Glucose and other reducing sugars can react nonenzymatically with the free amino groups of proteins to form reversible Schiff bases followed by Amadori rearrangement. These early glycation products undergo further complex reactions such as dehydration, condensation, and cross-linking to yield irreversible fluorescent derivatives termed advanced glycation end products (AGEs). Under hyperglycemic conditions in diabetes, AGEs are formed at an accelerated rate and are accumulated in blood and in tissue. Many studies revealed that AGEs have been implicated in the initiation and development of diabetic vascular complications primarily by interacting with their intracellular signal-transducing receptor for advanced glycation end products (RAGE).1-5 RAGE present on the surface of endothelial cells, smooth muscle cells, lymphocytes, and mononuclear phagocytes is a multiligand member of the immunoglobulin superfamily whose repertoire of ligands includes not only AGEs but also amyloid fibrils, amphoterins, and S100/calgranulins relevant to distinct pathological processes, and whose short, highly charged cytosolic tail is critical for RAGE-mediated intracellular signaling. The engagement of RAGE on endothelial cells by AGEs results in the induction of intracellular reactive oxygen species (ROS), which seems to be linked to the activation of the NADPH-oxidase system and the mitochondrial electron transport system.6,7 The increased ROS in turn activates a complex cascade of signal transduction pathways and subsequently enhances the expression of many genes that are highly relevant for inflammation, immunity, and atherosclerosis such as cell adhesion molecules, tissue factor, chemokines, and cytokines.8 Of those altered genes, endothelial cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) play a key role in the diabetic vascular perturbation. The enhanced expression of VCAM-1 and ICAM-1 is associated with the endothelial adherence and migration of leukocytes, which is 1 of the first steps in atherosclerosis, thus priming and sustaining vascular wall injury and inflammatory response and causing diabetic vascular lesions.
Because intracellular oxidative stress is crucial for the activation of signaling cascade and subsequent altered gene expression induced by AGEs-RAGE engagement, the authors are deeply convinced that some drugs with antioxidative abilities should have some inhibitory influences on AGEs-RAGE-mediated deleterious effects. Yamagishi et al reported that minodronate, a nitrogen-containing bisphosphonate, inhibited VCAM-1 expression in AGEs-exposed human umbilical vein endothelial cells (HUVEC) by suppressing ROS generation.9 Similar results were found in the presence of other antioxidants such as α-lipoic acid and N-acetylcysteine.10 In addition, nifedipine has been shown to significantly inhibit RAGE gene expression in endothelial cells through their antioxidant mechanism.11 Grape seed proanthocyanidin extracts (GSPEs) is a natural extract from grape seeds and possesses powerful free radical scavenging and antioxidant properties.12,13 A growing body of evidence shows that GSPEs have an effective cardiovascular protective ability. For instance, GSPEs could provide significant protection against myocardial ischemia-reperfusion injury and doxorubicin-induced cardiotoxicity,14-17 prevent low-density lipoprotein (LDL) oxidation,18 attenuate the development of atherosclerosis as demonstrated by reducing the formation of foam cells,19,20 decrease arterial pressure in estrogen-depleted, female, spontaneously hypertensive rats,21 and inhibit platelet aggregation and laser irradiation- induced thrombus formation.22,23 Moreover, we have recently found GSPEs possess anti-nonenzymatic glycation efficacy in diabetic rats. In this study, we evaluate whether GSPEs can effect adhesion molecule expression in AGEs-exposed endothelial cells by suppressing ROS generation.
MATERIALS AND METHODS
GSPEs (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, MA). VCAM-1 and β-actin primer sets were synthesized by Sangon Biotech (Shanghai, China).
AGEs used in this study were prepared by incubating 50 mg/mL BSA with 0.5 mol/L glucose in 0.2 mol/L 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 AGEs content in AGE-modified BSA was 81.8 U/mg proteins, whereas that in unmodified BSA was <0.9 U/mg proteins. The endotoxin content of these preparations was measured by the limulus amebocyte lysate assay (Endos), which revealed negligible values (<0.2 μg/L).
Endothelial Cell Isolation and Culture
HUVEC were obtained from freshly umbilical cords by collagenase digestion according to the method of Jaffe.24 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 min at 37°C. The resulting cellular suspension was supplemented with PBS, centrifuged, and cultured in Medium 199 containing 100 U/mL penicillin, 100 mg/mL streptomycin, 2.5 μg/mL fungizone, 50 μg/mL gentamycin, 2 mmol/L glutamine, 20 mmol/L HEPES, and 20% FBS. Culture was on gelatin-coated 25-cm2 flasks, and 6- or 24-well tissue culture plates at 37°C under humidified 5% CO2 in room air. The medium was replaced every 2 d until confluence (3-5 d). HUVEC purity was confirmed by the “cobblestone” morphology that is 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 of the concentrations of 5, 15, and 25 μg/mL were 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). Respective controls were treated with equal volumes of DMSO. Next, the HUVEC were stimulated with 200 μg/mL of AGEs or unmodified BSA for defined periods. Under all conditions, cell viability was >96% as judged by trypan blue exclusion.
Measurement of VCAM-1 and ICAM-1 Expression in HUVEC
The expression of adhesion molecules in HUVEC was measured by flow cytometry as described previously. Briefly, the cells were incubated with or without different concentrations of GSPE for 4 h followed by stimulation with 200 μg/mL of AGEs or unmodified BSA for 12 h. After stimulation, cells were washed with PBS twice, harvested by mild trypsinization, washed again twice with PBS, and treated for 60 min at 4°C with a saturating amount of fluorescein isothocyanate-conjugated mouse anti-VCAM-1 and anti-ICAM-1 monoclonal antibodies or immunoglobulin G1 (isotype control; Jingmei Biotech, San Diego, CA). Cells were next washed with fluorescent-activated cell sorter (FACS) buffer, fixed in 1% paraformaldehyde and analyzed (10,000 cells/sample) by FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). After correcting for unspecific binding (isotype control), mean specific fluorescence intensity was measured in each channel.
Evaluation of VCAM-1 and ICAM-1 Gene 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) following 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, reverse transcriptase (RT) was performed using 2 μg of total RNA in a 20-μL final volume of reaction mixture (5 × reaction buffer, 10 mmol/L dNTPmix, ribonuclease inhibitor, random hexamers, and Moloney murine leukemia virus RT by incubation at 25°C for 10 min and 42°C for 60 min. The reaction was stopped by incubation at 70°C for 10 min (RT kit from MBI Fermentas Canada, Burlington). Thereafter, polymerase chain reaction (PCR) was carried out using 5-μL cDNA obtained as templates in a 50-μL reaction volume containing 25 μL Premix Taq, 2 μL upstream primers (10 μmol/L), 2 μL downstream primers (10 μmol/L), and 16 μL double-distilled water (PCR kit from TaKaRa Bio, Otsu, Japan). The primers designed were as follows: VCAM-1 (sense) 5′-CCCTTGACCGGCTGGAGATT-3′, (antisense) 5′-CTGGGGGCAACATTGACATAAAGTG-3′; ICAM-1 (sense) 5′-GTCCCCCTCAAAAGTCATCC-3′, (antisense) 5′-AACCCCATTCAGCGTCACCT-3′; β-actin (sense) 5′-ACACTGTGCCCATCTACGAGGGG-3′, (antisense) 5′-ATGATGGAGTTGAAGGTAGTTTCGTGGAT -3′. The amplification profile consisted of an initial denaturation at 94°C for 4 min; followed by denaturation at 94°C for 50 s, annealing at 52°C (for VCAM-1) and 57°C (for ICAM-1) for 50 s and extension at 72°C for 1 min with a number of 35 cycles; and final extension at 72°C for 5 min. Of the PCR products obtained (VCAM-1 241 bp, ICAM-1 942 bp, β-actin 375 bp), 10 μL were visualized by ethidium bromide staining and photographed under UV radiation following 1.5% agarose gel electrophoresis. Identified signal intensities of specific VCAM-1 and ICAM-1 PCR product bands were normalized with that of the β-actin PCR product and expressed as a ratio.
Detection of ROS in HUVEC
According to the method of Bass et al,25 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 an impermeable membrane and is rapidly oxidized to the highly fluorescent DCF in the presence of intracellular ROS. After treatment, cells (2 × 106) plated in 6-well plates were treated with 5-μmol/L DCFH-DA in the dark for 30 min 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 (%).
Results were expressed as mean ± SD, based on at least 3 separate experiments. One-way ANOVA was used to determine the significance of differences among treatments. P < 0.05 was considered statistically significant.
Effect of GSPE on the Expression of VCAM-1 and ICAM-1 in AGEs-exposed HUVEC
The expression of VCAM-1 and ICAM-1 in the untreated HUVEC was 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 h induced strong increases in the surface expression of VCAM-1 and ICAM-1. Preliminary tests showed that such increases reached a peak at 12 to 24 h of AGEs treatment. Next, pretreatment of HUVEC with different concentrations of GSPE markedly reduced AGEs-stimulated VCAM-1 expression in a dose-dependent manner, but it did not influence the increase in ICAM-1 levels. The levels of VCAM-1 expression were reduced to 79.90% ± 8.49% for 5 μg/mL, 52.09% ± 5.19% for 15 μg/mL, and 20.53% ± 5.21% for 25 μg/mL, respectively (P < 0.001 vs AGE-BSA alone; Fig. 1).
Effect of GSPE on VCAM-1 and ICAM-1 mRNA levels in HUVEC Stimulated by AGE-BSA
To characterize the molecular mechanisms responsible for the selective inhibition of AGEs-induced endothelial cell adhesion molecule expression by GSPE, VCAM-1 and ICAM-1 mRNA levels were evaluated by RT-PCR. Stimulation of HUVEC with 200 μg/mL of AGE-BSA for 6 h led to a marked increase in specific VCAM-1 and ICAM-1 PCR products as compared to low levels in unstimulated cells. Pretreatment with different concentrations of GSPE significantly reduced the levels of VCAM-1 transcripts after AGEs stimulation in a dose-dependent manner, whereas the enhanced ICAM-1 mRNA levels were not altered. Reduction of VCAM-1 mRNA levels differed ranging from 0.388 ± 0.035 to 0.275 ± 0.015 for 5 μg/mL, to 0.146 ± 0.031 for 15 μg/mL, to 0.077 ± 0.009 for 25 μg/mL, respectively (P < 0.001 vs 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 also affect the transcription of VCAM-1 gene but not ICAM-1 (Figs. 2 and 3).
Effect of GSPE on Intracellular ROS Formation in AGEs-exposed HUVEC
Intracellular ROS was estimated by flow cytometry using DCFH as a probe. The levels of ROS were low in the unstimulated HUVEC and the stimulation of HUVEC with 200 μg/mL of AGE-BSA significantly enhanced the ROS formation, whereas pretreatment of GSPE in a dose-dependent manner apparently prevented AGEs-induced ROS generation (Fig. 4), which was reduced to 78.19% ± 8.80% for 5 μg/mL, to 59.66% ± 5.10% for 15 μg/mL, and to 41.58% ± 1.49% for 25 μg/mL, respectively (P < 0.001 vs AGE-BSA alone). These results suggest that GSPEs suppress AGEs-stimulated VCAM-1 expression at a gene and protein level probably through inhibition of ROS generation.
It is now well known that adhesion of mononuclear leukocytes to the vascular endothelium and subsequent migration of cells into the vessel wall are early cellular events in the development of an atherosclerotic lesion. Various cell adhesion proteins including VCAM-1 and ICAM-1 expressed on activated endothelial cells are responsible for mediating cell-cell interaction between mononuclear leukocytes and endothelial cells. These cell adhesion proteins are normally present on the endothelial cells at a lower level but are induced in the presence of various stimuli such as interleukin-1α, tumor necrosis factor-α (TNF-α), lipopolysaccharides, and ox-LDL. In particular, AGEs resulting from nonenzymatic glycation and oxidation of proteins or lipids have been reported to stimulate the expression of VCAM-1 and ICAM-1 on endothelial cells and to participate in the development of diabetic vascular lesions. Exposure of cultured human endothelial cells to AGEs prepared in vitro or derived from in vivo sources resulted in increased cellular surface expression of VCAM-1 and ICAM-1 and resulting endothelial adhesion of polymorphonuclear leukocytes in a manner suppressed by treatment with antioxidants or on blockade of RAGE.8,26-29 Nondiabetic rabbits treated intravenously with AGEs showed accumulation of AGEs, the development of atheromatous changes, and positive focal expression of VCAM-1 and ICAM-1.3 Otherwise, apoE null mice with type 2 diabetes mellitus displayed accelerated atherosclerosis and enhanced expression of VCAM-1, tissue factor, and matrix metalloproteinase-9.30 In the present study, AGEs were prepared by incubating BSA with a high concentration of glucose. Incubation of cultured HUVEC with 200 μg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the expression of VCAM-1 and ICAM-1. This result was consistent with previous data mentioned above.
GSPE is a naturally occurring polyphenolic compound from grape seeds with potent antioxidant and anti-inflammatory properties. We examined whether GSPE could have an influence on the expression of VCAM-1 and ICAM-1 mediated by AGEs in endothelial cells. The results of our study showed that GSPE concentration dependently downregulated AGEs-induced VCAM-1 but not ICAM-1 surface protein expression. The selective suppressive effect of GSPE on endothelial cell adhesion molecules correlated with the effect on their mRNA levels. Meanwhile, similar to its effect on VCAM-1 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 may be involved in its inhibitory effects on VCAM-1 expression. There is much evidence that redox-sensitive mechanisms regulate the activation of endothelial cells caused by various kinds of cell stimuli, which is a fundamental reason why some antioxidants can effectively inhibit agonist-induced endothelial cell dysfunction. Cayatte et al have reported that an other polyphenolic compound, S17834, reduced TNF-α-stimulated endothelial expression of VCAM-1, ICAM-1, and E-selectin, and explained that this drug acted by inhibiting NADPH oxidase activity of endothelial cell membranes and corresponding endogenous superoxide anion production.31 In fact, activation of NADPH oxidase is crucial for AGEs-RAGE-induced oxidative stress and subsequent cell adhesion molecule expression. In our study, we did not determine the activities of NADPH oxidase and other oxidases, but Al-Awwadi et al have demonstrated that administration of GSPE to high-fructose-fed rats significantly suppressed the increased expression of the p91phox NADPH oxidase subunit and overproduction of ROS.32 Hence, we speculate that inhibition by GSPE of ROS, like S17834, is probably associated with the interference of NADPH oxidase irrespective of the other mechanisms, thereby leading to the reduction in the expression of AGEs-stimulated VCAM-1 in endothelial cells.
Only a few studies have been conducted to investigate the GSPE regulation of cell adhesion molecules. Kalin et al have reported that administration of activin derived from the grape seed proanthocyanidins in patients with systemic sclerosis significantly lowered the levels of soluble adhesion molecule including ICAM-1, VCAM-1, and E-selectin in plasma and attenuated the formation of malonaldehyde, a marker for oxidative stress.33 An in vitro study has demonstrated that GSPE at low concentrations (1-5 μg/mL) selectively downregulated TNF-α-induced VCAM-1 expression but not ICAM expression in HUVEC without influencing NF-κB pathway and corresponding decreased adherence of T cells to HUVECs.34 Although AGEs, as a prevalent stimulating factor in diabetes, is different from TNF-α, our experimental result was in fundamental agreement with the latter. GSPE only dose dependently suppressed AGE-BSA-induced VCAM-1 expression in HUVEC by inhibiting the intracellular ROS production, whereas the increased ICAM-1 level was not affected by pretreatment with GSPE. This selective inhibition of agonist-induced VCAM-1 expression was also observed in in vitro studies for many other drugs with antioxidant properties such as PD098063 (a flavonoid), α-tocopherol, tea flavonoid epigallocatechin-3-gallate, and probucol.35-38 In addition, a clinical study demonstrated that chronic dietary administration of flavanol-rich cocoa in hypercholesterolemic postmenopausal women decreased plasma levels of soluble VCAM rather than ICAM.39 We presume that the effect of these drugs including GSPE on the selective inhibition of VCAM-1 expression may follow a similar pattern, but the exact mechanism is not fully understood. Because kinetics of VCAM-1 and ICAM-1 expression are different and involve distinct regulatory pathways, GSPE may affect only the signaling pathways that are specific for AGEs-stimulated induction of VCAM-1 expression. However, this result from our study may be desirable in that ICAM-1 is involved in the adhesion of various leukocytes, whereas VCAM-1 participates primarily in monocyte and lymphocyte adhesion and these cells are specifically found in atherosclerotic lesions. VCAM-1 but not ICAM-1 plays a dominant role in the initiation of atherosclerosis.40
In summary, GSPE could significantly inhibit the expression of VCAM-1 surface proteins and mRNA induced by AGEs in endothelial cells through its strong antioxidant ability, but AGEs-induced ICAM expression was not altered by pretreatment with GSPE. Hence, in conjunction with other properties of GSPE such as the suppression of LDL oxidation, thrombus formation, and nonenzymatic glycation of protein, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in patients with diabetes. Further studies are needed to elucidate the precise molecular mechanisms underlying the differential regulation of VCAM-1 and ICAM-1 by GSPE.
The authors acknowledge the support of Prof Ye-bin Zhou and the text revisions completed by Ms Kay Howitt. They also thank Ms Yan-li Liu, Ms Shu-ying Li, Ms Li Ma, and Mr Yong-liang Yin for their assistance.
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