined oral contraceptives induces a rise in serum C-reactive protein in the absence of a general inflammatory response, J. Thromb. Haemost. 4 (1) (2006) 77e82. [29] A. Chatzigeorgiou, V. Harokopos, C. Mylona-Karagianni, et al., The pattern of inflammatory/anti-inflammatory cytokines and chemokines in type 1 diabetic patients over time, Ann. Med. 42 (6) (2010) 426e438. [30] B. Glowinska, M. Urban, Selected cytokines (Il-6, Il-8, Il-10, MCP-1, TNF-alpha) in children and adolescents with atherosclerosis risk factors: obesity, hypertension, diabetes, Wiad. Lek. 56 (3e4) (2003) 109e116. [31] L. Gilardini, P.G. McTernan, A. Girola, et al., Adiponectin is a candidate marker of metabolic syndrome in obese children and adolescents, Atherosclerosis 189 (2) (2006) 401e407. [32] J. Svensson, S. Eising, D.M. Hougaard, et al., Few differences in cytokines between patients newly diagnosed with type 1 diabetes and their healthy siblings, Hum. Immunol. 73 (11) (2012) 1116e1126. [33] G. Dong, L. Liang, J. Fu, et al., Serum interleukin-18 levels are raised in diabetic ketoacidosis in Chinese children with type 1 diabetes mellitus, Indian Pediatr. 44 (10) (2007) 732e736. [34] C. Symeonidis, E. Papakonstantinou, A. Galli, et al., Matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetic patients: MMPs in tear samples from type 1 diabetes, Graefes Arch. Clin. Exp. Ophthalmol. 251 (3) (2013) 741e749. [35] C. Marchesi, F. Dentali, E. Nicolini, et al., Plasma levels of matrix metalloproteinases and their inhibitors in hypertension: a systematic review and meta-analysis, J. Hypertens. 30 (1) (2012) 3e16.

Inflammation in childhood type 1 diabetes; influence of glycemic control.

Patients with type 1 diabetes have increased mortality from cardiovascular disease, and inflammation is important in the development of atherosclerosi...
306KB Sizes 1 Downloads 5 Views