Introduction
Advanced glycation end products (AGEs) accumulate in long lived tissue during lifetime, which is regarded as a process of normal ageing. AGE accumulation results from a combination of hyperglycaemia, hyperlipaemia, oxidative/carbonyl stress and also decreased renal clearance of AGE precursors. Accelerated AGE accumulation is therefore, seen in diabetes mellitus and renal failure and contributes to long term complications and mortality [
1‐
3] AGEs also play a major pathogenetic role in atherosclerosis [
4‐
7]. Cross linking of AGEs with collagen and elastin within the vascular wall contributes to arterial stiffness [
1,
5,
6]. AGEs also alter the extracellular matrix and promote atheroma formation [
6‐
8]. Furthermore, activation of cell membrane receptors including the receptor for AGE (RAGE) leads to activation of several oxidative and inflammatory pathways [
1,
7,
8]. This cascade leads to endothelial dysfunction, vascular inflammation and production of reactive oxygen species [
1]. These mechanisms accelerate the formation of atherosclerosis [
1]. Finally, AGE accumulation and overexpression of RAGE within the plaque may promote plaque instability [
6].
Accumulation of AGEs in tissue can be assessed through illumination of the skin, a technique named skin autofluorescence (SAF), which has previously been validated by simultaneous measurements of SAF and contents of specific AGE assessments in skin biopsies [
9‐
11]. Although the fluorescent characteristics in this method are not specific for fluorescent AGE, multiple validation studies have shown convincingly and consistently that SAF has a strong correlation with specific AGE content in skin biopsies [
9‐
11]. The correlation between SAF with the fluorescent AGE pentosidine is very high:
r = 0.87. Surprisingly, not only fluorescent AGE (pentosidine) but also non fluorescent AGE (N-carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL)) in the skin biopsies showed great correlation with SAF. Skin AGE content explained the major part of the variance (up to 76%) in the SAF signal in a pooled analysis of three validation studies [
10]. We earlier reported increased SAF in several groups of patients with increased AGEs formation such as diabetes mellitus [
3,
12], decreased clearance of AGEs such as renal failure [
13] and overt atherosclerotic disease such as patients with stable coronary artery disease [
14]. Earlier studies have already demonstrated an elevated serum level of AGEs in patients with carotid disease; a positive association between intima media thickness (IMT) and serum levels of AGEs were found in population with renal insufficiency starting dialysis [
15]. Baumann et al. showed that the AGE N-epsilon-carboxymethyllysine (CML) is present in the subendothelial space of atherosclerotic human carotid artery material of normoglycaemic subjects with a mean age of 50 years [
16]. However, the level of SAF as a measurement of increased tissue accumulation of AGEs rather than plasma AGEs level has not yet been studied in patients with carotid artery stenosis. Therefore, the present study evaluates SAF in patients with atherosclerotic carotid artery stenosis with or without coexisting peripheral arterial disease. The possible value of SAF as a risk indicator in this specific cohort of patients is further discussed.
Discussion
In the current study we showed that SAF is significantly elevated in patients with carotid artery stenosis and PAOD compared to controls. Two findings were especially remarkable.
First, PAOD proved to be an important determinant of SAF. High SAF values were especially found in the group with carotid artery disease and coexisting PAOD. Even within the group of carotid artery disease, SAF was significantly higher when there was coexisting PAOD with a SAF of 3.36 versus 2.64 (P = 0.003). This could not be explained by differences in baseline characteristics between the patients with carotid artery stenosis with or without PAOD. Also in linear regression analysis, PAOD proved to be a strong determinant of SAF, even more so then diabetes. This finding is interesting and warrants further research. It suggests that SAF should primarily be seen as an indicator of widespread atherosclerotic disease. Currently, a study analysing SAF in patients with primary peripheral artery disease has been initiated.
The second remarkable finding was that SAF was specifically elevated for patients in the age group 50–60 years. Furthermore, the relation between age and SAF, as normally seen, was present in the control group but absent in the patients with carotid artery stenosis.
The marked elevation of SAF levels in carotid artery stenosis was demonstrated in non-diabetic patients as well as in diabetics. A high SAF in diabetes mellitus may therefore, be regarded as an indicator of widespread atherosclerotic disease and not only a manifestation of diabetes mellitus per se. Mulder et al. previously reported similar findings in a cohort with stable coronary artery disease [
14]. SAF was significantly increased in stable coronary artery disease compared with controls, irrespective of diabetes, current smoking and renal function. Earlier studies in patients with diabetes mellitus also showed that SAF is manifestly increased in these patients. The level of AGE accumulation correlated with the duration and the grade of complications of diabetes [
2,
3,
20].
Univariate and multivariate analysis of associations with SAF in the present study supports this interpretation in our study group of patients with carotid artery stenosis. SAF was univariately associated with age, smoking, diabetes mellitus, renal dysfunction, dyslipidaemia and the presence of carotid artery disease and peripheral arterial occlusive disease which is in concordance with earlier studies [
2,
3,
10,
13] Multivariately, SAF was determined by age, smoking, diabetes mellitus, renal dysfunction and peripheral arterial occlusive disease. Therefore, again, increased SAF may rather be regarded as an indicator of widespread atherosclerotic disease than as a specific identifier of carotid atherosclerotic disease.
This study however, has several limitations. At baseline, despite matching for sex and age in the control group, there were differences between the patient and control groups including presence of diabetes, smoking behaviour, hypertension, use of antihypertensive medication and use of statins. This may be considered a source of confounding. However, it does represent the expected increased presence of risk factors for cardiovascular disease in patients with carotid artery stenosis compared to healthy people.
In the control group asymptomatic atherosclerosis may have existed as no carotid ultrasound or other vascular tests were performed in the control group. If asymptomatic atherosclerosis was present in the control group this would result in an underestimation of the difference between the control group and patient group. The differences we found between the control group and the group with atherosclerotic carotid stenosis and peripheral artery disease therefore, would be even greater in a better selected control group.
Our study is also limited by the small number of patients. The fact that backward linear regression did not show carotid artery disease or coronary artery disease to be independent determinants of SAF may therefore, be caused by the lack of power. Mulder et al. has earlier shown that SAF indeed is elevated in stable coronary disease [
14]. A large study evaluating the relationship between SAF en intima media thickness is currently being executed to further clarify this issue. What may be the use of the present results? The correlation of SAF with traditional cardiovascular risk factors, the presence of diabetes, renal insufficiency and peripheral arterial occlusive disease could indicate that SAF may be an indicator of high cardiovascular risk patients. Moreover, since SAF represents end organ damage it may predict morbidity and mortality more than the classical risk factors for atherosclerosis separately. For patients with type 2 diabetes this has already been established since SAF added prognostic information to the UKPDS risk calculator in predicting mortality [
21]. Strong predictive results of SAF for cardiovascular mortality were also seen in patients with renal failure [
22]. The same may be true for patients with carotid artery stenosis and peripheral artery disease. Prospective follow-up studies are necessary to elucidate this. Moreover, spectroscopy techniques show promising results for imaging vulnerable plaques and the near future will tell whether they really shine light on unstable cardiovascular disease.
In conclusion, skin autofluorescence is increased in patients with carotid artery stenosis and PAOD compared to healthy controls. SAF is especially elevated in the age group of 50–60 years, suggesting an increased accumulation of tissue AGEs in these patients. The univariate and multivariate associations of SAF with age, smoking, diabetes mellitus, presence of renal insufficiency and peripheral arterial occlusive disease suggest that increased SAF should primarily be seen as an indicator of widespread atherosclerotic disease. These associations further underscore the important role of AGEs in the pathophysiology of atherosclerotic disease. SAF might therefore, be an indicator of an overall high burden of widespread atherosclerosis as a consequence of AGE accumulation. Future research should investigate the use of SAF as a superior predictor of cardiovascular events in this population.
Conflict of interest
A.J. Smit is founder of DiagnOptics B.V., Groningen, The Netherlands, manufacturer of the AGE Reader™, which has been used to perform skin autofluorescence measurements as reported in this manuscript.