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Rheumatology Advance Access originally published online on August 27, 2006
Rheumatology 2007 46(3):412-416; doi:10.1093/rheumatology/kel076
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Circulating levels of N{varepsilon}-(carboxymethyl)lysine are increased in systemic sclerosis

O. Kaloudi, G. Basta3, F. Perfetto, F. Bartoli, A. Del Rosso, I. Miniati, M. L. Conforti, S. Generini, S. Guiducci, R. Abbate1, A. Pignone, S. Castellani2, R. Livi, R. De Caterina3,4 and M. Matucci-Cerinic

Department of Medicine, Division of Rheumatology and Departments of 1Medical and Surgical Critical Care and 2Internal Medicine and Cardiology, University of Florence, Florence, 3CNR Institute of Clinical Physiology, University of Pisa, Pisa and 4Institute of Cardiology and Center for Excellence on Aging, ‘G. d’Annunzio’ University, Chieti, Italy.

Correspondence to: M. Matucci-Cerinic, Department of Medicine and Surgery, Division of Rheumatology, University of Florence, Viale G. Pieraccini, 18 – 50139, Florence, Italy. E-mail: cerinic{at}unifi.it


   Abstract
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 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Objective. Advanced glycation endproducts (AGEs), including N{varepsilon}-(carboxymethyl)lysine-protein adducts (CML) are involved in micro/macrovascular changes and are co-localized with adhesion molecules in inflamed tissues. Serum levels of CML were investigated in systemic sclerosis (SSc) characterized by microvascular modifications and correlated with indices of micro/macrovascular damage.

Methods. In 66 SSc patients (limited SSc, n = 55; diffuse SSc, n = 11) and 20 controls, CML serum levels were measured by enzyme-linked immunosorbent assay. Nailfold capillaroscopy, intima-media thickness (IMT) and the ankle-brachial index (ABI) were also recorded, to characterize micro/macrovascular involvement.

Results. CML levels were significantly higher in SSc (79.2 ± 39 mg/ml vs 49.6 ± 26.1 mg/ml, mean ± S.D.; P<0.01), without significant differences between SSc subsets. CML levels were significantly higher in all capillaroscopic patterns: the ‘early’ pattern showed higher levels than ‘active’ and ‘late’ patterns. IMT was significantly higher in SSc (P<0.01) than in controls, whilst ABI was no different from controls.

Conclusions. These data indicate that although both CML formation and macrovascular involvement are increased in SSc, there is no correlation between these two parameters. However, the characteristic early nailfold capillaroscopy changes of SSc are associated with proportionally greater CML formation, suggesting that AGEs are involved in SSc microangiopathy.

KEY WORDS: Systemic sclerosis, N{varepsilon}-(carboxymethyl)lysine (CML), Advanced glycation endproducts.


   Introduction
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 Introduction
Subjects and methods
 Results
 Discussion
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In systemic sclerosis (SSc), Raynaud's phenomenon (RP) and microcirculatory abnormalities are early features of microvascular involvement and endothelial injury [1, 2]. This may be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS), generated locally by inflammatory processes and by reperfusion injury [3]. Increased levels of antibodies against oxidized LDL, produced through ROS-induced lipid peroxidation, have been detected in SSc [4, 5].

Advanced glycation endproducts (AGEs) are bioreactive compounds formed during hyperglycaemic conditions by non-enzymatic glycation and oxidation of several short- and long-lived extra- and intracellular proteins, via the so-called Maillard reaction [6]. N{varepsilon}-(carboxymethyl)lysine (CML) adducts of proteins forming in a milieu characterized by increased oxidative stress are among the most abundant AGEs detected in vivo, and can alter the structure and function of other molecules in biological systems [7]. These compounds are implicated in the pathogenesis of diabetic macro/microangiopathy, in ageing, renal failure and smoking [8]. AGEs have been detected in normoglycaemic patients with atherosclerotic lesions, and a significant correlation between circulating AGE-modified apolipoprotein B and AGE levels in arterial tissue has been reported in the vessel wall of carotid arteries from non-diabetic patients with occlusive disease requiring endarterectomy [9].

Few studies have addressed the prevalence of large-vessel disease in SSc [10] and angiotensin-converting enzyme insertion/deletion polymorphism [11] and anti-endothelial cell antibodies (AECA) [12] have been addressed as potentially involved in vascular damage.

The aim of our study was to investigate the levels of AGEs in SSc, and to correlate them with micro- and macrovascular involvement.


   Subjects and methods
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Results
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Patients
Sixty-six white patients (59 female and 7 male) affected by SSc were consecutively recruited at the Department of Medicine, Section of Rheumatology, of the University of Florence. The mean age was 60.5 ± 12.2 yr (range 26–78 yr), and the mean disease duration, calculated from the onset of the first non-Raynaud's symptom, was 9.4 ± 7.8 yr (range 1–42 yr). Twenty healthy subjects (18 female and 2 male), aged 58 ± 12.3 yr (range 22–75 yr), served as controls. SSc patients were classified as limited SSc (lSSc) (55 patients; age 62 ± 12.1 yr) or diffuse SSc (dSSc) (11 patients, age 53 ± 12.8 yr) [13] (Table 1). All patients were diagnosed and assessed according to international consensus criteria [13].


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  		TABLE 1. Demographic characteristics of SSc patients and controls

 
Exclusion criteria for patients and controls were: the presence of diabetes (fasting glucose levels >7.8 mmol/l), cigarette smoking and renal failure.

Blood samples were collected in the morning, after overnight fasting, into vacuum tubes containing EDTA, and kept on ice until determination. Antinuclear antibodies were determined as previously reported [12]. Concomitant treatment included only calcium channel blockers, proton-pump inhibitors and topical glyceryl trinitrate. None of these drugs has been demonstrated to induce CML adducts or AGE products. Informed written consent was obtained from all subjects enrolled in this study. The ethical committee of our institutions gave their consent.

Capillaroscopy
The physician (LC) performing nailfold videocapillaroscopy (NVC) was blinded to patient data. Patients were allowed to adapt at room temperature (20–22°C) for at least 15 min before the examination. Nailfolds of all 10 fingers were analysed for the following parameters: presence of enlarged and giant capillaries, pericapillary oedema, haemorrhages, loss of capillaries, ramified/bushy capillaries, and disorganization of the vascular distribution. According to these features, patients were classified in ‘early’, ‘active’ and ‘late’ disease pattern [14].

Macrovascular involvement
Two techniques were used to evaluate macrovascular involvement.

Measurement of the intima-media thickness (IMT)
IMT is considered a surrogate marker for cardiovascular events [15] and in particular of the extent and progression of macrovascular involvement. IMT was performed by the same operator (FB) blinded to patient data, using an ultrasound ATL 3000 scanner equipped with a 7 MHz linear transducer with a 38 mm aperture. An ECG signal (lead II) was simultaneously recorded to synchronize the image capture at the top of the R wave to minimize variability related with the cardiac cycle. The left and right carotid arteries were scanned at the level of the bifurcation, and images for IMT measurements were recorded from the far wall in the common carotid artery. The software program gives the average thickness of the intima-media complex. IMT was defined as the distance from the leading edge of the lumen–intima interface to the leading edge of the media–adventitia interface of the far wall [16]. At the position of the thickest part of the wall (assessed visually), a frozen longitudinal image was captured and recorded on videotape. A short sequence of real-time images was also recorded on videotape to assist in the interpretation of the frozen images. The images were measured with an automated analysing system (Metris software), which performs automatic detection of the echo structures in the ultrasonic image but allows the operator the option of making manual corrections. For the present analysis, the average of the IMT measurements from the left and right common carotid arteries (common carotid artery and carotid bulb) was used. Carotid duplex scanning was performed by a vascular technician.

Measurement of the ankle-brachial index (ABI)
The ABI is used in the investigation of atherosclerotic peripheral obstructive disease [17]. Measurements of the ABI, bilateral ankle and brachial arterial systolic pressures were made using a portable Sonicaid Doppler probe and a Hawkley random zero sphygmomanometer after a 10 min rest in a comfortably warm room. The ABI was calculated as the posterior tibial artery pressure divided by the brachial pressure. Calculation of the index was performed at a later date, during the analysis of the data. The normal ABI is 1.0, and any value <1.0 is usually considered to be abnormal [18], with the severity of arterial disease being inversely proportional to the ABI [19]. We selected a definitive ABI cut-off point of <0.9 because this criterion has a 95% sensitivity and 100% specificity for detecting arterial disease using angiographically defined disease as the gold standard [19, 20], but also evaluated the presence of milder disease with ABI of <1.0 and <0.98.

Enzyme-linked immunosorbent assay for quantitative detection of CML adducts
A CML–bovine serum albumin (BSA) standard was prepared according to Reddy et al. [21]. Preparations of CML-BSA were characterized as the percentage modification of lysine residues compared with unmodified BSA, as determined by of the use of fluorescamine [22]. The CML standard preparation contained 20 CML-modified lysine residues per molecule of albumin, corresponding to 24% of all the available lysine residues, and was used both to coat the plates and as the standard for the assay.

Serum CML levels were measured in triplicate by a competitive enzyme-linked immunosorbent assay (ELISA) using the mouse F(ab')2 anti-AGE monoclonal antibody 6D12 (ICN Pharmaceuticals, Italy), which recognizes CML protein adducts [23]. Serum samples were thawed and diluted 1:4 with dilution buffer (PBS containing 0.02% Tween 20; PBST) just prior to assay. All serum samples were run in one batch.

The ELISA is similar to a previously reported one [24], with minor modifications. Briefly, CML–BSA in coating buffer (50 mmol/l of sodium bicarbonate, pH 9.6) was coated for 48 h at 4°C on 96-well ELISA plates (Nunc-Immuno Plates Maxisorp). Wells were washed three times with washing buffer (PBST 0.05%), and then blocked with 100 µl blocking buffer (PBS containing 1% BSA) at room temperature for 1 h. After three rinses with washing buffer, 50 µl of diluted sample were added, followed by 50 µl of anti-AGE antibody-peroxidase conjugate (0.1 µg/ml) in blocking buffer. Chemically synthesized CML–BSA was used as standard in a concentration range of 0.1–100 µg/ml. Plates were incubated at room temperature for 3 h with gentle agitation on a horizontal rotary shaker. After three rinses, 100 µl of tetramethybenzidine substrate (Sigma) was then added to each well. After 5 to 30 min, 50 µL of 2 mol/l sulphuric acid was added to stop the reaction and the optical density at 450 nm determined by an ELISA plate reader.

One CML unit was defined as the competitive activity of 1 µg CML–BSA standard, according to Makita et al. [25]. Intra- and interassay coefficients of variation of our assay were 3.2 and 8.7%, respectively.

Statistical analysis
The distribution of continuous variables in groups is expressed as means ± standard deviation (S.D.). Data were analysed by the Student's t-test (comparison between two means) and analysis of variance (comparison among three or more means) followed by post-hoc test (Student–Newman–Keuls, SNK), and linear and multiple regression analysis (performed with the Systat 5.2.1 and Stat-View 4.5 software for Macintosh). Statistical significance was set at a P value of <0.05.


   Results
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Patients and controls showed similar age and body mass index (BMI) values (Table 1). Ten SSc patients showed an early, 39 an active and 17 a late NVC pattern.

At baseline, 16 patient were hypertensive (defined by systolic or diastolic blood pressure >140 and >90 mmHg, respectively), and eight had hypercholesterolaemia (fasting plasma cholesterol levels >5.2 mmol/l) (Table 1).

Circulating levels of CML adducts
The levels of CML adducts were significantly higher in SSc patients than in controls (79.2 ± 39 vs 49.6 ± 26.1 mg/ml; P<0.0009). Significantly (P<0.05) higher levels of CML were found both in lSSc (80.1 ± 39.6 mg/ml) and in dSSc (75.2 ± 37.6 µg/ml), but were not different between the two SSc subsets (Fig. 1A).


Figure 1
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  		FIG. 1. (A) Circulating CML levels in controls and in different subsets of cutaneous involvement of systemic sclerosis patients, diffuse (dSSc) and limited (lSSc) are shown. ANOVA: P = 0.009. *Significantly different by Student-Newman-Keuls post-hoc analysis: P<0.05 versus controls. (B) Circulating CML levels in control groups and in three systemic sclerosis (SSc) subgroups with different capillaroscopy pattern (early, active and late). ANOVA: P<0.0001. Different by Student–Newman–Keuls post-hoc analysis: P<0.001 versus controls. Different by Student–Newman–Keuls post-hoc analysis: P<0.05 versus SSc active and SSc late capillaroscopy patterns. The upper and lower limits of the box plot represent 75th and 25th percentiles. The horizontal bar indicates the median values and the vertical bars indicate the inner adjacent value.

 
In all three NVC patterns, circulating CML adducts were significantly (P<0.001) higher than in controls (108.4 ± 37.9 mg/ml for the early, 72.1 ± 38.2 mg/ml for the active and 78.4 ± 35.6 mg/ml for the late pattern) (Fig. 1B). Furthermore, CML adducts were significantly (P<0.05) higher in early than in active and late patterns, while no difference was found between late and active patterns (Fig. 1B). None of the other parameters correlated with CML adducts. No difference between levels of CML adducts in hypertensive (77.2 ± 38.9 mg/ml) and normotensive (79.9 ± 39.4 mg/ml) or in hypercholesterolaemic (61.9 ±39.8 mg/ml) and normocholesterolaemic (81.6 ± 38.7 mg/ml) SSc patients was detected (P>0.2).

SSc patients with hypercholesterolaemia had IMT values similar to normocholesterolaemic patients (0.10 ± 0.05 vs 0.088 ± 0.034, respectively; P>0.1).

Macrovascular involvement
IMT values were significantly higher in SSc (0.90 ± 0.037 mm) than in controls (0.69 ± 0.013 mm; P<0.01). The two SSc subsets had similar IMT values (Table 1). In SSc, no significant IMT differences were found between early (0.82 ± 0.032 mm), active (0.97 ± 0.038 mm) and late (0.77 ± 0.035 mm) NVC patterns. Compared with controls, only the active pattern showed a significant difference (P<0.01).

Only three patients had ABI values below the cut-off point of 0.9, and none had clinical evidence of symptomatic peripheral artery disease. SSc patients and controls had similar ABI values and no significant difference was found between patients with lSSc and dSSc (Table 1). Furthermore, the values of ABI were no different in the NVC patterns and controls. Hypercholesterolaemic patients had a significantly higher ABI index than normocholesterolaemic patients (1.167 ± 0.258 vs 1.022 ± 0.106; P<0.007). No significant difference was found between hypertensive and normotensive SSc patients.

No correlation was found between IMT and ABI values and CML AGE levels.


   Discussion
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 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Circulating and urinary AGEs were elevated and correlated with disease activity in rheumatoid arthritis while levels of pentosidine, a specific AGE species, were elevated in synovial fluid of rheumatoid arthritis [26]. Until now, plasma AGE levels have never been investigated in SSc. Our data clearly demonstrate that AGEs, despite normal glycaemic levels, are significantly increased in SSc with early microcirculatory changes.

In diabetes, chronic hyperglycaemia leads to the formation of AGEs, which are involved in the initiation and progression of diabetic macro- and microvascular complications. Structural and dysfunctional glomerular changes during microangiopathy are paralleled by AGE accumulation [8].

In euglycaemic conditions, AGEs may form as a result of increased oxidative stress. In SSc, oxidative stress may favour disease progression by inducing a rapid loss of endothelial cell function [4]. CML adducts are AGEs considered as markers of increased oxidative stress [27]. In SSc, AGEs may be involved, due to their association with ROS, in the initiation of tissue damage, and may be directly or indirectly involved in disease pathogenesis. The accumulation of CML adducts might be the result of oxidative stress due to local and systemic inflammation, or to vascular tone dysfunction. Basta et al. [7] have reported that AGEs, through their main receptor (RAGE), may prime pro-inflammatory mechanisms in endothelial cells, thereby amplifying pro-inflammatory mechanisms in atherogenesis and chronic inflammatory disorders. The binding of AGEs to RAGE leads to endothelial activation and changes in expression of pro-coagulant tissue factor, vascular cell adhesion molecule-1 and increased diffusional transit of solutes across monolayers. Activation of RAGE by AGEs triggers signalling pathways in endothelial cells that may induce a sustained inflammatory response, underlying the vascular involvement reported in the pathogenesis of diabetes (macro- and microangiopathy) as well as in ageing, renal failure and smoking [8]. The disease duration of the SSc patients in this study is more than 8 yr, suggesting that inflammatory phase of the disease has subsided and the observations of the study are primarily due to an ischaemic–reperfusion mechanism.

A two-hit model has been proposed to explain vascular dysfunction involving AGEs [28]: an initial AGE–RAGE interaction results in cellular activation and inflammation, followed by lipoprotein accumulation leading to chronic inflammation and further accelerated atherosclerosis.

Our data show that SSc patients have significantly higher carotid IMT, in agreement with previous works [10]. Usually, the modification of IMT at the level of the carotid artery is associated with cardiovascular (CV) risk factors or overt CV disease and atherosclerosis in other vascular beds [29]. In particular, IMT reflects not only early atherosclerosis but also non-atherosclerotic intimal reactions such as intimal hyperplasia and intimal fibrocellular hypertrophy [30]. We accounted for the possibility of confounders by matching subjects for age and sex, and statistically adjusting for other CV risk factors except for hypertensive and hypercholesterolaemic lSSc patients who were enrolled in the study. This might be expected to result in a bias in favour of the lSSc group. Subjects who smoked and a patient with renal failure were excluded from our study because smoking and uraemia are a risk factor for atherosclerosis and may themselves be associated with increased AGE levels [31]. However, no significant relationship between IMT and CML adduct levels was found. In our patients there was only an association between early microvascular, but not macrovascular, involvement and AGE levels.

We evaluated the morphological modification of the microvasculature with the nailfold capillary examination. The limitation of this technique is the lack of a quantitative measurement of the microcirculatory modification, but it still allows the detection of vascular abnormalities [14].

Recent studies have disclosed that AGEs, after binding to their receptor RAGE, may stimulate smooth muscle cell production of transforming growth factor-beta (TGFß) [32], suggesting that AGEs may have a 2-fold action in SSc. The first one is on the vessels and the second is on fibroblasts, favouring proliferation and smooth muscle cell hypertrophy.

In conclusion, our findings support a possible role for AGEs as biochemical agents of vascular damage in patients with apparent early SSc microvascular involvement, although they cannot yet be considered a specific marker for SSc. The role of AGEs on tissues in SSc remains to be elucidated. Furthermore, our study confirms an increased risk of macrovascular disease in SSc that does not seem correlated to AGE levels. Since vascular lesions are crucial in determining the prognosis in patients with SSc, AGEs may promote vascular damage by determining platelet activation, endothelial dysfunction, hypercoagulability and vascular smooth muscle cell proliferation. Further studies should evaluate whether lowering the concentration of CML adducts (i.e. with antioxidants) might represent a new therapeutic strategy in SSc.

The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Submitted 12 August 2005; revised version accepted 10 February 2006.
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