Detection of free elastin-derived peptides among diabetic children

Atherosclerosis 192 (2007) 342–347

Detection of free elastin-derived peptides among diabetic children G. Nicoloff a,∗ , Ch. Petrova b , P. Christova c , A. Nikolov a a

Department of Biology & Pathological Physiology, Medical University, 1, St. Kliment Ohridski Street, 5800 Pleven, Bulgaria b Department of Pediatrics, Medical University, Pleven, Bulgaria c Department of Social Medicine, Medical University, Pleven, Bulgaria Received 12 May 2006; received in revised form 6 July 2006; accepted 1 August 2006 Available online 22 September 2006

Abstract Elastin breakdown products are found in the serum of all human subjects. The presence of these elastin-derived peptides (EDP) and the corresponding antibodies in circulation leads to formation of circulating immune complexes (CIC). The aim of this study was to determine if serum level of free-EDP (unbound in CIC) correlate with the development of microvascular complications in children with Type 1 (insulindependent) diabetes mellitus. To this end we used a method for detecting immune complexes (CIF-ELISA) in combination with an ELISA for detection of EDP. The levels of free EDP were studied in sera of 81 diabetic children (mean age 13.46 ± 3.51 years, diabetes duration 5.17 ± 4.21 years). Forty-two of the children had vascular complications (group 1) and 39 were without vascular complications (group 2). Twenty-one healthy children (mean age 12.6 ± 2.47 years) were used as controls. Diabetics showed significantly higher levels of free EDP (68.1 ± 25 ng/ml versus 51 ± 12.5 ng/ml; p = 0.003) compared to the control group. In group 1, free EDP showed significantly higher levels than controls (78.9 ± 25.6 ng/ml versus 51 ± 12.5 ng/ml; p = 0.0001). About 38 of 81 (47%) patients were positive for free EDP (30/42 – 71% in group 1 and 8/39 – 21% in group 2). Free EDP levels in all diabetics showed a correlation with insulin dose (r = 0.23; p = 0.041), and microalbuminuria (r = 0.57; p = 0.0001). Patients who had vascular pathology showed a correlation of free EDP with microalbuminuria (r = 0.41; p = 0.0081), retinopathy (r = 0.32; p = 0.041), insulin dose (r = 0.37; p = 0.02), HbA1c (r = 0.35; p = 0.03), systolic blood pressure (r = 0.30; p = 0.045) and total cholesterol (r = 0.36; p = 0.02). These findings suggest that elevated levels of free EDP are associated with the development of diabetic vascular complications in children. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Diabetes mellitus; CIF-ELISA; Free elastin-derived peptides; Microalbuminuria; Retinopathy

1. Introduction Patients with juvenile onset of Type 1 (insulin-dependent) diabetes mellitus are at high risk of diabetic microvascular complications due to alteration in the structure of the vascular proteins. Elastin is one of the major structural matrix proteins of the arterial wall [1–3]. Mature elastin is composed of soluble elastin subunits, which are intermolecularly crosslinked into a fibrous network (desmosine and isodesmosine formation) and thus construct a highly polymerized Abbreviations: EDP, elastin-derived peptides; AEAbs, antielastin antibodies; CIF, complement inhibition factor ∗ Corresponding author. Tel.: +359 64 884271; fax: +359 64 801 603. E-mail address: nicoloff [email protected] (G. Nicoloff). 0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.08.007

insoluble protein. Degradation of arterial wall elastin is a characteristic feature in atherogenesis. Serum concentration of elastin-derived peptides (EDP) is elevated in atherosclerotic patients and reflects elastin turnover [4]. Increased serum concentration of EDP is a potential indicator of advanced atherosclerosis such as plaque instability and is also a predictor of rupture in atherosclerotic aortic aneurysms [5]. Our previous studies have documented an increased degradation of elastin and production of antielastin antibodies (AEAbs) in diabetic children with microangiopathy [6,7]. These autoantibodies bind to their cognate antigen and thus form circulating immune complexes (CIC). Such CIC may have pathogenic potential since they can give rise to microangiopathy following deposition in small blood vessels. CIC containing LDL or elastin–antielastin has been identi-

G. Nicoloff et al. / Atherosclerosis 192 (2007) 342–347

343

Table 1 Free elastin-derived peptides (EDP) in sera of children with Type 1 diabetes mellitus with (n = 42) or without (n = 39) vascular complications (VC) Groups

Group 1 VC

Vascular complications (subgroups) M

M + HBP

M+R

N

R

Group 2 (without vascular complications)

Total

HBP

Positive Negative

30 12

12 3

2 0

3 1

1 1

6 6

6 1

8 31

38 43

Total

42

15

2

4

2

12

7

39

81

The table shows the number of positive or negative patients for free-EDP in each group. M, microalbuminuria; M + HBP, microalbuminuria and high blood pressure; M + R, microalbuminuria and retinopathy; N, neuropathy; R, retinopathy; HBP, high blood pressure.

fied in blood circulation of patients with diabetes mellitus and atherosclerosis [8–10]. The method for isolation and identification of CIC was developed in our laboratory [8]. It is based on the sequential polyethylene glycol precipitation on the CIC from the serum according to Eskinazi [11], followed by the dissociation of the precipitated immune complexes and the identification of the antigen incorporated in these by means of ELISA. Diabetic patients with vascular damage and healthy controls were tested by this method for detection and identification of elastin–antielastin CIC in human sera. Among different patients, the elastin–antielastin CIC varied in size and elastin content, showing some correlation between these two characteristics and the existence of diabetic microvascular complications. Our study showed the existence of a correlation between the condition of the blood vessel wall and the detection of elastin–antielastin CIC in the sera. Smallsized CIC with high elastin content seem to be characteristics for vascular damage. Elastin–antielastin CIC may be formed especially among diabetic and atherosclerotic patients with high levels of antigen (EDP) and low levels of specific antibodies. The elastin–antielastin CIC in the sera of these patients are small and have high elastin content. Such complexes can be formed when there is a great excess of antigen and inadequately low quantity of specific antibodies. In our previous study, a method based on C3 binding glycoprotein named CIF-ELISA (developed by [12]) was used for the detection of CIC (IgG, IgM and IgA) in sera of diabetic children [13]. A correlation was found between CIC from class IgG and development of vascular complications. In the present study, we used CIF-ELISA combined with ELISA for detection of elastin-derived peptides (EDP) to: (i) eliminate EDP, which are incorporated in CIC; (ii) measure the levels of free EDP (unbound in CIC); (iii) test the possible correlation between free EDP and development of diabetic vascular complications in children.

age 13.46 ± 3.51 years) diagnosed using the WHO definition. The control group consisted of 21 healthy children of similar age (12.6 ± 2.47 years) and sex with no family history of diabetes, atherosclerosis, and/or nephropathy. The patients are all diabetic children residing in the vicinity of the Pleven University Hospital and were all treated with a standard dose of human insulin obtained from Novo Nordisk Industri, Copenhagen, Denmark. None of the subjects were taking antihypertensive medication prior to the appearance of persistent microalbuminuria and none had a diagnosis of renal disease unrelated to diabetes. The mean duration of diabetes was 5.17 ± 4.21 years. All subjects were being administered 2–4 subcutaneous doses of insulin per day. Forty-two of 81 diabetics had vascular complications (group 1)—Table 1. All patients of group 1 had diabetic microangiopathy. It is the most common chronic complication of Type 1 diabetes mellitus and affects the small vessels (capillaries) as: thickening of basement membrane impaired (increased) vascular permeability and narrowing capillary lumen leads to ischemia. Target organs are the eyes, kidneys, peripheral and autonomic nervous system. Microalbuminuria was defined as a persistent urinary albumin excretion rate (AER) in the range of 20 and 200 ␮g/min in urine. Levels of hemoglobin A1c (HbA1c), total serum cholesterol, triglycerides and AER were measured as described earlier [7]. Ethical approval was obtained from the institutional research ethics committee and the parents of all subjects gave written informed consent prior to enrolment in the study. 2.2. Antigen

2. Materials and methods

Soluble ␣-elastin was prepared from human cadaver aortas of young healthy subjects (killed by accident) as described earlier [4]. Elastin purity was confirmed by amino acid analysis by Prof. R. Mecham (Washington University, St. Louis, USA).

2.1. Subjects

2.3. Immune sera

The baseline study population consisted of 81 patients (37 boys and 44 girls) with Type 1 diabetes mellitus (mean

Polyclonal immune sera to human ␣-elastin were developed in rabbits and sheep as described earlier [4].

344

G. Nicoloff et al. / Atherosclerosis 192 (2007) 342–347

2.4. Procedure Isolation of a new glycoprotein complement inhibition factor (CIF) from the parasitic plant Cuscuta europea seed, which appears to bind specifically to complement component C3 has been provided a unique tool for the measurement of immune complexes by means of ELISA-type techniques (CIF-ELISA). Free EDP were detected using a two-step method consisting of CIF-ELISA to remove immune complexes [12] followed by an elastin-specific ELISA for detection of EDP [6] • CIF-ELISA plates were prepared by incubating wells of polystyrene plates with CIF (20 ␮g/ml in 0.2 M carbonate–bicarbonate buffer pH 9.6 overnight at 4 ◦ C). CIF was isolated as described [14]. After several washes to remove unbound CIF, human samples (100 ␮l) were added to the plates and incubated for 60 min at 37 ◦ C. • Coating of new plates with 10 ␮g/ml rabbit antielastin IgG (in 100 ␮l of 0.05 M carbonate buffer, pH 9.6) for 3 h at 37 ◦ C and overnight at 4 ◦ C. • Blockade of the remaining “active” centers of the polystyrene wells was done by polystyrene plate incubation for 24 h with 1% solution of bovine serum albumin (BSA) (Sigma, USA) in phosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween 20. • At the end of the incubation period, the treated human samples (diluted 1:5 in PBS) were transferred into antielastin wells to be tested and incubated for 1 h at 37 ◦ C. • After washing, the plates were incubated with sheep antielastin IgG (10 ␮g/ml) for 1 h at 37 ◦ C. • Then an incubation with anti-sheep IgG peroxidase immunoconjugate (SIGMA, USA) diluted 1:10,000 was done. • The reaction was stopped with 50 ␮l of 4 M H2 SO4 . Absorbance readings were made using a Microelisa Reader 210 (Organon Teknika, Belgium) at a wavelength of 492 nm. Each experiment contained the following control: (i) substrate control—only substrate solution was added to the plates, coated with rabbit antielastin IgG. (ii) Immunoconjugate control—the immunoconjugate was added directly to the polystyrene wells, coated with rabbit antielastin IgG, and the wells were then incubated with substrate solution. (iii) Control of rabbit immune serum: rabbit antielastin IgG was replaced with normal rabbit IgG. (iv) CIF-ELISA control—after absorption of EDP CIC by CIF, 100 ␮l of rabbit or sheep immune sera of absorbed serum was tested for the presence of EDP CIC. (v) Negative controls of the specificity of the ELISA: the assay was carried out according to the usual protocol, but the tested samples were replaced with BSA (SIGMA, USA), standard collagen type IV from human placenta (type VI, SIGMA, USA), saline extracts from the following organs of a 15-year-old healthy subject (killed in an accident): aorta, heart, lung, brain, liver, spleen, kidney, testicle, thymus, skin, and cross-striated muscles (all with

concentration 1 ␮g/ml). (vi) Positive control of ELISA: the tested sample was replaced with human ␣-elastin with concentration 1 ␮g/ml diluting buffer. All the samples were tested in triplicate and peripheral wells were not used to avoid border effects. 2.5. Statistical analyses All values are expressed as mean ± S.D. Statistical analyses was performed using the computer programs Excel and Statgraphics plus for Windows. The Student’s t-test and ANOVA were used to assess differences between study groups (LSD, Tukey HSD, Scheffe, Bonferroni, Newman–Keuls, and Duncan). For non-parametric analysis was used Kruskal–Wallis test. For select data sets, correlation analysis was performed and data considered significant with a p value of less than 0.05. 3. Results First, we tested the specificity of the immune sera (and the specificity of the assay as a whole) by the negative controls. The standard human albumin solution gave a colour reaction, equivalent to that of the immunoconjugate control. The same occurred with the standard collagen preparation and with normal rabbit IgG. The controls with saline extracts from different organs gave a very weak reaction 4 ± 1.1 ng/ml. Results from the inhibition ELISA with human aortic ␣elastin showed that preincubation of the 10 human sera with the highest EDP levels with AEAbs resulted in almost complete inhibition of the ELISA signal. The values obtained were 10 ± 4.1 ng/ml. The difference can be clearly seen when comparing these data with the results obtained for samples that were not preincubated with AEAbs (Table 2). To determine whether EDP bound in CIC were successfully removed we tested the serum samples after absorption with CIF by CIF-ELISA for the presence of EDP CIC. There was no significant increase in EDP CIC levels—the mean level was 5.9%. The patients were divided into two groups based on the presence (group 1) or absence (group 2) of vascular complications. The mean free EDP value of the healthy persons was 51 ± 12.5 ng/ml. The cutoff point for positivity was set at the mean + 2 S.D. (i.e., 2 S.D. above the mean in healthy subjects). Patient’s values more than 76 ng/ml were considered positive. About 38 of 81 (47%) patients were positive for free EDP (30/42 – 71% in group 1 and 8/39 – 21% in group 2)—(Table 2). Six of 7 patients with HBP and 1 of 2 of neuropathy were positive for free-EDP. Twenty-one diabetic children had microalbuminuria of which 17 were positive for free-EDP. Of the 12 patients with retinopathy, 6 were positive for free-EDP. None of the healthy controls was positive. In group 1 (n = 42), free EDP showed significantly higher levels (Table 3) than group 2 (n = 39) and controls (p = 0.0001). However, in group 2 the level of free EDP was not significantly elevated compared to controls.

G. Nicoloff et al. / Atherosclerosis 192 (2007) 342–347

345

Table 2 Clinical characteristics of diabetic patients with or without vascular complications Clinical data

Group 1

Age (years) Mean diabetes duration (years) Mean glycated hemoglobin (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Triglycerides (mmol/l) Total cholesterol (mmol/l) Insulin dose (U/kg 24 h) Albumin excretion rate (␮g/min) Free EDP (ng/ml)

13.76 5.79 10.8 111.2 73.8 1.46 5.05 1.04 28.4 78.9

± ± ± ± ± ± ± ± ± ±

Group 2 3.54 4.1 2.7 14.3 9.3 1.3 1.41 0.28 18.22 25.6

13.15 4.51 9.24 110.6 72.1 1.26 4.39 1.03 14.24 56.7

± ± ± ± ± ± ± ± ± ±

p 3.5 4.27 1.39 10.9 7.2 0.88 1.16 0.23 6.47 19

NS NS 0.0015 NS NS NS 0.02 NS 0.00001 0.0001

Group 1, patients with vascular complications (n = 42); group 2, patients without vascular complication (n = 39).

Free EDP levels in all diabetics showed a correlation with insulin dose (r = 0.23; p = 0.041), and microalbuminuria (r = 0.57; p = 0.0001). Likewise, free EDP of patients who had vascular pathology showed a correlation with microalbuminuria (r = 0.41; p = 0.0081), retinopathy (r = 0.32; p = 0.041), insulin dose (r = 0.37; p = 0.02), HbA1c (r = 0.35; p = 0.03), systolic blood pressure (r = 0.30; p = 0.045) and total cholesterol (r = 0.36; p = 0.02). Significantly higher levels in group 1 than group 2 showed: HbA1c (p = 0.0015), total cholesterol (p = 0.02) and microalbuminuria (p = 0.0001)—Table 2. 4. Discussion Elastin is a fibrous protein constituent of the extracellular matrix, degradation of which may be detected by the presence of serum EDP in circulation. Presently, two major antigenic classes are recognized on the elastin molecule, one species specific and the other with broad species cross-reactivity [15]. ␣-Elastin is an oxalic acid solubilized product of insoluble mature elastin. The levels of circulating soluble elastin in healthy human subjects has been suggested as an indicator for aging [4], whereas some controversial result was also reported [16]. It was also reported that markers should be utilized for diagnosis of aneurysms [17]. EDP levels were significantly elevated in the patients with acute aortic dissection [18]. The soluble elastin levels in circulating blood may inform us of some clinical condition of vascular wall [19]. In previous longitudinal study we found a correlation between development of retinopathy and increased EDP con-

centrations in 10 diabetic children [6]. But it was impossible to distinguish free EDP from EDP bound up in immune complexes. The reason is that not all epitopes of EDP are engaged with cognate paratopes. That is why some of the nonengaged epitopes of EDP, incorporated in elastin–antielastin CIC, react with AEAbs during standard ELISA for detection of EDP. In the present study, the values of free EDP in serum samples of diabetics with vascular complications were markedly higher than healthy controls. Moreover, the number of positive patients with vascular pathology was much more than positive ones without vascular complications (71% versus 21%). The biological significance of this difference is that diabetics of group 1 are with pathologically increased elastin degradation and develop vascular complications. The data of eight patients of group 2 are over the normal limit of free EDP but they did not developed vascular complications. It could be that the relationship between the development of vascular complication and EDP are time dependent, i.e. just because we have EDP does not mean that will have vascular complications but with time and if the EDP continues to be present, the vascular complications are going to be detected later. It is important to follow if positive patients of this group will develop vascular complications before the other negative patients. The majority of our patients were negative (43 versus 38) for free EDP. From 43 negative subjects 31 belong to group 2. The evidence that elastin have antigenic properties has led to a hypothesis that EDP found in vivo, as a result of degradation or synthesis, may exert an autoimmune response [20–22]. It is possible some of free EDP detected by us form

Table 3 Free EDP obtained after testing of diabetic children (n = 81) and healthy controls (n = 21) Groups

Total diabetics Group 1 Group 2 Controls

Free EDP (ng/ml)

Comparison with other groups

Mean ± S.D.

Range

Group 1

Group 2

Total diabetics

± ± ± ±

12–124 19–124 12–92 25–68

– – p = 0.0001 p = 0.00001

– p = 0.0001 – NS

– – – p = 0.0031

68.1 78.9 56.7 51

25 25.6 19 12.5

Group 1, patients with vascular complications (n = 42); group 2, patients without vascular complication (n = 39); controls (n = 21).

346

G. Nicoloff et al. / Atherosclerosis 192 (2007) 342–347

specific AEAbs immune complexes. These complexes may form consequently to the association of modified elastin and antibodies against elastin epitopes. The elastin may becomes substrate for formation of both insoluble IC (it formed at tissue level) or soluble, CIC in the serum. It could be speculated that the positive patients of group 1 are in early active phase of development of vascular complications and increased elastin degradation. Probably, the rest 12 negative patients of the same group are also with increased degradation (because they developed vascular complications) but the majority of them are involved in the soluble or insoluble immune complexes. We confirmed our previous result [6] about correlation between EDP and diabetic retinopathy in more representative population. An association was found between free EDP and microalbuminuria. Such a correlation was not found in our previous longitudinal study [6]. In the current study, the levels of free EDP are lower than those found in our previous investigations. We speculate that the method used in this study results in the removal of elastin–antielastin CIC with active elastin epitopes. We interpreted the correlation between free EDP and HbA1c in group 1 as specific manifestation. The patients of group 1 had significantly higher level of HbA1c in comparison with the patients without vascular complications. The development of microvascular disease is associated with high percentage of glycated hemoglobin. An HbA1c of around 8% has been shown to increase the risk of microvascular disease [23]. In the present study we confirmed our previous result—a correlation between free EDP and systolic blood pressure [24]. Total cholesterol was above the normal limit in the group of patients with vascular complications. Free EDP showed a positive correlation with total cholesterol. The elevated level of total cholesterol correlate with the development of diabetic vascular complications as described [25]. It is well known that hypertriglyceridemia and hypercholesterolemia are the most common abnormality found in poorly controlled diabetes. EDP are markers for elastin degradation. Increased levels of EDP induce immune system to produce AEAbs. Recently, we found elevated levels of AEAbs in sera of diabetic children with microvascular complications [26]. Some EDP may be consumed by binding with AEAbs while others stay free in circulation. We observed lower free EDP in number of diabetic subjects with vascular complications, and postulated that antibodies against elastin form immune complexes in vivo, hampering their determination. The highest levels of free EDP may be present at the very beginning or at later activation of the degradation process. The production of free EDP, free AEAbs and elastin–antielastin immune complexes may play an important role in the onset and progression of late complications of diabetes. The evaluation of the role of EDP will require a number of studies. In conclusion, we suggest that elevated levels of free EDP are associated with the development of diabetic microvascular complications in children.

References [1] Rucker R, Tinker D. Structure and metabolism of arterial elastin. Int Rev Exp Pathol 1977;17:1–47. [2] Spina M, Garbisa S, Hinnie J, Hunter J, Serafini-Fracassini A. Agerelated changes in composition and mechanical properties of tunica media of the upper thoracic human aorta. Arteriosclerosis 1983;3: 64–76. [3] Dobrin P. Mechanics of normal and diseased blood vessels. Ann Vasc Surg 1988;2:283–94. [4] Baydanoff S, Nicoloff G, Alexiev Ch. Age-related changes in the level of circulating elastin-derived peptides in serum from normal and atherosclerotic human subjects. Atherosclerosis 1987;66:163–8. [5] Petersen E, Wagberg F, Angquist K. Serum concentrations of elastinderived peptides in patients with specific manifestations of atherosclerotic disease. Eur J Vasc Endovasc Surg 2002;24:440–4. [6] Nicoloff G, Baydanoff S, Stanimirova N, Petrova Ch, Christova P. Relationship between elastin-derived peptides and the development of diabetic microvascular complications—a longitudinal study in children with Type 1 (insulin-dependent) diabetes mellitus. Gen Pharmacol 2000;35:59–64. [7] Nicoloff G, Baydanoff S, Stanimirova N, Petrova Ch, Christova P. An association of anti-elastin IgA antibodies with development of retinopathy in diabetic children. Gen Pharmacol 2000;35:83–7. [8] Baydanoff S, Nicoloff G, Russev T, Alexiev Ch. Detection of elastin–antielastin circulating immune complexes (CIC) in diabetic patients with vascular damage. Cor et Vasa 1988;30:361–7. [9] Tertov V, Orekhov N, Kacharava G, et al. Low density lipoproteincontaining immune complexes and coronary atherosclerosis. Exp Mol Pathol 1990;52:300–8. [10] Baydanoff S, Nicoloff G. Determination of elastin–antielastin circulating immune complexes in serum of normal subjects and atherosclerotic patients. Cor et Vasa 1991;33:282–7. [11] Eskinazi D. Advantages of sequential polyethylene glycol (PEG) precipitation to isolate and analyse circulating immune complexes. Immun Lett 1984;7:221–8. [12] Stanilova S, Slavov E. Comparative study of circulating immune complexes quantity detection by three assays—CIF-ELISA, C1q-ELISA and anti-C3 ELISA. J Immunol Meth 2001;253:13–21. [13] Nicoloff G, Blazhev A, Petrova Ch, Christova P. Circulating immune complexes among diabetic children. Clin Dev Immunol 2004;11:61–6. [14] Zhelev Z, Stanilova S, Carpenter B. Isolation, partial characterization and complement inhibiting activity of a new glycoprotein from Cuscuta europea. Biochem Biophys Res Commun 1994;202:186–94. [15] Wrenn D, Mecham R. Immunology of elastin. Meth Enzymol 1987;144:246–59. [16] Fulop T, Wei S, Robert L, Jacob MP. Determination of elastin peptides in normal and atherosclerotic human sera by ELISA. Clin Physiol Biochem 1990;8:273–8. [17] Wei S, Erdei J, Fulop T, Robert L, Jacob M. Elastin peptide concentration in human serum: variation with antibodies and elastin peptides used for the enzyme-linked immunosorbent assay. J Immunol Meth 1993;164:175–87. [18] Shinohara T, Suzuki K, Okada M, et al. Soluble elastin fragments in serum are elevated in acute aortic dissection. Arterioscler Thromb Vasc 2003;23:1839–44. [19] Shinohara T, Okada M, Suzuki K, Ohsuzu F. Quantitative analysis for soluble elastin in circulation and cell culture fluids using monoclonal antibody-based sandwich immunoassay. J Immunoassay Immunochem 2005;26:189–202. [20] Peterszegi G, Mandet C, Texier S, Robert L, Bruneval P. Lymphocytes in human atherosclerotic plaque exhibit the elastin-laminin receptor & potential role in atherogenesis. Atherosclerosis 1997;135: 103–7. [21] Peterszegi G, Texier S, Robert L. Human helper and memory lymphocytes exhibit an inducible elastin-laminin receptor. Int Arch Immunol 1997;114:218–23.

G. Nicoloff et al. / Atherosclerosis 192 (2007) 342–347 [22] Colburn K, Langga-Shariffi E, Kelly G, et al. Abnormalities of serum antielastin antibodies in connective tissue diseases. J Invest Med 2003;51:104–9. [23] Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–86.

347

[24] Nicoloff G, Nikolov A, Dekov D. Serum AGE-elastin derived peptides among diabetic children. Vasc Pharmacol 2005;43:193–7. [25] Steiner G. Diabetes mellitus, dyslipoproteinaemias and atherosclerosis. Diabetologia 1997;40(Suppl 2):S147–8. [26] Nicoloff G, Blazhev A, Petrova Ch, et al. Detection of free antielastin antibodies among diabetic children. J Invest Med 2005;53:128– 34.