**4.1. AGEs and inflammation phase in a diabetic wound**

In this part, the interaction between AGEs and RAGE could not be neglected. Data support that it negatively affects various aspects of inflammatory response in diabetic wound. Increased RAGE expression has been found in wound tissues from diabetic mice in parallel with increased AGEs accumulation and increased inflammation [39]. In Shuliang Lu's study [40], compared with the controls, enhanced expression of RAGE and accelerated cell apoptosis were observed in the burned skin of diabetic rats. The altered expression pattern of inflammatory cytokines and oxidative markers between diabetic and control groups revealed delayed neutrophil chemotaxis and respiratory burst. Furthermore, the results in vitro showed that exposure to AGEs inhibited the viability of neutrophils, promoted RAGE production and cell apoptosis, which was consistent with the findings in vivo. Besides, the mice fed with a rich AGEs diet demonstrated an increased and sustained inflammatory phase compared with those fed with a low AGEs diet [41]. In vitro, human neutrophils were isolated and treated with AGE-human serum albumin. Cell viability and reactive oxygen species levels were increased [42].

In keratinocytes, AGEs decrease cell viability and migration and induce the expression of proinflammatory mediators as well [43]. Various growth factors or proteins significant for cellular functions may be glycated inhibiting their functions [44]. Furthermore, treatment of murine macrophages with AGEs resulted in increased levels of iNOS, which has been found to be increased in diabetic wounds [45]. Macrophages play a critical role in wound healing and can be activated to two distinctive phenotypes in vitro: M1 and M2 [46]. It demonstrated insufficient M1 in the early stage but excessive M2 in the later proliferative phase. The macrophage activation markers were correlated with the instructive T helper cell type 1 (Th1)/ Th2 cytokines in both groups. Other studies suggested that RAGE expression has been strongly linked to the expression of matrix metalloproteinases (MMP)-1, MMP-3, MMP-9, mainly through RAGE engagement by AGEs [47]. In addition, AGEs induced the production of oxygen-reactive intermediates from inflammatory and endothelial cells via NADPH activation probably through their receptors, promoting further cellular activation and proinflammatory cytokine expression [48].

### **4.2. AGEs and proliferation phase in the diabetic wound**

It has been reported that the presence of AGEs not only affected the interaction of the fibroblasts with the extracellular matrix but also reduced the amount of the extracellular matrix as well. This effect would influence almost all the cells involved in the proliferative process. In vitro incubation of human dermal fibroblasts with pentosidine or pyrraline resulted in reduction of the extracellular matrix content, which was collagen and proteoglycan [49]. In vitro study showed that type I collagen synthesis from fibroblasts was not affected in AGEs; however, the synthesis of hyaluronic acid was significantly reduced [50]. It also showed a direct effect of AGEs on fibroblast synthetic capacity and explained the decreased extracellular matrix in the diabetic wound. Because hyaluronic acid is associated with cellular locomotion, migration, and proliferation, decreased content in the matrix could result in disturbance of the proliferative phase of the healing process. Besides, histological evaluation of wound sections from diabetic rats demonstrated absence of actively migrating inflammatory cells toward the central region of the wound, reduced angiogenesis, a decrease in the secretion of extracellular matrix, and then poor granulation tissue formation [51].

AGEs may also change the action of the wound-associated cytokines and growth factors, by affecting the growth factors or their receptors. Glycation of bFGF, after its incubation with glucose-6-phosphate (G6P) or fructose, resulted in decreased heparin-binding capacity, which is necessary for the binding of bFGF to its receptor. A reduction in its mitogenic activity was also observed compared with the control bFGF group [45]. In addition, incubation of FGF2 with G6P resulted in glycation of FGF2. Bovine aortic endothelial cells incubated with the glycated FGF2 showed a reduction of proliferation, decreased mean capillary length and new blood vessel formation, a weaker increase in tyrosine-phosphorylated proteins, especially ERK-1 and ERK-2 [52] .While ECV304 cells were incubated with glyoxal proteins, a significant reduction in the free amino acid groups of the EGF receptors was found. It also showed that the EGF-induced recruitment and activation of the downstream effectors of the EGF receptor pathway PLCg1 and ERK1/2 was inhibited by AGEs [53]. Furthermore, an animal study using rats with subcutaneous implantation of sponge disks showed that pretreatment of animals with D-glucose resulted in a reduction in the angiogenesis measured by the hemoglobin content of the implanted disks and a reduction in the granulomatous response compared with control groups.

### **4.3. AGEs and remodeling phase in the diabetic wound**

Every stage of normal wound healing appears to be disrupted in the diabetic patients. A derangement in wound contraction and remodeling is expectable. A large body of evidence supports the effects of AGEs on the phenotype, invasiveness, behavior, and survival of the cells and cell membrane interactions with extracellular matrix. Animal data showed that diabetic mice with lower circulating and tissue-bound AGEs as a result of exposure to a diet low in AGEs, showed improved reepithelialization, granulation tissue formation and angiogenesis compared with the group fed with a diet high in AGEs [54].

Th2 cytokines in both groups. Other studies suggested that RAGE expression has been strongly linked to the expression of matrix metalloproteinases (MMP)-1, MMP-3, MMP-9, mainly through RAGE engagement by AGEs [47]. In addition, AGEs induced the production of oxygen-reactive intermediates from inflammatory and endothelial cells via NADPH activation probably through their receptors, promoting further cellular activation and proinflammatory

It has been reported that the presence of AGEs not only affected the interaction of the fibroblasts with the extracellular matrix but also reduced the amount of the extracellular matrix as well. This effect would influence almost all the cells involved in the proliferative process. In vitro incubation of human dermal fibroblasts with pentosidine or pyrraline resulted in reduction of the extracellular matrix content, which was collagen and proteoglycan [49]. In vitro study showed that type I collagen synthesis from fibroblasts was not affected in AGEs; however, the synthesis of hyaluronic acid was significantly reduced [50]. It also showed a direct effect of AGEs on fibroblast synthetic capacity and explained the decreased extracellular matrix in the diabetic wound. Because hyaluronic acid is associated with cellular locomotion, migration, and proliferation, decreased content in the matrix could result in disturbance of the proliferative phase of the healing process. Besides, histological evaluation of wound sections from diabetic rats demonstrated absence of actively migrating inflammatory cells toward the central region of the wound, reduced angiogenesis, a decrease in the secretion of extracellular matrix,

AGEs may also change the action of the wound-associated cytokines and growth factors, by affecting the growth factors or their receptors. Glycation of bFGF, after its incubation with glucose-6-phosphate (G6P) or fructose, resulted in decreased heparin-binding capacity, which is necessary for the binding of bFGF to its receptor. A reduction in its mitogenic activity was also observed compared with the control bFGF group [45]. In addition, incubation of FGF2 with G6P resulted in glycation of FGF2. Bovine aortic endothelial cells incubated with the glycated FGF2 showed a reduction of proliferation, decreased mean capillary length and new blood vessel formation, a weaker increase in tyrosine-phosphorylated proteins, especially ERK-1 and ERK-2 [52] .While ECV304 cells were incubated with glyoxal proteins, a significant reduction in the free amino acid groups of the EGF receptors was found. It also showed that the EGF-induced recruitment and activation of the downstream effectors of the EGF receptor pathway PLCg1 and ERK1/2 was inhibited by AGEs [53]. Furthermore, an animal study using rats with subcutaneous implantation of sponge disks showed that pretreatment of animals with D-glucose resulted in a reduction in the angiogenesis measured by the hemoglobin content of the implanted disks and a reduction in the granulomatous response compared with

Every stage of normal wound healing appears to be disrupted in the diabetic patients. A derangement in wound contraction and remodeling is expectable. A large body of evidence

cytokine expression [48].

228 Wound Healing - New insights into Ancient Challenges

control groups.

**4.2. AGEs and proliferation phase in the diabetic wound**

and then poor granulation tissue formation [51].

**4.3. AGEs and remodeling phase in the diabetic wound**

**Figure 2.** Immunohistochemical localization of AGE and RAGE proteins in dermis is shown. A, B, The distribution of AGE in normal skin tissue (A) and in diabetic skin tissue (B). AGE protein staining was expressed faintly at dermal matrices and cells in control skin but was prominent at the dermal matrices, cells, and basement membrane of vessels in the diabetic skin. C, D, The distribution of RAGE in normal skin tissue (C) and in diabetic skin tissue (D). RAGEpositive cells appear brown, and a light hematoxylin counter stain was used to visualize nuclei. More positive cells were detected in diabetic dermal layer than in control. [Original magnification, ×200 (A, B); original magnification, ×400 (C, D)]. (Illustrated from Ref. [55]).

The balance between proliferation and apoptosis of skin cells is responsible for the success of the wound healing process. Recent reports have shown that AGEs formation participates in dermatologic problems in diabetes. Shuliang Lu's group reported that effects of dermal microenvironment glycosylation. Histology and immunohistochemical staining were performed on type 2 diabetic and nondiabetic skin specimens to determine the distributions of proliferating cell nuclear antigen, apoptotic cells, AGEs and RAGE. Diabetic skin has degenerative, loosely arranged collagen and increased apoptotic cells compared with normal skin. Expression of AGEs and RAGE were increased in diabetic skin. Glycosylated matrix induced cell cycle arrest and apoptosis of cultured dermal fibroblasts, whereas application of RAGE-blocking antibodies redressed these changes [55] (**Figure 2**).

AGEs may alter the signaling of the wound cytokines and growth factors by disrupting the structure of either the growth factors or their receptors. Glycation of fibroblast growth factor (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 responsible for the derangement of the remodeling stage [42].

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 important part in the cell dysfunction [40].

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 promotion of MDA production were shown [63].
