Skin collagen advanced glycation endproducts (AGEs) and the long-term progression of sub-clinical cardiovascular disease in type 1 diabetes

Historical considerations

In a recent study we have shown that expansion of the original set of 6–10 skin collagen AGEs and parameters of collagen solubility enhances the association between the skin markers of long-term glycemic damage and the progression of microvascular complications [5]. Notably the full set of all 10 AGE markers, including fluorescence, was associated with the risk of retinopathy, nephropathy and neuropathy progression in spite of adjustments for mean DCCT and EDIC A1c. Collagen glycation, i.e. fructose-lysine measured as furosine acid conversion product, was most consistently associated with either past or future progression of all three microvascular complications in EDIC. Adjustment for DCCT A1c did not nullify these robust associations except for nephropathy. Similarly, the associations between glucosepane and retinopathy, and MG-H1 with neuropathy were not nullified by adjustment for DCCT A1c, respectively. Finally, the association between DCCT mean A1c and each microvascular complication was nullified by adjustment for AGEs, as a group or individually. The above results raised the compelling question of the extent to which skin AGEs measured up to 19 years prior to assessment of subclinical indicators of macrovascular disease were also associated with their progression in type 1 diabetes, and whether specific AGEs were associated with specific subclinical macrovascular disease.

Associations between early glycation, advanced glycation and CAC deposition

In this study, only furosine (early glycation) and mean DCCT A1c were associated with CAC. This latter association was nullified upon adjustment for furosine indicating a close relationship between glycemia at the tissue level and the process leading to calcium deposition in the coronary arteries of individuals with type 1 diabetes. In view of the fact that none of the specific advanced glycation end products predicted this association, it is difficult to envisage a direct mechanistic role in this process. However, van Eupen et al., found that plasma AGEs are associated with CAC deposition [13]. In that regard CML was found to accelerate calcification in diabetic rat, possibly by engaging RAGE in macrophages and induction of the BMP-2-cbfα1-ALP-calcification cascade [14]. However, since the association uncovered by van Eupen et al. was preferentially with pentosidine, a marker of increased ascorbic acid oxidation [15], a more likely explanation is that CAC is accelerated by hyperglycemia mediated oxidant stress and NADPH oxidase activity which triggers BMP activation and calcium deposition [15].

Possibly implicating indirect effects in which CML-rich proteins e.g. stimulate BMP deposition [16] into the arterial wall.

Association with intima-media thickness

In contrast to coronary artery calcium deposition, a causal relationship between IMT and glycation is strongly suggested from the observation that IMT is associated with multiple AGE crosslinks, such as glucosepane and pentosidine, as well as methylglyoxal hydroimidazolone MG-H1. Methylglyoxal itself is a source of methylglyoxal-derived imidazole (MODIC) crosslinks which, although not measured here, are also elevated in skin collagen from diabetic individuals [17]. Together, these three arginine-lysine AGE crosslinks may participate in decreasing collagen solubility, explaining the significant inverse relationship noted between pepsin solubility and IMT change between year 1 and 6. A role for glucosepane crosslinks in this process is strongly supported by the observation that glucosepane is the single major AGE and crosslink in skin collagen from individuals with diabetes [17]. Moreover, the Oslo Diabetes Study recently reported similar associations between skin glucosepane and both IMT and pulse wave velocity in 27 participants with type 1 diabetes of very long duration [18].

Somewhat puzzling is that the association between AGEs and IMT became non-significant at year 12, though both furosine and glucosepane were borderline significant reinforcing the potential role of matrix crosslinking in IMT. One explanation is the contribution of lipid abnormalities became with time less important as determinant of IMT progression as that the number of participants on statins increased from 2.94 to 13.2 and 49 % at EDIC year 1, 6 and 12, respectively. In addition, glycemia improved in the control group with mean A1c values decreasing concomitantly from 8.9 to 8.6 and 8.3 %, respectively. These two metabolic effects likely altered either the IMT progression rate and/or the collagen turnover rate, resulting in weakened ability for skin AGEs to predict the very long-term IMT progression rate.

Association with left ventricular mass and LV mass/end diastolic volume ratio

A similar relationship between collagen solubility and LV mass was observed, although neither furosine nor glucosepane were themselves associated with LV mass. This may suggest that cardiac hypertrophy is only indirectly influenced by hyperglycemia, perhaps via methylglyoxal levels, as suggested by the association between MG-H1 and LV mass. The observation that the ratio of LV mass/end diastolic volume was associated with furosine and MG-H1, as well as the glycoxidation product CML, is surprising because CML was not associated with any of the other subclinical complications. In contrast, numerous studies showed that serum or plasma CML is strongly associated with cardiovascular disease in diabetes and aging [19–21]. The potential mechanism and implication of these findings is that CML formation from fructose-lysine could be catalyzed by Cu²⁺ [22]. Indeed Cu²⁺ chelation was shown to ameliorate diabetic cardiomyopathy [23], thus supporting the proposed association between abnormal copper metabolism and diabetic cardiomyopathy [24].

Potential role of methylglyoxal in subclinical CVD complications

The observation that MG-H1 is associated with IMT independently of glycemia, LV mass and LV mass/EDV raises the question of whether methylglyoxal plays a mechanistic role in the pathogenesis of atherosclerotic complications. This proposition is supported by the lower levels of MG-H1 (and glucosepane!) in EDIC subjects with prior DCCT intensive control [5] and the findings of lower IMT levels in the intensive treatment group of the whole DCCT/EDIC cohort [1]. A direct role for methylglyoxal lysine dimer (MOLD) [25] or MODIC [17] crosslinks has been alluded to, though these are present in amounts 100 times lower than glucosepane in skin from diabetic individuals [17]. Blocking of arginine residues by MG-H1 together with crosslinking might contribute to lowering collagen turnover and thickening of the artery wall. Indeed Chong et al. found the methylglyoxal modified collagen binds less well phagocytes thought to play a role in collagen turnover [26]. However, there is also the possibility that higher methylglyoxal levels could stimulate collagen deposition. Several authors noted that cells incubated with methylglyoxal modified collagen induced collagen expression, TGF β1 expression [27] and promoted myofibroblast differentiation [28]. Methylglyoxal also exerts it’s toxicity by inhibiting cell binding to the modified matrix [29] resulting in anoikis [30]. Finally, a profibrotic effect was directly demonstrated when methylglyoxal given orally to mice lead to kidney collagen accumulation [31].

Finally, as noted above, this study’s findings parallel the recently observed glucosepane –IMT association in the Oslo study [18], and also the similar recent observation by the same investigators of an association between impaired left ventricular dysfunction, MG-H1 and glyoxal hydroimidazolone G-H1, with the caveat that MG-H1 was measured in the serum and G-H1 in skin [32]. Taken together, they raise the important question of whether a combination of glycation burden and altered cell signalling by the glycated extracellular matrix is the elusive mediator in the sclerotic process that underlies the pathogenesis of accelerated cardiovascular disease in diabetes.

Limitations in the current study

The major limitation of our study is that the demonstrated associations between skin AGEs and subclinical CVD outcomes cannot prove a cause and effect relationship. Moreover, none of the many specific skin AGE molecules correlated consistently with any specific CAC, carotid IMT or CMRI outcome, undoubtedly reflecting the complex pathogenesis of CVD in T1DM. Nonetheless, the false discovery rate was held to 0.05, minimizing the possibility that these are chance findings resulting from the multiple statistical tests that were conducted. Moreover, the positive association between plasma pentosidine and CAC has been found in T1DM [13] supports the proposition that AGEs are possibly involved in the development of CAC in individuals with T1DM. Similarly, specific AGE combinations, notably pentosidine and CEL, were strongly associated with micro- and macrovascular complications in the Medalist Study [33]. Thus, these data are in keeping with the present study findings and reinforce them, even though p values were not as strong as for microvascular disease outcomes [5]. Yet, in spite of these important findings, a major practical limitation is that biopsies for risk biomarker assessment are unlikely to be routinely implemented, and that therefore non-invasive methods will be needed to assay specific AGEs in skin.