**3. Potential implications for pharmacological modulation of AGE-RAGE axis activity**

In an attempt to counteract the inflammatory effects of AGE-HSA, we selected three RAGE inhibitors: a soluble form of RAGE (sRAGE; R&D systems), used at 0.25, 0.5 and 1 ng/mL; a monoclonal antibody against RAGE (anti-RAGE; R&D systems), used at 5, 10 and 20 μg/ mL; and the RAGE antagonist FPS-ZM1 (Calbiochem, Merck Millipore), used at 125, 250, 500 and 1000 nM. HUVECs were pre-treated with different concentrations of these inhibitors and 50 min later treated with 25 μg/mL AGE-HSA. The inhibitory effect of these agents on the expression of VCAM-1 and ICAM-1 in HUVECs was studied.

However, contrary to what we expected, blockade of RAGE by using sRAGE, anti-RAGE antibody and FPS-ZM1 was not sufficient to counteract the AGE-induced VCAM-1 and ICAM-1 up-regulation at any of the concentrations tested under our experimental conditions. Our results may suggest that on endothelium, other RAGE-independent mechanisms may also be acting to increase adhesion molecule expression and induce inflammation. Other possible explanation for these results is that the pharmacological tools actually available to block RAGE activity are not able to block the effects of AGEs at the endothelial level. However, the results obtained on *in vivo* models of disease are promising, as we comment below.

To investigate the effects of RAGE blockade in pathological conditions, many studies have used soluble forms of RAGE or anti-RAGE antibodies, which can antagonize RAGE-ligand interaction to competitively inhibit the activation of RAGE signaling [39, 44, 45]. Evidence from these studies has shown that RAGE blockade protected against various disease challenges. Soluble RAGE, which competes with cellular RAGE for ligand binding, has been able to reduce inflammatory responses in several models tested. Streptozotocin-induced diabetic apoE−/− mice treated with once daily injections of murine sRAGE showed suppressed acceleration of atherosclerotic lesions in a dose-dependent manner [46]. In parallel with decreased atherosclerotic lesion area and the complexity of the atheroma plaque composition, the levels of tissue factor, VCAM-1, AGEs, and nuclear translocation of NF-kB were decreased in the aortas of sRAGE-treated mice [42, 46]. In other work, sRAGE-treated mice displayed significant stabilization of the lesion area at the aortic root. Compared with diabetic mice receiving albumin (placebo), those receiving sRAGE had significantly diminished activity of monocyte chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX-2), VCAM-1 and matrix metalloprotease 9 (MMP-9) within aortic tissue [47]. Similarly, administration of sRAGE resulted in a highly significant decrease in atherosclerotic lesion area in parallel with decreased vascular expression of pro-inflammatory RAGE ligand S100/calgranulins and VCAM-1 and MMPs [48]. Moreover, sRAGE-treated non-diabetic mice displayed significantly decreased atherosclerosis and vascular inflammation [47, 48].

were incubated with PBMCs for 1 h. A slight but significant increase in PBMCs adhesion (measured as explained above) was observed with high-AGE HSA with respect to low-AGE HSA at 12.5 μg/mL (*p* < 0.05), but not with respect to healthy HSA (**Figure 8**; *p* < 0.05). A trend toward an increase in PBMCs adhesion was also observed after treatment with high-AGE HSA with respect to low-AGE HSA at 25 μg/mL (*p* = 0.06). This suggests that *in vivo* glycated albumin needs more time to induce PBMCs adhesion than highly *in vitro* glycated albumin (AGE-HSA).

least three independent experiments. #*p* < 0.05 with respect to low-AGE HSA (Student's *t* test).

220 Endothelial Dysfunction - Old Concepts and New Challenges

**Figure 8.** PBMCs adhesion to HUVEC monolayers treated with albumin from healthy volunteers (healthy HSA) or low-AGE HSA and high-AGE HSA from cardiovascular patients for 24 h. Columns represent the fold change of percentage of PBMCs adhered with respect to commercial HSA, expressed as mean values (columns) ± S.E.M. (vertical bars) of at

**3. Potential implications for pharmacological modulation of AGE-**

expression of VCAM-1 and ICAM-1 in HUVECs was studied.

In an attempt to counteract the inflammatory effects of AGE-HSA, we selected three RAGE inhibitors: a soluble form of RAGE (sRAGE; R&D systems), used at 0.25, 0.5 and 1 ng/mL; a monoclonal antibody against RAGE (anti-RAGE; R&D systems), used at 5, 10 and 20 μg/ mL; and the RAGE antagonist FPS-ZM1 (Calbiochem, Merck Millipore), used at 125, 250, 500 and 1000 nM. HUVECs were pre-treated with different concentrations of these inhibitors and 50 min later treated with 25 μg/mL AGE-HSA. The inhibitory effect of these agents on the

However, contrary to what we expected, blockade of RAGE by using sRAGE, anti-RAGE antibody and FPS-ZM1 was not sufficient to counteract the AGE-induced VCAM-1 and ICAM-1 up-regulation at any of the concentrations tested under our experimental conditions. Our results may suggest that on endothelium, other RAGE-independent mechanisms may also

**RAGE axis activity**

Further studies using anti-RAGE IgG fragments to block ligand binding to RAGE have confirmed these results, especially at the highest dose (up to 10 μg/mL) tested [49]. Exposure of HUVECs to AGE-bovine serum albumin induced expression of VCAM-1 and increased adhesiveness of the monolayer for T lymphoblast of the Molt-4 cell line, which was inhibited by addition of anti-RAGE IgG or sRAGE [40]. Activation of signaling pathway on endothelial cells by advanced oxidation products resulted in overexpression of VCAM-1 and ICAM-1 at both, gene and protein levels, something that was prevented by blocking RAGE with either anti-RAGE IgG or excess sRAGE [27]. Administration of anti-RAGE IgG or sRAGE strongly blocked the increase in vascular permeability in diabetic rats injected with human diabetic red blood cells [50]. Mice treated with sRAGE or anti-RAGE F(ab')2 fragments displayed significantly lower intima/media ratio (a marker of negative vascular remodeling after injury) compared to vehicle-treated animal models of femoral artery injury [51]. However, despite the fact that both, sRAGE and anti-RAGE IgG were able to reduce inflammatory responses in all models tested so far [42, 46, 50, 52], no significant decrease in ICAM-1 and VCAM-1 expression was observed after pre-treatment with soluble RAGE or anti-RAGE antibody, under our experimental conditions.

A recently developed high-affinity RAGE-specific inhibitor: FPS-ZM1 (N-benzyl-4-chloro-N-cyclohexylbenzamide; Calbiochem, Merck Millipore) [53] was also studied. This inhibitor was developed to interact with the ligand-binding domain of the receptor and block RAGE signaling. In our *in vitro* experimental conditions this approach was also unable to inhibit AGE-induced VCAM-1 and ICAM-1 up-regulation.

It is worth mentioning that, most of the above-mentioned works did not elucidate the precise AGE(s) that trigger signal transduction mechanisms upon interacting with RAGE. Kislinger et al. [54] studied the effect of CML-adducts and showed that CML-mediated VCAM-1 expression on HUVECs was also suppressed in the presence of excess sRAGE or anti-RAGE IgG. Nevertheless, they suggest that the findings presented in their work do not rule out other specific AGE products of glycation or oxidation, such as pentosidine, pyralline, methylglyoxal, and imidazolone [55–57], which are present in our modified albumins. Additionally, they also specified that their findings do not rule out either the presence of other receptors or cellular interaction sites for CML adducts, being possible that other receptors for AGE [58–60] may also engage CML- and AGE-modified adducts. These situations might explain why no reduction in the up-regulation of adhesion molecules is observed after pre-treatment with sRAGE and anti-RAGE antibody under our experimental conditions.

rats increased the renal expression of sRAGE and decreased the expression of renal full-length RAGE protein [74]. These investigators also showed that plasma sRAGE levels were significantly increased by inhibition of ACE in both diabetic rats and human subjects with type 1 diabetes [74]. Olmesartan, an angiotensin II type 1 receptor blocker, inhibited the AGE-evoked ROS generation and reduced the expression levels of monocyte chemoattractant protein 1 and ICAM-1 in endothelial cells, subsequently blocking T-cell adhesion to endothelial cells [75].

Impact of Advanced Glycation End Products on Endothelial Function and Their Potential Link…

http://dx.doi.org/10.5772/intechopen.73025

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Other potential agents that may affect circulating sRAGE include the thiazolidinediones [76, 77] and statins [78–80], both of which are known to modulate AGE-RAGE axis. Marx et al. [76] investigated the effects of the two thiazolidinediones available, rosiglitazone and pioglitazone, on RAGE expression in HUVECs. Exposure of HUVECs to thiazolidinedione resulted in a similar reduction in RAGE mRNA expression, via inhibition of NF-κB activation, and in RAGE cell surface expression, demonstrating how these drugs may influence RAGE expression and its deleterious inflammatory activity in subjects with DM [76]. Blockade of the interaction of S100A12 (an endogenous ligand of RAGE) with RAGE by statins at an early stage may prevent inflammation in atherosclerosis and counteract the harmful effects mediated by C reactive protein [81]. Finally, recent results testing new potential drugs have been reported. Curcumin, a polyphenolic natural compound is able to trap methylglyoxal, an important precursor of AGEs [82]. Added on endothelial cell cultures curcumin reduced the intracellular ROS levels and improved cell viability compared with the treatment of methylglyoxal alone. There was also a significant reduction in the expression levels of ICAM-1 [82]. Liquiritin, the 4'-O-glucoside of the flavanone liquiritigenin, reduced AGEs-induced apoptosis and ROS generation in HUVECs and also significantly increased AGEs-reduced SOD activity [83]. It even downregulated the RAGE protein expression and significantly blocked NF-κB activation [83].

Oxidative stress induction by AGEs at endothelium triggers molecular signaling pathways that produce an inflammatory response or even endothelial dysfunction. Adhesion molecules expression at the membrane surface of endothelial cells as a consequence of this response or induced by AGEs by other mechanisms mediates the adhesion of leukocytes to endothelium. This adhesion is a key step in the atherogenesis process and the possible involvement of AGE-RAGE axis in it should be considered as potential therapeutic target. Finally, possible pharmacological modulation of AGE-RAGE axis activity at the endothelium is suggested, but specific pharmacological tools available nowadays are not efficient enough; momentarily, drugs used for cardiovascular and metabolic problems could be helpful in modulating the

This study was supported by the *Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016* and the *Instituto de Salud Carlos III* (grant number PI14/01140), co-financed by European Regional Development Fund. *Axudas para a consolidación e estructuración de* 

**4. Conclusions**

AGE-RAGE axis.

**Acknowledgements**

Additionally, Amadori-modified albumin stimulates adhesion of monocytes to endothelial cells through enhanced transcription of the cell surface adhesion molecules E-selectin, VCAM-1 and ICAM-1 [61], implicating an initial endothelial cell activation occurring at atherosclerosis-prone vascular sites [62, 63]. However, Amadori products do not compete with AGE-albumin for binding to AGE receptors such as RAGE [64]. Aortic endothelial cells express specific receptors for Amadori-modified albumin [37, 65]. Although less information is available for the receptor for Amadori products and signaling through Amadori-modified albumin receptors remains obscure, calnexin [66] and nucleophosmin [67, 68] have been reported to be the fructosyl-lysine specific binding proteins [66–68]. Binding of Amadori-modified albumin to calnexin-like receptors may participate in degradation and/or activation of signal transduction processes involved in mediating the biologic activities of Amadori-modified albumin [66]. The E-selectin expression induced by Amadori-modified albumin was 10 or 20 times higher than that induced with three types of AGEs-HSAs and was not suppressed by anti-RAGE antibody [69]. This would explain why RAGE antagonism would not counteract the increase in adhesion molecules expression.

In agreement with this hypothesis, Esposito et al. [70] found that anti-RAGE antibody completely prevented leukocyte adhesion to endothelial cells grown for 8 weeks in high-glucosecontaining media, but it did not reduce the adhesion at 24 h. These results demonstrate that AGEs are important mediators of high-glucose-induced endothelial dysfunction after longterm exposure, whereas the same changes in acute exposure occur with the action of mediators other than AGEs. As the formation of Amadori products is highly probable after 24 h incubation in high glucose medium, but not the formation of AGEs, the effects on the inflammation parameters observed by Esposito et al. [70], and not prevented by anti-RAGE antibodies, might be due to the effect of the early glycated products, and not AGEs.

Besides from directly blocking RAGE, alternative pharmacological approaches might turn out to be more promising. Namely, it has been shown that both RAGE and sRAGE can be regulated by currently available pharmacological agents [71]. Other drugs currently in use for diabetic complications have been shown to have an effect on AGE accumulation. These include the antihypertensive angiotensin-converting enzyme inhibitor (ACEI) ramipril [72] and the glucose-lowering drug metformin [73], which both reduce AGE. Forbes et al. [74] demonstrated that compared with placebo, the ACEI perindopril increased human plasma sRAGE levels and reduced plasma AGE concentrations, suggesting an additional mechanistic effect of ACE inhibition in the treatment and prevention of vascular disease. The inhibition of ACE in rats increased the renal expression of sRAGE and decreased the expression of renal full-length RAGE protein [74]. These investigators also showed that plasma sRAGE levels were significantly increased by inhibition of ACE in both diabetic rats and human subjects with type 1 diabetes [74]. Olmesartan, an angiotensin II type 1 receptor blocker, inhibited the AGE-evoked ROS generation and reduced the expression levels of monocyte chemoattractant protein 1 and ICAM-1 in endothelial cells, subsequently blocking T-cell adhesion to endothelial cells [75].

Other potential agents that may affect circulating sRAGE include the thiazolidinediones [76, 77] and statins [78–80], both of which are known to modulate AGE-RAGE axis. Marx et al. [76] investigated the effects of the two thiazolidinediones available, rosiglitazone and pioglitazone, on RAGE expression in HUVECs. Exposure of HUVECs to thiazolidinedione resulted in a similar reduction in RAGE mRNA expression, via inhibition of NF-κB activation, and in RAGE cell surface expression, demonstrating how these drugs may influence RAGE expression and its deleterious inflammatory activity in subjects with DM [76]. Blockade of the interaction of S100A12 (an endogenous ligand of RAGE) with RAGE by statins at an early stage may prevent inflammation in atherosclerosis and counteract the harmful effects mediated by C reactive protein [81].

Finally, recent results testing new potential drugs have been reported. Curcumin, a polyphenolic natural compound is able to trap methylglyoxal, an important precursor of AGEs [82]. Added on endothelial cell cultures curcumin reduced the intracellular ROS levels and improved cell viability compared with the treatment of methylglyoxal alone. There was also a significant reduction in the expression levels of ICAM-1 [82]. Liquiritin, the 4'-O-glucoside of the flavanone liquiritigenin, reduced AGEs-induced apoptosis and ROS generation in HUVECs and also significantly increased AGEs-reduced SOD activity [83]. It even downregulated the RAGE protein expression and significantly blocked NF-κB activation [83].
