**5. Measurement of AGEs**

(bFGF), after its incubation with intracellular sugars resulted in decreased heparin-binding capacity, which is essential for the ligation of bFGF to its receptor [56]. Bovine aortic endothelial cells incubated with the glycated FGF-2 showed a reduction in the proliferation, decreased mean capillary length and overall new blood vessel formation as well as a clearly weaker increase in tyrosine phosphorylated proteins, particularly ERK-1 and ERK-2 [57]. It has also been shown that the epidermal growth factor (EGF)-induced recruitment and activation of the downstream effectors of the EGF receptor pathway, the serine-threonine kinases ERK1/2, was inhibited by glyoxal and methylglyoxal [58]. Moreover, diabetic rats exhibited poor TGF-β1 expression in fibroblasts [20]. The effects of TGF-β1 on extracellular matrix synthesis and cellular phenotypes are crucial for the final stage of wound healing, suppression of MMP secretion, differentiation of fibroblasts into contractile myofibroblasts, and cellular programmed death. The increased levels of MMPs and proinflammatory cytokines in the context of a vicious self-perpetuating cycle of an inappropriately inflammatory response may be respon-

Literature data also support that in the presence of AGEs, not only the interaction of the fibroblasts with the extracellular matrix is affected, but also the amount of the extracellular matrix constituents normally secreted by cells is reduced. This effect would deprive almost all the cells involved in the proliferative process of the extracellular scaffold [59]. In vitro, studies showed a direct effect of AGEs on fibroblast survival and synthetic capacity, which might partially explain the decreased extracellular matrix density in the diabetic non-healing wounds. Human adult primary skin fibroblasts treated with CML-collagen (glycated collagen) showed a time- and dose-dependent apoptosis, which was threefold compared with that of control collagen-treated fibroblasts [60]. An insight has been gained into the mechanisms that underlie the AGEs-promoted cell apoptosis. The proapoptotic intracellular signaling consists of involving a chain of events, such as the generation of intracellular reactive oxygen species, which cause the activation of mitogen-activated protein kinase (MAPK) pathways and finally the induction of transcription factor FOXO1 and caspase-3. In addition, keratinocytes pretreated with glycoaldehyde and type I collagen exhibited reduced migration and an impaired adhesive capacity [61]. These effects were caused by conformational changes on the glycated collagen, which altered the effective receptor binding [62]. Furthermore, increased AGE/RAGE expression has been found in the diabetic skin. The apoptotic effects could be reversed by the application of RAGE antibodies, suggesting that AGEs and RAGE interaction played an

Another study of Shuliang Lu's group demonstrated that thickness of abdominal dermis from diabetic patients was reduced with obscured multilayer epithelium and disorganized collagen fibrils, as well as with chronic inflammatory cell infiltration. It was also shown that the prominent accumulation of AGEs in the diabetic skin induced an oxidative damage of fibroblasts and thus contributed to the thinner thickness of diabetic abdominal dermis. In vivo, less hydroxyproline, higher myeloperoxidase activity, and increased malondialdehyde (MDA) content were found in the diabetic skin. In vitro, the time- and dose-dependent inhibitory effects of AGE-bovine serum albumin (BSA) on fibroblast viability and the promo-

sible for the derangement of the remodeling stage [42].

230 Wound Healing - New insights into Ancient Challenges

important part in the cell dysfunction [40].

tion of MDA production were shown [63].

Since the biochemistry of AGEs has been widely discussed, the effort to develop the measurement has been made as well. Blood is more accessible for repeated measurements of AGEs than tissue-requiring biopsies, but plasma AGEs assays have not yet been shown to be directly related to tissue AGEs content [64]. As tissue accumulation of AGEs proves a long-term course with low reversibility, the AGEs accumulated in long-lived tissue proteins like skin collagen may be a carrier of metabolic memory over a long period, even years [65]. Because certain AGEs have intrinsic fluorescence properties, tissue AGEs accumulation can be assessed as skin autofluorescence (SAF) by the AGE Reader™ (Diagn-Optics, Groningen, the Netherlands) easily and noninvasively [66], instead of other traditional invasive techniques (**Figure 3**). Several studies demonstrated that the SAF value obtained from the skin of the lower arm correlated with content of both fluorescent and nonfluorescent AGEs measured from skin biopsy specimens on the same site [67]. It is strongly related to AGEs accumulation in healthy subjects, and diabetic and hemodialysis patients over a broad age range [68]. There is no surprise that SAF values of the diabetic patients were significantly higher than the healthy population [69, 70], and SAF values of diabetes with complications were elevated compared with those without complications [71, 72]. So far, there have been SAF referential values and its influential factors for healthy Dutch, Slovakian, and Chinese people, and this offers the baseline values to further analyze diabetes and its chronic complications [73–75].

However, studies of SAF on predicting the diabetic vascular complications were of considerable clinical heterogeneity, and different experimental results were adjusted in different conditions [76, 77]. Recent publications have suggested that SAF serves as a marker of vascular damage [78], as well as a predictor of cardiac mortality in patients with type 1 and 2 diabetes. In Shuliang Lu's group, Liu Chuanbo et al. did a cross-sectional survey consisting of 118 consecutive hospitalized diabetic foot patients. The diabetic microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular referring to coronary heart disease (CHD), cerebrovascular disease (CVD), or peripheral artery disease (PAD) complications were evaluated .The mean SAF value was 2.8 ± 0.2 AU. SAF was significantly associated with diabetes duration and blood urea nitrogen (R2 = 62.8%; P < 0.01). Moreover, in logistic regression analysis, SAF was significantly associated with retinopathy (odds ratio [OR] = 40.11), nephropathy (OR = 8.44), CHD (OR = 44.31), CVD (OR = 80.73), and PAD (OR = 5.98 × 109). Therefore, SAF, reflecting tissue accumulation of AGEs is independently associated with the presence of micro-and macro-vascular complications in diabetic foot ulcer (DFU) patients [79]. Similarly, SAF values were significantly higher in type 1 diabetic patients with microvascular complications, like neuropathy, compared to those without complications [80]. Lisanne et al. reported that SAF was independently associated with all-cause mortality and fatal or non-fatal major adverse cardiovascular events in patients with peripheral artery disease after a 5-year follow-up [81].

**Figure 3.** (A) The autofluorescence reader illuminates a skin surface with an excitation light source between 300–420 nm. Only light from the skin is measured with a spectrometer. (B) Various fluorescence spectrum results from different subjects: healthy subject (black line), diabetic patient without cardiovascular complications (blue line), diabetic patient with peripheral artery occlusive disease (green line), hemodialysis patient with recent myocardial infarction (red line). I = intensity (a.u.). (Illustrated from Meerwaldt R, van der Vaart MG, van Dam GM, et al. Clinical relevance of advanced glycation end products for vascular surgery. Eur J Vasc Endovasc Surg. 2008;36(2):125–31).

The use of SAF in the diabetic wound was also discussed. Meerwaldt et al. showed that SAF was increased and correlated with the Wagner score in DFU with neuropathy. SAF correlated inversely with nerve conduction velocity and amplitude [82]. Lapolla et al. found that AGEs were higher in type 2 diabetics with PAD compared to those without PAD; AGEs were correlated inversely to ABPI, even after correction for other cardiovascular risk factors [83]. SAF is independently associated with diabetic foot ulcerations. It might be a useful screening method for foot ulceration risk of diabetic patients [77].

Use of SAF measurement to assess foot vulnerability and to predict DFU events in high-risk patients seems to be promising. Yet, Vouillarmet et al.'s study in a subgroup of patients with an active DFU showed a nonsignificant correlation (P = 0.06) between SAF and the incidence of healing at 2 months, but the magnitude of effect is still high. Therefore, researchers deemed that the small number of patients may be the reason for the lack of statistical power. SAF method deserves attention because of its prognostic value for healing [84].

However, long-term studies validating both the specificity and sensitivity of this investigation, and its link to certain AGEs, remain to be confirmed. The importance of its use in the followup of DFU is not reported. Thus, AGEs might have some value as a screening tool for DFU, but there is no strong evidence for other clinical use in diabetic wound, and AGEs measurements should not be considered a replacement for HbA1c as a marker of glycemic control.
