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Originally published online as doi:10.1189/jlb.0603265 on March 12, 2004

Published online before print March 12, 2004
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(Journal of Leukocyte Biology. 2004;75:1062-1069.)
© 2004 by Society for Leukocyte Biology

Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants

J. C. Mamputu and G. Renier1

CHUM Research Centre, Notre-Dame Hospital, Department of Medicine, University of Montreal, Quebec, Canada

1 Correspondence: CHUM Research Centre, Notre-Dame Hospital, J-A. de Seve Pavilion, Room Y-3622, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1. E-mail: genevieve.renier{at}umontreal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence indicates a role for advanced glycation end-products (AGEs) in the development of diabetic retinopathy. In the present study, we examined the in vitro effect of AGEs on human monocyte adhesion to bovine retinal endothelial cells (BRECs) and the molecular mechanisms involved in this effect. Treatment of cultured BRECs with AGEs led to a significant increase in monocyte adhesion and intercellular cell adhesion molecule-1 (ICAM-1) expression. These effects were inhibited by antioxidants including gliclazide and vitamins C and E. On the basis of the stimulatory effect of AGEs on vascular endothelial growth factor (VEGF) secretion by retinal endothelial cells, the role of this growth factor as mediator of AGE-induced monocyte adhesion to BRECs was next investigated. Incubation of BRECs with VEGF increased monocyte adhesion to these cells and enhanced ICAM-1 expression. Treatment of BRECs with an anti-VEGF antibody abrogated AGE-induced monocyte adhesion and ICAM-1 expression. Finally, incubation of BRECs with protein kinase C (PKC) and nuclear factor (NF)-{kappa}B inhibitors suppressed monocyte adhesion and ICAM-1 expression elicited by AGEs and VEGF. Taken together, these data indicate that AGEs increase monocyte adhesion to BRECs and that this effect is mediated through VEGF-induced ICAM-1 expression. They also demonstrate that this effect is oxidative stress-sensitive and involves PKC and NF-{kappa}B-dependent signaling pathways.

Key Words: AGE • VEGF • adhesion molecules • gliclazide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing evidence indicates that retinal leukocyte stasis (leukostasis) is a key event in the development of diabetic retinopathy (DR). Indeed, earlier reports have shown that occurrence of this event coincides with the development of several retinal abnormalities associated with the pathogenesis of DR, including vascular permeability, capillary occlusion, and endothelial injury [1 2 3 ]. The pathogenic mechanisms mediating diabetic retinal leukostasis include increased expression of cell adhesion molecules [3 4 5 ], activation, and decreased deformability of diabetic leukocytes, oxidative stress, and protein kinase C (PKC) activation [6 ]. Convincing data identify vascular endothelial growth factor (VEGF) as one major stimulus for retinal leukostasis associated with DR [7 ].

Advanced glycation end-products (AGEs) are believed to play an important role in the development of DR by inducing blood-retinal barrier (BRB) dysfunction [8 , 9 ]. Although the mechanisms underlying AGE-induced retinal vascular leakage are still poorly understood, several observations suggest a role for VEGF in this process [10 , 11 ]. Supporting this possibility, we and others have demonstrated that AGEs enhance VEGF expression in retinal cells [12 13 14 15 ] and that this growth factor is operative in the pathogenesis of vascular leakage [16 ]. Evidence that BRB breakdown elicited by VEGF is leukocyte-dependent [4 , 7 ] further suggests that VEGF-induced leukostasis may be involved in the effect of AGEs on retinal vascular permeability. Despite their well-established role in leukocyte adhesion to macrovascular cells [17 , 18 ], the role of AGEs in retinal leukostasis has not been investigated. In the present study, we sought to examine the effect of AGEs in monocyte adhesion to retinal endothelial cells in vitro and the role of VEGF in this process. Based on our previous results demonstrating that gliclazide, a sulfonylurea that decreases oxidative stress [19 20 21 22 ], inhibits AGE-induced VEGF expression in retinal endothelial cells [12 ], we also examined the effect of this drug on monocyte adhesion to retinal cells. Our results demonstrate that AGEs enhance retinal leukocyte adhesion through oxidative stress and that this effect involves VEGF-induced intercellular cell adhesion molecule-1 (ICAM-1) expression. These data along with our previous finding that AGEs stimulate retinal VEGF expression identify VEGF as a potential key autocrine regulator of leukostasis elicited by AGE accumulation in the retina.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Plasma-derived horse serum, fibronectin, glucose, immunoglobulin (Ig)-free bovine serum albumin (BSA), vitamin E, dianisidine dihydrochloride, vitamin C, heparin, o-phenylediamine dihydrochloride (OPD), and glyburide were obtained from Sigma Chemical Co. (St. Louis, MO). Endothelial basal medium (EBM) was obtained from Clonetics (San Diego, CA). Bovine endothelial cell growth factor (bECGF) was purchased from Roche Molecular Biochemicals (Laval, QC, Canada). Fetal bovine serum (FBS) was obtained from Wisent (St-Bruno, QC, Canada). Penicillin–streptomycin and phosphate-buffered saline (PBS) were obtained from Invitrogen (Burlington, ONT, Canada). GF10923X and BAY 11-7085 were purchased from Calbiochem (La Jolla, CA). Les Laboratoires Servier (Neuilly, France) supplied sodium salt gliclazide. Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN). Polyclonal antibody to VEGF was purchased from Peprotech (Rock Hill, NJ). Antibodies to PKCß1 and nuclear factor (NF)-{kappa}Bp65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of AGEs
Ig-free BSA was subjected to nonenzymatic glycation by incubation with glucose 0.5 mol/L in 0.4 mol/L sodium phosphate buffer containing 0.5 mmol/L EDTA. The solution was sterile-filtered by passage through a 0.2-µm Gelman filter and then incubated at 37°C for 4 weeks under aerobic conditions. Nonglycated albumin was obtained by incubating BSA in the same reaction mixture in the absence of glucose. At the end of the incubation period, samples were dialyzed extensively against 10 mmol/L PBS (pH 7.4) at 4°C to remove unreacted glucose. The presence of AGEs was confirmed by the typical absorption and fluorescent spectra patterns of these proteins. Endotoxin content of the AGE preparations (100 µg/ml) was determined by the Limulus amebocyte lysate assay (Sigma Chemical Co.) and was consistently found to be lower than 6 pg/ml.

Bovine retinal endothelial cell (BREC) culture
BRECs were kindly provided by Dr. Lloyd P. Aiello (Harvard Medical School, Cambridge, MA) and were isolated as described previously [23 ]. The cells were grown in EBM supplemented with 10% plasma-derived horse serum, 3% FBS, 50 µg/ml heparin, 50 µg/ml bECGF, and 1% (vol/vol) penicillin–streptomycin on fibronectin-coated dishes at 37°C in 5% CO2/95% air atmosphere. At confluence, the cells were trypsinized and subcultured in 96-well culture plates or 100 x 20 mm tissue-culture dishes according to the appropriate assay conditions. Cells were used in all experiments at passages 5–10. AGE treatment was performed in cells cultured in serum-free EBM supplemented with 1% (vol/vol) penicillin–streptomycin.

Isolation of human monocytes
Human monocytes were isolated from fresh, heparinized blood (100 ml), collected from nonsmoker, healthy male and female donors as described previously [24 ]. First, peripheral blood mononuclear cells were obtained by density centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Baie d’Urfé, QC, Canada). The cells collected from the interface were washed three times with Hanks’ balanced saline solution (HBSS) and were allowed to aggregate in the presence of FBS. After further purification by rosetting technique and density centrifugation, recovery of highly purified monocytes (85–90%), as assessed by fluorescence-activated cell sorter analysis, was obtained. Human monocytes were resuspended in serum-free RPMI-1640 medium, supplemented with 1% (vol/vol) penicillin–streptomycin.

Monocyte adhesion assay
BRECs were grown to confluence in 96-well plates at 37°C. On the day of treatments, cells were washed twice with HBSS and cultured in serum-free EBM supplemented with 1% (vol/vol) penicillin–streptomycin. The cells were then incubated for various times with AGEs or VEGF in the presence or absence of appropriate agents. At the end of the incubation period, cells were washed twice with HBSS, and 230,000 highly purified human monocytes were added to each well. After a 2-h incubation at 37°C, nonadherent monocytes were removed by washing the cells with PBS (pH 6.0). Measuring monocyte myeloperoxidase (MPO) activity quantitated monocyte adhesion to BRECs as described previously [25 ].

Determination of cell-associated adhesion molecule expression
Endothelial cell surface expression of ICAM-1, VCAM-1, and E-selectin was determined by cellular enzyme-linked immunosorbent assay (ELISA) method. After treatment with the appropriate agents, confluent BRECs were washed with PBS. To block nonspecific binding, endothelial cells were treated for 1 h at room temperature with PBS–3% BSA. Monoclonal antibodies (10 µg/ml) against ICAM-1, VCAM-1, E-selectin, and control IgG (R&D Systems) were then added to the cells for 2 h at 37°C. After washing, endothelial cells were incubated for 90 min with horseradish-conjugated anti-mouse IgG (1/1000; Bio-Rad, Mississauga, Canada). The peroxidase substrate, OPD (Sigma Chemical Co.), was then added to the cells. The reaction was stopped by addition of 50 µl H2SO4 (0.5 M), and the optical density was read at 490 nm.

Adhesion blockade assay
BRECs were incubated with AGEs or VEGF in the presence or absence of antibodies against VEGF or ICAM-1 for 12 h. At the end of this incubation period, the cells were washed, and monocyte adhesion was measured as described previously.

Determination of cell viability
Cellular toxicity of all the agents under study was assessed by trypan blue exclusion. Cell viability was consistently found to be higher than 90%.

Western blot analysis
Cytosolic, membrane, or nuclear proteins (15 µg) were separated by electrophoresis through a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane using a trans-blot cell system (Bio-Rad). The membrane was blocked for 1 h at room temperature with PBS containing 3% BSA. After three washes with PBS/Tween 20 0.1%, the membrane was incubated overnight at 4°C with antibodies to PKCß1 or NF-{kappa}Bp65 in PBS/Tween. The membrane was next washed with PBS/Tween and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-mouse or donkey anti-rabbit IgG (1:5000). Antigen detection was performed with an enhanced chemiluminescence detection system (Amersham Biosciences).

Statistical analyses
Data were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test for multiple comparisons or by the unpaired Student’s t-test for pairwise comparisons. Results are expressed as mean ± SEM values. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of AGEs on human monocyte adhesion to BRECs: inhibitory of antioxidants
Incubation of BRECs with increasing concentrations of AGEs for 6 h enhanced monocyte adhesion in a dose-dependent manner (Fig. 1A ). Maximal effect was seen at concentrations between 100 and 200 µg/ml AGEs (Fig. 1A) . This effect was still observed at 12 h, declining toward baseline after 24 h of stimulation (Fig. 1B) . The presence of gliclazide (1–10 µg/ml) during the incubation period of BRECs with AGEs inhibited AGE-induced monocyte adhesion in a concentration-dependent manner (Fig. 1C) . In contrast, exposure of these cells to equimolar concentrations of glyburide, a sulfonylurea without antioxidant activity (5 mg glyburide equivalent to 80 mg gliclazide) did not affect this parameter (Table 1 ). The effect of gliclazide on monocyte adhesion to BRECs was mimicked by established antioxidants such as vitamin C (10 µM) and vitamin E (50 µM; Table 1 ). Taken collectively, these data support the possibility that the antioxidant properties of gliclazide might account for its inhibitory effect on AGE-induced monocyte adhesion.



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  		Figure 1. Effect of AGEs on human monocyte adhesion to BRECs. Inhibitory effect of gliclazide. Confluent BRECs were incubated with (A) increasing concentrations of AGEs for 6 h, (B) 100 µg/ml AGEs for 6–24 h, or (C) 100 µg/ml AGEs in the presence or absence of increasing concentrations of gliclazide for 12 h. At the end of the incubation period, cells were washed twice with HBSS, and 230,000 freshly isolated human monocytes were added to each well. Monocyte adhesion to BRECs was measured by the MPO assay as described in Materials and Methods. Data represent the mean ± SEM of four independent experiments. **, P < 0.01, versus control (CTL); *, P < 0.05, versus CTL; #, P < 0.05, versus AGEs.

 

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  		Table 1. Effect of Vitamin C, Vitamin E, and Glyburide on AGE-Induced Monocyte Adhesion to BRECs

 
Effect of AGEs on retinal endothelial cell adhesion molecule expression: effect of oxidative stress
To determine the molecular mechanisms involved in AGE-induced monocyte adhesion to BRECs, the effect of AGEs on retinal endothelial cell adhesion molecule expression was next examined. Treatment of BRECs with increasing concentrations of AGEs for 6 h significantly enhanced ICAM-1 expression. Maximal effect was observed at a concentration of 100 µg/ml AGEs (Table 2 ). AGE (100 µg/ml)-induced ICAM-1 expression was still evident at 12 h (data not shown). In contrast, exposure of BRECs to AGEs for 6 (Table 2) or 12 h (data not shown) did not increase VCAM-1 or E-selectin expression. To evaluate whether increased expression of ICAM-1 was involved in AGE-induced monocyte adhesion, BRECs were treated with AGEs in the presence of an antibody to ICAM-1 (10 µg/ml) before adhesion experiments. Anti-ICAM-1 antibody totally inhibited AGE-induced monocyte adhesion (Fig. 2 ). As regulation of endothelial adhesion molecule expression involves oxidative stress [26 ], the effect of antioxidants on AGE-induced ICAM-1 expression was next determined. Incubation of BRECs with gliclazide (2.5 µg/ml) or vitamin E (50 µM) led to a marked suppression of AGE-induced ICAM-1 expression (Table 2) .


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  		Table 2. Effect of AGEs on Cell Adhesion Molecule Expression in BRECs: Inhibitory Effect of Gliclazide and Vitamin E (% of Control Values)

 


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  		Figure 2. Effect of anti-ICAM-1 or anti-VEGF antibodies on AGE-induced monocyte adhesion. Confluent BRECs were incubated for 12 h with AGEs (100 µg/ml) in the presence or absence of an antibody against ICAM-1 (10 µg/ml) or VEGF (2 µg/ml). At the end of this incubation period, cells were washed twice with HBSS, and 230,000 freshly isolated human monocytes were added to each well. Monocyte adhesion to BRECs was determined by the MPO assay as described in Materials and Methods. Data represent the mean ± SEM of five independent experiments. **, P < 0.01, versus CTL; ##, P < 0.01, versus AGE.

 
Involvement of VEGF in AGE-induced monocyte adhesion to BRECs
On the basis of our previous results showing that AGEs induce VEGF expression in BRECs [12 ], we evaluated the role of this growth factor as mediator of the stimulatory effect of AGEs on monocyte adhesion. To do so, we first evaluated the direct effect of VEGF on monocyte adhesion to BRECs and the mechanisms underlying this effect. Our results demonstrated that incubation of BRECs with VEGF (20 ng/ml) for 6 h significantly increased monocyte adhesion to these cells (169% over control values; P<0.01). This treatment also increased the cell-associated expression of ICAM-1, whereas VCAM-1 and E-selectin antigens remained unchanged (Fig. 3A ). Exposure of BRECs to anti-ICAM-1 antibody (10 µg/ml) inhibited VEGF-induced monocyte adhesion (Fig. 3B) , thus supporting a role for this antigen in VEGF-induced monocyte adhesion to BRECs.



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  		Figure 3. VEGF increases ICAM-1 expression on BRECs. (A) Confluent BRECs were treated with VEGF (20 ng/ml) for 12 h. Endothelial cell-surface expression of ICAM-1, VCAM-1, and E-selectin was determined by the cellular ELISA method as described in Materials and Methods. Data represent the mean ± SEM of four independent experiments. *, P < 0.05, versus CTL. (B) Confluent BRECs were incubated with VEGF (20 ng/ml) for 12 h in the presence or absence of an antibody against ICAM-1 (10 µg/ml). At the end of this incubation period, cells were washed twice with HBSS, and 230,000 freshly isolated human monocytes were added to each well. Monocyte adhesion to BRECs was determined by the MPO assay as described in Materials and Methods. Data represent the mean ± SEM of four independent experiments. **, P < 0.01, versus CTL.

 
To evaluate the possibility that VEGF could mediate the stimulatory effect of AGEs on monocyte adhesion to BRECs, we next assessed the effect of VEGF immunoneutralization on AGE-induced monocyte adhesion and ICAM-1 expression. Incubation of BRECs with AGEs in the presence of an anti-VEGF antibody (2 µg/ml), before adhesion experiments, inhibited the increase in monocyte adhesion (Fig. 2) and ICAM-1 expression in response to AGEs [ICAM-1 expression (% of control values), control: 100±9; AGE: 145±12; anti-VEGF+AGE: 93±13]. To determine whether oxidative stress is involved in VEGF-induced monocyte adhesion and ICAM-1 expression, we further assessed the effect of gliclazide and vitamin E on these parameters. Incubation of BRECs in the presence of gliclazide (2.5 µg/ml) or vitamin E (50 µM) abrogated the stimulatory effect of VEGF on monocyte adhesion and ICAM-1 expression, respectively (Fig. 4A and 4B ).



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  		Figure 4. Effect of gliclazide and vitamin E on VEGF-induced monocyte adhesion and ICAM-1 expression on BRECs. Confluent BRECs were incubated with AGEs (100 µg/ml) in the presence or absence of gliclazide (2.5 µg/ml) or vitamin E (50 µM) for 12 h. Monocyte adhesion to BRECs (A) and endothelial cell-surface expression of ICAM-1 (B) were measured by the MPO assay and the cellular ELISA method, respectively. Data represent the mean ± SEM of four independent experiments. *, P < 0.05, versus CTL; **, P < 0.01, versus CTL; #, P < 0.05, versus VEGF; ##, P < 0.01, versus VEGF.

 
Effect of PKC and NF-{kappa}B inhibitors on AGE-induced monocyte adhesion and ICAM-1 expression
As we previously demonstrated that PKC and NF-{kappa}B activation mediates AGE-induced VEGF expression in BRECs [12 ], we next examined whether pharmacological inhibition of these signaling pathways may affect AGE-induced monocyte adhesion and ICAM-1 expression. Results from Western blot analysis demonstrated that addition of specific inhibitors of PKC (GF10923X, 20 nM) or NF-{kappa}B (BAY117086, 40 µM) during the incubation period of BRECs with AGE or VEGF reduced, at the time of adhesion, PKCß1 expression as well as NF-{kappa}B activation elicited by these agents (data not shown). Inhibition of PKC and NF-{kappa}B also suppressed AGE-induced monocyte adhesion (Fig. 5A ) and ICAM-1 expression (Fig. 6A ). Likewise, culture of BRECs in the presence of PKC and NF-{kappa}B inhibitors completely abrogated the stimulatory effect of VEGF on these parameters (Figs. 5B and 6B) .



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  		Figure 5. PKC and NF-{kappa}B activation is involved in AGE- and VEGF-induced monocyte adhesion to BRECs. Confluent BRECs were treated for 12 h with AGEs (100 µg/ml; A) or VEGF (20 ng/ml; B) in the presence or absence of inhibitors of PKC (GF10923X, 20 nM) or NF-{kappa}B (BAY117086, 40 µM). At the end of this incubation period, cells were washed, and monocyte adhesion to BRECs was measured by the MPO assay as described in Materials and Methods. Data represent the mean ± SEM of four independent experiments. **, P < 0.01, versus CTL; ***, P < 0.001, versus CTL; ##, P < 0.05, versus AGE; ###, P < 0.001, versus VEGF.

 


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  		Figure 6. AGEs and VEGF induce ICAM-1 expression on BRECs through PKC and NF-{kappa}B activation. Confluent BRECs were treated for 6 h with AGEs (100 µg/ml; A) or VEGF (20 ng/ml; B) in the presence or absence of inhibitors of PKC (GF10923X, 20 nM) or NF-{kappa}B (BAY117086, 40 µM). Endothelial cell-surface expression of ICAM-1 was determined by the cellular ELISA method as described in Materials and Methods. Data represent the mean ± SEM of four independent experiments. **, P < 0.01, versus CTL; ***, P < 0.001, versus CTL; ##, P < 0.05, versus AGE or VEGF; ###, P < 0.001, versus AGE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have demonstrated that AGEs increase monocyte adhesion to macrovascular endothelial cells and that this effect involves oxidative stress and the up-regulation of endothelial cell adhesion molecules, including ICAM-1, VCAM-1, and E-selectin [18 , 27 ]. The present work demonstrates for the first time that AGEs at concentrations similar to those found in diabetic patients [28 ] increase human monocyte adhesion to retinal microvascular endothelial cells, thus suggesting a causal role for these products in retinal leukostasis associated with DR.

Considerable evidence indicates a pivotal role for AGEs in the pathogenesis of DR. A major mechanism by which accumulation of AGEs in the retina may contribute to this vasculopathy is the induction of VEGF expression by retinal cells [10 , 11 ]. Indeed, it has been shown that this growth factor mediates the deleterious effects of AGEs on vascular permeability and blood flow changes [16 ]. Recent in vivo data demonstrate that increased leukocyte adhesion to the retinal capillaries is operative in the pathogenesis of vascular leakage [3 , 29 ] and that this pathological process is triggered by VEGF [7 ]. In line with these observations, our results demonstrate that VEGF exerts a direct stimulatory effect on human monocyte adhesion to cultured retinal endothelial cells. In accordance with previous data showing that VEGF increases retinal vascular ICAM-1 expression in vivo [30 ], we found that this growth factor induces the selective expression of ICAM-1 at the retinal endothelial cell surface. This finding, along with our results demonstrating that immunoneutralization of ICAM-1 totally abrogates the stimulatory effect of VEGF on monocyte adhesion to retinal endothelial cells, clearly identify ICAM-1 as the key mediator of the effect of VEGF on retinal leukostasis. This conclusion is supported by previous observations showing that up-regulation of ICAM-1 and VEGF coincides, in experimental diabetes, with retinal leukostasis [2 , 3 ] and that inhibition of VEGF suppresses ICAM-1 expression and leukostasis [4 ]. The selective effect of VEGF on ICAM-1 expression in microvascular cells is in contrast with the documented effect of this growth factor on ICAM-1, VCAM-1, and E-selectin in macrovascular cells [31 ], thus suggesting that expression of adhesion molecules in response to VEGF differs between large vessel and microvascular endothelial cells.

Among the upstream stimuli for retinal VEGF expression are AGEs. Indeed, these factors have been shown to induce VEGF expression in several retinal cells, including epithelial, Muller, and endothelial cells [12 13 14 15 ]. Based on these findings and on the documented effect of VEGF and AGEs on retinal leukostasis and monocyte adhesion to macrovascular cells, respectively [3 , 8 , 27 ], we hypothesized that VEGF could mediate the stimulatory effect of AGEs on monocyte adhesion to retinal cells. Our results, showing that AGEs induce monocyte adhesion to retinal endothelial cells and that immunoneutralization of VEGF totally suppresses this effect, indicate a new role for VEGF, that of mediating the stimulatory effect of AGEs on retinal leukostasis. Moreover, our data showing that immunoneutralization of VEGF totally inhibits ICAM-1 expression in AGE-treated BRECs clearly identify VEGF as the key determinant of ICAM-1 induction in these cells. As our data were generated with isolated retinal endothelial cells, we conclude that VEGF released by AGE-treated retinal endothelial cells acts as an autocrine-activating stimulus for retinal endothelial cells leading to increased ICAM-1 expression and monocyte adhesion.

Several arguments suggest that oxidative stress-induced PKC and NF-{kappa}B activation may be involved in the stimulatory effect of AGEs on monocyte adhesion to retinal endothelial cells. First, we and others have demonstrated that AGEs and VEGF activate PKC and NF-{kappa}B in BRECs [12 , 32 , 33 ] and that AGEs induce VEGF expression in BRECs through activation of these signaling pathways [12 ]. Second, evidence has been provided that regulation of cell adhesion molecule expression implicates oxidative stress-induced activation of PKC and NF-{kappa}B [34 35 36 ]. Third, vitamin E, {alpha}-lipoic acid, and the PKCß isoform inhibitor LY333531 have been shown to prevent enhanced leukostasis in diabetic rats [37 ]. Fourth, inhibition of retinal NF-{kappa}B activation has been shown to coincide with reduction of retinal ICAM-1 expression and leukostasis in a rat model of DR [38 ]. Consistent with a major role of oxidative stress in AGE-induced leukostasis, we found that antioxidants such as vitamins C and E as well as gliclazide, a sulfonylurea with antioxidant activity [19 20 21 22 ], suppress AGE-induced retinal endothelial cell ICAM-1 expression and monocyte adhesion. These results are in line with previous data showing that gliclazide and the antioxidant {alpha}-lipoic acid reduce, through inhibition of adhesion molecule expression, monocyte adhesion to macrovascular endothelial cells induced by AGEs [18 , 27 ]. Previous studies have demonstrated that reactive oxygen species are important in VEGF signaling in vascular cells, including human vein endothelial cells [39 ] and smooth muscle cells [40 ], and that induction of ICAM-1 by VEGF involves the activation of stress-sensitive pathways, including NF-{kappa}B and PKC [31 ]. In accordance with these results, we have demonstrated that agents with antioxidant properties inhibit VEGF-induced ICAM-1 expression and monocyte adhesion to retinal endothelial cells and that pharmacological inhibition of PKC and NF-{kappa}B abolishes retinal ICAM-1 expression and monocyte adhesion in AGE- and VEGF-treated retinal endothelial cells. On the basis of our previous results showing that gliclazide and antioxidants decrease AGE-induced PKC and NF-{kappa}B activation in BRECs [12 , 41 ], it seems reasonable to postulate that oxidative stress-dependent activation of these signal transduction pathways is involved in monocyte adhesion to retinal endothelial cells elicited by AGEs.

Although the relevance of our in vitro findings remains to be determined, several observations suggest that AGEs may promote leukostasis through VEGF induction by retinal cells in vivo. First, a close association has been established between AGE accumulation and VEGF expression in human retinas [11 , 42 ], and a direct stimulatory effect of AGEs on retinal VEGF expression has been documented [12 13 14 15 ]. Second, AGE inhibition has been shown to attenuate retinal microvascular lesion formation in experimental diabetes [43 ]. Third, evidence has been provided that blocking VEGF suppresses retinal leukostasis in diabetic rats [4 ].

In summary, this study demonstrates that AGEs are potent stimuli in inducing ICAM-1-mediated monocyte adhesion to retinal endothelial cells. This effect involves VEGF expression by these cells, oxidative stress, and activation of PKC and NF-{kappa}B signaling pathways. Further work is required to determine whether targeting retinal VEGF expression by pharmacological agents, which decrease oxidative stress or inhibit PKC activity, may reduce AGE-induced diabetic retinal leukostasis and BRB breakdown in experimental diabetes.


   ACKNOWLEDGEMENTS
 
This study was supported by Servier Canada. The authors thank Dr. Lloyd P. Aiello (Joslin Diabetes Centre, Harvard Medical School) for providing BRECs.

Received June 11, 2003; revised December 17, 2003; accepted February 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Miyamoto, K., Ogura, Y. (1999) Pathogenetic potential of leukocytes in diabetic retinopathy Semin. Ophthalmol. 14,233-239[Medline]
  2. Joussen, A. M., Murata, T., Tsujikawa, A., Kirchhof, B., Bursell, S-E., Adamis, A. P. (2001) Leukocyte-mediated endothelial cell injury and death in the diabetic retina Am. J. Pathol. 158,147-152[Abstract/Free Full Text]
  3. Miyamoto, K., Khosrof, S., Bursell, S. E., Rohan, R., Murata, T., Clermont, A. C., Aiello, L. P., Ogura, Y., Adamis, A. P. (1999) Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition Proc. Natl. Acad. Sci. USA 96,10836-10841[Abstract/Free Full Text]
  4. Joussen, A. M., Poulaki, V., Qin, W., Kirchhof, B., Mitsiades, N., Wiegand, S. J., Rudge, J., Yancopoulos, G. D., Adamis, A. P. (2002) Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo Am. J. Pathol. 160,501-509[Abstract/Free Full Text]
  5. Barouch, F. C., Miyamoto, K., Allport, J. R., Fujita, K., Bursell, S. E., Aiello, L. P., Luscinskas, F. W., Adamis, A. P. (2000) Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes Invest. Ophthalmol. Vis. Sci. 41,1153-1158[Abstract/Free Full Text]
  6. Miyamoto, K., Ogura, Y., Kenmochi, S., Honda, Y. (1997) Role of leukocytes in diabetic microcirculatory disturbances Microvasc. Res. 54,43-48[CrossRef][Medline]
  7. Miyamoto, K., Khosrof, S., Bursell, S. E., Moromizato, Y., Aiello, L. P., Ogura, Y., Adamis, A. P. (2000) Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1) Am. J. Pathol. 156,1733-1739[Abstract/Free Full Text]
  8. Stitt, A. W., Bhaduri, T., McMullen, C. B., Gardiner, T. A., Archer, D. B. (2000) Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats Mol. Cell. Biol. Res. Commun. 3,380-388[CrossRef][Medline]
  9. Leto, G., Pricci, F., Amadio, L., Iacobinic, C., Cordone, S., Diaz-Horta, O., Romeo, G., Barsotti, P., Rotella, C. M., di Mario, U., Pugliese, G. (2001) Increased retinal endothelial cell monolayer permeability induced by the diabetic milieu: role of advanced non-enzymatic glycation and polyol pathway activation Diabetes Metab. Res. Rev. 17,448-458[CrossRef][Medline]
  10. Segawa, Y., Shirao, Y., Yamagishi, S., Higashide, T., Kobayashi, M., Katsuno, K., Iyobe, A., Harada, H., Sato, F., Miyata, H., Asai, H., Nishimura, A., Takahira, M., Souno, T., Segawa, Y., Maeda, K., Shima, K., Mizuno, A., Yamamoto, H., Kawasaki, K. (1998) Upregulation of retinal vascular endothelial growth factor mRNAs in spontaneously diabetic rats without ophthalmoscopic retinopathy. A possible participation of advanced glycation end products in the development of the early phase of diabetic retinopathy Ophthalmic. Res. 30,333-339[CrossRef][Medline]
  11. Murata, T., Nagai, R., Ishibashi, T., Inomuta, H., Ikeda, K., Horiuchi, S. (1997) The relationship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas Diabetologia 40,764-769[CrossRef][Medline]
  12. Mamputu, J. C., Renier, G. (2002) Advanced glycation end products increase, through a protein kinase C-dependent pathway, vascular endothelial growth factor expression in retinal endothelial cells. Inhibitory effect of gliclazide J. Diabetes Complications 16,284-293[CrossRef][Medline]
  13. Lu, M., Kuroki, M., Amano, S., Tolentino, M., Keough, K., Kim, I., Bucala, R., Adamis, A. P. (1998) Advanced glycation end products increase retinal vascular endothelial growth factor expression J. Clin. Invest. 101,1219-1224[Medline]
  14. Hirata, C., Nakano, K., Nakamura, N., Kitagawa, Y., Shigeta, H., Hasegawa, G., Ogata, M., Ikeda, T., Sawa, H., Nakamura, K., Ienaga, K., Obayashi, H., Kondo, M. (1997) Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Muller cells Biochem. Biophys. Res. Commun. 236,712-715[CrossRef][Medline]
  15. Hoffmann, S., Friedrichs, U., Eichler, W., Rosenthal, A., Wiedemann, P. (2002) Advanced glycation end products induce choroidal endothelial cell proliferation, matrix metalloproteinase-2 and VEGF upregulation in vitro Graefes Arch. Clin. Exp. Ophthalmol. 240,996-1002[Medline]
  16. Ido, Y., Chang, K. C., Lejeune, W. S., Bjercke, R. J., Reiser, K. M., Williamson, J. R., Tilton, R. G. (2001) Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C Microcirculation 8,251-263[CrossRef][Medline]
  17. Basta, G., Lazzerini, G., Massaro, R., Simoncini, T., Tanganelli, P., Fu, C., Kislinger, T., Stern, D. M., Schmidt, A. M., De Caterina, R. (2002) Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses Circulation 105,816-822[Abstract/Free Full Text]
  18. Desfaits, A. C., Serri, O., Renier, G. (1999) Gliclazide reduces the induction of human monocyte adhesion to endothelial cells by glycated albumin Diabetes Obes. Metab. 1,113-120[CrossRef][Medline]
  19. Desfaits, A. C., Serri, O., Renier, G. (1997) Gliclazide decreases cell-mediated low-density lipoprotein (LDL) oxidation and reduces monocyte adhesion to endothelial cells induced by oxidatively modified LDL Metabolism 46,1150-1156[CrossRef][Medline]
  20. Noda, Y., Mori, A., Paker, L. (1997) Gliclazide scavenges hydroxyl, superoxide, and nitric oxide radicals: an ESR study Res. Commun. Mol. Pathol. Pharmacol. 96,115-124[Medline]
  21. O’Brien, R. C., Luo, M., Balazs, N., Mercuri, J. (2000) In vitro and in vivo antioxidant properties of gliclazide J. Diabetes Complications 14,201-206[CrossRef][Medline]
  22. Nomura, H., Naito, M., Kodama, M., Kato, Y., Iguchi, A. (2000) Effects of gliclazide on low-density lipoprotein oxidizability and atherosclerosis in cholesterol-fed rabbits J. Atheroscler. Thromb. 7,104-109[Medline]
  23. Aiello, L. P., Northrup, J. M., Keyt, B. A., Takagi, H., Iwamoto, M. A. (1995) Hypoxic regulation of vascular endothelial growth factor in retinal cells Arch. Ophthalmol. 113,1538-1544[Abstract]
  24. Mentzer, S. J., Guyre, P. M., Burakoff, S. J., Faller, D. V. (1986) Spontaneous aggregation as a mechanism for human monocyte purification Cell. Immunol. 101,312-319[CrossRef][Medline]
  25. Bath, P. M. W., Booth, R. F. G., Hassal, D. G. (1989) Monocyte-lymphocyte discrimination in a new microtitre-based adhesion assay J. Immunol. Methods 118,59-65[Medline]
  26. Kunsch, C., Medford, R. M. (1999) Oxidative stress as a regulator of gene expression in the vasculature Circ. Res. 85,753-766[Abstract/Free Full Text]
  27. Kunt, T., Forst, T., Wilhelm, A., Tritschler, H., Pfuetzner, A., Harzer, O., Engelbach, M., Zschaebitz, A., Stofft, E., Beyer, J. (1999) {alpha}-Lipoic acid reduces expression of vascular cell adhesion molecule-1 and endothelial adhesion of human monocytes after stimulation with advanced glycation end products Clin. Sci. 96,75-82[Medline]
  28. Makita, Z., Vlassara, H., Cerami, A., Bucala, R. (1992) Immunochemical detection of advanced glycosylation end products in vivo J. Biol. Chem. 267,5133-5138[Abstract/Free Full Text]
  29. Miyamoto, K., Hiroshiba, N., Tsujikawa, A., Ogura, Y. (1998) In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats Invest. Ophthalmol. Vis. Sci. 39,2190-2194[Abstract/Free Full Text]
  30. Lu, M., Perez, V. L., Ma, N., Miyamoto, K., Peng, H. B., Liao, J. K., Adamis, A. P. (1999) VEGF increases retinal vascular ICAM-1 expression in vivo Invest. Ophthalmol. Vis. Sci. 40,1808-1812[Abstract/Free Full Text]
  31. Kim, I., Moon, S. O., Kim, S. H., Kim, H. J., Koh, Y. S., Koh, G. Y. (2001) Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-{kappa} B activation in endothelial cells J. Biol. Chem. 276,7614-7620[Abstract/Free Full Text]
  32. Sone, H., Deo, B. K., Kumagai, A. K. (2000) Enhancement of glucose transport by vascular endothelial growth factor in retinal endothelial cells Invest. Ophthalmol. Vis. Sci. 41,1876-1884[Abstract/Free Full Text]
  33. Marumo, T., Schini-Kerth, V. B., Busse, R. (1999) Vascular endothelial growth factor activates nuclear factor-{kappa}B and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells Diabetes 48,1131-1137[Abstract]
  34. Zhang, W. J., Frei, B. (2001) {alpha}-Lipoic acid inhibits TNF-{alpha}-induced NF-{kappa}B activation and adhesion molecule expression in human aortic endothelial cells FASEB J. 15,2423-2432[Abstract/Free Full Text]
  35. Lum, H., Roebuck, K. A. (2001) Oxidant stress and endothelial cell dysfunction Am. J. Physiol. Cell Physiol. 280,C719-C741[Abstract/Free Full Text]
  36. Pueyo, M. E., Gonzalez, W., Nicoletti, A., Savoie, F., Arnal, J. F., Michel, J. B. (2000) Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-{kappa}B activation induced by intracellular oxidative stress Arterioscler. Thromb. Vasc. Biol. 20,645-651[Abstract/Free Full Text]
  37. Abiko, T., Abiko, A., Clermont, A. C., Shoelson, B., Horio, N., Takahashi, J., Adamis, A. P., King, G. L., Bursell, S. E. (2003) Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation Diabetes 52,829-837[Abstract/Free Full Text]
  38. Joussen, A. M., Poulaki, V., Mitsiades, N., Kirchhof, B., Koizumi, K., Dohmen, S., Adamis, A. P. (2002) Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-{alpha} suppression FASEB J. 16,438-440[Free Full Text]
  39. Ushio-Fukai, M., Tang, Y., Fukai, T., Dikalov, S. I., Ma, Y., Fujimoto, M., Quinn, M. T., Pagano, P. J., Johnson, C., Alexander, R. W. (2002) Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signalling and angiogenesis Circ. Res. 91,1160-1167[Abstract/Free Full Text]
  40. Wang, Z., Castresana, M. R., Newman, W. H. (2001) Reactive oxygen and NF-{kappa}B in VEGF-induced migration of human vascular smooth muscle cells Biochem. Biophys. Res. Commun. 285,669-674[CrossRef][Medline]
  41. Mamputu, J. C., Renier, G. (2004) Signalling pathways involved in retinal endothelial cell proliferation induced by advanced glycation end products. Inhibitory effect of gliclazide Diabetes Obes. Metab. 6,95-103[CrossRef][Medline]
  42. Endo, M., Yanagisawa, K., Tsuchida, K., Okamoto, T., Matsushita, T., Higuchi, M., Matsuda, A., Takeuchi, M., Makita, Z., Koike, T. (2001) Increased levels of vascular endothelial growth factor and advanced glycation end products in aqueous humor of patients with diabetic retinopathy Horm. Metab. Res. 33,317-322[CrossRef][Medline]
  43. Stitt, A., Gardiner, T. A., Alderson, N. L., Canning, P., Frizzell, N., Duffy, N., Boyle, C., Januszewski, A. S., Chachich, M., Baynes, J. W., Thorpe, S. R., Anderson, N. L. (2002) The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes Diabetes 51,2826-2832[Abstract/Free Full Text]



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