Our study had three main findings. First, we found higher plasma levels of the AGEs pentosidine, CML and THP in individuals with T1DM. Second, the AGE pentosidine was positively associated with CAC, an early marker of CVD. Third, the association of pentosidine with CAC was not explained by markers of LGI or ED.
Plasma AGEs in type 1 diabetes
This is the first larger study that has quantified multiple plasma AGEs with state-of-the-art ultra-performance liquid chromatography (UPLC) in combination with tandem mass spectrometry or, in case of pentosidine, with high-performance liquid chromatography (HPLC) and fluorescence detection. These techniques are considered to be the most accurate techniques for the measurement of AGEs at this moment. Unfortunately, because of acid instability of THP, it was not possible to measure THP with these techniques and therefore THP was measured with an ELISA. We investigated four out of many different AGEs and found that plasma levels of the AGEs pentosidine, CML and THP were significantly higher in individuals with T1DM as compared to controls, independent of age, sex, systolic and diastolic blood pressure, BMI, WHR, smoking, LDL and HDL cholesterol, triglycerides and eGFR. Levels of CEL were not statistically different. These results are in agreement with previous studies which were confined to small study populations [10, 11] and/or have used non-specific immunological techniques for the detection of AGEs [12, 13].
We did not find any association of plasma AGEs with HbA1c in the individuals with T1DM. These findings are consistent with many other studies that reported no association of plasma AGEs with HbA1c [31–35]. An explanation for this lack of association could be that AGEs can also be formed through other pathways, for example lipid peroxidation, besides glucose metabolism. Moreover, HbA1c and AGEs presumably reflect different pathways following hyperglycaemia and different timeframes of hyperglycaemia.
Associations between plasma AGEs and CAC in type 1 diabetes
We and others have previously shown that the prevalence of CAC is increased in T1DM [16, 17]. In individuals with kidney disease, the AGE pentosidine and the AGE-RAGE axis have been associated with arterial calcification [14, 18]. This is the first study that has examined the association of plasma AGEs with CAC in T1DM. We found higher levels of the plasma AGE pentosidine, but not CML, CEL and THP, in individuals with T1DM with a moderate to high compared to a low CAC score. These results are in line with a previous study that reported an independent positive association of pentosidine with CAC in individuals undergoing hemodialysis , while no association was found between CML and CAC. Moreover, Conway et al. and Orchard et al. showed that skin autofluorescence, as a possible reflectance of tissue AGE accumulation, was associated with CAC severity in T1DM [36, 37].
Potential mechanisms underlying the associations between AGEs and CAC
In contrast to the positive association of pentosidine with CAC, CML, CEL and THP were not associated with CAC. This could indicate that plasma pentosidine is a better reflection of total AGE formation than these other AGEs or that it is more precisely quantified with current methods. Alternatively, pentosidine differs from the other AGEs measured because it is known as a cross-linking AGE [4, 38], and it might be that cross-linking AGEs in particular are linked to CAC. THP was borderline significantly associated with CAC (p = 0.09). THP is one of the AGEs, next to CEL, which is formed from the reaction of the reactive dicarbonyl MGO with arginine or lysine, respectively, predominantly formed from intracellular glycation . Little is known about THP, but it could represent a better reflection of intracellular MGO-AGEs than CEL. Moreover, auto-antibodies against MGO-modified apolipoprotein B100 have been found to be inversely associated with CAC in patients with type 2 diabetes , also indicating a role of MGO in CAC. Therefore, if our results indeed reflect a causal link between AGEs and CAC, this might mean that cross-linking or intracellular glycation via MGO may stimulate the process of arterial calcification. The fact that we did find an association of pentosidine and THP with CAC in individuals with T1DM who were relatively young and had an early stage of CAC may indicate that increased levels of these AGEs are associated with early development of CAC. However, overall, our numbers of individuals with substantial calcification in the study are low, probably partially due to the relatively young age of the participants, which is why these analyses should be replicated in larger cohorts.
AGEs are able to induce LGI and ED [3, 5, 40], and markers of these processes are associated with coronary or carotid artery disease in T1DM [41, 42]. We demonstrated that the association of pentosidine with CAC was not explained (i.e. mediated) by LGI or ED. Other studies that investigated the mediating effect of LGI and ED in the association of AGEs with CVD in T1DM [3, 43] also found no mediating effect of either of these potential mechanisms. Therefore, other mechanisms besides LGI and ED might be involved in the association of AGEs with CAC. Recent publications show that AGEs-induced vascular calcification in rat vascular smooth muscle cells (VSMCs) is mediated by oxidative stress in vitro [44, 45], and oxidative stress may thus provide an additional mechanism explaining the association of AGEs with CAC.
The association between pentosidine and CAC might be causal since experimental studies have demonstrated a direct link of AGEs with calcification . In response to AGEs, aortic VSMCs differentiate into cells that exhibit an osteoblast-like phenotype characterized by the deposition of calcium into the extracellular matrix [19, 20]. These findings support a direct role of AGEs in the stimulation of CAC. Furthermore, the AGE-RAGE axis has been associated with arterial calcification in animal studies [22, 23]. However, in our study, we did not find an association between CML, a known ligand for RAGE, and CAC. Another pathway by which AGEs are thought to contribute to atherosclerosis is by the stimulation of apoptosis of endothelial progenitor cells (EPCs) and by the impairment of EPC functions [46, 47]. Indeed, skin autofluorescence, an estimate of tissue AGE accumulation, has been negatively associated with circulating EPCs . Furthermore, low levels of EPCs have been shown to be an independent determinant of carotid intima media thickness (cIMT) in young individuals with T1DM . Interestingly, in individuals with compared to those without coronary atherosclerosis, it was found that a higher percentage of EPCs express the osteoblastic marker osteocalcin (OCN) , which has been shown to correlate with markers of bone formation . Therefore, a particular subset of EPCs has been suggested to mediate abnormal vascular repair and vascular calcification .
Limitations of our study
Our study had a cross-sectional design; therefore we cannot draw any conclusions about causality in the association of AGEs with T1DM and CAC. Because not all atherosclerotic plaques contain calcium, the CAC score does not take non-atherosclerotic plaques into account. Despite this caveat, it has been shown that CAC is highly associated with total coronary atherosclerotic plaque burden . Additionally, individuals with diabetes are known to have a higher prevalence of medial calcification of the peripheral vessels. However, medial calcification of the coronary tree, not caused by atherosclerosis, is not very common in diabetes . It therefore seems likely that the CAC score measured in our study indeed reflects intima calcification associated with atherosclerosis.
We cannot discard the possibility that the use of a single or a selection of markers representing LGI and ED, respectively, may have led to an underestimation of their mediating effects in the association of AGEs with CAC. However, hsCRP is one of the most studied and best validated markers and is thought to represent overall LGI. Furthermore, hsCRP is known to be associated with coronary heart disease . sVCAM-1 and vWF are well known markers of ED .