**6. Anti-AGEs strategies**

Since AGEs were considered as an important factor in diabetes, the development of strategies against AGEs has been of interest. Substances, which can prevent or inhibit the formation of AGEs, as well as agents that can break AGEs or antagonize their signaling have been identified.

### **6.1. Inhibit the formation of AGEs**

**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 ad-

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

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

vanced glycation end products for vascular surgery. Eur J Vasc Endovasc Surg. 2008;36(2):125–31).

method deserves attention because of its prognostic value for healing [84].

method for foot ulceration risk of diabetic patients [77].

232 Wound Healing - New insights into Ancient Challenges

The first approach is to reduce the formation of AGEs by intervention at one of the steps involved such as aminoguanidine [85]. Aminoguanidine was one of the first substances identified limiting the formation of AGEs [86]. It is a highly reactive nucleophilic reagent that prevents the formation of AGEs by reacting with the carbonyl groups as well as alpha- and beta-dicarbonyl compounds such as methylglyoxal, glyoxal, and 3-deoxyglucosone. Particularly, long-term aminoguanidine treatment improved the nerve conduction deficit and myelinated fiber pathology in diabetic rats in vivo [87]. A double-blinded, multiple-dose, placebo-controlled, randomized clinical trial of aminoguanidine in diabetic patients with overt diabetic nephropathy (ACTION) was completed in 1998; ACTION I involved 690 type 1 diabetic patients and ACTION II involved 599 type 2 diabetic patients. These studies were designed to evaluate the safety and efficacy of aminoguanidine in slowing the rate of renal disease progression in patients with overt diabetic nephropathy. However, ACTION II was terminated prematurely due to safety concerns and apparent lack of efficacy. Reported side effects included gastrointestinal disturbance, liver function abnormalities, flu-like symptoms, and a rare vasculitis [88]. Its use in clinical practice is limited due to adverse effects in clinical trials with diabetic patients. Despite the earlier promising results, aminoguanidine is unlikely to be used for therapeutic purpose due to safety concerns and lack of efficacy [89]. Studies on topical application of aminoguanidine on the skin are still lacking.

Metformin that is routinely used in the treatment of type 2 diabetic patients has some structural similarities to aminoguanidine and it was shown that in type 2 diabetes, treatment with metformin reduced levels of methylglyoxal [90]. Pyridoxamine is a natural intermediate of vitamin B6 metabolism and a potent inhibitor of the formation of AGEs [91]. Pyridoxamine traps reactive carbonyl intermediates and scavenges ROS. In addition, it inhibits post-Amadori stages of AGEs formation. Marked effects of pyridoxamine such as delayed development of nephropathy and retinopathy have been demonstrated in diabetic rats. Its oral intake could result in potent inhibition of skin collagen CML formation in diabetic rats as well [92].

### **6.2. Anti-RAGE**

RAGE is the most studied receptor for advanced glycation end products. AGER1 has been shown to counteract AGEs-induced oxidative stress via inhibition of RAGE signaling [93]. sRAGE is a truncated splice variant of RAGE containing the ligand-binding domain but not the transmembrane domain and has been found in plasma. sRAGE is a soluble extracellular protein without signaling properties and it is considered as a natural decoy receptor of RAGE [30].

Blockage of RAGE by sRAGE may be a new target for therapeutic intervention in diabetic disorders. Potential protective effects of sRAGE have been shown in various diabetes and inflammatory models [94]. Interestingly, sRAGE could also attenuate impaired wound healing in diabetic mice. Other promising effects in various systems have been shown in vitro and in vivo with neutralizing anti-RAGE antibodies [31]. Possible approaches include gene knockdown of RAGE by siRNA or anti-sense and antagonism of RAGE with putative small molecular inhibitors against RAGE-induced signaling [95].

### **6.3. AGEs breakers**

Chemical substances and enzymes that are able to recognize and break the Maillard reaction crosslinks have been identified. Such chemical AGEs breakers are dimethyl-3-phenaylthiazolium chloride (ALT-711) [64], N-phenacylthiazolium and N-phenacyl-4,5-dimethylthiazolium. Promising results against diabetic cardiovascular complications have been reported, though their actual ability to cleave existing protein crosslinks in tissues has been questioned [96]. However, treatment with ALT-711 for 2 weeks had no effects on motor nerve conduction deficit, C-fiber-mediated nociceptive dysfunction, or impaired pressure-induced vasodilation in diabetic mice [97].

Interference with intrinsic AGE-detoxifying enzymes like fructosyl-amine oxidases (FAOXs), fructosamine-3-kinase (FN3K), and the enzymatic system of glyoxalase I is another interesting strategy to remove AGEs, because enzymes could recognize specific substrates [60]. It is reported that overexpression of glyoxalase I significantly inhibits hyperglycemia-induced intracellular formation of AGEs in bovine aortic endothelial cells and in mouse mesangial cells by reduction of intracellular oxidative stress and apoptosis [98]. The pharmacological induction of such enzymes could represent a novel future strategy against AGEs.

Other anti-AGE agents, including the thiazolidine derivative named OPB-9195, have been investigated [99]. OPB-9195 has been shown to prevent the progression of diabetic nephropathy in rats. It has also been demonstrated to improve motor nerve conduction slowing without affecting body weight and blood glucose levels. The improvement was associated with reduced serum AGEs levels and peripheral nerve expression of AGEs and immunoreactive 8-hydroxy-2-deoxyguanosine, which is a marker for oxidative stress-related DNA damage as well as an increase in peripheral nerve (Na+, K+)-ATPase activity [100].

Diabetic rats were found to have increased mesenteric vascular AGEs accumulation and mesenteric vascular hypertrophy, both of which were prevented by treatment with N- phenacylthiazolium bromide (PTB) [101]. A more recent study has demonstrated that although AGE-breakers such as PTB and N-phenacyl-4,5-dimethylthiazolium cleave model crosslinks in vitro, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats [59].

Benfotiamine, a lipophilic analogue of thiamine, is a transketolase activator that inhibits three of the four major biochemical pathways implicated in the pathogenesis of hyperglycemiainduced vascular damage: the hexosamine pathway, PKC activation, and AGEs formation [102]. In diabetic rats, nearly normalized nerve conduction velocity and inhibition of neural imidazole-type AGEs and CML formation after 6 months of benfotiamine treatment were observed [103]. In both nondiabetic and diabetic rats, benfotiamine also reduced inflammatory and neuropathic nociception [104].

### **6.4. Nutrient substance**

**6.2. Anti-RAGE**

234 Wound Healing - New insights into Ancient Challenges

**6.3. AGEs breakers**

in diabetic mice [97].

inhibitors against RAGE-induced signaling [95].

[30].

RAGE is the most studied receptor for advanced glycation end products. AGER1 has been shown to counteract AGEs-induced oxidative stress via inhibition of RAGE signaling [93]. sRAGE is a truncated splice variant of RAGE containing the ligand-binding domain but not the transmembrane domain and has been found in plasma. sRAGE is a soluble extracellular protein without signaling properties and it is considered as a natural decoy receptor of RAGE

Blockage of RAGE by sRAGE may be a new target for therapeutic intervention in diabetic disorders. Potential protective effects of sRAGE have been shown in various diabetes and inflammatory models [94]. Interestingly, sRAGE could also attenuate impaired wound healing in diabetic mice. Other promising effects in various systems have been shown in vitro and in vivo with neutralizing anti-RAGE antibodies [31]. Possible approaches include gene knockdown of RAGE by siRNA or anti-sense and antagonism of RAGE with putative small molecular

Chemical substances and enzymes that are able to recognize and break the Maillard reaction crosslinks have been identified. Such chemical AGEs breakers are dimethyl-3-phenaylthiazolium chloride (ALT-711) [64], N-phenacylthiazolium and N-phenacyl-4,5-dimethylthiazolium. Promising results against diabetic cardiovascular complications have been reported, though their actual ability to cleave existing protein crosslinks in tissues has been questioned [96]. However, treatment with ALT-711 for 2 weeks had no effects on motor nerve conduction deficit, C-fiber-mediated nociceptive dysfunction, or impaired pressure-induced vasodilation

Interference with intrinsic AGE-detoxifying enzymes like fructosyl-amine oxidases (FAOXs), fructosamine-3-kinase (FN3K), and the enzymatic system of glyoxalase I is another interesting strategy to remove AGEs, because enzymes could recognize specific substrates [60]. It is reported that overexpression of glyoxalase I significantly inhibits hyperglycemia-induced intracellular formation of AGEs in bovine aortic endothelial cells and in mouse mesangial cells by reduction of intracellular oxidative stress and apoptosis [98]. The pharmacological induc-

Other anti-AGE agents, including the thiazolidine derivative named OPB-9195, have been investigated [99]. OPB-9195 has been shown to prevent the progression of diabetic nephropathy in rats. It has also been demonstrated to improve motor nerve conduction slowing without affecting body weight and blood glucose levels. The improvement was associated with reduced serum AGEs levels and peripheral nerve expression of AGEs and immunoreactive 8-hydroxy-2-deoxyguanosine, which is a marker for oxidative stress-related DNA damage as well

Diabetic rats were found to have increased mesenteric vascular AGEs accumulation and mesenteric vascular hypertrophy, both of which were prevented by treatment with N-

tion of such enzymes could represent a novel future strategy against AGEs.

as an increase in peripheral nerve (Na+, K+)-ATPase activity [100].

An increasing list of natural antioxidants and chelating agents such as ascorbic acid, αtocopherol, niacinamide, pyridoxal, sodium selenite, selenium yeast, trolox, rivoflavin, zinc, and manganese has been shown to inhibit glycation of albumin in vitro [105]. Many spices and herbs could inhibit glycation of albumin in vitro as well, such as ginger, cinnamon, cloves, rosemary, and tarragon [106]. Besides, green tea, vitamins C and E, and a combination of Nacetylcystein with taurine and oxerutin could inhibit skin collagen glycation in mice [107]. In healthy human subjects, supplementation of vitamin C significantly decreased serum protein glycation [108].

Alpha-lipoic acid could reverse tail tendon collagen glycation in fructose-fed rats, an effect which was attributed to its endogenous antioxidant action, its ability to recycle ascorbic acid and GSH, as well as to its positive influence on glucose uptake and glycemia [109]. Blueberry extract, an AGE-inhibitor and C-xyloside, was tested for 12 weeks in female diabetic subjects. This treatment resulted in significant improvement of skin firmness, wrinkles, and hydration. However, it failed to show a significant decrease in the cutaneous content of AGEs [110].

### **6.5. Molecular chaperones**

Molecular chaperones like carnosine have shown promise in improving skin appearance in part by reducing the amounts of skin AGEs [111]. Yet, more studies are needed to address the accumulation of AGEs in diabetic wound.

In conclusion, AGEs are a heterogeneous group of molecules that form from the non-enzymatic addition of sugar moieties onto arginine and lysine residues of proteins, free amino groups on lipids, or guanine nucleic acids. The AGE-RAGE interactions play an important role in the diabetic wound healing process. The measurement of AGEs on the skin, namely, skin autofluorescence might have some value as a screening tool for diabetic foot ulcer, but until now, there is no strong evidence for other clinical use in diabetic wound. In addition, substances which can prevent or inhibit the formation of AGEs, as well as agents that can break AGEs or antagonize AGE/RAGE signaling have been identified.
