**2.1. AGEs-induced ROS production in the endothelium**

**1. Introduction**

212 Endothelial Dysfunction - Old Concepts and New Challenges

Cardiovascular diseases resulting from atherosclerosis have become the most important cause of mortality and morbidity in the general population [1]. Although atherosclerosis develops as a consequence of multiple risk factors such as hypertension, dyslipidemia, diabetes, aging and smoking, the common pathway for its development is endothelial dysfunction and vascular inflammation [2]. In the last two decades, the role of advanced glycation end products (AGEs) in the development of endothelial dysfunction has gained increasing interest [3–5], initially as a possible molecular mechanism of diabetic cardiovascular complications [3], and,

AGEs are products of non-enzymatic molecular modifications of proteins and lipids that affect the structure and function of the target molecule. They are produced endogenously by spontaneous reactions, but pathophysiological conditions may accelerate their formation and

AGEs comprise a heterogeneous group: the most studied are pentosidine and Nε-carboxymethyllysine (CML) and quantitatively, the most important in the tissues are the hydroimidazolones like CML [7]. AGEs are formed by a combination of glycation, oxidation, and/or carbonylation reactions both in the extra- and in the intracellular space. Other processes involving lipid peroxidations in the cell membranes lead to the formation of advanced lipid end products, as for example, malondialdehyde [8]. The classical mechanism of AGE formation is the slow Maillard reaction between glucose or reducing sugars and proteins [9]. The interaction between the carbonyl groups of reducing sugars and amino groups of proteins results in the formation of a Schiff base within a few hours. Intramolecular rearrangement of the Schiff base results in more stable Amadori products [9]. An example of these types of products is glycated hemoglobin or glycated albumin, the former is widely used in clinical practice for diagnosis and follow-up of diabetes mellitus and the last could be regarded as a smart alternative to modified hemoglobin for the same purposes, with less dependence on hematological diseases and intracellular conditions. Finally, the process of oxidation of the Amadori products leads to reactive carbonyl compounds and subsequently to the formation of AGEs within weeks to months. AGEs can also be formed intracellularly. Glucose is altered into reactive carbonyl compounds during glycolysis pathway, of which the best-known is methylglyoxal. The chemical reaction between

Absorption of exogenous AGEs also contributes to their accumulation in tissues. Tobacco smoke contains highly reactive glycation products which rapidly form AGEs *in vitro* and *in vivo* and therefore, increase the serum AGEs levels in smokers compared to non-smokers [11]. The content of AGEs in food depends on the temperature at which food products are prepared, with oven frying being the most severe inducer [12]. Approximately 10% of the ingested AGEs are absorbed from the gastrointestinal tract into the blood [13]. The final level of AGEs accumulation depends on their clearance and the metabolic mechanisms by the kidney and liver, respectively. Increased level of AGEs can be found in patients with either renal [13] or liver failure [14]. The role of AGEs in cardiovascular diseases is a matter of interest in the last years [15], and the strong association between the axis of action of AGEs and their receptor (RAGE) and atherosclerosis or cardiovascular ischemic disease [3, 16, 17] has attracted increased attention.

in the last years, as an independent risk factor of vascular injury [6].

they also contribute to disease by different mechanisms.

these carbonyl compounds and proteins can result in AGEs [10].

One of the first and best studied actions of AGEs on endothelial cells is the induction of ROS. The suggested mechanisms for this action are several and range from the activation of ROS-producing enzymes to the reduction of ROS-neutralizing enzymes. In the first group of enzymes or enzyme complexes are nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [21] and mitochondria [22], whereas in the second, there are endothelial nitric oxide (NO) synthase (eNOS) [23], superoxide dismutase (SOD) and glutathione peroxidase [24, 25]. The molecular mechanisms of these actions have been related to the activation of NF-κB via RAGE [26, 27].

ROS production in endothelial cells has important consequences on endothelial activation. In brain microvascular endothelial cells, AGEs-induced ROS production enhances vascular endothelial growth factor (VEGF) expression, which mediates an increase in cell permeability [28], and platelet tissue factor up-regulation [29]. Other mechanisms of AGEs on endothelial cells promoting endothelial activation or dysfunction are the generation of asymmetric dimethylarginine (ADMA, a metabolic by-product of natural protein modification processes in the cytoplasm of cells, that acts as a competitive inhibitor of NOS) [30], or impaired calcium signaling [31].

It is important to note that the effects of AGEs' precursors (i.e. Amadori products or glycated proteins) on endothelial cells, differ from the effects of AGEs themselves. Several works have focused on this issue (see, for a review, [32]). Amadori products modify eNOS activity and gene expression, promoting apoptosis of endothelial cells [33, 34]. A recent study performed by our group has highlighted the important molecular and functional differences between early glycated human serum albumin (gHSA) and advanced glycated albumin (AGE-HSA), obtained commercially or by glucose incubation during 4 weeks at 37°C in aseptic conditions, respectively [35]. The respective control molecules of these treatments were unmodified commercial HSA and HSA incubated for the same time than AGE-HSA, but without glucose (Ct-HSA). Molecular characterization of the early and advanced glycation products formed on each modified albumin (gHSA and AGE-HSA) were studied by matrix assisted laser desorption/ionization—time of flight (MALDI-TOF)-mass spectrometry. Once characterized, the effects on ROS production of human umbilical vein endothelial cells (HUVECs) under the stimuli of gHSA or AGE-HSA were compared [35]. Low concentrations of gHSA enhanced long-lasting ROS production in HUVECs, whereas AGE-HSA induced extracellular ROS production after short time of incubation and at lower concentrations than gHSA. Extracellular ROS production of HUVEC was measured by the cytochrome C reduction method, whereas intracellular ROS production of HUVEC was measured by 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (cDCF-DA; Sigma-Aldrich), an intracellular dye for that purpose [36].

Interestingly, at 25 μg/mL, gHSA significantly enhanced the intracellular ROS production,

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Therefore, differences in the induction of ROS production were observed between gHSA (a low glycated product) and AGE-HSA (a high glycated product). Although the effects of AGE-HSA are accepted to be mediated by RAGE, the receptor that mediates the effects of gHSA has not

RAGE-ligands interaction induces a series of signal transduction cascades and lead to the activation of transcription factor NF-κB as well as increased expression of cytokines, chemokines, and adhesion molecules [39]. Expression of inducible adhesion molecules is a final common pathway in the development of vascular inflammation and pathology, rendering the vascula-

A number of studies have demonstrated induction of vascular cell adhesion molecule-1 (VCAM-1) expression in a RAGE-dependent manner when endothelial cells are exposed to AGEs [18]. Moreover, engagement of RAGE by AGEs results in enhanced expression of other adhesion molecules, such as E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) [40–42]. High expression of adhesion molecules in endothelial cells may promote adhesive interactions of circulating monocytes with the endothelial surface, resulting, eventually, in transendothelial migration [43]. We confirmed that AGE-HSA up-regulated ICAM-1 and VCAM-1 expression more than gHSA, in terms of mRNA quantitative changes, measured by total messenger RNA retrotranscription and quantitative real-time polymerase chain reaction (qPCR) [35]. Even while

**Figure 2.** Intracellular ROS production in HUVECs after 4 h of treatment with different concentrations of gHSA (white columns) and AGE-HSA (black columns), as indicated in the x-axis. Results are shown as the ratio modified HSA/control HSA, expressed as mean, in columns, ± S.E.M. (vertical bars) of at least four independent experiments. Comparisons

were made between each ratio level and the unit (\**p* < 0.05; Student's *t* test).

whereas AGE-HSA only showed a trend to slightly increase it (**Figure 2**).

been revealed yet [37], since, the effects of gHSA are not mediated by RAGE [38].

**2.2. Expression of adhesion molecules mediating leukocyte adhesion to** 

ture a selective target for circulating peripheral blood cells [27, 40].

**endothelium**

Treatment of HUVECs with gHSA (25–100 μg/mL) for different times (4–12 h) induced significant increments of extracellular ROS production with respect to treatment with the same concentration of un-modified albumin (HSA, used as control) [36]. The maximal response (i.e. the quantity of ROS) was obtained with 25 μg/mL gHSA after 4 h of treatment (**Figure 1a**). The effects of AGE-HSA were studied under the same conditions. AGE-HSA increased the extracellular ROS production at lower concentrations (12 μg/mL) and after shorter time of exposure than gHSA (2 h). Another important difference is that, at long incubation periods, the ROS-inducing effects of gHSA were maintained, whereas no significant increases on ROS production were observed with AGE-HSA at 4–8 h (**Figure 1b**).

Similar experiments were designed to measure the intracellular ROS production by using cDCF-DA after 4 h of treatment the HUVECs with gHSA or AGE-HSA (12–50 μg/mL).

**Figure 1.** Extracellular ROS production in HUVECs after treatment with different concentrations of (a) gHSA (12–50 mg/ mL) or (b) AGE-HSA (12–50 mg/mL), for periods of time indicated on each graph. Columns represent the ratio of ROS produced by treatment/HSA and are expressed as mean ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 with respect to the control values (Student's *t* test).

Interestingly, at 25 μg/mL, gHSA significantly enhanced the intracellular ROS production, whereas AGE-HSA only showed a trend to slightly increase it (**Figure 2**).

Therefore, differences in the induction of ROS production were observed between gHSA (a low glycated product) and AGE-HSA (a high glycated product). Although the effects of AGE-HSA are accepted to be mediated by RAGE, the receptor that mediates the effects of gHSA has not been revealed yet [37], since, the effects of gHSA are not mediated by RAGE [38].
