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Inhibitory Effect of GSPE on RAGE Expression Induced by Advanced Glycation End Products in Endothelial Cells

Zhang, Feng-Lei PhD; Gao, Hai-Qing PhD; Shen, Lin PhD

Journal of Cardiovascular Pharmacology: October 2007 - Volume 50 - Issue 4 - p 434-440
doi: 10.1097/FJC.0b013e3181342bfa
Original Article
Free

Advanced glycation end products' (AGEs) engagement of a cell-surface receptor for AGEs (RAGE) has been causally implicated in the pathogenesis of diabetic vascular complications via induction of reactive oxygen species (ROS) and subsequent alteration of many gene expressions, including RAGE itself. Grapeseed proanthocyanidin extract (GSPE), which is a naturally occurring polyphenolic compound, has been reported to possess potent radical-scavenging and antioxidant properties and to display significant cardiovascular protective action. In this study, we investigated whether GSPE could inhibit AGE-induced RAGE expression through interference with ROS generation in human umbilical-vein endothelial cells (HUVECs). AGE-modified bovine serum albumin (AGE-BSA) was prepared by incubating BSA with high-concentration glucose. Stimulation of cultured HUVECs with 200 μg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the protein and mRNA expression of RAGE; unmodified BSA and GSPE alone had no effect. However, GSPE preincubation markedly downregulated AGE-induced surface expression of RAGE in a time- and concentration-dependent manner. In AGE-stimulated HUVECs, GSPE also dose-dependently decreased RAGE mRNA levels and inhibited AGE-induced ROS generation at defined time periods. These results demonstrate that GSPE can inhibit enhanced RAGE expression in AGE-exposed endothelial cells by suppressing ROS generation, thereby limiting the AGE-RAGE interaction. Hence, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in diabetic patients.

From the Department of Geriatrics, Qilu Hospital of Shandong University, Jinan, China.

Received for publication March 16, 2007; accepted June 5, 2007.

Reprints: Hai-Qing Gao, PhD, Department of Geriatrics, Qilu Hospital of Shandong University, Jinan 250012, China (e-mail address: gaohaiqing99@yahoo.com.cn).

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INTRODUCTION

Glucose and other reducing sugars can react nonenzymatically with the free amino groups of proteins to yield irreversibly cross-linked protein derivatives, termed advanced glycation end products (AGEs). Under hyperglycemic conditions in diabetes, AGEs are formed at an accelerated rate and accumulate in blood and tissue. Many studies have revealed that AGEs have been implicated in the initiation and development of diabetic vascular complications, mainly through interactions with their receptor, RAGE (receptor for advanced glycation end products), which is expressed on the surface of a variety of cell types, including endothelial cells, smooth-muscle cells, lymphocytes, and monocytes. RAGE on endothelial cells may function as an adhesive receptor that directly interacts with leukocyte β2-integrins, thereby being directly involved in inflammatory cell recruitment.1 Furthermore, RAGE has already been identified as the best-characterized signal transducer for AGEs. The engagement of RAGE on endothelial cells by AGEs activates a complex cascade of signal-transduction events via the induction of intracellular reactive oxygen species (ROS), which seems to be linked, at least in part, to the activation of the NADPH oxidase system and the mitochondrial electron-transport system.2,3 Subsequently, the expression of many genes, the majority of which are highly relevant for inflammation, immunity, and atherosclerosis-such as cell-adhesion molecules, tissue factor, chemokines, and cytokines-is enhanced,4 thereby leading to the deterioration of vascular functions. Importantly, the expression of RAGE itself is also upregulated in this pathological procedure. Indeed, at sites of accumulated AGEs in the vascular lesions, there is increasing expression of the endothelium RAGE.5 This positive-feedback activation by AGE-RAGE interaction is thought to lead to prolonged cellular dysfunction and vascular injury.

Multiple in vivo studies have demonstrated that blockage of RAGE by administration of anti-RAGE IgG or soluble RAGE (sRAGE, the extracellular ligand-binding decoy of RAGE) reversed the enhanced vascular hyperpermeability, attenuated the accelerated early lesion expansion, and suppressed the progression of established atherosclerotic lesions and the development of complexity in diabetic rats or diabetic apolipoprotein (apo)E null mice.6-9 Moreover, in diabetic rats subjected to artery injury, administration of sRAGE resulted in remarkable inhibition of neointimal expansion.10 Similar results were also found in both RAGE null mice and transgenic mice with cytosolic domain-deleted RAGE.11 Otherwise, RAGE blockade has a striking, beneficial effect on diabetic microvascular complications such as nephropathy, neuropathy, and retinopathy.12-14 These findings further suggest that RAGE imparts a critical impact on diabetic vascular lesions and that it may be an important therapeutic target. Yamagishi and Takeuchi15 found that nifedipine significantly inhibited RAGE gene expression in endothelial cells through their antioxidant mechanism. These data provide us new insight on whether many antioxidants derived from the plants can also affect RAGE expression in the vasculature, thereby interrupting the adverse effects and vicious circle caused by AGE-RAGE interaction. GSPE is a natural extract from grapeseeds; it possesses powerful free radical-scavenging and antioxidant properties,16,17 and it has been reported to have an effective cardiovascular protective ability. We also have recently found that GSPE possesses antinonenzymatic glycation efficacy in diabetic rats. In this study, we evaluate whether GSPE can inhibit RAGE overexpression in AGE-exposed endothelial cells by suppressing ROS generation.

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MATERIALS AND METHODS

GSPE (the proanthocyanidin content exceeds 96%) were provided by Jianfeng, Inc. (Tianjin, China). Bovine serum albumin (BSA), D-glucose, collagenase, trypsin/EDTA solution, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, Mo). Fetal bovine serum (FBS) and Medium 199 were obtained from GIBCO (Grand Island, NY). Tissue-culture flasks and plates were supplied by Costar (Cambridge, Mass). RAGE and β-actin primer sets were synthesized by Sangon Biotech (Shanhai, China).

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AGE-BSA Preparation

AGEs used in this study were prepared by incubating 50 mg/mL of BSA with 0.5 M glucose in 0.2 M phosphate-buffered saline (PBS pH 7.4) in the dark at 37°C for 3 months. Before incubation, the solution was sterile-filtered by passing it through a 0.2-mm filter. Unmodified BSA was treated under the same conditions without glucose as a control. At the end of the incubation period, extensive dialysis was performed against PBS to remove unincorporated sugars. The identification of AGEs was performed by fluorescence spectrophotometry (Hitachi V-2001, Japanese; 390-nm excitation wavelength, 450-nm emission wavelength) because of its typical absorption and fluorescent spectra patterns. As a result, the AGE content in AGE-modified BSA was 81.8 U/mg of protein, whereas that in unmodified BSA was <0.9 U/mg of protein. The endotoxin content of these preparations was measured by the Limulus amebocyte lysate assay (Cambrex, USA), which revealed negligible values (<0.2 μg/L).

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Endothelial Cell Isolation and Culture

HUVECs were obtained from freshly umbilical cords by collagenase digestion, according to the method of Jaffe,18 which was approved by the ethics committee of our institution. In brief, the veins of umbilical cords were perfused with PBS to remove blood cells, filled with 0.1% collagenase (type Ia), and then left for 10 minutes at 37°C. The resulting cellular suspension was supplemented with PBS, centrifuged, and cultured in Medium 199 containing 100 U/mL of penicillin, 100 mg/mL of streptomycin, 2.5 μg/mL of fungizone, 50 μg/mL of gentamycin, 2 mM glutamine, 20 mM HEPES, and 20% FBS. Culture was on gelatin-coated 25-cm2 flasks, 6- or 24-well tissue-culture plates at 37°C under humidified 5% CO2 in room air. The medium was replaced every 2 days until confluence (3-5 days). HUVEC purity was confirmed by the “cobblestone” morphology typical for quiescent endothelial cells and by the presence of von Willebrand factor antigen. All of the experiments were performed with cells at passages 2 to 3. At cell confluence, FBS concentration in the medium was reduced to 5%, and GSPE in concentrations of 5, 15, and 25 μg/mL was added for preincubation. GSPE stock solutions for cell treatment were prepared fresh in DMSO at concentrations such that the final concentration of the solvent in cell suspension never exceeded 0.1% (vol/vol). The respective controls were treated with equal volumes of DMSO. Next, the HUVECs were stimulated with 200 ug/mL of AGEs or unmodified BSA for defined periods. Under all conditions, cell viability was greater than 96%, as judged by trypan blue exclusion.

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Measurement of RAGE Surface Expression in HUVEC

The expression of RAGE on HUVECs was measured by flow cytometry, as previously described. Briefly, the cells were preincubated in the absence and presence of GSPE for the indicated periods, followed by stimulation with 200 μg/mL of AGEs or unmodified BSA for 12 hours. After stimulation, cells were harvested by mild trypsinisation, treated with a polyclonal rabbit antihuman RAGE antibody or IgG1 (isotype control) for 30 minutes, and then stained with an FITC-conjugated goat antirabbit antibody for 30 minutes (Beijing Bioss Biotech Co, China). Next, cells were thoroughly washed with fluorescent-activated cell sorter buffer, fixed in 1% paraformaldehyde, and analyzed (10,000 cells per sample) by FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). After correction for nonspecific binding (isotype control), mean specific fluorescence intensity (MFI) was measured in each channel.

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Evaluation of RAGE mRNA Expression in HUVEC

According to the manufacturer's instructions, total RNA was extracted from cells using the UNIQ-10 Trizol total RNA extract kit (Sangon Biotech, Shanghai) after stimulation. The concentration of total RNA was determined by spectrophotometry at a 260-nm wavelength. The integrity of RNA samples was checked by gel electrophoresis in 1% agarose gel stained with ethidium bromide. To synthesize the first strand of cDNA, RT was performed using 2 μg of total RNA in a 20-μL final volume of reaction mixture (5 × reaction buffer, 10 mM dNTPmix, ribonuclease inhibitor, random hexamers, and MMLV reverse transcriptase) by incubation at 25°C for 10 minutes and 42°C for 60 minutes. The reaction was stopped by incubation at 70°C for 10 minutes (RT kit from MBI Fermentas Canada, Burlington, Canada). Thereafter, polymerase chain reaction (PCR) was carried out using 5 μL of cDNA obtained as templates in a 50-μL reaction volume containing 25 μL of Premix Taq, 2 μL of upstream primers (10 μM), 2 μL of downstream primers (10 μM), and 16 μL of double-distilled water (PCR kit from TaKaRa Bio, Otsu, Japan). The primers designed were as follows: RAGE (sense) 5′-CACCTTCTCCTGTAGCTTCA-3′, (antisense) 5′-TGCCACAAGATGACCCCAA-3′ β-actin (sense) 5′-ACACTGTGCCCATCTACGAGGGG-3′, (antisense) 5′-ATGATGTTGAAGGTAGTTTCGTGGAT-3′. The amplification profile consisted of an initial denaturation at 94°C for 4 minutes, denaturation at 94°C for 50 seconds, annealing at 55°C for 50 seconds, extension at 72°C for 1 minute with 35 cycles, and then final extension at 72°C for 5 minutes. Of the PCR products obtained (RAGE 480 bp, β-actin 375 bp), 10 μL were visualized by ethidium bromide staining and photographed under UV radiation after 1.5% agarose gel electrophoresis. Identified signal intensities of specific RAGE-PCR product bands were normalized with that of the β-actin-PCR product and expressed as a ratio.

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Detection of ROS in HUVEC

According to the method of Bass et al,19 the production of intracellular ROS was estimated fluorometrically using 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, sigma). DCFH-DA is a nonpolar compound that is converted into a nonfluorescent polar derivative (DCFH) by cellular esterases after incorporation into cells. DCFH is membrane impermeable and is rapidly oxidized to the highly fluorescent DCF in the presence of intracellular ROS. After treatment, cells (2 × 106) plated in six-well plates were treated with 5 μM DCFH-DA in the dark for 30 minutes at 37°C. The formation of DCF was determined by flow cytometry at a level of 10,000 events for each test. An argon-ion laser was used at an excitation wavelength of 488 nm, and green fluorescence collected through a 530-nm band-pass filter was measured on a logarithmic scale. The formation of ROS was expressed as relative fluorescence intensity (%).

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Statistical Analysis

Results were expressed as means ± SD and were based on at least three separate experiments. One-way ANOVA was used to determine the significance of differences among treatments. P < 0.05 was considered statistically significant.

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RESULTS

Effect of GSPE on RAGE Surface Expression in AGE-Exposed HUVEC

The surface expression of RAGE in the untreated HUVEC was very low. Both unmodified BSA and GSPE did not affect these basal expressions. The incubation of HUVEC with 200 μg/mL of AGE-BSA for 12 hours induced strong increases in the RAGE surface expression. Interestingly, pretreatment of HUVEC with different concentrations of GSPE for 4 hours markedly reduced AGE-stimulated RAGE expression in a dose-dependent manner. The levels of RAGE expression were reduced to 78.62 ± 7.01% for 5 μg/mL, to 48.16 ± 7.82% for 15 μg/mL, to 29.45 ± 5.76% for 25 μg/mL, respectively (P < 0.001 versus AGE-BSA alone; Fig. 1); otherwise, 25 μg/mL of GSPE preincubation for 30 minutes, 1 hours, and 4 hours before AGE stimulation also significantly downregulated the RAGE expression in a time-dependent manner. The levels of RAGE expression were reduced to 85.88 ± 7.29% for 30 minutes (P < 0.05 versus AGE-BSA alone), to 62.08 ± 10.02% for 1 hour, to 29.45 ± 5.76% for 4 hours (P < 0.001 versus AGE-BSA alone; Fig. 2).

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

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Effect of GSPE on RAGE mRNA Levels in HUVEC Stimulated by AGE-BSA

To characterize the molecular mechanisms responsible for the downregulation of AGE-induced RAGE expression by GSPE, RT-PCR was performed to evaluate the expression of RAGE genes. RAGE mRNA levels were very low in the untreated HUVEC. Likewise, both unmodified BSA and GSPE did not alter their basal expressions, whereas stimulation of HUVEC with 200 μg/mL of AGE-BSA for 6 hours led to a marked increase in specific RAGE-PCR products. However, pretreatment with different concentrations of GSPE for 4 hours significantly reduced the level of RAGE transcripts after AGE stimulation in a dose-dependent manner. Reductions differed, ranging from 1.12 ± 0.15 to 0.75 ± 0.11 for 5 μg/mL, to 0.48 ± 0.08 for 15 μg/mL, to 0.27 ± 0.10 for 25 μg/mL, respectively (P < 0.001 versus AGE-BSA alone). In contrast, the levels of β-actin mRNA expressed under these conditions remained the same. Consistent with the protein expression, GSPE may affect the transcription of RAGE gene (Fig. 3).

FIGURE 3

FIGURE 3

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Effect of GSPE on Intracellular ROS Formation in AGE-Exposed HUVEC

Intracellular ROS was estimated by flow cytometry using DCFH as a probe. The levels of ROS were low in the unstimulated HUVEC. Whereas both unmodified BSA and GSPE alone did not influence ROS generation, the stimulation of HUVEC with 200 μg/mL of AGE-BSA did significantly enhance ROS formation. On the other hand, pretreatment of GSPE, in a dose-dependent manner, apparently prevented AGE-induced ROS generation (Fig. 4), which was reduced to 78.45 ± 6.05% for 5 μg/mL, to 55.67 8.33% for 15 μg/mL, to 28.82 ± 2.03% for 25 μg/mL, respectively (P < 0.001 versus AGE-BSA alone). These results suggest that GSPE suppresses AGE-stimulated RAGE expression, probably through inhibition of ROS generation.

FIGURE 4

FIGURE 4

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DISCUSSION

AGE formation occurs ubiquitously and irreversibly in patients with diabetes mellitus. A growing body of evidence has shown that AGEs play a major role in the initiation and development of diabetic vascular complications, such as atherosclerosis and diabetic microangiopathy. As for its pathomechanisms, in addition to direct cross-linking and irreversible modification of vessel-wall structure, AGEs exert their detrimental effects on the vasculature primarily by interacting with their receptor, RAGE. RAGE belongs to the immunoglobulin superfamily, having three immunoglobulin-like domains in the N-terminal extracellular segment, one transmembrane region, and a short, highly charged C-terminal cytosolic tail, which is essential for RAGE-mediated intracellular signaling. Because of its properties as a pattern-recognition receptor (PRR), RAGE possesses multiple ligands including not only AGEs but also amyloid fibrils, amphoterins, and S100/calgranulins relevant to distinct pathological processes.20 The AGEs' engagement of RAGE triggers the generation of intracellular ROS and subsequent transcriptional activation of a variety of genes, such as cell-adhesion molecules, tissue factor, chemokines, and cytokines, thus helping to promote vascular-wall inflammation and procoagulant activity and, thereby, causing the deterioration of vascular functions. Importantly, the expression of RAGE itself is induced by AGE-RAGE interaction. This positive-feedback activation is thought to lead to prolonged cellular dysfunction. Therefore, limiting RAGE expression might be an intriguing method for prevention and treatment of diabetic vascular complications. In the present study, AGEs were prepared by incubating BSA with high-concentration glucose. Incubation of cultured HUVEC with 200 μg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the surface protein and gene expression of RAGE. Both unmodified BSA and GSPE alone had no effect. This result was fundamentally consistent with previous findings.

Intracellular oxidative stress plays a key role in the activation of signaling pathways and subsequent alteration of gene expression mediated by the AGE-RAGE interaction. Some antioxidants have been reported to modulate this pathological procedure through suppression of intracellular ROS generation. For example, N-acetylcysteine inhibited the enhanced VCAM-1 expression and blocked the induction of specific DNA-binding activity for NF-κB in the VCAM-1 promoter in AGE-treated endothelial cells.21 Alpha-lipoic acid also reduced the expression of VCAM-1 and endothelial adhesion of human monocytes after stimulation of HUVEC with AGEs.22 Similarly, minodronate, a nitrogen-containing bisphosphonate, prevented AGE-induced NF-κB activation and subsequent VCAM-1 gene expression in HUVEC by suppressing NADPH oxidase-derived ROS generation.23 Moreover, gliclazide, by its antioxidative properties, suppressed AGE-induced VEGF expression, PKC-NF-κB activation, and cell proliferation in bovine retinal endothelial cells.24 Also, the work from Yamagishi et al has shown that nifedipine downregulated gene expression of RAGE in AGE-exposed endothelial cells by suppressing ROS generation.15

GSPE with potent antioxidant and antiinflammatory properties, a naturally occurring polyphenolic compound from grapeseeds, has been reported to possess powerful cardiovascular protective abilities, as demonstrated by antiatherosclerotic, antithrombotic, and antihypertensive effects, reducing myocardial ischemia/reperfusion injury, inhibiting doxorubicin-induced cardiotoxicity, and preventing LDL oxidation.25-31 Our previous works have shown GSPE inhibited nonenzymatic glycation reaction of proteins and resulting AGE formation in diabetic rats. We also have found that GSPE could selectively suppress AGE-induced VCAM-1, but not ICAM-1, expression at the surface protein and mRNA levels in endothelial cells, in a concentration-dependent manner.32 In the present study, we have examined whether GSPE might influence the gene expression of RAGE, mediated by AGEs in endothelial cells. The results of our study demonstrate that GSPE preincubation markedly downregulated AGE-induced RAGE surface expression in a time- and concentration-dependent manner. In AGE-exposed HUVEC, GSPE also dose-dependently decreased RAGE mRNA levels. Meanwhile, similar to its effect on RAGE protein and mRNA expression, GSPE also inhibited intracellular ROS generation in a dose-dependent fashion at defined time periods, suggesting that the reactive oxygen-scavenging properties of GSPE might involve its inhibitory effects on RAGE expression at the protein and mRNA levels.

Puiggros et al33 have found that H2O2 treatment of the hepatocarcinoma cell line HepG2 led to a significant decrease in total GST (glutathione S-transferase) content and GPx/GR (glutathione peroxidase/glutathione reductase) mRNA levels, with significant increases in MDA and GR activity. Moreover, GPx and GST activity increased only slightly, and GST mRNA levels did not change. GSPE preincubtion prevented the decline of GST and maintained its content at the same level as in controls, significantly increased the mRNA levels of GPx and GR, and slightly increased GST mRNA level, but it did not modify all the enzyme activities. This suggests that GSPE probably improves the cellular redox status via the glutathione-synthesis pathways instead of regulation of the GPx and/or GR activities, which protect against oxidative damage.33 The work from Al-Awwadi et al34 also shows that administration of a grapeseed extract enriched in procyanidins to high-fructose-fed rats prevented insulin resistance, hypertriglyceridemia, and overproduction of ROS, and it normalized the increased expression of cardiac p91phox NADPH oxidase subunit.34 In our study, we have shown, for the first time, that GSPE time- and dose-dependently suppressed AGE-BSA-induced RAGE protein and gene expression in HUVEC by inhibiting intracellular ROS production. Although we did not determine the expression of intracellular antioxidant enzymes and NADPH oxidase or their activities, we presume that the inhibitory effect of GSPE on ROS formation and subsequent endothelial RAGE expression probably results from the same mechanism as those reported by Puiggros et al33 and Al-Awwadi et al.34 Hence, further studies are needed to elucidate the exact molecular mechanisms underlying the GSPE regulation of RAGE expression.

sRAGE and anti-RAGE IgG, though effectively intercepting the AGE-RAGE interaction, are not suitable as routine therapies. In the present study, we have found that GSPE markedly downregulates RAGE expression in AGE-exposed endothelial cells, thereby preventing the AGE-RAGE interaction and interrupting the vicious circle that perpetuates the atherogenic process. In conjunction with other properties of GSPE, such as suppressing LDL oxidation, thrombus formation, and nonenzymatic glycation of protein, antiatherosclerotic effects, redundant resource, and lack of toxicity, GSPE may have therapeutic potential in the prevention and treatment of diabetic vascular complications in patients with diabetes.

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ACKNOWLEDGMENTS

We greatly acknowledge the support of Prof Ye-bin Zhou and Jian-min Wu. We also thank Miss Yan-li Liu, Miss Shu-ying Li, Mr Bei-an You, and Mr Yong-liang Yi for their friendly help.

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Keywords:

proanthocyanidin; glycation; receptor; endothelial cells; reactive oxygen species

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