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ADVMS 41 1–5 Advances in Medical Sciences xxx (2014) xxx–xxx

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Original Research Article

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells? Luzak a,*, Anna Kosiorek a, Kamila Syska a, Marek Rozalski b, Michal Bijak c, Anna Podsedek d, Ewa Balcerczak b, Cezary Watala a, Jacek Golanski a

Q1 Boguslawa


Department of Haemostasis and Haemostatic Disorders, Medical University Hospital No. 2, Medical University of Lodz, Lodz, Poland Department of Pharmaceutical Biochemistry, Molecular Biology Laboratory, Medical University of Lodz, Lodz, Poland Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland d Institute of Technical Biochemistry, Department of Biotechnology and Food Sciences, Lodz University of Technology, Lodz, Poland b c



Article history: Received 13 May 2013 Accepted 11 February 2014 Available online xxx

Purpose: Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. Material/methods: After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. Results: Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 mg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 mg gallic acid equivalent/ml. Conclusions: The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.

Keywords: Grape seed extract Endothelial cells Platelet reactivity Cardiovascular disease

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1. Introduction

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Cardiovascular disease (CVD) is a leading cause of death in many economically developed nations. Although some of the major risk factors for CVD are not ‘‘modifiable’’ – age, sex, genetic predisposition – aspects of diet and lifestyle are recognized as major risk factors modulating the outcome of CVD [1]. Recent findings have highlighted the importance of oxidative stress, vascular inflammation and endothelial dysfunction in the development of CVD [2]. Under physiological conditions, the endothelial cells prevent blood coagulation in vessels via production of nitric oxide and prostacyclin (PGI2), both of which are potent inhibitors of platelet activation. The endothelial cells also produce an ectoADPase (also known as CD39 or NTPDase-1), which degrades

* Corresponding author at: Department of Haemostasis and Haemostatic Disorders, Medical University Hospital No. 2, Medical University of Lodz, Z˙eromskiego 113, 90-549 Lodz, Poland. Tel.: +48 42 639 3471; fax: +48 42 678 7567. E-mail address: [email protected] (B. Luzak).

extracellular adenosine 50 -diphosphate (ADP) resulting in decreased platelet reactivity [3]. Homeostasis of the vascular endothelium, in terms of both metabolic and physiological activities, is subject to fine-tuning by individual nutrients or nutrient derivatives [4,5]. Grape skin or seeds are very rich source of polyphenols, especially flavanols with proanthocyanidins [6], and other compounds with antioxidant and anti-inflammatory activities resembling those of resveratrol [7,8]. Many studies confirm the cardioprotective effects of grape seeds, demonstrating reduction of blood pressure, lower levels of oxidized low-density lipoprotein (ox-LDL), and enhanced nitric oxide (NO) production [9–12]. Recent studies have provided evidence that red grape components are a source of antioxidant compounds that ameliorate the viability and function of endothelial progenitor cells (EPC) [13]. Extracts from purple grape skins and seeds inhibit platelet function and platelet-dependent inflammatory responses [14], but the mechanism involved in these beneficial effects of grape extracts on platelet activation remains unresolved. Vitseva et al. [15] report a decrease in aggregation, a marked decrease in

http://dx.doi.org/10.1016/j.advms.2014.02.005 1896-1126/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.

Please cite this article in press as: Luzak B, et al. Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells? Adv Med Sci (2014), http://dx.doi.org/10.1016/j.advms.2014.02.005

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superoxide release, a significant increase in radical-scavenging activity, and enhanced platelet NO when platelets were incubated with seed or skin extract. The present study examines the influence of the polyphenolrich grape seed extract (GSE) on the viability and antiplatelet properties of human endothelial cells under in vitro conditions. Seed extract at lower concentration of polyphenolic compounds was found to increase the antiplatelet action of human umbilical vein endothelial cells (HUVECs) after stimulation with ADP. HUVECs incubated with higher concentrations of GSE did not demonstrate altered platelet reactivity, and up to 10 mg/ ml GSE (as gallic acid equivalent) significantly reduced HUVEC proliferation.


2. Material and methods


2.1. Reagents

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DMSO, (+)catechin, gallic acid, vanillin, ADP, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma (St. Louis, MO, USA). All other chemicals were reagent-grade products purchased from POCh S.A. (Gliwice, Poland). Materials for flow cytometry (anti-CD61PerCp, antiCD62PE, PAC-1-FITC, CellFix and others) were obtained from Becton Dickinson (San Diego, CA, USA).


2.2. Characteristics of GSE

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Grape (Vitis vinifera L.) seed extract (OMNIVIR1) was purchased from C.E. Roeper GmbH, Germany. For chemical analysis, the extract was diluted to 10 mg/ml with 10% DMSO. The total phenolic content of the extract was determined colorimetrically according to Folin– Ciocalteau [16] and expressed as milligrams gallic acid equivalent per gram of extract (mg GAE/g). Total flavanol content was estimated using the vanillin–H2SO4 method [17] and was expressed as milligrams (+)catechin equivalents per gram of extract (mg CE/g). Total proanthocyanidin content was determined after acid depolymerisation to the corresponding anthocyanidins as described by Rosch et al. [18]. The proanthocyanidin content (mg cyanidin/g of extract) was calculated using the molar extinction coefficient of cyanidin (e = 17,360 L mol1 cm1) and its molar mass (287 g mol1). The total flavonoid concentration was measured using a colorimetric assay [19] and was expressed as milligrams of (+)-catechin equivalent per gram of extract. The composition of the GSE was determined spectrophotometrically (Table 1). The extract was found to comprise 41% phenolic compounds by weight, 81% of which were flavonoids. In total, flavanols (monomers and proanthocyanidins) constituted 27.2% of the extract by weight and proanthocyanidins (oligomers and polymers of flavanols) only 6.9% [6,10]. HPLC analysis of the same GSE samples published in an earlier report revealed that the extract contained monomers such as (+)-catechin and ()epicatechin, dimers such as procyanidin B1 and B2, and a trimer: procyanidin C1 [20].

Table 1 Composition of grape seed extract. Component

Concentration (mg/g)

Total phenolics Flavonoids Flavanols Proanthocyanidins

410  2 333  3 272  6 69  1

Data presented as mean  SE, n = 5. The composition of grape seed extract was determined by spectrophotometric methods.

2.3. Maintenance of HUVEC culture


HUVECs and all reagents needed for cell culture were purchased from Cascade Biologics (Portland, Oregon, USA). The HUVECs were cultured according to the manufacturer’s instructions and the cells underwent 2–6 passages. In flow cytometry studies, HUVECs were first transferred to 24well plates at 75  103 cells/well and grown further at 37 8C for 24 h under a humidified atmosphere of 5% CO2 in air. For measurement of eNOS expression, HUVECs (1  106) were placed in 75 cm2 culture bottles (NUNC, Roskilde, Denmark) and grown for 48 h. The culture medium was then replaced and stock solutions of polyphenol extract from grape seeds were added to give final concentrations of 0, 0.25, 1, 2.5, 5 and 10 mg gallic acid equivalent/ml. After 24 h the culture medium was discarded and the HUVEC monolayer was rinsed four times with PBS. The cells in PBS were then scraped and centrifuged (250  g, 6 min, +4 8C). Finally, 1 ml of fenozole was added to the cell pellet for RNA isolation [21].

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2.4. Effect of polyphenol extract from grape seeds on HUVEC proliferation

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HUVECs were seeded on 96-well plates (10,000 cells/well) and grown further at 37 8C for 24 h in a humidified atmosphere of 5% CO2 in air. They were then exposed to polyphenol extract at final concentrations of 0.1, 0.5, 1, 5, 10, 25, 50, 100 and 200 mg gallic acid equivalent/ml grape extract for 24 h. After this incubation, the polyphenol-containing culture medium was carefully removed and replaced with fresh medium, and the cells were cultured for a further 24 h. Proliferative activity was then determined by MTT assay [22]. The cells were treated with the MTT reagent for 2 h. The MTT-formazan crystals were dissolved in 20% SDS, 50% DMF, pH 4.7 and the absorbance at 570 nm was measured using a Victor3 multifunctional ELISA-plate reader (Perkin-Elmer, MA, USA).

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2.5. Determination of eNOS expression


RNA was isolated using a Total RNA Prep Plus Minicolumn Kit (A&A Biotechnology, Poland) based on the RNA isolation method developed previously [21]. For real-time PCR normalization, UV absorbance was used to determine the amount of RNA added to a cDNA reaction. PCRs were then set up using cDNA derived from the same amount of input RNA; isolated RNA has an A260/280 ratio of 1.6–1.8. For the reverse transcriptase (RT) reaction, an Enhanced Avian HS RT-PCR Kit (Sigma, St. Louis, MO, USA) was used according to the manufacturer’s protocol. The cDNA was used immediately or stored at 20 8C. Before the quantitative analysis of gene expression by real-time PCR, the parameters were checked using qualitative PCR. The reaction mixture for PCR amplification consisted of a cDNA template, 0.5 mM of each primer, 10 AccuTaq Buffer, 0.5 U of AccuTaq LA DNA Polymerase Mix, 0.2 mM each dNTP, and water to a final volume of 20 ml. A negative control (sample without a cDNA template) was included in each experiment. The primer sequences for both genes, target-eNOS and reference GAPDH, were designed using Primer3 online software (Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/ other/primer3.html). Real-time PCR was performed on the corresponding cDNA synthesized from each sample. For real-time PCR the MX3005PTM System (STRATAGEN, Santa Clara, CA, USA) was used. The gene eNOS and a reference gene GAPDH were amplified in parallel for each sample in separate wells during the same PCR procedure. GAPDH was utilized as an internal positive control and as a normalizer for correcting the expression data. For

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Please cite this article in press as: Luzak B, et al. Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells? Adv Med Sci (2014), http://dx.doi.org/10.1016/j.advms.2014.02.005

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each PCR run, a reaction mixture was prepared consisting of 12.5 ml SYBR1 Green JumpStartTM Taq ReadyMixTM (Sigma, St. Louis, MO, USA), 0.5 ml forward primer (final concentration 0.2 mM), 0.5 ml reverse primer, 9 ml nuclease-free water and 2.5 ml template cDNA. The thermal cycling conditions comprised an initial denaturation step at 95 8C for 2 min, 35 cycles at 94 8C for 30 s, 59 8C for 30 s and 72 8C for 30 s and a final extension step at 72 8C for 3 min. After the reaction, a melting curve was obtained to confirm reaction specificity. Experiments for all samples were performed in triplicate. The relative level of eNOS expression was calculated as described previously [23].


2.6. Measurement of platelet reactivity

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Blood was collected from 16 healthy donors (8 men and 8 women, mean age 23.6  3.1 yrs) into vacuum tubes containing buffered 0.105 M sodium citrate and was centrifuged for 6 min at 1100 rpm to obtain platelet-rich plasma (PRP). The platelets were isolated by BSA–Sepharose 2B gel filtration [24]. Monolayers of HUVECs were preincubated (24 h) with GSE (1, 2.5, 5, 10 mg/ml). The cells incubated with 10% DMSO was as a control. Platelets in suspension (1.5  108 ml–1) were incubated with HUVECs on 24well cluster dishes (10 min, 37 8C). After 10 min, the platelets were activated with 20 mM ADP, aspirated from the wells, mixed, immediately incubated with antibodies (anti-CD61PerCp, antiCD62PE, PAC-1-FITC) and fixed with CellFix (2 h at room temperature or overnight at 4 8C). The BD LSR II System (Becton Dickinson, San Diego, CA, USA) was then applied to samples of 5000 platelets to determine the expression of surface membrane P-selectin (CD62P) and the activated form of integrin aIIbb3 (monitored as the fraction of platelets that bound PAC-1). The study was performed under the guidelines of the Helsinki Declaration for Human Research and approved by the committee on the Ethics of Research in Human Experimentation at the Medical University of Lodz (No . RNN/13/07/KB).


2.7. Statistical analysis

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Mean  SE or median and interquartile range (IQR: from lower quartile, Q1 to upper quartile, Q3) are given for all parameters. The Shapiro–Wilk test was used to verify whether the data was normally distributed. The significance of differences was determined using ANOVA for repeated measures followed by the paired t test or Wilcoxon signed-rank test with Bonferroni’s correction for multiple comparisons. To analyze the effect of the seed extract on HUVEC proliferation, the single sample test was used in comparison to untreated control cells, which were considered as 100%. The nonlinear regression approach was used to calculate EC50 and EC80 values (GraphPad Prism v. 5 for Windows, GraphPad Sofware, San Diego, CA, USA, www.graphpad.com).


3. Results


3.1. The effect of polyphenolic GSE on HUVEC proliferation

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In order to evaluate the effect of GSE on HUVEC proliferation, endothelial cell cultures were exposed to GSE over a wide concentration range (0.01–200 mg/ml gallic acid equivalent) for 24 h. The results showed that concentrations lower than 5 mg/ml GSE had not a significant effect on HUVEC proliferation, but concentrations of 10 mg/ml and higher resulted in cell proliferation rates lower than those of untreated cells (p < 0.0001) (Fig. 1). The dose-dependent inhibition of proliferation was also assessed by calculating the concentrations of GSE that give 80% (EC80) and 50% (EC50) of the maximum HUVEC proliferation rate (Fig. 1): the EC80 was 14.0  0.5 mg/ml and the EC50 was 24.8  0.4 mg/ml GSE. The


Fig. 1. The effect of grape seed extract on HUVEC proliferation estimated with the MTT assay. Data is presented as percentage of control (untreated cells) for different concentrations of GSE (log of concentration values shown on x axis). The estimated EC80 was 14.0  0.5 mg of gallic acid equivalent/ml; EC50 was 24.8  0.4 mg of gallic acid equivalent/ml.

maximal polyphenol concentration used in further experiments was 10 mg/ml, expressed as gallic acid equivalent.

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3.2. GSE modulates platelet–endothelium interactions


An evaluation was made of the influence of 1, 2.5, 5 or 10 mg/ml (gallic acid equivalent) GSE on the inhibition of platelet reactivity by HUVECs. The expression of the activated form of GPIIbIIIa (estimated by PAC-1 binding) and CD62P on the platelet surface was measured in the presence of 20 mM ADP. The basal PAC-1 level in platelets incubated with HUVECs was found to be 20% lower than in platelets without HUVEC (p < 0.002). This effect was further intensified when the HUVECs had been preincubated with GSE. This measurement of surface antigen expression revealed that GSE significantly enhanced the antiplatelet action of HUVECs, and that this effect was dependent on extract concentration: despite being significant at 1 mg/ml GSE, it was not present at higher concentrations (Fig. 2).

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3.3. The influence of polyphenolic extract from grape seeds on the expression of eNOS mRNA in HUVECs

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Human endothelial cells incubated for 24 h with GSE at final concentrations of 0, 1, 2.5, 5 and 10 mg/ml showed no significant

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Fig. 2. The effect of GSE on the expression of the activated form of GPIIb/IIIa (PAC-1) in ADP-activated platelets in the presence of HUVEC. Data shown as median and interquartile range (LQ: UQ; n = 16). A significant decrease was observed in the fraction of PAC-1-positive platelets in the presence of HUVEC treated with 1 mg/ml GS extract, *p < 0.05 compared to control (platelets incubated with untreated HUVECs).

Please cite this article in press as: Luzak B, et al. Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells? Adv Med Sci (2014), http://dx.doi.org/10.1016/j.advms.2014.02.005

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Table 2 The effect of GSE and resveratrol on eNOS gene expression in HUVECs. GSE (mg/ml), n = 7 0 1 23.3  1.9 24.9  1.8

2.5 26.4  1.2

5 24.9  1.0

Resveratrol (mg/ml), n = 3 0 0.25 38.7  2.0 46.1  3.9

1 63.7  4.6

10 88.0  2.6*

10 26.3  2.5

Data shown as mean  SE. The expression of the eNOS gene is presented as 2deltadelta values compared to the reference gene (GAPDH: glyceraldehyde 3phosphate dehydrogenase). Resveratrol significantly increased the expression of eNOS gene at the concentration of 10 mg/ml. * p < 0.05.

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changes in eNOS expression (Table 2). The basal level of eNOS mRNA was 23.75  2.76 2-deltadelta in untreated cells, and did not change after incubation with polyphenols. However, the application of 10 mg/ml of resveratrol resulted in eNOS expression doubling compared to untreated cells, according to eNOS mRNA analysis.


4. Discussion

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The last few years have seen increasing interest in screening substances, especially plant natural products, that can stimulate vascular endothelial cells to produce antiplatelet modulators (prostacyclin (PGI) and nitric oxide (NO)) [10–12,25]. Most published studies suggest that vascular protection by GSE might be due to the direct action of polyphenols on endothelial cells, resulting in enhanced NO synthase (eNOS) expression [10,12]. The present study is the first one testing the indirect effects of GSE on platelet inhibition dependent on endothelial cells. Our aim was to assess whether the natural extract of grape seeds can further attenuate platelet reactivity beyond the endothelial cell-mediated inhibition of platelet function. By monitoring the expression of activated fibrinogen receptor to measure reduced platelet reactivity, it was found that GSE increases the antiplatelet action of human endothelial cells with no increase in eNOS mRNA level. We observed that the ability of GSE to increase the efficacy of platelet inhibition by endothelial cells was highly dependent on concentration: while significant at low concentration (1 mg/ml), it was not present at higher concentrations. Similar results were obtained using the extract of Aronia melanocarpa fruits, which at low concentration (5 mg/ml) potentiated the inhibition of platelet aggregation in the presence of endothelial cells [26]. Sen and Bagchi [27] report that low concentrations of GSE (1–5 mg/ml) significantly inhibited the TNFa-induced adhesion of Jurkat cells to human endothelial cells by inhibiting the expression of inducible VCAM-1 at the transcriptional or post-transcriptional level. Feng et al. [12] found that grape seeds enhance eNOS expression and NO production in hydrogen peroxide-treated HUVECs via increased AKT phosphorylation. Indeed, many investigators have reported direct evidence that GSE stimulates NO synthase (eNOS) by increased gene expression or activation and subsequent endothelial NO release [10,28], but GSE was found to have no effect on eNOS expression. These observed discrepancies may be due to differences in experimental protocol. The stimulation of vascular endothelial cells was longer in the present study (24 h) and only the effect of resveratrol, a potent stimulator, was evaluated on eNOS expression. Supplementation with GSE is sufficient to provide the in vivo concentrations of polyphenols identified in this study to increase the antiplatelet properties of endothelial cells. Previous studies show that plasma flavanol concentrations in the range 1.5–2 mg/ ml can be achieved in this way [29,30]. The results of our study indicate that flavan-3-ols are probably responsible for the increased antiplatelet properties of endothelial cells treated with

low concentrations of GSE. A study of the antioxidant properties of GSE showed that ()-epicatechin protects against the toxic action of peroxynitrite [31]. The possible mechanisms by which GSE exerts antiplatelet effects, including improved vascular endothelial function and inhibition of platelet aggregation, may depend on ecto-ADPase. Grape products such as grape seeds are known to have beneficial health effects against CVD and cancer, including inflammation, cell proliferation and apoptosis. Indeed, GSE at 25–50 mg/ml caused significant inhibition of cell growth in some cancer cell lines [32]. The results of the present study indicate, that GSE at concentrations over 10 mg/ml gallic acid equivalent significantly reduces human endothelial cell proliferation after 24 h incubation. Agarwal et al. [33] report that GSE, comprising 95% proanthocyanidins, exhibits anti-angiogenic potential by inhibiting the growth and survival of HUVECs. They show that GSE significantly reduces HUVEC growth (

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells?

Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endoth...
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