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

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

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

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.

production were observed with AGE-HSA at 4–8 h (**Figure 1b**).

\**p* < 0.05 with respect to the control values (Student's *t* test).

dye for that purpose [36].

214 Endothelial Dysfunction - Old Concepts and New Challenges

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

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).

the effects of gHSA seemed to be limited to 4 h- treatment, AGE-HSA up-regulated VCAM-1 and ICAM-1 expression for longer periods of time (from 2 to 6 h). Differences on the active concentrations of both glycation products were also observed: whereas gHSA was only active at 25 μg/mL, AGE-HSA was also effective at 12 and 100 μg/mL (**Figure 3**).

To further confirm the increase in the expression of these adhesion molecules, protein levels of VCAM-1 and ICAM-1 were analyzed by western blot analysis after the treatment of HUVECs with two relevant concentrations of gHSA and AGE-HSA: 25 and 100 μg/mL, in comparison with the same concentrations of unmodified HSA and Ct-HSA, respectively. There was a significant elevation of VCAM-1 and ICAM-1 levels caused by the effect of both AGE-HSA concentrations tested. On the other hand, only the concentration of 25 μg/mL gHSA (but not 100 μg/mL) enhanced the ICAM-1 protein levels (**Figure 4**).

The functional translation of VCAM-1 and ICAM-1 up-regulation was analyzed by the adhesion of peripheral blood mononuclear cells (PBMCs) to HUVEC monolayers after treatment with both types of modified albumins for 4 h (**Figure 5**). After these treatments, the adhesion of calcein-AM-stained PBMCs to HUVEC monolayers after 1 h of incubation and washing of non-adhered

PBMCs was quantified by fluorescence. In these conditions, gHSA (25 μg/mL) induced no significant effect in PBMCs adhesion in comparison with the control HSA. However, AGE-HSA (25 μg/

**Figure 4.** The expression levels of VCAM-1 and ICAM-1 measured by western blot in HUVEC protein extracts obtained after 4 h of treatment with gHSA or AGE-HSA (25 or 100 μg/mL, as indicated). TNF-α (10 ng/mL) was used as a positive inducer control. (a) Representative blots for VCAM-1, ICAM-1 and β-actin. Columns represent the fold change of protein expression for (b) VCAM-1 and (c) ICAM-1 calculated by optical densitometry with respect to β-actin and expressed as mean values (columns) ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 with respect to

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The effects of gHSA and AGE-HSA on PBMCs transmigration through HUVEC monolayers were studied in comparison to the ICAM-1 and VCAM-1 changes of expression. For these experiments HUVEC with transfected green fluorescent protein were grown until confluence onto transwells with 5 μm of pore size (Millipore). After treatment with AGE-HSA (25 and 100 μg/mL) for 4 h PBMCs were layered over the HUVECs and incubated at 37°C. TNF-α (10 ng/mL) was used as a positive control because it induces endothelial cell activation and promotes PBMCs transmigration through the endothelial monolayer. The number of transmigrated PBMCs were estimated by quantification of nuclei acids content with CyQUANT® GR dye (Molecular probes, Invitrogen) at the end of the experiment. Unless for the case of TNF-α, no changes were observed for any of the stimuli after 3 h of treatment. However, after 24 h of HUVEC incubation with 25 μg/mL AGE-HSA, a significant increase in the migration of PBMCs was observed as compared to control (**Figure 6**). On the contrary, higher concentration of AGE-HSA (100 μg/mL), showed no effect in the transmigration of PBMCs. The positive control with TNF-α increased the migration of PBMCs even more than after 3 h (**Figure 6**).

mL) induced a significant increase in the adhesion of PBMC to HUVEC monolayers.

unmodified HSA or Ct-HSA for gHSA and AGE-HSA, respectively (Student's *t* test).

**Figure 3.** The expression levels of mRNA of VCAM-1 (a and c) and ICAM-1 (b and d) after treatment with gHSA or AGE-HSA at the concentrations and times indicated on HUVEC cultures. Results are shown as the ratio treatment/respective control, expressed as mean (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) and between AGE-HSA and gHSA treated experiments ate the same time of incubation (a and b) or concentration (c and d; #*p* < 0.05; Student's *t* test).

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the effects of gHSA seemed to be limited to 4 h- treatment, AGE-HSA up-regulated VCAM-1 and ICAM-1 expression for longer periods of time (from 2 to 6 h). Differences on the active concentrations of both glycation products were also observed: whereas gHSA was only active

To further confirm the increase in the expression of these adhesion molecules, protein levels of VCAM-1 and ICAM-1 were analyzed by western blot analysis after the treatment of HUVECs with two relevant concentrations of gHSA and AGE-HSA: 25 and 100 μg/mL, in comparison with the same concentrations of unmodified HSA and Ct-HSA, respectively. There was a significant elevation of VCAM-1 and ICAM-1 levels caused by the effect of both AGE-HSA concentrations tested. On the other hand, only the concentration of 25 μg/mL gHSA (but not

The functional translation of VCAM-1 and ICAM-1 up-regulation was analyzed by the adhesion of peripheral blood mononuclear cells (PBMCs) to HUVEC monolayers after treatment with both types of modified albumins for 4 h (**Figure 5**). After these treatments, the adhesion of calcein-AM-stained PBMCs to HUVEC monolayers after 1 h of incubation and washing of non-adhered

**Figure 3.** The expression levels of mRNA of VCAM-1 (a and c) and ICAM-1 (b and d) after treatment with gHSA or AGE-HSA at the concentrations and times indicated on HUVEC cultures. Results are shown as the ratio treatment/respective control, expressed as mean (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) and between AGE-HSA and gHSA treated

experiments ate the same time of incubation (a and b) or concentration (c and d; #*p* < 0.05; Student's *t* test).

at 25 μg/mL, AGE-HSA was also effective at 12 and 100 μg/mL (**Figure 3**).

100 μg/mL) enhanced the ICAM-1 protein levels (**Figure 4**).

216 Endothelial Dysfunction - Old Concepts and New Challenges

**Figure 4.** The expression levels of VCAM-1 and ICAM-1 measured by western blot in HUVEC protein extracts obtained after 4 h of treatment with gHSA or AGE-HSA (25 or 100 μg/mL, as indicated). TNF-α (10 ng/mL) was used as a positive inducer control. (a) Representative blots for VCAM-1, ICAM-1 and β-actin. Columns represent the fold change of protein expression for (b) VCAM-1 and (c) ICAM-1 calculated by optical densitometry with respect to β-actin and expressed as mean values (columns) ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 with respect to unmodified HSA or Ct-HSA for gHSA and AGE-HSA, respectively (Student's *t* test).

PBMCs was quantified by fluorescence. In these conditions, gHSA (25 μg/mL) induced no significant effect in PBMCs adhesion in comparison with the control HSA. However, AGE-HSA (25 μg/ mL) induced a significant increase in the adhesion of PBMC to HUVEC monolayers.

The effects of gHSA and AGE-HSA on PBMCs transmigration through HUVEC monolayers were studied in comparison to the ICAM-1 and VCAM-1 changes of expression. For these experiments HUVEC with transfected green fluorescent protein were grown until confluence onto transwells with 5 μm of pore size (Millipore). After treatment with AGE-HSA (25 and 100 μg/mL) for 4 h PBMCs were layered over the HUVECs and incubated at 37°C. TNF-α (10 ng/mL) was used as a positive control because it induces endothelial cell activation and promotes PBMCs transmigration through the endothelial monolayer. The number of transmigrated PBMCs were estimated by quantification of nuclei acids content with CyQUANT® GR dye (Molecular probes, Invitrogen) at the end of the experiment. Unless for the case of TNF-α, no changes were observed for any of the stimuli after 3 h of treatment. However, after 24 h of HUVEC incubation with 25 μg/mL AGE-HSA, a significant increase in the migration of PBMCs was observed as compared to control (**Figure 6**). On the contrary, higher concentration of AGE-HSA (100 μg/mL), showed no effect in the transmigration of PBMCs. The positive control with TNF-α increased the migration of PBMCs even more than after 3 h (**Figure 6**).

**Figure 5.** Quantification of the adhesion of PBMCs to a HUVEC monolayer, after treatment of HUVECs during 4 h with gHSA (25 μg/mL) or AGE-HSA (25 μg/mL), compared with HSA (25 μg/mL) or ct-HSA (25 μg/mL), respectively. The graph represents the mean percentage of adhesion (columns) ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 between the columns indicated (ANOVA followed by Tukey's test).

The whole study and protocols were approved by the Ethics Committee for Human Studies at Galicia (Spanish region) in accordance to the 1975 Declaration of Helsinki. Particularly, we analyzed the effect of HSAs categorized in healthy or nonglycated (from healthy volunteers), low-AGE or high-AGE (from cardiovascular patients), according to their content in AGE adducts. Glycation level was estimated by the molecular weight increment of isolated HSAs, due to the incorporation of different glycation products to the molecule. This was measured by mass spectrometry with a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems). On this basis, attending to the increase of HSA molecular weight with respect to non-modified HSA, three stocks of HSA were prepared and categorized as healthy-nonglycated HSA, low-, and high-AGE HSA (molecular weights of 66,481, 66,665 and 66,778 Da for healthy, low-, and high-AGE HSA, respectively). HUVECs were incubated with a range of concentrations of these types of HSAs (12–200 μg/mL) for 4 h. In these conditions, the treatment with high-AGE HSA significantly increased the mRNA expression of ICAM-1 at concentrations of 12.5 and 25 μg/mL with respect to healthy-nonglycated HSA (**Figure 7a**; *p* < 0.001). An increase was also observed at 100 μg/mL concentration with respect to healthy HSA (*p* = 0.046). Moreover, high-AGE HSA was able to induce a significant

**Figure 7.** The expression of mRNA for ICAM-1 (a) and VCAM-1 (b) after the treatment of HUVECs with HSA isolated from healthy volunteers (healthy HSA) or with low-AGE HSA and high-AGE HSA from cardiovascular patients for 4 h. Columns represent the fold increase of mRNA expression for each gene and are expressed as mean values (columns) ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 with respect to healthy HSA.

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increase with respect to low-AGE HSA at 12.5 and 25 μg/mL (**Figure 7a**; *p* < 0.05).

trigger chronic endothelial dysfunction.

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

In the case of VCAM-1 expression, high-AGE HSA only induced an increase in the mRNA expression at 12.5 μg/mL with respect to healthy HSA and low-AGE HSA (**Figure 7b**; *p* < 0.05). At this concentration, low-AGE HSA also induced an increase in the expression of VCAM-1 with respect to healthy HSA (**Figure 7b**; *p* < 0.05). Finally, at a concentration of 50 μg/mL, high-AGE HAS induced a reduction in the expression of VCAM-1 with respect to healthy HSA (*p* < 0.05). This reduction in the expression of VCAM-1 was only transient as the mRNA levels recovered again at higher concentrations. Altogether, these results suggest that *in vivo* glycation of albumin could have a pro-inflammatory effect in endothelial cells, which would

PBMCs adhesion to HUVECs was also studied with *in vivo* glycated albumins at 12.5, 25 and 100 μg/mL. HUVECs were treated with these concentrations for 24 h. After that, HUVECs

**Figure 6.** Transmigration of PBMCs through HUVEC monolayers after 3 h (white columns) or 24 h (black columns). Columns represent the mean (columns) ± S.E.M. (in vertical bars) of the increase of PBMCs transmigration after treatment compared to untreated control. \**p* < 0.05 with respect to untreated control (Student's *t* test).

Given the results obtained in the adhesion molecules expression in HUVECs, another approach was performed repeating the study with *in vivo* glycated albumin obtained from healthy volunteers and from cardiovascular patients, which donated their blood after signing informed consent.

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**Figure 7.** The expression of mRNA for ICAM-1 (a) and VCAM-1 (b) after the treatment of HUVECs with HSA isolated from healthy volunteers (healthy HSA) or with low-AGE HSA and high-AGE HSA from cardiovascular patients for 4 h. Columns represent the fold increase of mRNA expression for each gene and are expressed as mean values (columns) ± S.E.M. (vertical bars) of at least three independent experiments. \**p* < 0.05 with respect to healthy HSA. #*p* < 0.05 with respect to low-AGE HSA (Student's *t* test).

The whole study and protocols were approved by the Ethics Committee for Human Studies at Galicia (Spanish region) in accordance to the 1975 Declaration of Helsinki. Particularly, we analyzed the effect of HSAs categorized in healthy or nonglycated (from healthy volunteers), low-AGE or high-AGE (from cardiovascular patients), according to their content in AGE adducts. Glycation level was estimated by the molecular weight increment of isolated HSAs, due to the incorporation of different glycation products to the molecule. This was measured by mass spectrometry with a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems). On this basis, attending to the increase of HSA molecular weight with respect to non-modified HSA, three stocks of HSA were prepared and categorized as healthy-nonglycated HSA, low-, and high-AGE HSA (molecular weights of 66,481, 66,665 and 66,778 Da for healthy, low-, and high-AGE HSA, respectively). HUVECs were incubated with a range of concentrations of these types of HSAs (12–200 μg/mL) for 4 h. In these conditions, the treatment with high-AGE HSA significantly increased the mRNA expression of ICAM-1 at concentrations of 12.5 and 25 μg/mL with respect to healthy-nonglycated HSA (**Figure 7a**; *p* < 0.001). An increase was also observed at 100 μg/mL concentration with respect to healthy HSA (*p* = 0.046). Moreover, high-AGE HSA was able to induce a significant increase with respect to low-AGE HSA at 12.5 and 25 μg/mL (**Figure 7a**; *p* < 0.05).

In the case of VCAM-1 expression, high-AGE HSA only induced an increase in the mRNA expression at 12.5 μg/mL with respect to healthy HSA and low-AGE HSA (**Figure 7b**; *p* < 0.05). At this concentration, low-AGE HSA also induced an increase in the expression of VCAM-1 with respect to healthy HSA (**Figure 7b**; *p* < 0.05). Finally, at a concentration of 50 μg/mL, high-AGE HAS induced a reduction in the expression of VCAM-1 with respect to healthy HSA (*p* < 0.05). This reduction in the expression of VCAM-1 was only transient as the mRNA levels recovered again at higher concentrations. Altogether, these results suggest that *in vivo* glycation of albumin could have a pro-inflammatory effect in endothelial cells, which would trigger chronic endothelial dysfunction.

PBMCs adhesion to HUVECs was also studied with *in vivo* glycated albumins at 12.5, 25 and 100 μg/mL. HUVECs were treated with these concentrations for 24 h. After that, HUVECs

Given the results obtained in the adhesion molecules expression in HUVECs, another approach was performed repeating the study with *in vivo* glycated albumin obtained from healthy volunteers and from cardiovascular patients, which donated their blood after signing informed consent.

**Figure 6.** Transmigration of PBMCs through HUVEC monolayers after 3 h (white columns) or 24 h (black columns). Columns represent the mean (columns) ± S.E.M. (in vertical bars) of the increase of PBMCs transmigration after treatment

compared to untreated control. \**p* < 0.05 with respect to untreated control (Student's *t* test).

**Figure 5.** Quantification of the adhesion of PBMCs to a HUVEC monolayer, after treatment of HUVECs during 4 h with gHSA (25 μg/mL) or AGE-HSA (25 μg/mL), compared with HSA (25 μg/mL) or ct-HSA (25 μg/mL), respectively. The graph represents the mean percentage of adhesion (columns) ± S.E.M. (vertical bars) of at least three independent

experiments. \**p* < 0.05 between the columns indicated (ANOVA followed by Tukey's test).

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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

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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].

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

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

experimental conditions.

AGE-induced VCAM-1 and ICAM-1 up-regulation.

results obtained on *in vivo* models of disease are promising, as we comment below.

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

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).
