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Original Article

Grape Seed Proanthocyanidin Extracts Inhibit Vascular Cell Adhesion Molecule Expression Induced by Advanced Glycation End Products Through Activation of Peroxisome Proliferators-Activated Receptor γ

Ma, Li; Gao, Hai-qing MD, PhD; Li, Bao-ying MD; Ma, Ya-bing MD; You, Bei-an MD; Zhang, Feng-lei MD

Author Information
Journal of Cardiovascular Pharmacology: May 2007 - Volume 49 - Issue 5 - p 293-298
doi: 10.1097/FJC.0b013e31803c5616
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Prolonged hyperglycemia, dyslipidemia, and oxidative stress in diabetes result in the production and accumulation of advanced glycation end products (AGEs). There is growing evidence that formation and accumulation of AGEs progress during normal aging and at an extremely accelerated rate in diabetes, thus being involved in the pathogenesis of diabetic vascular complications, such as arthrosclerosis. AGEs are thought to act through receptor-independent and -dependent mechanisms to promote vascular damage, fibrosis, and inflammation associated with accelerated arthrosclerosis. Interaction of AGEs with their receptor, RAGE, activates numerous signaling pathways leading to activation of proinflammatory and procoagulatory genes and is responsible for endothelial cell dysfunction observed in diabetic rats.1,2 RAGE is present on the surface of vascular cells.3 The ligands of RAGE include AGEs, amyloid fibrils, amphoterins, and S100/calgranulins. Binding of AGEs to RAGE activates intracellular signaling processes, thus mediating proinflammatory AGE effects. In endothelial cells, AGEs increase RAGE expression, generate oxidative stress, and activate the transcription factor nuclear factor-κB (NF-κB), subsequently leading to increased expression of proatherogenic mediators such as vascular cell adhesion molecule-1 (VCAM-1).4 Experiments have shown that AGEs upregulated endothelial cell adhesion molecules such as E-selectin, VCAM-1, and vascular endothelial growth factor (VEGF).5 In vitro experiments6 suggest limiting RAGE expression might be a promising target to modulate vascular disease in patients with diabetes.

Grape seed proanthocyanidin extracts (GSPE), derived from grape seeds, polymerized by monomers of catechin or epicatechin have been reported to possess a variety of potent properties. GSPE could provide significant protection against myocardial ischemia-reperfusion injury and doxorubicin-induced cardiotoxicity,7-10 prevent LDL oxidation,11 attenuate the development of atherosclerosis by reducing the formation of foam cells,12,13 decrease arterial pressure in estrogen-depleted and spontaneously hypertensive female rats,14 and inhibit the platelet aggregation and the laser-irradiation induced thrombus formation.15,16 GSPE could help prevent oxidative DNA damage in human cultured cells and isolated DNA by decreasing the formation of 8-oxodG.17 In vitro data suggested that AGEs downregulated the expression of peroxisome proliferator-activated receptor γ (PPAR γ), a nuclear hormone receptor regulating gene expression in response to specific ligands.18 Data established PPAR γ expression in human endothelial cells, and PPAR γ-activating thiazolidinediones (TZDs) have been shown to modulate gene expression in these cells.4,19,20 TZDs are a group of antidiabetic agents that increase insulin sensitivity, thus lowering blood glucose levels in treated patients with type 2 diabetes. On a molecular level, TZDs activate the transcription factor PPAR γ. Our previous experiments showed that GSPE displayed antinonenzymatic glycation, reducing RAGE protein expression, subsequently leading to decreased expression of high-level VCAM-1 induced by AGEs.21,22 However, hitherto, nothing is known about the effect of GSPE on endothelial PPAR γ expression.

Given the key role of RAGE for diabetic vascular complications, we examined whether GSPE might modulate endothelial RAGE expression via affecting PPAR γ expression with subsequent decreased proinflammatory mediator VCAM-1 expression and low-level Von Willebrand factor (vWF).



Grape seed proanthocyanidin extracts (56% dimeric proanthocyanidins, 12% trimeric proanthocyanidins, 6.6% tetrameric proanthocyanidins, and small amounts of monomeric and high-molecular-weight oligomeric proanthocyanidins and flavanols; Lot No G050412) were purchased from Jianfeng Inc (Tianjin, China). The components of GSPE were analyzed using high-performance liquid chromatography with gas chromatography-mass spectrometry detection. GSPE stock solutions for treatment were prepared fresh in phosphate buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 6 mM Na2HPO4•12H2O, 2 mM KH2PO4, pH 7.4). Medium 199, fetal bovine serum (FBS), and trypsin were purchased from Gibco (USA). Bovine serum albumin (BSA), D-glucose, and collagenase were obtained from Sigma (USA). Rabbit anti-PPAR γ polyclonal antibody and rabbit antihuman VCAM-1 polyclonal antibody were from Upstate (USA), peroxidase conjugated Affinity purified Goat Anti-Rabbit IgG (H+L) was from KLP (USA), rabbit anti-RAGE was purchased from Biosynthesis Biotechnology Co (Beijing, China), and actin was from Santa Cruz (USA). The vWF test kit was obtained from Corgenix (USA).

AGE-BSA Preparation

AGEs used in the current study were prepared by incubating 50 mg/mL BSA with 0.5 M glucose in 0.02 M PBS (pH 7.4) in the dark at 37°C for 12 weeks. The solution was filtered through a 0.22-μm filter before incubation. Unmodified BSA was treated under the same conditions without glucose as a control. At the end of the incubation, extensive dialysis was done 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 AGEs content in AGE-modified BSA was 81.8 U/mg proteins, whereas that in unmodified BSA was less than 0.9 U/mg proteins. The endotoxin content of these preparations was measured by the Limulus amebocyte lysate assay (Endos, USA), which revealed negligible values (<0.2 μg/L).

Endothelial Cell Culture

Human umbilical vein endothelial cells (HUVEC) were available from fresh umbilical vein cords, according to Jaffe.23 First, the umbilical vein cords were perfused with PBS to remove blood cells, then they were filled with 0.05% collagenase (type Ia) and left for 15 minutes at 37°C. The cellular suspension was supplemented with PBS, centrifuged, and cultured in medium 199 containing 100 mg/mL streptomycin, 100 U/mL penicillin, 2 mM glutamine, 2.5 μg/mL fungizone, 50 μg/mL gentamycin, 2 mM glutamine, 20 mM HEPES, and 20% FBS. Gelatin-coated flasks were used for incubation at 37°C under 5% CO2 in 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. At passage 2-3, the confluent HUVEC were used for experiments. The FBS concentration in the medium 199 was reduced to 5%, and GSPE of the concentrations of 10 mg/L, 50 mg/L, and 100 mg/L were added for preincubation for 4 hours, respectively. The control group was treated with complete medium 199 with 1% FBS only.

Western Blot Analysis

For Western blot analysis of RAGE and PPAR γ expression, HUVECs were stimulated with or without different concentrations of GSPE for 4 hours before stimulation with 200 mg/L of AGEs or unmodified BSA for 24 hours. After treatment, the cells were washed with ice-cold PBS 3 times, lysed with 0.80 mL of cell lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% [v/v] glycerol, 10% TritonX 100M lysis) with freshly prepared 1 mM PMSF (phenylmethyl sulfonylfluoride), 10 mg/L aprotinin, 1 mg/L leupetin, 10 mg/L pepstatin A, and 1 mM sodium orthovanadate; then they were scraped with a cell scraper. The lysate was transferred to a 1.5-mL microcentrifuge tube. After fresh PMSF was added, the lysate was incubated on ice for 5 minutes. The cell lysate was centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant fluid was used as the total cell lysate. Tris-glycine SDS (sodium dodecyl sulfate) sample buffer (5×) was added into the cell lysate and then boiled for 5 minutes to denature the protein. The concentration of total protein amount was measured spectrophotometrically using Bradford kit (Pierce) following manufacturer's instructions. Electrophoresis was performed. To ensure equal loading of protein, actin was used. Equally, 70 μg of protein were fractioned on the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to pretreated polyvinylidene fluoride (PVDF) membranes. After membrane transferring, membranes were blocked with 5% nonfat dried milk in PBS with 0.1% Tween 20 (TPBS) at 37°C for 1 hour with gentle shaking. Rabbit polyclonal primary antibody was diluted 1:350 in 5% nonfat milk in TPBS, and antiactin was diluted 1:500. The filters were incubated at 4°C overnight and washed 3 × 10 minutes in TPBS. The secondary antibody, peroxidase conjugated Affinity purified Goat Anti-Rabbit IgG (H+L), was diluted 1:5000 in TPBS. The filters were incubated for 1 hour at 37°C and washed in TPBS 6 × 10 minutes. A DAB kit was used for color reaction following the manufacturer's protocol.

Flow Cytometry Analysis

To examine the effect of reduced RAGE expression on the proinflammatory AGE effect, the expression of adhesion molecules VCAM-1 on HUVEC was measured by flow cytometry as previously described.24 HUVEC were stimulated as described before. After stimulation, cells were washed with PBS (NaCl 8.00 g/L, KCl 0.20 g/L, Na2HPO4•12H2O 2.08g/L, KH2PO4 0.20 g/L, pH 7.4) twice, harvested by 0.25% trypsin, and stained with a polyclonal rabbit antihuman VCAM-1 antibody. After washing twice with PBS, cells were stained with fluorescein isothiocyanate-conjugated antirabbit antibody (Jingmei Biotech Co, China). Controls used isomatched immunoglobulin G. Cells were next washed with FACS buffer, fixed in 1% paraformaldehyde at 4°C, and analyzed (10,000 cells per sample) by FACScan flow cytometer (Becton Dickinson, USA) within 24 hours.

vWF Enzyme-Linked Immunosorbent Assay

After treatment according to the method described earlier, an enzyme-linked immunosorbent assay (ELISA) microplate reader was used to read the absorbance of each group at 450 nm according to the manufacturer's protocol.

Statistical Analysis

Results were expressed as means ± standard deviation (SD). The data were analyzed by SPSS10.0 software. One-way analysis of variance was used to determine the significant differences among treatments. The P value (<0.05) was considered statistically significant.


Effect of GSPE on RAGE Protein Expression Induced by AGEs in HUVEC

The effect of GSPE on RAGE expression in HUVEC surface was assessed by Western blot analysis. AGEs are known to increase endothelial RAGE expression. We next examined the effect of GSPE on AGEs-induced RAGE protein expression (Fig. 1A). Stimulation of endothelial cells with AGEs (200 mg/L) for 24 hours led to a 2.96-fold increase in cell RAGE protein expression (data not shown), whereas pretreatment with GSPE for 4 hours significantly reduced AGEs-induced RAGE protein expression in a concentration-dependent manner with reduction to 66.88%, 40.21%, and 25.85% at 10 mg/L, 50 mg/L, and 100 mg/L, respectively (Fig. 1B). Unmodified BSA-treated HUVEC did not have high expression of RAGE.

GSPE-reduced RAGE protein expression. A, HUVEC were stimulated for 24 hours with AGEs (200 mg/L) in the absence or presence of GSPE at the concentrations indicated (10 mg/L, 50 mg/L, 100 mg/L) before Western blotting was performed. Equal loading of intact protein was confirmed by staining for actin. Three separate similar experiments were performed. B, Densitometric analysis of 3 independent Western blot experiments showed the effect of GSPE on AGEs-induced RAGE protein expression. Results are expressed as percent of AGEs-stimulated cells (100%) and are given as mean ± SD from 3 separate experiments. *P < 0.01 compared with AGEs-stimulated cells; ▴P < 0.05 compared with the untreated cells; ▴▴P < 0.01 compared with the untreated cells.

Effect of GSPE on PPAR γ Protein Expression Induced by AGEs in HUVEC

GSPE-upregulated PPAR γ protein expression was also assessed by Western blot analysis (Fig. 2A). GSPE increased AGEs-induced PPAR γ protein expression in a concentration-dependent manner. Pretreatment of HUVEC with GSPE (10 mg/L, 50 mg/L, 100 mg/L) for 4 hours before AGEs stimulation led to a 1.63 ± 0.10-fold, 2.36 ± 0.06-fold, and 2.78 ± 0.26-fold significant increase in PPAR γ expression, respectively (P < 0.01, compared with AGEs-treated cells) (Fig. 2B).

GSPE-upregulated PPAR γ protein expressions. A, HUVEC were stimulated for 24 hours with AGEs (200 mg/L) in the absence or presence of GSPE at the concentrations indicated (10 mg/L, 50 mg/L, 100 mg/L) before Western blotting was performed. Three separate similar experiments were performed. B, Densitometric analysis of 3 independent Western blot experiments was performed. Results are expressed as percent of AGEs-stimulated cells (100%) and are given as mean ± SD. *P < 0.01 compared with AGEs-stimulated cells.

Effect of GSPE on the Expression of VCAM-1 Induced by AGEs in HUVEC

Activation of RAGE has previously been reported to upregulate the expression of proinflammatory mediators such as VCAM-1 in human endothelial cells. The expression of VCAM-1 in the control was very low. Unmodified BSA did not affect the basal expression of VCAM-1. Stimulation of HUVEC with AGEs for 24 hours significantly increased VCAM-1 expression. A 4-hour pretreatment of cells with different concentrations of GSPE significantly reduced AGE-mediated VCAM-1 release in a concentration-dependent manner. The levels of VCAM-1 expression were reduced to 64.20% ± 7.48% for 10 mg/L GSPE, to 23.69% ± 6.49% for 50 mg/L GSPE, and to 8.89% ± 4.58% for 100 mg/L GSPE, respectively (P < 0.001 for all, compared with AGEs-treated cells; n = 5) (Fig. 3). These data suggested that GSPE-mediated reduction of RAGE expression limited the cells susceptibility toward proinflammatory AGEs effect.

Effect of GSPE on VCAM-1 expression induced by AGEs in HUVEC. HUVEC were preincubated with or without GSPE (10 mg/L, 50 mg/L, 100 mg/L) for 4 hours before treated with AGEs (200 mg/L) or with unmodified BSA (200 mg/L) for 24 hours. After treatment, cells were stained for VCAM-1 and analyzed by flow cytometry. Data were expressed as percent of AGEs-stimulated cells (100%) and are given as mean ± SD. *P < 0.001 compared with AGEs-stimulated cells.

Effect of GSPE on the Content of vWF in Culture Fluid

The dysfunction or activation induced by a variety of stimulating factors to endothelial cells lead to increased levels of vWF, which is a marker of endothelial cells dysfunction.25-28 In our experiments, AGEs were strong stimulating factors and the level of vWF in the AGEs-treated group was higher than that of the control (0.185 ± 0.007 vs 0.164 ± 0.006, P < 0.01). The preincubation with GSPE of 10 mg/L, 50 mg/L, and 100 mg/L reduced AGEs-induced high level of vWF in a dose-dependent manner (0.155 ± 0.006, 0.114 ± 0.011, 0.072 ± 0.006, vs 0.185 ± 0.007, n = 6). The pretreatment of 10 mg/L GSPE decreased vWF level, but there was no significant difference compared with the control (P > 0.05). Fifty mg/L and 100 mg/L GSPE significantly reduced vWF (P < 0.01). Unmodified BSA did not affect vWF content (Fig. 4). These data revealed that GSPE protected endothelial cells against activation and cellular damage via reducing VCAM-1 expression.

GSPE decreased the high level of vWF induced by AGEs in HUVEC. The HUVEC were incubated with or without GSPE (10 mg/L, 50 mg/L, 100 mg/L) for 4 hours before treated with 200 mg/L AGEs or 200 mg/L unmodified BSA for 24 hours. The data were expressed as mean ± SD (n = 4). **P < 0.01 compared with the untreated cells. ▴P < 0.01 compared with each other.


To our knowledge, this is the first report to find a reduction of RAGE expression via upregulating PPAR γ expression in human endothelial cells by antioxidant GSPE, with subsequently reduced endothelial susceptibility toward proinflammatory AGEs effects and inhibited cellular activation and dysfunction.

Antioxidants such as vitamins C and E have the property of inhibiting the AGEs-mediated signal transduction, protecting people with diabetes against the double injuries induced by free radicals and nonenzymatic glycation products.29 Our study extended the understanding of GSPE's action by demonstrating reduction of AGEs-induced RAGE expression and increasing of PPAR γ expression in human endothelial cells. In vitro experiment data suggested that AGEs decreased PPAR γ protein expression in cultured endothelial cells.30 Activating PPAR γ reduced cell surface RAGE protein expression.4,20 In this study, we found that GSPE treatment could reverse the AGEs-inhibited PPAR γ expression as well as AGEs-stimulated RAGE expression in a concentration-dependent manner. Resveratrol, naturally occurring polyphenolic compounds derived from grape seeds, have similar biological activity to GSPE. In vitro data revealed that resveratrol could dose-dependently activate PPAR γ and increase the expression of PPAR γ in a cell culture model of colon cancer.31 In a mice model on a high-calorie diet, resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulinlike growth factor-1 (IGF-I) levels, increased adenosine monophosphate (AMP)-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α).32 Our Western blot data suggested that GSPE may be an activator of PPAR γ. This deduction needs to be confirmed by further experiments.

The reduction of RAGE expression by GSPE decreased the cells' susceptibility toward proinflammatory AGE effects, as shown by impaired VCAM-1 release after GSPE pretreatment. These data were consistent with previous reports showing that antioxidants inhibit TNF-induced VCAM-1 messenger ribonucleic acid and protein expression in HUVEC.33 In our experiment, pretreatment with GSPE inhibited AGEs-induced vWF secretion from endothelial cells. This suggested that GSPE decreased cellular damage and activation, which may contribute to reduced VCAM-1 expression directly.

The high-risk population of people with diabetes exhibits increased AGE levels on one hand and enhanced RAGE expression in the vasculature on the other hand. Indeed, AGEs elicit oxidative stress generation in vascular wall cells through an interaction with their receptor (RAGE), thus playing an important role in vascular inflammation and altered gene expression of growth factors and cytokines. In endothelial cells, cyclic AMP and intracellular calcium are involved in the AGE-signaling pathway34,35 and AGE-RAGE interaction leads to a short-lasting cellular activation in sustained cellular dysfunction with subsequent long-lasting expression of proatherogenic mediators, such as VCAM-1, which furnishes lesion development by facilitating leukocyte recruitment with subsequent activation of these cells in the vessel wall.36,37 The lasting expression of RAGE is the key component element that leads to dysfunction and injury of vascular endothelial cells in diabetic chronic vascular complications.38 Animal data demonstrating that interruption of the AGE-RAGE interaction decreased lesion size in mouse models of arteriosclerosis39 led to the consideration that limiting RAGE activation might be a key target for therapeutic intervention in cardiovascular disease. The reduction of RAGE expression by GSPE might represent such a way to limit RAGE-mediated cell activation, thus potentially modulating atherogenesis in patients with diabetes. We demonstrate that GSPE's reduction of AGEs-induced RAGE expression is mediated by a promotion of PPAR γ activation. The reduction of RAGE expression by GSPE via PPAR γ activation, as shown here, might represent such a way to limit RAGE-mediated cell activation in the vessel wall, thus potentially modulating atherogenesis in patients with diabetes.

Nowadays, GSPE are already in use as a dietary supplement for people's multiple health benefits.40 The data in our studies displaying beneficial effects of GSPE treatment on reduction of RAGE expression and on activation of PPAR γ expression in patients with diabetes suggested that antioxidant GSPE, protecting endothelial cells against cellular damage induced by AGEs, may have the same important position as antidiabetic agents TZDs in treatment of patients with type 2 diabetes. Our findings may contribute to the wide use of GSPE in the prevention and treatment of diabetic vascular complications.


This work in part was supported by grants HW067.

We wish to thank Professor Ling Xu for her revision. We also wish to thank the Institute of Basic Medical Sciences of Qi-Lu Hospital, Shandong University.


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proanthocyanidin; glycation; receptor; endothelial cells; peroxisome proliferators-activated receptor γ

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